Natural Resource Ecology and Management
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Natural Resource Ecology and Management
8-2014
Unexpected Effects of Chitin, Cellulose, and Lignin
Addition on Soil Dynamics in a Wet Tropical
Forest
Ann E. Russell
Iowa State University, [email protected]
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This is a manuscript of an article from Ecosystems 17 (2014): 918, doi:10.1007/s10021-014-9769-1. Posted with permission.
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ArticleTitle
Unexpected Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics in a Wet Tropical Forest
Article Sub-Title
Article CopyRight
Springer Science+Business Media New York
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Journal Name
Ecosystems
Corresponding Author
Family Name
Russell
Particle
Given Name
Ann E.
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Schedule
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Department of Natural Resource Ecology and Management
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Iowa State University
Address
Ames, Iowa, 50011, USA
Email
[email protected]
Received
9 October 2013
Revised
Accepted
20 March 2014
Abstract
Decades of studies on the role of decomposition in carbon (C) and nitrogen (N) cycling have focused on
organic matter (OM) of plant origin. Despite potentially large inputs of belowground OM from fungal cell
walls and invertebrate exoskeletons, studies of the decomposability of their major constituent, chitin, are
scarce. To explore effects on soil C dynamics of chitin, in comparison with two plant-derived chemicals,
cellulose and lignin, I conducted a field-based chemical-addition experiment. The design contained three
chemical treatments plus a control, with four replicates in each of two species of tropical trees grown in
plantations. The chemicals were added in reagent-grade form at a rate that doubled the natural detrital C inputs
of 1000 g C m−2 y−1. Despite its purported recalcitrance, chitin was metabolized quickly, with soil respiration
(R soil) increasing by 64% above the control within days, coupled with a 32% increase in soil extractable
ammonium. Cellulose, which was expected to be labile, was not readily decomposed, whereas lignin was
rapidly metabolized at least partially in one of the forest types. I examined effects of stoichiometry by adding
to all treatments ammonium nitrate in a quantity that adjusted the C:N of cellulose (166) to that of chitin (10),
using both field and in vitro experiments. For cellulose, CO2 release increased more than five- to eightfold
after N addition in root-free soil incubated in vitro, but only 0–20% in situ where roots were intact. By the
end of the 2-year-long field experiment, fine-root biomass tended to be higher in the chitin treatment, where
R soil was significantly higher. Together these findings suggest that soil N availability limited cellulose
decomposition, even in this Neotropical forest with high soil N stocks, and also that trees successfully
competed for N that became available as chitin decomposed. These results indicate that the major constituent
of cell walls of soil fungi, chitin, can decompose rapidly and release substantial N that is available for plant
and microbial growth. As a consequence, soil fungi can stimulate soil OM decomposition and N cycling, and
thereby play a disproportionate role in ecosystem C and N dynamics.
Keywords: (separated by '-')
cellulose - chitin - decomposition - lignin - nitrogen - organic matter stoichiometry - fungi
Footnote Information
Electronic supplementary material The online version of this article (doi:10.1007/s10021-014-9769-1)
contains supplementary material, which is available to authorized users.
Metadata of the article that will be visualized in OnlineAlone
Electronic supplementary
material
Below is the link to the electronic supplementary material.
MOESM1: Supplementary material 1 (DOCX 28 kb).
Ecosystems
DOI: 10.1007/s10021-014-9769-1
Ó 2014 Springer Science+Business Media New York
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Ann E. Russell*
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ABSTRACT
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Decades of studies on the role of decomposition in
carbon (C) and nitrogen (N) cycling have focused
on organic matter (OM) of plant origin. Despite
potentially large inputs of belowground OM from
fungal cell walls and invertebrate exoskeletons,
studies of the decomposability of their major constituent, chitin, are scarce. To explore effects on soil
C dynamics of chitin, in comparison with two
plant-derived chemicals, cellulose and lignin, I
conducted a field-based chemical-addition experiment. The design contained three chemical treatments plus a control, with four replicates in each of
two species of tropical trees grown in plantations.
The chemicals were added in reagent-grade form at
a rate that doubled the natural detrital C inputs of
1000 g C m-2 y-1. Despite its purported recalcitrance, chitin was metabolized quickly, with soil
respiration (Rsoil) increasing by 64% above the
control within days, coupled with a 32% increase
in soil extractable ammonium. Cellulose, which
was expected to be labile, was not readily decomposed, whereas lignin was rapidly metabolized at
least partially in one of the forest types. I examined
effects of stoichiometry by adding to all treatments
Received 9 October 2013; accepted 20 March 2014
Electronic supplementary material: The online version of this article
(doi:10.1007/s10021-014-9769-1) contains supplementary material,
which is available to authorized users.
*Corresponding author; e-mail: [email protected]
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ammonium nitrate in a quantity that adjusted the
C:N of cellulose (166) to that of chitin (10), using
both field and in vitro experiments. For cellulose,
CO2 release increased more than five- to eightfold
after N addition in root-free soil incubated in vitro,
but only 0–20% in situ where roots were intact. By
the end of the 2-year-long field experiment, fineroot biomass tended to be higher in the chitin
treatment, where Rsoil was significantly higher.
Together these findings suggest that soil N availability limited cellulose decomposition, even in this
Neotropical forest with high soil N stocks, and also
that trees successfully competed for N that became
available as chitin decomposed. These results indicate that the major constituent of cell walls of soil
fungi, chitin, can decompose rapidly and release
substantial N that is available for plant and microbial growth. As a consequence, soil fungi can
stimulate soil OM decomposition and N cycling,
and thereby play a disproportionate role in ecosystem C and N dynamics.
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Key words: cellulose; chitin; decomposition; lignin; nitrogen; organic matter stoichiometry; fungi.
