Published 2013

Chapter 20

Organic Matter Mineralization

and Decomposition
Scott D. Bridgham* and Rongzhong Ye

Abstract
Organic matter mineralization is the transformation of organic C and
nutrients into inorganic forms or into smaller, simpler organic compounds
if mineralization is not complete. Organic matter mineralization is of
central importance in many ecosystem functions. Methods to measure
organic matter decomposition and mineralization are discussed in this
chapter. The litterbag technique is the most widely used method for
determining short-term decomposition rates and nutrient mineralization–
immobilization dynamics of fine fresh litter (leaves, plant reproductive
parts, fine roots, and small-diameter wood). Soil heterotrophic respiration
is measured as the production of CO2 and CH4. The alkali absorption
method for CO2 production and the gas chromatography method
for CO2 and CH4 production are described. A method to measure the
photodegradation of dissolved organic matter to CO2 is also described.

Abbreviations: AA, alkali absorption; DOM, dissolved organic matter; GC,

gas chromatography.

S.D. Bridgham and R. Ye, Environmental Sciences Institute and Institute of Ecology and Evolution, Univ.
of Oregon, Eugene, OR 97403-5289; R. Ye, current address: Dep. of Land, Air and Water Resources,
Univ. of California, PES 3316, Davis, CA 95616. *Corresponding author ([email protected]).

doi:10.2136/sssabookser10.c20

Copyright © 2013 Soil Science Society of America, 5585 Guilford Road, Madison, WI 53711-5801, USA.
Methods in Biogeochemistry of Wetlands. R.D. DeLaune, K.R. Reddy, C.J. Richardson, and J.P. Megonigal,
editors. SSSA Book Series, no. 10.

385
386 Bridgham & Ye

O
rganic matter mineralization is the transformation of organic C and nutri-
ents into inorganic forms or into smaller, simpler organic compounds if
mineralization is not complete (Bridgham and Lamberti, 2009). Organic
matter mineralization has been widely studied in wetlands and other ecosystems
because of its central importance in the cycling of C and nutrients, sequestration
of C, release of trace gases such as CH4, production of dissolved organic matter
(DOM), and relationship to most soil physiochemical properties. A large suite of
soil organisms are involved in organic mineralization (Bridgham and Lamberti,
2009). Soil invertebrates are particularly important in the fragmentation of detritus
and in soil food web interactions. The microbial groups bacteria, archaea, and fun-
gi are largely responsible for the final mineralization of organic C to the gaseous
end products of heterotrophic respiration, i.e., CO2 under aerobic conditions and
CO2 and CH4 under anaerobic conditions. Hence, heterotrophic soil respiration is
often construed to be a more narrow measure of microbial respiration.
Organic matter mineralization studies are typically done in two ways: (i)
decomposition studies determine mass loss and the accompanying nutrient
dynamics of serial cohorts of detritus (or “litter”) starting with fresh plant mate-
rial and followed for several years, and (ii) soil heterotrophic respiration studies
determine the gaseous end products CO2 and CH4 that result from the respira-
tion of soil organisms. Both methods are actually measuring decomposition, but
there is a long history in the scientific literature of this distinction in terminology.
Photodegradation of DOM is the photolysis of the high-molecular-weight
compounds of DOM, primarily fulvic and humic acids, by sunlight and thus is
an abiotic process. It is done primarily by the lower wavelength, higher energy
ultraviolet portion of the solar spectrum (Larson et al., 2007). Wetlands are major
sources of DOM to downstream aquatic ecosystems, where DOM has major
effects on light and ultraviolet radiation attenuation, water chemistry, and trophic
dynamics (Johnston et al., 2008). The lower molecular weight organic products
of photodegradation are often more biodegradable than DOM that has not been
exposed to sunlight (Bertilsson and Tranvik, 2000).

