Transport of Carbon and Nitrogen Between (1)
Transport of Carbon and Nitrogen Between Litter and Soil Organic Matter in a Northern Hardwood
Forest
1 1 Timothy J. Fahey, 1 Joseph B. Yavitt, * Ruth E. Sherman,
2 3 Peter M. Groffman, 4 Melany C. Fisk, and John C. Maerz
1 Department of Natural Resources, Cornell University, Ithaca, New York 14583, USA; 2 Cary Institute of Ecosystem Studies, Millbrook, New York 12545, USA; 3 Department of Zoology, Miami University, Oxford, Ohio 45056, USA; 4 Warnell School of Forestry and Natural
Resources, University of Georgia, Athens, Georgia 30602, USA
A BSTRACT
We used sugar maple litter double-labeled with 13 C the structural and non-structural labeled litter. By and 15 N to quantify fluxes of carbon (C) and nitro-
October the percentage recovery of litter 13 C in soil gen (N) between litter and soil in a northern hard-
was much lower (16%). The C released from litter wood forest and the retention of litter C and N in
and remaining in soil organic matter (SOM) after
soil. Two cohorts of litter were compared, one in - 1 year represented over 30 g C m 2 y 1 of SOM which the label was preferentially incorporated into
accumulation. Recovery of litter 15 N in soil was non-structural tissue and the other structural tissue.
much higher than for C (over 90%) and in May 15 N Loss of 13 C from this litter generally followed dry
was mostly in organic horizons whereas by October
mass and total C loss whereas loss of 15 N (20–30% in
it was mostly in 0–10 cm mineral soil. A small pro-
1 year) was accompanied by large increases of total portion of this N was recovered as inorganic N (2– N content of this decaying litter (26–32%). Enrich-
6%). Recovery of 15 N in microbial biomass was ment of 13 C and 15 N was detected in soil down to 10–
higher in May (13–15%) than in October (about
15 cm depth. After 6 months of decay (November– 5%). The C:N ratio of the SOM and microbial bio-
mass derived from the labeled litter was much recovered in the soil, with no differences between
May) 36–43% of the 13 C released from the litter was
higher for the structural than the non-structural litter and for the forest floor than mineral SOM, illustrating the interactive role of substrates and microbial activity in regulating the C:N stoichiom- etry of forest SOM formation. These results for a forest ecosystem long exposed to chronically high
atmospheric N deposition (ca. 10 kg N ha - 1 y - 1 )
suggest possible mechanisms of N retention in soil:
Received 15 July 2010; accepted 10 December 2010;
published online 3 February 2011
increased organic N leaching from fresh litter and
Author Contributions: Fahey coordinated project, primary responsi-
reduced fungal transport of N from soil to decaying
bility for writing. Yavitt assisted in project coordination, data analysis and
litter may promote N stabilization in mineral SOM
interpretation and writing. Sherman coordinated field data collection and data analysis. Groffman—project conceptualization and coordination and
even at a relatively low C:N ratio.
writing primarily for microbial aspects. Fisk—project conceptualization, field and laboratory analysis and writing. Maerz—project conceptualiza-
Key words: carbon; forest soil; litter decay;
tion, field and laboratory analysis.
nitrogen; sugar maple; isotope tracer.
Transport of Carbon 327
I NTRODUCTION
microbial C:N support this hypothesis, the mecha- nistic underpinnings are not fully explored
A principal source of carbon (C) and nitrogen (N) (Cleveland and Liptzin 2007 ). For example, how supply to forest soil is from transformation of
does variation in the biochemistry of detrital sub- aboveground litter, including dissolution and
strates affect the C:N ratio of stabilized SOM? leaching of organic C and N, fragmentation and
The objective of the present study was to quan- particle transport (either biotic or abiotic) and
tify the co-transport of C and N between leaf litter biological mineralization of N to inorganic soluble
and an acid forest soil. Using dual labeled ( 13 C and forms. Filamentous soil fungi promote N transport
15 N) leaf litter, we traced C and N flux during the via extensive hyphal networks that connect litter
first-year of litter decay. Two types of litter were and soil (Lindahl and others 2001 ; Frey and others
compared, one in which the 13 C was incorporated 2003 ). In many acidic forest soils litter accumulates
preferentially into non-structural leaf tissue and on the soil surface because of limited invertebrate
the other structural components. We also quanti- activities capable of fragmentation, particle trans-
fied N transport from older litter and soil to fresh port and soil mixing. These processes are crucial to
leaf litter. We hypothesized that (1) the C:N ratio of forest soil formation and fertility and may be al-
organic matter derived from plant litter and accu- tered by changes in soil biota (for example, invasive
mulating in soil is lower than the C:N of the litter species such as earthworms; Alban and Berry 1994 )
substrate because during stabilization in SOM C is or environment (for example, N deposition, tem-
lost as CO 2 whereas N is conserved; (2) the 13 C perature, and so on). Although the process of leaf
from the non-structural label is more readily uti- litter decay has been characterized in great detail
lized by soil microbes than the structural label; and over the years, the transport of C and N between
(3) fungal transport of N to fresh litter is supplied litter and soil and its sensitivity to environmental
from older litter as well as mineral soil. change are not fully understood.