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INTRODUCTION
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Decomposition of organic matter (OM) and its accrual and persistence in soil are processes that
influence a variety of ecosystem services, including
soil fertility, water availability, resistance to erosion,
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Department of Natural Resource Ecology and Management, Iowa State University, Ames, Iowa 50011, USA
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Unexpected Effects of Chitin,
Cellulose, and Lignin Addition
on Soil Dynamics in a Wet
Tropical Forest
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simultaneously. Potentially confounding factors
include quantity and allocation of detrital inputs,
nutrient availability unrelated to plant tissue
quality, and unknown or unmeasured chemical
constituents, other litter traits such as toughness,
and plant–mycorrhizal associations that influence
plant access to limiting nutrients, as hypothesized
by Hättenschwiler and others (2011). Also, this
plant-based focus ignores effects on SOM decomposition of the potentially large OM inputs from
mycorrhizal fungi and their decomposibility.
The goal of this study was to address the question
of whether different carbon compounds influenced
soil C dynamics differently. To examine the effect
of OM chemistry alone on soil, I applied a set of
pure chemicals that vary in C:N and structural
complexity and then measured the effects on soil C
dynamics. Decomposition has been described as a
two-stage process: comminution, the breakdown of
organic matter into smaller pieces by detritivores;
and chemical breakdown by bacteria and fungi
(Aerts 1997). I focused on this latter stage by
applying the chemicals in powder or flake form and
selected three compounds that are abundant in
nature.
Cellulose and lignin are the second and third
most abundant classes of biochemicals in plants.
Chitin is the basic unit of cell walls of many fungi
and exoskeletons of arthropods. Cellulose and
chitin are homopolymers of glucose in which the
glucose units are linked by b-(1-4)-glycosidic
bonds, but chitin differs in that one hydroxyl group
on each monomer is replaced with an acetyl amine
group. This allows for increased hydrogen bonding
between adjacent polymers, giving the chitin–
polymer matrix increased strength. The hydrolytic
enzymes involved in decomposition of cellulose
and chitin are highly substrate specific in that their
catalytic efficacy is based on the structural
arrangement/chemical bonds in the substrates
(Caldwell 2005). In contrast structurally, lignin is a
high molecular, three-dimensional macromolecule
consisting of phenyl propane units (Kögel-Knabner
2002). The oxidoreductases involved in their mineralization, such as lignin peroxidase, tend to be
less substrate specific. The reactivity of these heteropolymers is determined by the identity of their
heterogeneous monomers and the linkages connecting these monomers (Nierop and others 2006;
Schweitzer and others 2008). Lignin’s structure is
thus generally considered to be the basis for its
recalcitrance. However, its macromolecular structure and cross-linkages determine the redox potential within the macromolecule, which in turn
influences its decomposability (Wong 2009). Thus,
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and mitigation of climate change. The effect of the
chemistry of OM inputs is integral to our conceptual framework for understanding how plant species composition influences soil organic matter
(SOM) dynamics. It has long been recognized that
plant species differ in various aspects of their litter
chemistry, which in turn may influence the decay
process (Waksman 1929; Minderman 1968). Litter
stoichiometry, especially C:N, is expected to drive
decomposition, owing to trade-offs in delivery of an
energy source (C compounds) and nutrients,
especially N. Because the C:N of plant litter can
vary widely, from 14 to 87, and it is generally
higher than the C:N of bacteria and fungi, 5–17,
plants can vary in the extent to which they limit
nutrients needed for growth and activity of
microbial decomposers (Cleveland and Liptzin
2007). Fungi are relatively under-studied, but
Koide and Malcolm (2009) found that decomposition in ectomycorrhizal (EM) fungi was also correlated with tissue N concentration.
Decomposition is not always predicted from
stoichiometric ratios of plant litter (Aerts 1997;
Cornwell and others 2008); however, secondary
compounds such as lignin or phenol, as well as
specific leaf area, are often correlated with
decomposition (Meentemeyer 1978; Hättenschwiler and Vitousek 2000; Cornwell and others 2008).
Thus, molecular complexity of plant litter is also
expected to influence decomposition. These concepts, based on studies of decomposition of fresh
litter from plant species that differ in tissue chemistry, have informed our historical view of soil C
cycling. That is, stabilized SOM consists mainly of
decomposed plant litter and microbially synthesized humic substances possessing a chemical
complexity and composition that is resistant to
decomposition by microbes (Schmidt and others
2011). Decomposition of fresh plant litter is not
always correlated with these plant traits (for
example, Hättenschwiler and others 2011; Freschet
and others 2012); however, recent advances have
revealed that persistence of SOM involves a complex interplay among an array of biological and
environmental factors (Schmidt and others 2011).
Part of the problem in evaluating the role of
compound chemistry alone lies in the fact that
multiple traits vary simultaneously within a single
plant species. As such, comparison of litter
decomposition among multiple species—the main
in vivo approach which has informed our theoretical constructs to date—constitutes an uncontrolled
observational study with respect to plant traits and
their control over decomposition. That is, plant
factors other than the measured factor may co-vary
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(1) Rapidity of degradation would range from
fastest in cellulose to slowest in lignin, by the
rationale that structural complexity regulates
decomposition. Therefore, soil respiration
(Rsoil) was expected to be highest in cellulose
and lowest in lignin. Added lignin that did not
decompose would therefore accrue as soil C to
a greater extent than additions of cellulose or
chitin. Thus, soil C would increase the most
under lignin addition by the historical concepts
of soil OM persistence described above.
(2) Lignin would degrade faster in Pentaclethra then
in Vochysia. With higher lignin concentrations
in Pentaclethra tissues, microbial communities
more specialized in lignin decomposition were
expected to be present in this species.