LITTER DECOMPOSITION
Decomposition experiments typically focus on measuring the mass loss of soil
detritus with time. We focus here on the decomposition of fine litter, which includes
leaves, reproductive parts, fine roots, and small diameter wood. For methods for
determining the decay rates of large woody debris, see Harmon et al. (1999).
The litterbag technique is described in detail in this chapter as the preferred
technique for measuring relatively short-term (i.e., up to several years) decom-
position rates of fine fresh plant material. In this widely used technique, detritus
enclosed within mesh bags is placed in the field and successive cohorts are removed
with time to determine the degree of mass loss. The largest limitation of this
approach is that there is a tradeoff between having a fine enough mesh to enclose
the material without any leakage of material vs. exclusion of macroinvertebrates.
Typically, a fine enough mesh is used (?0.4–1.5 mm) that macroinvertebrates are
 Organic Matter Mineralization and Decomposition 387

excluded, which can be an important component of decomposition through shred-

ding, although this is probably of minimal importance under anaerobic conditions.
Litterbags can also increase the moisture concentration relative to unenclosed
detritus (Witkamp and Olson, 1963).
Where these limitations of litterbags are thought to be problematic, other
decomposition methods can be used. The “litter sandwich” method is very useful
for determining long-term decomposition rates (Binkley, 2002). In this technique,
a mesh screen is placed over each new annual cohort of litter in the field, which
allows horizontal movement of macroinvertebrates into the samples. Subsam-
ples can be taken from the screens to determine annual decomposition rates for
many years. In the mass balance approach to estimating annual decomposition
rates, the decomposition rate is equal to the amount of annual litterfall divided
by the mass of the litter layer (i.e., the O horizon) (Karberg et al., 2008; Olson,
1963). This method is less direct than the litterbag method and it assumes steady-
state conditions from year to year for litterfall and the O horizon, which is untrue
in many ecosystems. Decomposition rates are also sometimes determined with
detritus that is tethered in the field without any enclosure mechanism. While
this method avoids the problems of litterbags in terms of exclusion of macroin-
vertebrates and changes in microclimate, inflated decomposition rates are often
measured because of fragmentation and subsequent loss of the detritus (Wit-
kamp and Olson, 1963).
Soil decomposers are constantly taking up exogenous nutrients from their
environment to build their biomass (termed immobilization) and releasing nutrients
from the breakdown of organic matter and the turnover of their biomass (termed
mineralization) (Bridgham and Lamberti, 2009). When litter begins the decay pro-
cess, there is typically a period from months to several years of net immobilization
of nutrients (i.e., gross immobilization > gross mineralization), followed by the
net mineralization of nutrients from the litter (i.e., gross mineralization > gross
immobilization). Because this process is of considerable importance in soil nutri-
ent dynamics, it is often followed during decomposition experiments.
Many investigators are interested in how the initial substrate quality of the
litter determines its decomposition rate and how the substrate quality changes
during the course of decomposition. Typical substrate quality variables of interest
are lignin, soluble C, soluble phenolics, various cellulose fractions, and nutrient
content. For methods for quantification of litter quality, see Graca et al. (2005), Har-
mon and Lajtha (1999), Palm and Rowland (1997), and Wieder and Starr (1998).

Equipment and Instrumentation

• Muffle furnace or CHN analyzer
• Balance with precision to 0.01 g
• Drying oven
• Incubator or refrigerator
388 Bridgham & Ye

Materials and Reagents

• Air-dried litter
• Material to make litterbags (1.5-mm mesh fiberglass window screen recom-
mended)
• Vessel for weighing and oven drying
• Crucibles for determination of ash-free dry mass
• Tweezers
• Scissors
• Aluminum or plastic tags
• Nondegradable colored string to mark litterbags in the field