Atmospheric pollution associated with fossil fuel combustion and agriculture results in high levels of
M ETHODS
nitrogen deposition in downwind areas (Holland and others 1997 ) that could lead to an overabun-
Study Site
dance of N in soils and ecosystems, a condition The research was conducted at Cornell University’s known as N saturation (Aber and others 1989 ). In
Arnot Forest located in Tompkins County, central this condition availability of mineral N exceeds
New York State (42°15¢N, 76°40¢W) and situated on plant N demand, resulting in high rates of nitrifi-
the northern Allegheny Plateau. For a detailed site cation and consequent nitrate leaching and soil
description see Fain and others ( 1994 ). Briefly, the acidification. However, despite prolonged high N
study plots were located at 600–620 m elevation in deposition, many forests in the northeastern US do
forests dominated by Acer saccharum Marsh. The not exhibit high nitrate leaching (Martin and oth-
stands are mature, 2nd-growth forests originating ers 2000 ; Goodale and others 2003 ), presumably in
following clear-cut harvest in the 1870s. Basal area part because of a high capacity to retain N in soil
ranges from about 30 to 35 m 2 /ha and canopy organic matter (SOM). For example, it is well
height 23–25 m. Soils are acidic Dystrochrepts (pH known that decaying leaf litter acts as a strong sink
4.5–5.0) derived from glacial till overlying Upper for N (Bocock 1964 ) including N from atmospheric
Devonian shales. Clay content of the less-than-2- deposition (Micks and others 2004 ). Moreover,
mm fraction ranges from 24–28% in the 0–10 cm
soil and sand content from 13–17%. Soils are stony short-term immobilization of added N in microbial
isotopic tracer experiments using 15 N illustrate
averaging 22% by volume coarse fraction (>2 mm) biomass and SOM (Zogg and others 2000 ). How-
in the 0–10 cm soil. Although invasive earthworms ever, the forms and mechanisms of N retention in
are common in the Arnot Forest (Bohlen and others forest SOM are not well understood. There is par-
2004 ), the study plots were chosen in earthworm- ticular uncertainty about how N retention interacts
free sites with a well-developed organic horizon (ca. with the stabilization of soil C derived from plant
4 cm thick) overlying mineral soil. The climate is detritus. According to the reasoning of Waksman
temperate continental with mean temperature of and Starkey ( 1931 ) and elaborated in the Redfield
- 4°C in January and 22°C in July, and mean an- ratio hypothesis (Redfield 1958 ), the C:N ratio of
nual precipitation of 90 cm, evenly distributed the soil medium should approach that of the soil
through the year. The region receives moderately
microbial community as C is lost as CO -
high annual N deposition (ca. 10 kg N ha 1 y 1 ; retained. Although general patterns of soil and
2 but N is
Fahey and others 1999 ).
328 T. J. Fahey and others
Isotopic Labeling of Leaf Litter
subsamples of the initial litter following McLeod Sugar maple leaf litter was labeled with 13 C and 15 and others ( 2007 N ). Briefly, litter tissue was ex-
2 and applied to 0.5 m tracted with 10% formic acid to remove loosely quadrats in the field study bound polysaccharides; with phosphate buffer to
plots. The process for labeling leaf litter is described dissolve and remove loosely bound pectin; with in detail in Horowitz and others ( 2009 ). Briefly,
cyclohexanediamine tetra acetic acid (CDTA) to during summer 2006, seven 2.5 m tall 9 3 m
remove calcium, which releases bound pectin; with diameter aluminum chambers were positioned
urea to break hydrogen bonds and separate cellu- over dense sapling stands of sugar maple that had
lose from hemicelluloses; with sodium carbonate to been released by heavy overstory thinning in 2000.
remove remaining pectin; with sodium hydroxide The litter layer (O i ) was removed from the soil
to dissolve hemicelluloses; and finally with formic surface, and the root systems of the saplings inside
acid to remove all remaining non-structural sugars. the chambers were isolated by trenching to about
0.5 m. Saplings were labeled with 15 N by applying A subsample of the residual solid phase was re-
15 0.25 g - Nm 2 15 as 99 atom % enriched tained after each step for isotope analysis, as de- NH
scribed below. Also, lignin concentration of each chamber soon after leaf expansion on 22 May
4 Cl to
2006. To ensure even distribution the 15 N was ap-
structural and non-structural litter was analyzed by
the standard detergent extraction method (Van plied in solution (8 l per chamber) immediately
Soest 1963 ) by Dairy One, Inc. (Ithaca, NY). after a large rain event.
The saplings were labeled with 13 C after enclosing
Field Plot Establishment and Sampling
the chambers with polyethylene sheeting. Labeled
2 (40 atom % enriched) was added to each In each of three sugar maple-dominated stands 16 chamber on 13 sunny days between 1 September
13 CO
0.5 m 2 quadrats were established in fall 2007. Half and 20 September 2006. To ensure maximum uti-
of the quadrats were assigned randomly to the lization of the label, the procedure involved scrub-
structural-litter treatment and half to the non- bing ambient CO 2 (to ca. 50–60 ppm) after sealing
structural treatment. Fresh litter from 2007 was
removed from each quadrat and a coarse-mesh centration reached about 500 ppm, and re-opening 2 nylon screen (hole size = 6 cm ) was positioned on
the chambers, injecting 13 CO 2 until chamber con-
the underlying forest floor and anchored at the duced to nearly constant levels (typically after
the chambers when CO 2 concentrations were re-
corners. About 200 g (weighed to ±0.01 g) of iso- 0.5–1 h). The procedure was successful in enriching
tope-labeled litter was added to each quadrat to foliage to d 13 C over 300 per mil (Horowitz and
roughly match leaf litterfall in the study area. A others 2009 ).