(3) Stoichiometry, in particular the C:N, would not
have a significant effect on decomposition of
the model compounds, by the reasoning that
soil N stocks are very high and rates of N cycling are extremely rapid in this site (Russell
and Raich 2012). Thus, N was not expected to
limit decomposition in any of the compounds,
given the substantial N release from the natural
background of decomposing detritus, which
includes both litter and SOM.
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METHODS
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Site Description
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I conducted two chemical-addition experiments in
subplots within experimental plantations situated
at La Selva Biological Station in northeastern Costa
Rica (10°26¢N, 83°59¢W). The mature forest on this
land had been felled in 1955, after which pasture
was established and grazed until abandonment in
1987. In 1988 mono-dominant plantations of 11
tree species were planted in this abandoned pasture
in a randomized complete block design with four
blocks (Fisher 1995). Thus, only the dominant tree
species differs among the experimental plantations,
which all have similar parent material, soil type,
climate, and topography, and thus minimal differences in other state factors sensu Jenny (Amundson and Jenny 1997).
The study site has a mean annual temperature of
25.8 °C. Mean annual rainfall is 4000 mm, with
rainfall generally greater than 100 mm in all
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In comparing the control with three model
compounds applied as treatments (cellulose, chitin,
and lignin), with and without N addition, I tested
the following alternate hypotheses:
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peroxidases could have substrate-specific catalytic
abilities, depending on how the redox potential is
influenced by the macromolecular structure.
The objective of this study was to compare the
effects of these three compounds on soil C dynamics
and to gain insight into the mechanisms driving
these effects. To that end, I applied the compounds
in their pure forms, with and without NH4NO3,
both in vitro and in a field experiment conducted
for 2.5 years within an established long-term
experiment at La Selva Biological Station in Costa
Rica. This wet lowland tropical forest site offered
several advantages for achieving the objectives. (1)
Conditions are ideal for plant growth and OM
decomposition, given the site’s warm climate with
abundant rainfall. Thus, results for decomposition
studies come quickly, given that both C and N cycling are rapid and soil C and N stocks are very high
(Russell and others 2007; Raich and others 2009;
Russell and others 2010; Russell and Raich 2012).
(2) Tropical Oxisols are widely believed to be depleted in phosphorus (P), and soil extractable P in
this particular site is low (Russell and others 2007).
Thus, I anticipated that P, rather than N, would be
more limiting to decomposition, such that differences in the C:N of the model compounds would
not have an impact. (3) The tropical setting could
provide a broader perspective on lignin decomposition, given that the expected higher diversity of
microbes would increase the likelihood of lignin
specialists that may not occur in higher latitudes.
(4) The pre-existing experimental setting allowed
for comparisons of the model compounds within
two forest types which differed only in the dominant tree species. Set within the context of a larger
randomized complete block experiment, edaphic
factors were similar across the experimental units of
subplots used for chemical additions that were situated within the main plots. The two native tree
species in this study differ in the chemistry of their
detrital inputs, but not in the quantity (see
Appendix 1 in Supplementary material). Litter and
foliar N concentrations are relatively high and
similar in the two species, Pentaclethra macroloba
(Willd.) Kunth. (Leguminosae, nodulated) and Vochysia guatemalensis Donn. Sm. (Vochysiaceae, not
nodulated) (Raich and others 2007). Foliar d15N
data indicate that both species promote biological N
fixation however (Russell and Raich 2012); thus,
neither species was expected to be N limited. Newly
senesced leaves and fine roots of these species differ
primarily in their carbon chemistry, with lignin
higher in Pentaclethra and water-soluble C compounds higher in Vochysia (Raich and others 2007;
Russell and others 2007).
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The concept of these chemical-addition experiments was to apply model compounds in sub-plots
within the pre-existing experiment, with the
quantity of compounds applied large enough to
create measurable changes in response variables.
Doubling the OM inputs was thus the goal, to be
achieved by adding 1000 g C m-2 y-1 of the model
compound to the natural background detrital inputs of approximately 1000 g C m-2 y-1 (mean
across species, Russell and others 2010). The
quantity of chemical required for delivering
1000 g C m-2 y-1 to the soil was calculated using
the compound’s molecular formula. The response
variables measured included Rsoil, change in soil C
and N stocks, extractable inorganic soil N, and fineroot biomass; sampling depth was 0–15 cm.
The experimental design of Experiment 1 consisted of four treatments, three chemical amendments plus a control:
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The formulae, C:N, and Sigma-Aldrich catalog # of
the compounds added were the following: cellulose: [C12H8O10], S3504; chitin: [C8H13O5N1],
C7170; and lignin: [C10H12O2], 471003. Their
respective C:N values were 165.7, 9.6, and 244.6, as
determined from analyses of the purchased chemicals by dry combustion (Thermo-Finnigan EA
Flash Series 1112, CE Elantech, Lakewood, NJ).
Experiment 1 was conducted for two years, after
which Experiment 2 was initiated in which an N
treatment was added within each subplot of the
four ongoing chemical treatments in Experiment 1,
for n = 64 units. Ammonium nitrate (NH4NO3)
was added to all four chemical treatments in the
quantity required to adjust cellulose C:N to 9.6,
that of chitin. This is hereafter referred to as the
‘‘+N’’ treatment, whereas treatments not receiving
N are referred to as ‘‘0 N.’’