Many different materials may be used for the litterbags, such as nylon, polypro-
pylene, and fiberglass. The three most important considerations are (i) that the
material maintains its integrity throughout the period of deployment in the field,
(ii) that it be inert in terms of its biological effects and any effect on the nutrients of
interest, and (iii) mesh size. Most plastics will eventually photodegrade, so fiber-
glass mesh is a good choice for any time interval of placement.
Determining the mesh size for litterbags often involves a tradeoff between
a fine enough mesh to minimize the loss of the litter material from the bag and
exclusion of macroinvertebrates. A mesh diameter of at least 2 mm is necessary
for macroinvertebrate access (Coleman et al., 1999; Swift et al., 1979), but such a
coarse mesh may allow extensive loss of detritus from the bags. We recommend
that fiberglass window screen with a mesh of 1.5 mm be used for most decompo-
sition studies. This is a reasonable compromise between minimizing the loss of
detritus, allowing access to most soil organisms, and maintaining approximately
ambient environmental conditions. This material is relatively inexpensive and
available at hardware stores.

Sample Preparation
Litter Material
It is essential for biologically meaningful results that the litter be reflective of the
decomposition processes and detrital stages that the researcher wants to actually
test. Senescent aboveground litter is obtained typically by either physically shak-
ing branches with leaves and/or reproductive parts about ready to fall naturally
or using litter traps. If litter traps are used, it is important to retrieve the mate-
rial frequently from the trap because it will be undergoing decomposition and
leaching due to any precipitation events. For herbaceous species, the aboveground
standing senescent material usually is used (i.e., standing thatch). Certain events
can cause the input of large amounts of nonsenescent litter into an ecosystem, e.g.,
large windstorms; that litter has very different properties than senescent litter.
It is very difficult to determine the belowground plant biomass, such as roots,
that is senescing but has not yet undergone extensive decomposition. Therefore,
fresh roots are typically used for litterbags. Usually researchers choose to use
litterbags with a single type of detritus from a single species, but mixed-species
litter bags can be used. Interestingly, decay rates in single-species litterbags are
 Organic Matter Mineralization and Decomposition 389

often nonadditive in terms of predicting decay rates and nutrient dynamics in

mixed-species litterbags (Gartner and Cardon, 2004), which suggests limitations
in extrapolating single-species litterbag data to natural conditions. Because of the
importance of the source material of the litter, it is essential that the researcher
report collection methods in any publication.
Any collected litter should be brought immediately to the laboratory and
spread out to air dry. The material should be completely air dry without major
changes in moisture content before being used to make litterbags. A subsample
should be oven dried at 60°C for determination of an initial oven dry/air dry
ratio. Oven-dried subsamples should be archived for any determination of initial
organic matter concentration, nutrient concentrations, and C quality analyses.

Litterbags
Litterbag material of appropriate dimensions is doubled over and the three open
sides sewn closed with nylon thread (polyester thread is sensitive to photodegra-
dation; Harmon et al., 1999) or Monel staples (a noncorrosive alloy). Litterbags are
typically about 20 by 20 cm in size.
About 10 g of litter is placed in each bag, and the actual amount of litter in
each bag is recorded to 0.01-mg precision. Approximately the same amount of
detritus is put into each bag.
Bags must be identified as to treatment with aluminum or plastic tags attached
with a UV-resistant material.

Procedure
It is advisable to make a subset of bags with the litter of interest and to transport
them to and from the field to test for any loss due to handling. An appropriate
correction can be made, but this loss term should be minimized in the first place.
Another advisable procedural control is to place litterbags filled with an inert
material in the plots. These litterbags can be retrieved to determine the amount
and type of any exogenous material that may have entered the normal litterbags.
Harmon et al. (1999) suggested polyester fiberfill as the inert substance for this
purpose. Corrections can be made if necessary, as discussed below.
Samples are normally placed in the field when the litter would normally fall,
e.g., autumn. Enough litterbags need to be placed in the field initially so that rep-
licate bags can be retrieved in a predetermined sequence and decay rates can be
calculated. A minimum of three litterbags should be retrieved at each time point,
but a more robust design is to retrieve four to six bags per time point. The sam-
pling interval will depend on the goals of the experiment and the climate of the
research site.
Litterbags should be placed in the appropriate environment where the detri-
tus type would normally decay. This is especially important in wetlands, where
water-table levels can change dramatically with time, which can have large effects
on decomposition rates (Day, 1983). Leaves, fine wood, and reproductive parts
are normally placed on top of the O horizon. Roots should be placed within the
soil profile at a depth reflecting the maximum root distribution in a vertical or
390 Bridgham & Ye