second coarse-mesh screen was positioned on the All fresh leaf litter was collected from each
added litter and anchored to confine the litter and chamber in September and October 2006, returned
prevent subsequent addition of litterfall. to the laboratory, and air-dried to constant mois-
Fungal ingrowth bags were installed at approxi- ture content. Litter was thoroughly mixed and
mately 5 cm depth in the mineral soil in January subsamples taken for chemical analysis. In addi-
2008, to quantify incorporation of label into fungal tion, litter was again collected from the chambers
hyphae. Bags were placed adjacent to 2 quadrats in September–October 2007. We expected the
and inside 2 quadrats per litter type in each plot. biochemical nature of the isotopic label to differ
Ingrowth bags were constructed of nylon mesh markedly between the two cohorts of litter: the 13 C (50 lm mesh size, ca. 5 cm diameter). Each bag
should be primarily in non-structural components was filled with 27 g of acid-washed, autoclaved (polysaccharides) of the 2006 litter because the
sand, and sealed with a solder iron. Ingrowth bags label was added at the end of the growing season,
were collected in September 2008, and refrigerated
whereas in the 2007 leaves the 13 C primarily would
prior to hyphal extraction.
be in structural components. The 2007 litter was The field quadrats were destructively sampled on processed in the same way as for 2006; however,
21 May and 10 October 2008. Two quadrats from the drying and storage period were brief as litter
each litter type treatment and each stand were was added to the field plots about 1 month after the
chosen randomly for harvest on each date. First, end of the litterfall period in the chambers.
the corner anchors on the screens were removed To characterize the differences in the biochemi-
and all the litter remaining between the two cal distribution of the isotope labels between the
screens was collected. Next, the underlying forest structural and non-structural litter, a sequential
floor horizons (O e +O a ) were collected by exca- extraction procedure was conducted on several
vating with hand spades to the top of the mineral
Transport of Carbon 329 soil. Finally, mineral soil was cored to 20 cm depth
analyzer. Microbial biomass C and N content were using 5 cm diameter, sharpened split-PVC corers.
measured using the chloroform fumigation-incu- Soil samples from several cores (generally 6–8)
bation method (Jenkinson and Powlson 1976 ). The were composited for each quadrat by 5 cm depth
flush of carbon dioxide was measured by thermal increment. All samples were returned to the labo-
conductivity gas chromatography, and a propor- ratory for processing the same day as collected.
tionality constant (0.45) was used to calculate To quantify N transport from 1-year-old litter to
biomass C from the CO 2 flush. Inorganic N flush fresh litter the remaining (unharvested) quadrats of
data were not corrected with a proportionality each litter treatment type (structural and non-
constant. KCl-extracted samples were prepared for structural label) in each stand were used. In fall
15 N analysis by diffusing inorganic N onto acidified 2008, fresh sugar maple litter was collected in the
disks (Stark and Hart 1996 ) which were subse-
quently analyzed at the University of California was added to each quadrat and secured in place
stands and about 200 g m - 2 of this unlabeled litter
Davis Stable Isotope Lab on a Europa Integra iso- with a third coarse-mesh screen, as above. In mid-
tope ratio mass spectrometer with an integral July and mid-November 2009, subsamples of this
combustion unit. CO 2 flush samples were analyzed leaf litter were collected from each plot taking great
for 13 C at the same facility.
care to avoid any of the labeled older litter. These Fungal hyphae were extracted from ingrowth litter samples were processed for isotope analysis, as
bags by suspending sand from each bag in 100 ml described below.
deionized water and shaking for 5 min on a rotary shaker at 120 rpm. Hyphae floated out of the sand
and were collected by filtration (25 mm Isopore Lab Processing of Samples TM
membranes, 0.22 lm pore size). Successive ali- Litter and forest floor samples were weighed moist quots of water were added until no more hyphae and a subsample was taken for moisture determi- could be retrieved. Hyphae were rinsed from filter nation by re-weighing after oven-drying to con- membranes, frozen and lyophilized. Organic C and stant mass at 70°C. The subsample was stored for
13 N content, 15 C, and N of fungal hyphae were chemical analysis as described below. A second
quantified by mass spectroscopy at the University subsample of forest floor was taken for microbial
of California, Davis.
biomass and related measurements and stored at about 2°C. For mineral soil cores, coarse fragments (>1 cm) were removed and the rest of the bulk
Reference Soils and Isotopes
sample was weighed moist. Subsamples were taken Calculation of isotope pools and fluxes requires for moisture determinations, isotope analysis, and
accurate and precise estimates of reference (pre- microbial biomass (and related) measurements.
treatment) soil mass and bulk density, element The subsamples were either stored at 2°C (for
contents, and isotope natural abundance (Nadelh- microbial biomass) or dried to constant mass at
offer and Fry 1994 ). Soil mass, bulk density, and 70°C (for moisture determination) and sieved to
coarse fragment content were determined in each remove the less-than-2 mm fraction.
stand by the soil pit excavation method (Rowell Samples of litter, forest floor, and soil were finely
1994 ). Four soil pits (0.2 9 0.2 m) were excavated ground and homogenized for isotope analysis. The
to 20 cm depth in 5 cm depth increments at ran-
dom locations in each stand. The fine (<2 mm) these samples was measured on a Finnigan isotope
elemental and isotopic ( 13 C, 15 N) composition of
fractions were stored and processed for elemental ratio mass spectrometer at the Cornell Stable Iso-
and isotope analysis, as described earlier. tope Laboratory with appropriate standards for
Pools of 13 C and 15 N in litter on each plot were normalization correction, instrument linearity, and
calculated at time zero and at the time of plot col- precision purposes. Samples were run in batches
lection as the product of dry weight, carbon con- with expected similarity of isotope enrichment to
centration, and isotopic atom % ( 13 C and 15 N). The avoid sample carryover errors.
release of the isotope from each plot during decay For inorganic N and microbial biomass mea-
was estimated as the difference between initial and surements some pooling of samples was conducted:
final isotope pools in litter; these values were used mineral soil samples were pooled into 0–10 and 10–
to estimate percentage of isotope recovered in
20 cm depth increments and samples were pooled
underlying soil.