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4 Replicates ¼ 32 units ðsubplotsÞ:
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Chemical Application
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Following a 4-month calibration period after
establishment of the subplots for Experiment 1, the
chemical amendments began. The calibration period was 3 months in Experiment 2. The same
application procedure was used in both experiments. First, the litter layer was removed and
stored temporarily on a plastic sheet nearby. The
litter layer within the soil respiration collar was
stored separately for its return there. The preweighed dose of chemical was spread evenly over
the litter-free subplot, with a pre-weighed dose
allocated for the soil respiration collar, and another
dose for the remainder of the plot. The compound
was worked into the soil to a depth of about 1 cm
briefly with a fork. The litter layer was then replaced, with the collar litter returned to its appropriate collar. The control was given this same
treatment, except that no chemical was added. Half
of the annual chemical addition was added
approximately every six months, except for a delay
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Approach
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Within the main 50 9 50 m plot of each tree species in each replicate, four 1 9 1 m subplots were
randomly located within the relatively flat areas of
the plots on hilltops. To avoid complications
resulting from overland flow or flooding, sloping
areas and toes of slopes were excluded from consideration in siting the subplots. Chemical treatments were randomly assigned among the four
subplots which were situated within a 10-m radius
of each other within the main plot. In January
2008, each subplot was framed with PVC tubing,
with the corners firmly anchored with metal stakes.
Each subplot was equipped in the center with one
20-cm diameter PVC soil respiration collar, for a
total of 32 collars. The sharpened collar edge was
inserted to a depth of only about 1 cm, deep enough to maintain a seal, but not to impede fineroot growth substantively. Comparisons of fineroot biomass in the main part of the plot (outside
collars) confirmed this. The structure of this forest
soil derived from volcanic parent material is such
that this soil is exceptionally well-drained on hilltops. Thus, despite high rainfall, flooding did not
occur in the collars in this experiment.
For Experiment 2, a second soil respiration collar
was installed within each of the 32 subplots for
addition of the model compound plus NH4NO3 (+N
treatment), whereas the remainder of the subplot
received only the former (0 N treatment). Thus
there were n = 64 soil respiration collars. This
experiment lasted 6 months.
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Field and Laboratory Methods
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Subplot Establishment
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months (Sanford and others 1994). The soils in this
site are deep, volcanically derived, and classified as
Oxisols (Mixed Haplic Haploperox, Kleber and
others 2007). Despite the assumptions that La Selva
soils are greater than 1 Ma old and were formed
in situ, these soils have relatively higher levels of
rock-derived Sr and macronutrient concentrations
(including P), in comparison with similarly aged
Hawaiian and other continental soils (Porder and
others 2006).
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Fine-Root Biomass
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Within each subplot, a single soil core, 15-cm deep,
was collected from the center of each soil collar at
the end of the first experiment. Soil samples were
extracted using a hand-operated slide-hammer
metal corer, 5.37 cm inner diameter. Initial separation of roots from soil was done within 24 h of
collection, using a hydropneumatic elutriation
system (mesh size of 530 lm; Smucker and others
1982), as in Russell and others (2004). Roots were
separated from detritus by hand and live roots were
distinguished from dead roots, based on morphological characteristics. Samples were dried at 65°C.
Only live roots were included in biomass totals.
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Lab Incubation Experiment
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To assess CO2 respired from soil without live roots, I
conducted an in vitro experiment, measuring potential C mineralization in field-moist soil (0–5 cm)
from the control subplots of Vochysia and Pentaclethra in March 2010. One 50-g sample from Block 1
in each of the two species was collected and
transported to ISU on ice. Roots and detritus were
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the calibration period, daily for the first week
immediately following addition of chemicals; 1–2
times per week for the week following the chemical
addition, and every 2 weeks thereafter. Cumulative
annual Rsoil was calculated by multiplying the mean
value between one time point and the next by the
time elapsed between the two times and summing
these values over an annual time period.
Extractable inorganic N was measured at the end
of Experiment 1, in February 2010 on soil samples
(0–15 cm) also collected for measurement of SOC
and total N. As soon as samples were collected, they
were placed in Ziploc bags, brought to the lab on
ice, and processed immediately. Extractions of nitrate (NO3-) and ammonium (NH4+) were conducted with 2 M potassium chloride (KCl). Soil
solutions with a 1:5 dry soil/KCl ratio were shaken
for 30 min, allowed to settle for 30 min, and then
filtered through No. 42 Whatman paper. Two
blanks were also extracted at every sample time to
account for contaminant nitrate and ammonium in
the filters and vial. Filtrates were kept frozen and
transported on dry ice to Iowa State University
where they were analyzed colorimetrically for
NO3-–N and NH4+–N using an automated ion
analyzer (QuickChem 4100, Lachat Instruments
Division, Zellweger Analytics, Inc., Milwaukee,
WI). Field-moist subsamples of 5 g were dried at
105°C for 48 h to convert measurements to a dryweight basis.
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due to inclement weather in Nov–Dec 2008. To test
the effect of dose size, chemicals were added at 1-,
2- and 3-month intervals, with the doses adjusted
accordingly, during the last 6 months (that is,
Experiment 2). Thus, chemicals were added on
May 21, 2008, Jan 14, 2009, and April 22, 2009,
May 24–25, 2010, June 28–29, 2010, and August
30–31, 2010. The masses of chemicals added in
total were: cellulose 40 kg; chitin, 38 kg; and lignin, 24 kg.
Soil organic carbon (SOC) and total N were
sampled in the 0–5 and the 0–15 cm layers before
chemicals were applied using a 3.2-cm push-tube
sampler at randomly selected locations within the
subplots. Eight sub-samples per subplot were
bulked to provide a single sample per subplot. To
determine changes in soil C and N stocks (DSOC,
DSoil N, 0–15 cm), I re-sampled soil in February
2010 at the end of Experiment 1. Carbon and N
stocks were determined from these concentration
data in combination with bulk density data as in
Russell and others (2007). Changes in stocks were
calculated as the difference in SOC (or soil N) between final and initial values. Soil samples were
analyzed for total C and N by dry combustion as
described above for the model compounds. These
soils do not contain carbonates, so SOC equals total
soil C.