45° configuration unless the research is focused on a particular depth. A shovel

is used to make a narrow slit in the ground, the root litterbag is inserted, and
the ground tamped to ensure good contact between the soil and bag (Harmon
et al., 1999). Herbaceous material undergoes decomposition while in the “stand-
ing thatch” state before becoming part of the O horizon, so a decision needs to be
made which of these processes is to be studied, or separate sets of bags can be used
to study both.
It is important to adequately mark litterbags on the surface in a manner where
the marking material will not degrade or be consumed by animals (typical flag-
ging tape is inadequate for this purpose) so that individual bags can be found for
many years. Detailed site maps of bag placement relative to permanent fixtures in
the plot can be very helpful.
When litterbags are retrieved at each time point, they should be individually
placed in Ziploc or similar bags in the field. Take care to not dislodge material
from the bag, especially if larger mesh has been used.
Store samples in the laboratory at approximately 4°C before processing. Gen-
tly remove any material adhering to the outside of the bag. Cut open the bag and
place the contents, along with any material adhering to the inside of the bag, into
an appropriate container. Remove any living roots, and if the detritus was not
roots to begin with, also remove any dead roots because that is obviously external
organic matter that has gotten into the bags. Oven dry this material at 60°C for 48
h (or until constant moisture content) and weigh to the nearest 0.01 g.
If the litterbag material is heavily contaminated with mineral soil, then one
or more measures need to be taken. Simply scraping or rinsing the soil off of the
organic material is normally not sufficient. Sieving of the oven-dried material may
be attempted, but some soil contamination probably will remain. If litterbags with
inert material were placed in the field, these can be used to correct both decay rates
and mineralization–immobilization calculations for soil contamination, but there
may be high variability in this among litterbags. Therefore, for litterbag samples
with soil contamination, we recommend that calculations for decay rates use an
ash-free dry-mass correction (see calculations below). A similar correction can be
made for mineralization–immobilization rates in samples with heavy soil contam-
ination. Take the subsamples from the detritus before and after field incubation
to determine the ash-free dry mass percentage (= organic matter percentage) by
combustion at 450°C for 4 h (Cambardella et al., 2001) and the concentration of
any one of a number of numerous potential nutrients of interest using methods
described in Sparks (1996).
To correct for soil contamination, take several soil samples in the immediate
vicinity and at the same soil depth as the original litterbag placement at the time of
litterbag retrieval. Oven dry the soil sample at 60°C, and pass the sample through
a 2-mm mesh sieve. Determine the organic matter percentage and total nutrient
concentrations using the same methods as used for the litterbag material above.
 Organic Matter Mineralization and Decomposition 391

Comments
There is a rapid initial leaching of water-soluble components of the litter in the
first several weeks to months of deployment, followed by decay of the most
labile organic components during the next months to several years, followed by a
much slower loss of more complex organic compounds such as lignin, cutin, etc.,
over many years (Berg and Laskowski, 2006; Swift et al., 1979). If the goal of the
research is to examine the seasonal losses of the labile or soluble fractions, then
replicate bags should be retrieved at three to six time points during the first year,
e.g., 2 wk and 1, 3, 6, 9, and 12 mo. Subsequent time intervals can be spaced further
apart, with one to three time points per year. If only annual decay rates are to be
calculated, then samples need to be retrieved only on an annual basis, but then it
will not be known if the typical exponential decay model adequately fits the data.
The total organic C percentage may be used in place of the organic matter
percentage for both detritus and soil by using a commercial CHN analyzer. This
is particularly convenient because both N and organic C are measured in the
same procedure.