Isotopic enrichment of forest floor, mineral soil, NO - 3 ) was extracted from soil with 2 M KCl fol-
by litter type within plots. Inorganic N (NH + 4 and
inorganic N pools, and microbial biomass was esti- lowed by colorimetric analysis on a flow injection
mated in reference to the mean natural abundance
330 T. J. Fahey and others
in samples from the four soil pits in each stand. weight loss from the fresh litter followed the same (Note: The use of separate reference samples for the
time course as observed in 2008–2009 (Figure 1 ). three stands proved to be necessary for detecting
The increase in atom % 15 N and total N concen- small quantities of the litter-derived isotopes in the
tration were used to estimate the transport from mineral soil because of significant—though minis-
labeled litter, assuming that 15 N from unlabeled cule—differences in isotope natural abundance
sources (older litter, soil, atmospheric deposition) among the three stands.) We calculated the initial
carried the same reference atom % 15 N as unla-
beled litter; this assumption would likely result in stand from the mean isotope natural abundance,
total pool of 13 C and 15 N in each soil layer in each
minor error as local atmospheric deposition (del mean element (C or N) concentration and dry
15 N = 0.86&, Goodale and others 2009 ) and older weight (based on bulk density) of the fine soil
litter are only very slightly higher in 15 N than fresh fraction (<2 mm) for each depth. We calculated
litter (del 15 N = -1.79&).
the final isotope pool for each plot at the time of collection from isotope enrichment and element concentration and assuming bulk density and fine
R ESULTS
fraction content was equivalent to the stand-level
Reference Soils
values. Similarly, isotopic enrichment of microbial Soils in the study area were very stony with coarse biomass and fungal hyphae was calculated in ref-
fraction volume of about 30% in the upper 20 cm. erence to control samples collected within each
An organic horizon comprising about 1 kg dry plot. The differences between initial and final iso-
weight/m 2 covered the mineral soil surface topic pool estimates for each depth, component and
(Table 1 ). Bulk density of the less-than-2 mm quadrat were used to calculate % recovery of excess
fraction increased with depth in mineral soil from isotopes released from litter.
0.51 to 0.77 g cm - 3 . SOM content (and % C and % % recovery
N) decreased sharply from the 0–5 to 5–10 cm layer. The C:N ratio declined with depth from 17 in
¼ ð initial litter isotope pool - final litter isotope pool Þ
forest floor to 13.5 at 0–20 cm depth. The natural
where Soil isotope pool ¼ soil isotope atom%
½ This approach assumes no changes over time in
the natural abundance of 13 C and 15 N in the soil
pools considered. Because this percent recovery parameter accounts for between-plot variation in litter decay (that is,
13 C, 15 N changes) and litter treatment differences in isotope signatures, we used % recovery for statistical
comparisons between plots, litter types, and depths. These data were not normally distributed, nor could they be normalized by transformation; hence, non- parametric Mann–Whitney and Kruskal Wallis tests were used to test for differences in percent recovery
between litter types. For microbial biomass 13 C and
15 N where n = 3 (due to sample pooling) a relaxed threshold of P = 0.10 was used to evaluate signifi-
cant treatment effects. Finally, differences in dry weight, 13 C and 15 N loss from decaying litter were analyzed using Student’s t statistic. Transport of 15 N from the isotopically labeled 1- Figure 1. Decomposition of sugar maple litter, (upper) dry weight and 13 C loss and (lower) total N content and year-old litter to overlying fresh litter in 2009–2010
15 N loss, for structural (S) and non-structural (NS) la- was calculated on the assumption that overall dry
beled litter. Error bars indicate standard errors.
Transport of Carbon 331 Table 1. Selected Physical and Chemical Properties of Soil (<2 mm fraction) from the Study Area in Arnot
Forest, Central New York Soil layer
Bulk density
Mass/area
(g/cm 3 )
(kg/m 2 )
d 13 C d 15 N Forest floor
17.0 - 27.330 (0.174) 0.385 (0.133) 0–5 cm
15.9 - 25.941 (0.128) 7.988 (0.567) 5–10 cm
14.1 - 25.583 (0.107) 12.206 (0.815) 10–15 cm
14.4 - 25.391 (0.091) 12.587 (0.558) 15–20 cm
Standard errors in parentheses (n = 12).
in the structural litter and low in this fraction in the depth in soil and background variation was rela-
abundance of 13 C and 15 N increased steadily with
non-structural litter. Conversely, the hemicellulose tively low (Table 1 ).
fraction was much more highly labeled in the non- structural litter. Surprisingly, the lignocellulose
Litter Chemistry
fraction was highly labeled in the non-structural litter even though the labeling was conducted only
The isotopically labeled sugar maple leaf litter was
strongly enriched in both 13 C and 15 N (Table 2 ).
a few weeks before leaf abscission. The differences
Enrichment of 13 in 15 C was much greater in the structural N label distribution between litter types were relatively minor, and the label was relatively uni- (d 13 C = 417.8& or ‘‘per mil’’) than the non-struc-
tural litter (d 13 C = 142.1&) whereas the reverse was formly distributed among the extractive fractions. The most notable 15 N enrichment was in the urea- true for 15 N (d 15 N = 188.0 vs. 337.1&), so that the
C: 15 N ratio was higher in structural litter (62.5) than most notable 15 N depletion was in the lignocellu- the non-structural litter (33.6), compared with total lose residue, which also had low total N concen- C:N of 40–42.
soluble fraction (hemicellulose complexes) and the
13 The distribution of tration (0.35%N). C isotopes among the extractive biochemical fractions differed markedly
Weight Loss and Nitrogen Content
between the ‘‘structural’’ and ‘‘non-structural’’
Changes in Decaying Litter
litter for several fractions. The isotope distributions
are expressed as a percentage difference from the Litter decay followed a typical weak exponential
pattern through the first-year of decay (Figure 1 ). was particularly high in the lignocellulose fraction
bulk label in Table 2 . Most notably, the 13 C label
Over the first 6 months weight loss was signifi- Table 2. Chemistry of Isotope-labelled Sugar Maple Leaf Litter and Percent Enrichment (+) or Depletion (-)
of 13 C in Six Biochemical Extractive Fractions of Structural and Non-structural Litter Relative to the Bulk Litter
Fraction
% Structural
Non-structural % Carbon
1.2608 (0.0041) Atom % 15 N
Atom % 13 C 1.6330 (0.0209)
1.5823 (0.0409) Lignin
24.6 23.2 Free sugars
+5.9 (1.4) Weakly-bound polysac/pectin
- 9.9 (3.1) Strongly-bound polysac/pectin
+20.8 (4.2) Inaccessible sugars
Standard errors in parentheses.