To evaluate whether overland flow of the added
chemicals had occurred, soil was re-sampled in the
0–5 cm layer 6 weeks after the first chemical
addition. Carbohydrates, including cellulose-derived glucose, were measured through extraction of
soil with methanesulfonic acid, separation by anion
exchange chromatography, and detection by
pulsed amperometry using a Dionex DX 500 anion
chromatograph equipped with a CarboPac PA-10
column (2 mm 9 250 lm). Soil glucose measurements corroborated Rsoil findings and visual
inspection of the white cellulose powder; all indications were that the added cellulose was still retained in the soil six weeks after its addition. Thus,
loss of chemicals via erosion was deemed negligible.
Soil respiration (Rsoil) was measured with an
LI-8100 automated soil CO2 flux system and 8100102 (20-cm diameter) chamber (LI-CORÒ Biosciences, Lincoln, Nebraska, USA). Chambers were
monitored for 240 s after closure; fluxes were calculated based on the final 150 s of measurements.
Soil respiration was measured at various frequencies; the timing was determined with the goal of
capturing peak degradation of the model compounds, but also avoiding over-sampling so as not to
compact the soil. We sampled every 2 weeks during
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Statistical analyses were conducted using the SAS
System (Version 9.3, SAS Institute 2002–2010).
Differences in responses were tested using a mixed
model in PROC MIXED, with tree species, chemical
compound and N-addition treated as fixed and the
block or replicate as random effects (Littell and
others 1996). A split-plot design was used for
chemical treatments (subplots) applied within tree
species (main plots) (n = 32), and a split–split–plot
design for N addition (sub-subplots) within subplots
of chemical treatments (n = 64). I tested for homogeneity of variances and normality of distributions.
For the response variable in the laboratory incubation experiment (CO2–C respired), a natural log
transformation was required and statistical test results reflect analyses of the transformed variable.
Differences among individual means were compared
using least squares means with a ‘‘pdiff’’ statement.
Pearson’s correlation analysis was used to evaluate
the relationship between Rsoil and fine-root biomass.
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Chemical Addition Effects
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Soil Respiration
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During the 4-month-calibration period at the
beginning of the experiment, Rsoil averaged 6.12
(±0.12) and 4.67 (±0.09) lmol C m-2 s-1 in Vo-
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The effects of the added chemicals on changes in
soil C and N stocks (0–15 cm) were similar in the
two tree species, with no significant species or
species 9 chemical interaction effects (Appendix 1
in Supplementary material). Both responses, D soil
C and D soil N, were significantly higher in the
chitin treatment (Figure 3). The chemicals also had
similar effects in both species with respect to
extractable inorganic soil N. Mean extractable soil
NH4-N in chitin was significantly higher than in the
other three treatments, by 32% (Figure 4).
Extractable soil NO3–N was highly variable, such
that so significant differences were detected.
Fine-root biomass tended to be higher in Vochysia
than in Pentaclethra, and within species it was
generally higher in the chitin treatment (Figure 5).
At the end of Experiment 1, the positive correlation
of fine-root biomass with the cumulative annual
Rsoil was significant (P = 0.0046).
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Nitrogen Addition Effects
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In laboratory incubations, the response to N addition was complex, with significant chemical 9 N
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The soil plus chemical amendment(s) were placed
in 2.5 cm-diameter incubation tubes and sealed
tightly with butyl rubber septa. Incubated samples
were kept at 21°C. Over a 30-day-period, the rate
of CO2–C released was measured periodically (before CO2 concentrations reached 4%) by flushing
the flask headspace through an infrared gas analyzer (LI-820, LI-COR Biosciences, Inc., Lincoln,
NE). The incubation-tube atmosphere was kept
hydrated by piping the air supply through water.
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2 Species 4 Chemical Treatments
2N Treatments 3 Replicates ¼ 48:
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chysia and Pentaclethra, respectively (Figure 1A).
With the addition of the first dose of chemicals, Rsoil
responded immediately to chitin and lignin, but not
to cellulose addition (Figure 1, right panels provide
magnified view). Within one day of lignin addition,
Rsoil was 69 and 33% higher in the two species,
peaking at 10.37 (±0.51) and 6.23 (±0.42) lmol
C m-2 s-1 in Vochysia and Pentaclethra, respectively.
The response tapered off quickly in the lignin
treatment to fall below rates in the control. With
chitin addition, soil respiration peaked at 30 days in
Vochysia when Rsoil was 16.76 (±1.32) lmol C m-2
s-1 and in Pentaclethra at 9.66 (±0.86) lmol C m-2
s-1 17 days after addition. By the third addition, Rsoil
under chitin addition peaked at 26.87 (±1.23) and
23.94 (±3.01) lmol C m-2 s-1 in Vochysia and Pentaclethra, respectively.
Cumulative annual Rsoil was highest in chitin,
but the extent of this response differed between the
two species, such that the species 9 chemical
interaction was significant (P < 0.0001). In Vochysia, Rsoil of 4604 (±290) g C m-2 y-1 in chitin
was significantly higher than 2363 (±330) g C m2 -1
y
in the control and all other treatments
(P = 0.0001) (Figure 2). In Pentaclethra, Rsoil of
2748 (±186) in chitin was greater than 2200
(±318) g C m-2 y-1 in the control and the other
treatments (P = 0.0002). Lignin’s Rsoil of 1482
(±81) was significantly lower than in the control.
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removed and each sample was homogenized well.
Gravimetric moisture content was measured and
found to be similar in the two samples, so no
moisture adjustments were made. From each of the
two homogenized samples, 24 2-g subsamples were
weighed and mixed with the model compound(s),
with treatment assignments randomized and the
chemical treatments the same as in field Experiment 2. With three lab replicates for each treatment, the design was:
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Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics
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Figure 1. Soil respiration response to cellulose, chitin, and lignin addition. A Data over entire 2-year-period for plantations of Vochysia guatemalensis (upper panel) and Pentaclethra macroloba (lower panel). Arrows indicate date of chemical
addition. B Detailed data immediately following chemical application on three dates within a species (three panels across) for
each of the two species. Data are normalized by subtracting values within a plot from the Control (red line = 0). In all
figures, error bars represent standard errors of the mean.
interactions and differing trends between the two
species (see Appendix 2 in Supplementary materials). Nevertheless, there was a clear response in
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Figure 2. Cumulative annual soil respiration under four
chemical addition treatments in tree plantations of the
two species, Vochysia guatemalensis and Pentaclethra macroloba. Lowercase letters denote differences among treatments.