Calculations
The following calculations may be used for litterbag samples with little soil con-
tamination, such as would occur in samples incubated in peatlands or that have
remained in the O horizon in a mineral soil wetland. Decomposition is commonly
expressed either as loss of dry mass or ash-free dry mass, and calculations to derive
both units are presented below. Of course, the weight of any weighing vessel needs
to be accounted for, and that is assumed implicitly in the equations below.
Calculate the initial dry mass of detritus (DM0) in the litterbag as

SDM 0
DM 0 = AM 0 [1]
SAM 0

where AM0 is the air-dry mass of the detritus put into the litterbags, SDM0 is the
oven-dry mass of the subsample of that detritus that was used to determine the
moisture content, and SAM0 is the air-dry mass of that subsample.
Determine the percentage of organic matter in the litter (%LOM) as

æ SDM – Ash ö÷
%LOM = çç ÷÷100 [2]
çè SDM ÷ø

where Ash is the mass of the ash after combustion of an oven-dry subsample
of detritus.
Calculate the initial (LOM0) and final (LOMt) organic dry mass of litter in the
litterbag as

%LOM 0
LOM 0 = DM 0 [3]
100
392 Bridgham & Ye

and
%LOM t
LOM t = DM t [4]
100

where %LOM0 and %LOMt are the percentages of organic matter in the detritus
initially and at time t, respectively, and DMt is the measured dry mass of the litter
bag detritus when retrieved from the field at time t. As mentioned above, organic
C may be used in lieu of organic matter in these calculations.
The total mass loss due to decomposition is typically modeled as an exponen-
tial decay function (Olson, 1963):

DM t LOM t
= exp(-kt ) or = exp(-kt ) [5]
DM 0 LOM 0

where k is the decay rate [T−1], with year being the typical time interval reported.
The equation typically is solved by taking the natural logarithm of both sides
of the equation and graphing ln(DMt/DM0) or ln(LOMt/LOM0) on the y axis vs.
time on the x axis, in which case the slope equals the decay constant k.
The underlying assumption in the single exponential decay model is that the
absolute rate of decay decreases linearly with time, or a constant fraction of the
remaining detritus is decayed with time (Wieder and Lang, 1982). Decomposition
data do not always fit the exponential equation, and a number of other equations
have been proposed (Wieder and Lang, 1982).

Decomposition for Litterbag Samples with Substantial Soil Contamination

Litterbag samples sometimes have substantial contamination with mineral soil,
which obviously can cause erroneous decomposition estimates if appropriate
corrections are not made. The following calculations should be used for litterbag
samples with substantial soil contamination (Harmon et al., 1999):

%LOM t - %SOM t [6]

% Lt =
%LOM 0 - %SOM t

and
% Lt
LM t = DM t [7]
100

where %Lt is the percentage of litterbag material that is actually detritus and not
contaminating soil, %SOMt is the percentage of organic matter in the soil collected
at the same time that the litterbags were retrieved from the field, and LMt is the
mass of detritus in the litterbag at the time of retrieval, corrected for soil contami-
nation. This method assumes that the organic matter content of the litter remains
constant during decomposition. Although this may not be strictly true, it is prob-
ably much less problematic than the lack of any correction for soil contamination
of litterbags.
 Organic Matter Mineralization and Decomposition 393

Nutrient Mineralization or Immobilization

The net nutrient mineralization or immobilization percentage (%NM or %I) is
determined as
DM t % N t
%NM or %I = 100 [8]
DM 0 % N 0

where %N0 and %Nt represent the nutrient concentrations initially and at time
t, respectively. If this percentage is greater than 100%, then net immobilization is
being observed; if it is less than 100%, net mineralization is being observed.