332 T. J. Fahey and others
cantly higher for the non-structural than the quadrats (Table 4 ). Similarly, microbial biomass structural-labeled litter, but this difference was not
was significantly enriched in 13 C in forest floor and
0–10 cm mineral soil (Table 5 ) but not in 10–20 cm tracked dry weight loss. In contrast, N concentration
significant after 1 year. Loss of C and 13 C generally
soil. For purposes of comparison between litter in leaf litter increased dramatically as decomposi-
types and depths, we analyzed the recovery of 13 C tion proceeded, nearly doubling over the 1-year
as a percentage of the measured 13 C released from period. As a result, the total N content of the litter
the litter on each individual plot; this calculation increased to 26–32% of the initial over the first-year
utilizes stand-level measurements of reference 13 C of decay (Figure 1 ). No significant differences were
and bulk density (<2 mm fraction mass per depth) observed between the structural and non-structural
together with experimental quadrat-level mea- litter. Despite this apparent addition of N, significant
surements of litter mass and 13 C and soil % C. In release of 15 N occurred during the first-year of de-
May, we recovered about 43% of the 13 C released
from the structural litter in soil, with the highest (P < 0.01) higher for the non-structural litter than
cay (Figure 1 ). The loss of 15 N was significantly
proportion of recovery in 0–5 cm in mineral soil the structural litter, and about 20–30% of the initial
(Figure 2 ). The corresponding value for non-
structural litter appeared to be lower (36%), but observed both during winter/spring (especially for
15 N was lost over the first year. Release of 15 N was
this difference was not significant (P = 0.25). A less non-structural litter) and during the growing sea-
distinct depth pattern was observed for the non-
structural litter with no measurable recovery below Fresh litter that was added to the unharvested
son (both litter types; Figure 1 ).
10 cm depth.
quadrats in Fall 2008 increased significantly in both Percent recovery of 13 C in microbial biomass total N and d 15 N by July 2009 and further in-
ranged from 1.8% to 2.8% with roughly equal
amounts recovered in forest floor and 0–10 cm differences were observed between the structural
creased by November 2009 (Table 3 ). No significant
mineral soil (Table 5 ). Fungal hyphae collected on 1 and non-structural plots which were pooled for
September 2008 from bags in the upper mineral soil subsequent calculations. Because the only source 13 were enriched in
C and enrichment was signifi- of 15 N enrichment of this unlabeled litter was the
cantly higher in structural (9 ng 13 C/g soil ± 3.3) labeled litter from the previous year, we can esti-
than non-structural (3 ng 13 C/g soil ± 0.6) quad- mate the proportion of N transport to first-year
rats. The 13 C in the respirable carbon pool in soil litter derived from second-year litter. This value
(10-day incubation) averaged about twice as averaged 11.6% by July and 17.7% (cumulative)
large in the structural versus non-structural litter by November. Presumably, the rest of the N accu-
quadrats (Table 6 ), but this difference was not
mulating in first-year litter (Figure 1 ) is derived
statistically significant.
from atmospheric deposition, older litter, and SOM. By October 2008 both the 13 C enrichment in soil (Table 4 ) and the % recovery of 13 C released from
litter (Figure 2 Isotope Recovery in Soil and Microbial ) were significantly lower than in Biomass May, and little enrichment was detected below
10 cm depth for either structural or non-structural litter. Percent recovery of In May 2008, 6 months after addition of labeled 13
C was similar for both
13 litter types. Microbial biomass litter, we were able to detect 13 C enrichment of soil
C was similar in down to the 10–15 cm depth layer; no significant
October as in May but again % 13 C recovery in
13 C enrichment was observed at 15–20 cm depth microbial biomass was lower in October (Table 5 ). for either the structural or non-structural litter
The 13 C in the respirable 13 C pool also appeared to
Table 3. Estimated Translocation of Nitrogen from Decaying 1-Year-Old Sugar Maple Litter into Overlying Fresh Litter at Arnot Forest Plots, Based on Changes in N Concentration and d 15 N of Fresh Litter and Assuming Decay Rates Observed in Figure 1
Date
% N from old litter November (initial)
Standard errors in parentheses.