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Figure 3. Change in soil C and N stocks (0–15 cm) after
doubling C inputs with additions of CEllulose, CHitin,
and LIgnin in comparison with no additions (COntrol).
Positive change denotes accumulation and negative denotes loss. Effect of tree species was not significant so
values are means within treatments across two tree
species for N = eight replicates per chemical. Lowercase
letters denote differences among treatments for both DSoil
C and DSoil N because results were the same for both
variables.
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Figure 6. Soil CO2–C respired over 30 days during
in vitro chemical-addition experiment. Ammonium-nitrate N (denoted by ‘‘+’’ and cross-hatched bars) was added
to each of the four chemicals, in comparison with no
addition (denoted by ‘‘0’’). Chemicals are identified by
their first two letters: COntrol; CEllulose; CHitin; and
LIgnin. Means are for N = three lab replicates per
chemical within each of the two tree species, Vochysia
guatemalensis and Pentaclethra macroloba. Different lowercase
letters signify differences in responses among the four
chemicals with no N addition; uppercase letters are for the
+N treatment; asterisks denote differences between N
treatments within chemicals.
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Figure 4. Soil extractable inorganic N (ammonium,
NH4–N and nitrate, NO3–N) under the different chemical
treatments. Chemical treatments were: COntrol; CEllulose; CHitin; and LIgnin. N = four replicates per chemical
within each plantation. Soil data are for the 0–15 cm
depth. Effect of tree species was not significant so values
are means within treatments across two tree species for
N = eight replicates per chemical. Different lowercase letters
signify differences in NH4–N among the four chemicals.
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both species on one aspect: N addition stimulated
mineralization of cellulose more than eightfold in
Vochysia and more than fivefold in Pentaclethra,
increasing CO2–C release rates relative to those in
the chitin treatment (Figure 6). Nitrogen also significantly depressed degradation of lignin in both
species. Although N addition increased respiration
in the Vochysia control, the effect was not significant in Pentaclethra.
In the field experiment, the effect of N addition
was also complex (Appendix 2 in Supplementary
materials). The results differed from the in vitro
experiment in the lack of a strong response when N
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Figure 5. Soil respiration increased significantly with
fine-root biomass. All data are shown, with N = four
replicates per chemical within each plantation. Chemicals added were: COntrol; CEllulose; CHitin; and LIgnin.
Pema Pentaclethra macroloba, Vogu Vochysia guatemalensis,
DM dry matter.
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Figure 7. Soil respiration following additions of four
chemicals in the field, with and without ammonium nitrate. Chemicals are identified by their first two letters:
COntrol; CEllulose; CHitin; and LIgnin. Nitrogen-amended treatment is denoted by ‘‘+’’ (cross-hatched bars) and
unamended by ‘‘0.’’ Letters denote significant differences
among chemical treatments. There were four replicates
per treatment within each tree plantations of the two
species, Vochysia guatemalensis and Pentaclethra macroloba.
was added to cellulose (Figure 7). In the field, added N significantly increased Rsoil in all chemical
treatments in Vochysia, by 20% in chitin, whereas
there was no significant effect in Pentaclethra in any
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treatment. As in the first field experiment, Rsoil
continued to be highest in the chitin treatment in
both species, and significantly lower in lignin in
Pentaclethra.
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Compound Chemistry and
Decomposition
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In general, the two aspects of compound chemistry
considered—structural complexity and stoichiometry—were not very predictive of the model compound’s decomposition. Cellulose, the most
structurally simple compound, was not readily
metabolized either in the laboratory or the field.
This is consistent with Kögel-Knabner’s (2002)
observation that cellulose degrades slowly. Chitin,
also a long-chain homopolymer, was expected to
be less labile than cellulose as a result of its increased hydrogen bonding between adjacent polymers, but it degraded rapidly. In contrast, the most
structurally complex compound—lignin—was expected to decompose slowly, but instead very
quickly stimulated Rsoil in the field after all applications in Vochysia and in two of three in Pentaclethra (Figure 1, see normalized values magnified at
right). The soil-respiration response to lignin was
one fourth that of chitin addition, but nevertheless
some decomposers responded rapidly to lignin.
Interestingly, the response to lignin was minimal in
Pentaclethra, even though this species has higher
foliar and fine-root lignin concentrations, such that
decomposers adapted for lignin decomposition
were expected to be present. Apparently, Vochysia
has modified soil conditions for decomposers, such
that Rsoil responses were higher under all treatments, including lignin. A limited group of
decomposers, the white-rot fungi, are purported to
be capable of complete decomposition of lignin,
although soft rot and brown rot fungi may
accomplish partial decomposition (Kögel-Knabner
2002; Floudas and others 2012). A consortium of
microbial decomposers, however, is believed to
mediate decomposition within soil (Haider 1992).
Given the structural similarity between cellulose
and chitin, the results of the in vitro experiment
indicate that stoichiometry explained differences in
decomposition of these two compounds. This is
inferred because C mineralization in the cellulose + N treatment (in which the C:N of 9.6
equaled that of chitin) was similar to rates in the
chitin treatment and represented a five- to eightfold increase over rates in cellulose alone. Addition
of N to lignin did not stimulate its decomposition;
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Role of Fine Roots in Soil C and N
Dynamics
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Fine roots have the potential to regulate decomposition via two main effects on the microbial and
fungal production of extracellular enzymes that
decompose and release nutrients from SOM. First,
fine roots influence substrate availability, a major
constraint for microbial production of the enzymes
in soil that depolymerize N from proteinaceous
compounds, and thus increase N availability
(Schimel and Weintraub 2003). Detrital inputs of
aboveground litter and dead fine roots both supply
substrates to microbes, but isotopic and biomarker
studies reveal the dominance of root-derived substrates in soil (Mendez-Millan and others 2010)
and soil microorganisms (Kramer and others 2010).
In addition, live root exudates supply energy-rich
and easily accessible C compounds that stimulate
microbial breakdown of SOM (Fontaine and others
2003; Kuzyakov and others 2000; Blagodatsky and
others 2010).
Second, fine roots influence soil nutrient concentrations and can successfully compete with
microorganisms for these nutrients, owing to the
capacity of fine roots to grow, form associations
with mycorrhizal fungi, and intercept and take up
nutrients over space and time (Schimel and Bennett 2004). These plant-driven effects on soil
nutrients influence microbial dynamics because
microbial enzyme-producing potential is linked to
the stoichiometry of their nutrient demands (Sinsabaugh and others 2008). Differences among plant
species in these root dynamics, coupled with their
whole-plant sink for nutrients and promotion of
biological N fixation, drive differences among species in their regulation of soil nutrients. I hypothesize that these differences drive variability among
species in the capacity to reduce soil nutrient
availability for microbes and thereby regulate
decomposition. Hättenschwiler and others (2011)
hypothesized that poor litter quality in tropical
forests enforces starvation of decomposers. These
two hypothesized mechanisms for regulation of
decomposition by tropical plants are not mutually
exclusive; rather, both poor litter quality and
reduction of available nutrients in soil could operate simultaneously to slow decomposition. Weintraub and others (in press) found that another
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however, this suggests that reducing the C:N of
lignin does not trump its structural complexity and
thus expose it to decomposition by new groups of
microbes, at least not to groups that were present in
this study site.
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Chitin Chemistry and its Implications for
N Cycling
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The tissues of fungi may contain high concentrations of N (Clinton and others 1999; Langley and
Hungate 2003), as a result of the high content of
chitin and proteins in their cell walls (Cooke and
Whipps 1993). Both ectomycorrhizal (EM) and
arbuscular mycorrhizal (AM, phylum Glomeromycota) fungi can have significant belowground
biomass. In a Swedish conifer forest, Wallander and
others (2004) found that EM biomass was the same
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which would in turn reduce fine-root biomass,
thereby decreasing Rsoil and soil C accrual. Thus in
the cellulose and lignin treatments, the lack of
positive DSOC reflects that the experimental additions of 1000 g C y-1 were offset by reductions in
OM inputs from fine roots. Unfortunately, the d15N
and d13C of the purchased model compounds did
not differ significantly from natural background
values, but in future experiments if a distinctive
isotopic label or biomarker could be incorporated
into chitin, cellulose, lignin, and ammonium nitrate, the fate of the N constituents, whether to
roots or microbes, could be traced.
The role of N dynamics in the degradation of the
model compounds is supported by the finding that
N addition increased cellulose decomposition
in vitro, without roots present (Figure 6), indicating that N was relatively limiting for cellulose
decomposition. Similarly, Barantal and others
(2012) found in a P-poor Neotropical forest that
decomposition increased with N availability because N was required for a positive P effect. They
also found that energy limitations of microbial
decompositions were co-limited by nutrient availability.
The question in this study becomes: why was the
response to N addition in the field less than that in
the laboratory (Figures 6, 7)? The most likely
explanation is that despite large soil N stocks in this
site, fast-growing trees were not saturated with N,
such that N demand was high and roots were able
to out-compete microbes for the added N. If increased uptake by roots of added N did not have
immediate metabolic costs, then Rsoil would not
increase immediately, as found in this study. A
sustained increase in Rsoil in response to N addition
would be expected later; however, as root biomass
accrued. Unfortunately, the length of this second
experiment did not allow for the longer-term
measurements. Together, these results highlight
the importance of considering all aspects of fine
root dynamics in relation to soil C and N dynamics.
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microbially mediated process, nitrous oxide (N2O)
production, was negatively correlated with fineroot production in this site at La Selva, with fastergrowing trees having reduced NO3 substrate available and lower N2O emissions.
Fine roots also influence soil C and N stocks as a
result of their detrital inputs, with C and N stocks
changing in direct proportion to fine-root productivity if decomposition remains unchanged. Fineroot productivity would also be expected to influence Rsoil, given that the majority of CO2 respired in
forest soils comes from roots (Raich and others
2014). In this site, mean Rsoil across tree species was
1920 g C m-2 y-1, of which 55% was attributed to
root + rhizosphere respiration (Rrrh) (Russell and
others 2010).
These various effects of fine roots on soil C and N
dynamics provide insight into the results presented
here. With the addition of 1500 g C m-2 into each
of the three chemical treatments in Experiment 1
over an 18-month-period, one might expect to
detect measurable effects on Rsoil and soil C stocks.
That is, degradation of the compounds was expected to result in increased soil metabolism and/or
accrual of the microbial remains and/or the unmetabolized compound in soil. The results were
surprising in that only one treatment—chitin—resulted in significant accrual of soil C
(647 ± 145 and 526 ± 219 g C m-2 in Vochysia
and Pentaclethra, respectively) (Figure 3). Furthermore, Rsoil in the chitin treatment in Vochysia surpassed that in the control by 2241 g C m-2 y-1,
thus exceeding the amount of C added experimentally by more than 1200 g (Figure 2)! In contrast, in the lignin treatment in Pentaclethra, Rsoil
was significantly lower than the control, by 15% or
343 g C m-2 y-1, with no significant change in soil
C stocks. The most plausible explanation for these
findings is that addition of the model compounds
influenced fine-root dynamics, which in turn
influenced both soil respiration and soil C stocks.