Transport of Carbon 333 Table 4. Mass Excess of 13 C and 15 N (Enrichment above Background) in Soils Collected from Quadrats
Amended with 13 C and 15 N Labeled Leaf Litter in Arnot Forest, NY
Depth Excess 13 C mg/m 2 Excess 15 N mg/m 2
Oct 2008 A. Structural
0 0 B. Non-structural Oa
A. Structural label; B. non-structural label (standard errors in parentheses).
Table 5. Carbon and Nitrogen Content of Microbial Biomass, Excess 13 C and 15 N in Microbial Biomass in Forest Floor and 0-10 cm Mineral Soil, and % Recovery of Isotopes Released from Litter
Soil Litter Microbial biomass
% Recovery in depth (cm)
Isotopic enrichment of
type (mg per g soil)
microbial biomass
microbial biomass
(lg per g soil)
Microbial 13 C 15 N
13 C 15 N
C:N
A. May Forest floor S
0.002 (0.001) 1.23 (0.91) 4.1 (1.4) B. October Forest floor S
Sugar maple litter labeled with 13 C and 15 N in structural (S) and non-structural (NS) tissue was added to quadrats in November 2007 with soil collections in A. May 2008 and B. October 2008. Standard errors in parentheses.
decline by October, especially for the non-struc- between May and October as enrichment was ini-
tural litter quadrats (Table 6 ).
tially higher for the forest floor horizon and later
Not surprisingly, recovery of 15 N was much
for 0–5 cm mineral soil (Figure 3 ). Most of the
higher than 15 C. On both collection dates over 90% excess N recovered was in organic form. In May, of the 15 N released from litter was recovered in the
the proportion of inorganic 15 N recovered ranged upper 10 cm of soil. Significantly higher soil pool
from about 2% to 6% of total N with significantly enrichment was observed in the non-structural
higher values in mineral soil than in forest floor than the structural litter plots; however, no differ-
horizons.
ences in % recovery were observed between Microbial biomass also was enriched in 15 N in the
forest floor and upper mineral soil (Table 5 ). enrichment was detected below 10 cm depth (Ta-
structural and non-structural litter. No 15 N
Average enrichment was higher (but not signifi-
ble 4 ). The depth pattern of 15 N recovery changed
cantly) in the non-structural than the structural
334 T. J. Fahey and others
Figure 3. Recovery of 15 N from decaying sugar maple
litter in three soil depth layers, expressed as a percentage litter in three soil depth layers, expressed as a percentage
Figure 2. Recovery of 13 C from decaying sugar maple
of 15 N lost from structural and non-structural labeled of 13 C lost from structural and non-structural labeled
litter in May 2008 and October 2008. Error bars indicate litter in May 2008 and October 2008. Error bars indicate
standard errors.
standard errors. (Table 5 ). Fungal hyphae in the upper mineral soil
litter plots in May but values were more similar by also were significantly enriched in 15 N (2 ng 15 15 N/g October. Percent recovery of N in microbial bio-
soil) with no difference between structural and mass (10–20 % in May) was much higher than for
non-structural quadrats.
C and generally higher in May than October The ratio of 13 C: 15 N in the SOM formed from the (about 5%), especially for the forest floor horizons
decaying litter was generally much lower than for Table 6. Respirable Carbon in Soil (10 Day Aerobic Incubation), Excess Respirable 13 C (Relative to Refer-
ence 13 C) and the Ratio of Microbial C to Soil C for Total C and Excess 13 C in Structural (S) and Non-structural (NS) Litter Plots at Arnot Forest
Depth Litter type
Respirable
Excess respirable
Microbial C: Microbial 13 C:
Soil 13 C A. May
13 C (lg/g day)
13 C (ng/g day)
Soil C
Forest floor S
4.4% B. October Forest floor
A. May 2008 and B. October 2008.
Transport of Carbon 335 compared isotope movement between litter cohorts
in which either structural or non-structural tissue components were preferentially labeled. Our overall goal was to better understand this key process of soil formation and nutrient recycling and to provide insights into the role and mechanisms of anthropogenic N deposition in altering this pro- cessing.
Structural versus Non-Structural 13 C Labeling We observed large differences in the preferential
13 C enrichment of litter tissue fractions between the structural and non-structural litter, especially
for lignocellulose, hemicellulose-pectin complexes, and free sugars (Table 2 ). The labeling of the non- structural litter was conducted immediately before leaf abscission and senescence and considerable
retranslocation of 13 C was observed within 1 week of the end of the labeling period (Horowitz and others 2009 ). The structural litter acquired its label
almost entirely from the 13 C stored in perennial Figure 4. C:N ratio of soil organic matter and microbial
tissues the previous fall and remobilized during leaf biomass derived from decaying sugar maple litter in or-
expansion and shoot elongation (Horowitz and ganic and surface mineral soil in May 2008 and October
others 2009 ); hence, growing cell walls were 2008. Error bars indicate standard errors.
strongly labeled. The non-structural litter was especially strongly labeled in the hemicellulose
the litter itself (2–12 vs. 34–63), and significant fraction (urea extractable; McLeod and others (P < 0.01) differences between structural and
2007 ), 21% stronger labeling than for the bulk non-structural litter plots reflected the higher
C: 15 N in the former litter type (Figure 4 ). Patterns
litter tissue (Table 2 ). This hemi-cellulose fraction
is associated with pectins and not with lignin of differences among litter types and soil depths in
(Popper and Fry 2005 ). Therefore, in late summer the C: 15 N ratio of new SOM consistently reflected the labeled sugars apparently coalesced quickly into
13 C: 15 hemi-cellulose (Hoch 2007 ) and pectins that bind N ratio of microbial biomass was consistently
those of the soil microbial biomass (Figure 4 ). The
to hemicelluloses. Presumably this fraction is more much lower in the non-structural than the struc-
labile than the lignocellulose in the cell walls tural litter and this difference was statistically sig-
(Cosgrove 2005 ) that dominated the labeling in the nificant (P < 0.10) in the forest floor on both
13 15 collection dates (Figure structural litter (20% stronger label than bulk litter, 4 ). Similarly, the C: N Table 2 ). Not surprisingly, the free sugar fraction
ratio in new fungal hyphae was significantly lower was also much more strongly labeled in the non- in the non-structural (1.4 ± 0.3) than the struc-
structural than the structural litter. tural litter quadrats (4.4 ± 0.5).