In another wet tropical forest in Costa Rica,
Cleveland and Townsend (2006) also found that
soil respiration responses to N fertilization were
likely driven by effects on fine-root biomass. Similarly, I hypothesize that the model compounds in
my experiment had different effects on N availability, and thus differed in their influence on fineroot productivity. By this hypothesis, the concomitant release of N from chitin decomposition (Figure 4) stimulated fine-root growth, resulting in the
observed higher biomass (Figure 5), and thereby
increased root and rhizosphere respiration and OM
inputs to soil C stocks. In contrast, the high C:N of
cellulose and lignin would reduce N availability,
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CONCLUSIONS
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In this first experimental test of pure chitin addition
in a lowland wet tropical forest, the surprising result was that this compound was readily metabolized, despite its purported recalcitrance in some
studies. More unexpected were the results that
cellulose was not readily decomposed in either
forest type, and that lignin was rapidly metabolized
at least partially in one of the forest types. The latter
finding suggested that lignin decomposition can be
rapid in the presence of lignolytic decomposers,
despite its structural complexity and high C:N. Soil
N stocks and N cycling in this site rank among the
highest published values for a non-agricultural site
(Russell and Raich 2012), so the most unanticipated result was that N limited decomposition of
cellulose and apparently also limited fine-root
growth. I hypothesize that fast-growing trees with a
large whole-plant sink for N and high fine-root
growth can regulate decomposition by reducing
available N pools, thereby starving decomposers.
These results highlight the importance of soil fungi
in N cycling in this Neotropical forest, owing to
high concentrations of chitin in fungal cell walls,
the low C:N and lability of chitin, and thus its
capacity to release N available for uptake by plants.
ACKNOWLEDGMENTS
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Jim Raich provided advice throughout all stages of
this study and bench space for the laboratory
incubations, for which I am very grateful. I also
thank: Ricardo Bedoya, Flor Cascante, Eduardo
Paniagua, and Marlon Hernández for assistance in
the field; Amy Morrow for conducting soil C and N
analyses; Terry Grimard for the carbohydrate
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the same time, its structural arrangement and types
of chemical bonds would promote catalytic efficacy
for these enzymes.
Chitin cycling, accompanied by its release of N, is
thus hypothesized to play an important role in the
rapid N cycling and SOC accrual. In this tropical
study site, these effects of N cycling were mediated
via effects of N on fine roots. The findings in this
study and that of Fernandez and Koide (2012)
suggest that where soil fungi are abundant, fast
decomposition of their major constituent—chitin—provides a mechanism for promoting rapid N
cycling, which feeds back to stimulate C cycling.
The results from this experiment in which the OM
inputs were pure pinpoint more precisely the role
that the chemistry of chitin plays in the ability of
soil fungi to stimulate C and N cycling.
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order of magnitude as root biomass. Arbuscular
mycorrhizal fungi are estimated to act as a sink for
3–20% of host plant photosynthate (Jakobsen and
Rosendahl 1990; Johnson and others 2002a, b).
Nottingham and others (2010) found that AM
fungi contributed 1.4 ± 0.6 t C ha-1 y-1 or
14 ± 6% of total soil respiration in a moist tropical
forest, and were thus an important component of
the tropical forest C cycle. Hodge and Fitter (2010)
discovered that AM fungi have a high demand and
capacity to acquire N, despite the fact that as obligate biotrophs, they lack the saprotrophic capability. Thus, given their biomass and N acquisition
capacity, fungi represent a global N pool on par
with fine roots. In wet tropical forests, fungal
communities vary both spatially and temporally,
conferring variability in their effects on nutrient
availability (Lodge and Cantrell 1995). The binding
of leaf litter by fungal connections in Basidiomycetes can also influence nutrient cycling on tropical
forest slopes by reducing export of OM downslope
(Lodge and Asbury 1988).
The capacity of fungi to drive N dynamics would
also hinge on their decomposability. Fungal tissues
have been considered to be recalcitrant (for
example, Treseder and Allen 2000; Steinberg and
Rillig 2003; Driver and others 2005). Others,
however, have found fungal tissues to be relatively
labile (Okafor 1966; Gould and others 1981). Fernandez and Koide (2012) also found that chitin
within the walls of EM fungi was relatively labile
compared with other cell-wall constituents, such
that species-specific differences in decomposition
were positively related to chitin concentration. In
experimental studies, Cheng and others (2012)
found that elevated CO2 provided a C source that
stimulated degradation of SOC by AM fungi. In
contrast, Langley and others (2006) demonstrated
that colonization by EM fungi slowed decomposition of fine roots in a Pinus edulis system. Perhaps
confounding factors play a role in the disparity of
these findings.
Based on chitin’s molecular structure and C:N,
one would expect it to be labile in an N-limited
setting. In my experiment, despite high rates of N
cycling and high soil N stocks in this setting (Russell
and Raich 2012), inputs of pure chitin influenced
soil respiration within one day after its addition,
increasing annual Rsoil by 64% above the control,
and extractable NH4–N by 32% (Figure 1). This
indicates that chitin is inherently extremely labile
and that it supplies plant-available N (Figure 4).
This supports the expectation based in chitin’s
chemistry. That is, its low C:N would stimulate
microbial production of hydrolytic enzymes and at
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analyses; and Dan Olk for interpretation of the
carbohydrate data and comments on a previous
version of this manuscript. This material is based on
work supported by the U.S. National Science
Foundation (Grants DEB 0703561 and 1119223).
Haider K. 1992. Problems related to the humification processes
in soils of the temperate climate. In: Bollag J-M, Stotky G, Eds.
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Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics
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