Litter and Dissolved Organic Carbon
D ISCUSSION
It has long been known especially from studies of Leaf litter is a principal source of organic matter in
stream ecosystems that a considerable proportion of forest soils. In many cold and acidic forest soils
initial leaf litter weight loss is associated with where physical mixing of litter into soil is minimal
leaching by water (Meyers and Tate 1983 ). The because of limited soil macroinvertebrate commu-
same is true for terrestrial litter; for example, King nities, a thick organic horizon develops on the soil
and others ( 2001 ) noted that over 111 days of surface, and transport of organic matter to mineral
laboratory incubation, 78% of weight loss from soil occurs primarily by leaching (Park and Matzner
Populus litter was by DOC versus only 22% from 2003 ). We double labeled sugar maple litter with
microbial respiration. Not only soluble and labile
13 C and 15 N and traced these isotopes to soil. We sugars but also more recalcitrant tannins are lea-
336 T. J. Fahey and others
ched from decomposing leaf litter (Tiarks and oth- the first-year of decay remained in SOM, and this ers 1992 ). Hence, much of the weight loss observed
process could contribute significantly to the accu- from November to May in the present study (Fig-
mulation of C in forest soils. For example, given leaf - 2 - ure 1 1 ) probably resulted from leaching of soluble litterfall C flux at Arnot Forest (200 g C m y ;
organic matter. We observed differences in over- Bohlen and others 2004 ), this process could add winter weight loss between ‘‘structural’’ and ‘‘non-
about 32 g C m - 2 y - 1 to the SOM pool, including
15–20 g C m - 2 y - 1 in mineral soil. The process of that the non-structural litter was stored for a year
structural’’ litter (Figure 1 ), likely due to the fact
stabilization of soil C is reflected by the fact that before use. However, after 1 year, weight loss and
the ratio of microbial biomass 13 C:soil 13 C was sev-
13 C release from litter were not significantly dif- eral-fold higher than microbial biomass C:soil C ferent between the litter types, and the rate of
(Table 5 ). Additional processing of 13 C in SOM by weight loss was comparable to previous observa-
microbes would be expected to cause a reduction in tions for sugar maple in northern hardwood forests
the former ratio, but that was not clearly evident (Gosz and others 1973 ).
over the first-year of litter decay.
The proportion of 13 C released from litter be-
tween November and May that was recovered in
Interactions of Carbon and Nitrogen
soil was surprisingly high (36–43%, Figure 2 ),
indicating that much of the organic matter was not Carbon and nitrogen are intimately related in both rapidly utilized by soil microorganisms. Using a
the leaching of organic matter from plant litter and similar approach, Rubino and others ( 2010 ) ob-
the decomposition and mineralization of litter served that about two-thirds of C released from
(McGill and Cole 1981 ). In general, the C:N ratio of litter was recovered in soil, the rest being released
forest SOM decreases with depth (Table 1 ), but the as CO 2 . Qualls and Haines ( 1992 ) demonstrated
evolution of soil C:N is not completely understood that only a small proportion of DOC in forest soils
and undoubtedly involves both differential supply was likely utilized by microbes during the leaching
and removal processes. For example, Schoenau and process with most DOC removal by adsorption to
Bettany ( 1983 ) attributed declining C:N with depth the soil solid phase. Hence, we suspect that much of
to preferential leaching of N-rich DOM, whereas the overall 13 C loss occurred after DOC was
Qualls and Haines ( 1992 ) noted the role of hydro- deposited in the underlying soil.
lysis of DON linked to mineralization of DOC, ra-
ther than selective adsorption of DON, in evolution higher for structural than non-structural litter
Recovery of litter 13 C in soil in May was slightly
of forest soil C:N. Microbial processes clearly play a (Figure 2 ), perhaps reflecting the higher lability of
key role in soil organic N retention; for example, the label in the non-structural litter. Don and
Perakis and Hedin ( 2001 ) observed rapid transfor- Kalbitz ( 2005 ) argued that the most labile organic
mation of inorganic N into SOM in unpolluted, old- matter (for example, soluble sugars) is released first
growth forest. Our dual labeling of litter with 13 C followed by an increasing proportion of more re-
and 15 N provided further insights into the interac- calcitrant DOM as microbial activity generates
tions of these elements in SOM formation. additional soluble compounds in the decaying lit-
It has long been known that decaying litter of ter. Hence, a sequence in which an increasing
high initial C:N can be a strong sink for N (Bocock proportion of the DOC released from litter is sta-
1964 ) apparently transported from soil to litter by bilized in SOM could be hypothesized. However,
fungal hyphae (Wesse´n and Berg 1986 ; Hart and
Firestone 1991 ). In our study, this process is indi- May to November even though some additional
we observed decreasing recovery of 13 C in soil from
cated by the large increase in N content of decaying leaching of DOC from litter undoubtedly occurred.
sugar maple litter over the first-year of decay Apparently, the DOC that was added to soil from
(Figure 1 ), matching previous observations of Gosz litter between November and May was subse-
and others ( 1973 ). However, these measurements quently utilized by microbes at a much higher rate
indicate only the net flux of N into litter; others during the summer (probably in part because of
have noted that considerable loss of native litter N higher soil temperature) than any additional litter
can accompany the process of incorporation of organic matter that was added to soil by summer
exogenous N into litter (Blair and others 1992 ; leaching. Hence, the seasonal dynamics of C stor-
Zeller and others 2000 ). Our observation of 15 N loss age in temperate forest soils are influenced by
from decaying litter illustrates that the gross N flux seasonality in the supply from litter, its lability, and
into first-year litter is nearly twice as great as the microbial activity. A moderately large proportion
net flux. Hence, gross N transport to decaying leaf (<16%, Figure 2 ) of the C lost from litter during
litter is among the largest N flux pathways in these
Transport of Carbon 337 forests. Several potential sources of this exogenous
depolymerization of pectins and hemicelluloses may N transport to decaying litter have been identified,
produce high amounts of simple carbohydrates. If N including atmospheric deposition (Micks and oth-
availability is limiting to microbial growth as these ers 2004 ), asymbiotic N fixation in litter (Russell
substrates are being utilized, they may be respired to and Vitousek 1997 ), and fungal transport from
CO 2 with little growth and conservation of biomass, older litter and soil (Fahey and others 1985 ; Hart
in so-called overflow metabolism (Schimel and and Firestone 1991 ). We were able to estimate that
Weintraub 2003 ). An alternative but less likely the previous cohort of litter apparently supplies
explanation is that breakdown of products of non- about 18% of this N, with the remainder from
structural C are preferentially allocated to respira- these other potential sources.
tory pathways whereas those of structural C are
allocated to biosynthesis. In any case, the similarity decaying litter in the form DON because the
Presumably, most of the 15 N was leached from
in soil and microbial 13 C: 15 N supports the idea that immobilization potential for inorganic N in litter
stabilized SOM reflects (is derived from) microbial apparently is very high (Micks and others 2004 ).
organic matter (Six and others 2004 ; Jastrow and This N was strongly retained in the forest floor and
others 2007 ; Simpson and others 2007 ) and upper 5 cm of soil over the winter/spring. Our
emphasizes the importance of microbial processing double labeling of the litter allowed us to estimate
to soil N storage.
the C:N ratio of the litter-derived organic matter The increasing C:N values of litter-derived, re-
tained SOM with increasing soil depth for both much lower for non-structural than structural litter
that was retained in soil (Figure 4 ). This ratio was
litter types could represent either/both processes of plots and in spring it was much lower in mineral
dissolved organic matter adsorption or its sub- soil than forest floor. The lower value in the non-
sequent mineralization. For example, lower C:N structural litter plots could result in part from the
compounds could be preferentially adsorbed in lower 13 C/ 15 N ratio in the litter itself (33.6 vs.
surface horizons leaving DOM of higher C:N to be
transported more deeply; however, previous of SOM derived from the non-structural versus
62.5); however, the difference in the 13 C: 15 N ratio
observations suggest the reverse should be true, at structural litter was much greater (four to eight-
least for mineral soil horizons (Qualls and Haines fold) than the less than two-fold difference in the
1992 ; Kaizer and others 1996 ), because of a higher litter itself (Figure 4 ). Hence, the more labile C
affinity of hydrophobic DOC with lower N con- from the non-structural litter was probably utilized
centration for adsorption to mineral surfaces. to a greater extent and perhaps accompanied by
Notably, the behavior of surface organic horizons is more effective microbial N immobilization.
poorly understood because they are both a sink and The idea that non-structural material is more
source of DOC (Kalbitz and others 2000 ). labile than structural material is further supported
The fate of the C and N adsorbed to forest soil has
by the observation that soil 13 C: 15 N and microbial
received limited study. We observed large declines
13 C: 15 N showed similar patterns, with much lower in C:N of the retained SOM in the mineral soil from ratios in the non-structural than structural litter
spring to fall 2008 (Figure 4 ), suggesting that plots (Figure 4 ). A similar pattern also was ob-
mineralization of C was accompanied by stabiliza- served in fungal hyphae collected in soil bags
tion of N-rich compounds, at least within the
timeframe of our study. Apparently, the mineral not be mistaken as reflecting the C:N of the new
(C:N = 1.4 vs. 4.4). These low 13 C: 15 N ratios should
soil OM derived from litter DOM was not a major microbial tissue but rather the C:N of what was
source of N for plant root uptake as 15 N in this pool assimilated from the labeled litter; most of the C
actually increased during the growing season (Ta- and N in the new microbial tissues would be de-
ble 4 ). In contrast, the C:N of retained organic rived from unlabeled sources. The patterns in Fig-
matter in the forest floor increased slightly over the ure 4 support a strict Redfield ratio interpretation
growing season (Figure 4 ), and the 15 N recovery of the C:N stoichiometry of SOM: the DOM derived
declined substantially, presumably reflecting min- from litter and adsorbed to soil presumably had a
eralization and transport from this pool. It is also much higher C:N ratio than what was eventually
notable that the proportion (relative to total N) of assimilated by the microbial community, and the
inorganic 15 N in the forest floor was much higher SOM that accumulated in soil after 1 year carried
in fall (about 6%) than spring (2%) whereas the the C:N signature of that assimilated organic mat-
reverse was true in surface mineral soil (1% vs. ter.
5%). Clearly, surface organic horizons behave very One possible explanation for the relatively high
differently from surface mineral horizons regarding lability of the non-structural material is that the
the processing of litter-derived C and N.
338 T. J. Fahey and others
The C:N of retained SOM derived from 1 year of from atmospheric deposition. Moreover, presumed the structural label litter decay (7 to 13) was
higher C:N of fresh litter under pristine conditions somewhat lower than the overall average SOM (14
would favor higher microbial demand for N and to 17) but it exhibited the same pattern of decrease
higher flux into first-year litter (Frey and others
2000 ). Hence, it seems likely that decreased de- This litter-derived organic matter is probably a
from forest floor to mineral soil (Table 1 ; Figure 4 ).
mand for fungal translocation to first-year litter significant source of stabilized forest SOM and
could contribute to continuing N retention in soil eventually provides a small supply of N for sub-
under high N deposition. Forest floor and mineral sequent biological cycling in the ecosystem. Other
soil horizons exhibit highly contrasting behavior sources of forest SOM include particulate material
and presumably mechanisms of DOC and DON from the aboveground detrital–microbial complex,
dynamics (Figure 4 ). Mineral soil appears to pro- and root-derived organic matter including rhizo-