DECOMPOSITION OF STANDARD SUBSTRATES

Decomposition is controlled by both the endogenous quality of the litter and exog-
enous environmental variables, often in complicated interactions (Bridgham and
Lamberti, 2009; Bridgham and Richardson, 2003). Standard litter substrates are
often used as an “index” to examine the role of environmental factors and/or dis-
turbance in controlling decomposition while holding litter quality constant. This
can be done using natural detritus with the litterbag technique discussed above
(e.g., Bridgham and Richardson, 2003) or using a standard manufactured sub-
strate such as cellulose (e.g., Bridgham et al., 1991).
Cellulose is often chosen as a standard substrate because of its importance as
an organic constituent and its very low nutrient concentration, which presumably
makes it more sensitive to exogenous nutrient availability (Goodman et al., 2010).
Cellulose filters can be placed in the field without any enclosure and retrieved
over time to determine mass loss. The cotton-strip assay has also been used exten-
sively, where the loss in tensile strength of standardized cotton cloth (typically
Shirley soil burial test fabric, Shirley Institute) is determined with time (Harrison
et al., 1988). Tensile-strength loss is typically much faster than the mass loss of cot-
ton strips, allowing rapid assessment of the decomposition function (Tiegs et al.,
2007). Use of the method was described extensively in Harrison et al. (1988).

SOIL HETEROTROPHIC RESPIRATION

Emissions of CH4 and CO2 are routinely measured in the field, but for CO2 this
does not represent soil heterotrophic respiration because of the large respiratory
contribution from plant roots. Hence, soil heterotrophic respiration is normally
determined by bringing fresh soil samples into the laboratory. If determination
of microbially mediated activities is to be reflective of in situ conditions, samples
should have minimal storage time, field conditions of temperature and moisture
during the incubation, and short incubation times.
Alkali absorption (AA) with subsequent titration to measure microbial
respiratory CO2 production requires minimal equipment, but it may over- or
underestimate respiration because of a decrease in the absorption efficiency of the
alkaline solution with time, slow absorption of CO2, or an increase in flux rates
394 Bridgham & Ye

from the soil due to low headspace CO2 concentrations (Bekku et al., 1997; Freijer
and Bouten, 1991; Zibilske, 1994). Moreover, the static trapping method may not
be suitable for soils with a pH > 7 because of increasing dissolution of CO2 into
solution as HCO3− under alkaline conditions (Bailey et al., 2008). Additionally, the
AA method cannot measure CH4 production, which is often of interest in wetland
soils. In well-equipped laboratories, the CO2 concentration is typically quantified
by either gas chromatography (GC) or infrared gas analysis (IRGA), and CH4 is
quantified by GC because of the sensitivity and accuracy of these instruments. We
discuss the AA and GC methods of measuring soil heterotrophic respiration in
static (i.e., nonflowing) systems, but the IRGA method is similar to the GC method
except for the difference in the instrumentation for measuring CO2.
Flow-through systems are sometimes used to measure heterotrophic soil respi-
ration because they prevent the buildup of CO2 in the headspace, which can inhibit
metabolic activity and reduce gas diffusion (Zibilske, 1994); however, flow-through
systems are significantly more difficult to operate than static systems, especially for
many simultaneous samples. Moreover, the AA method does not allow a buildup of
headspace CO2 because of its continuous absorption by alkali, and the GC method
accounts for dissolved CO2 and CH4 in solution (see calculations below).

Alkali Absorption Method

As the name suggests, the AA method is based on absorption of CO2 by alkali
in either solid or solution forms. A mass-based approach with soda lime was
described by Zibilske (1994). Here we describe a titration method using a KOH
solution to trap evolved CO2 from respiration (a NaOH solution is also commonly
used) by the following reaction:

2KOH + CO 2 ® K 2CO 3 + H 2O [9]

An excess supply of KOH is necessary for an accurate estimation of the CO2

production so that absorption efficiency does not decrease significantly during
the incubation.
At the end of the incubation, an excess of BaCl2 is added to the KOH solu-
tion to precipitate the carbonate as BaCO3. The amount of remaining KOH is
determined by titration with 1 mol L−1 HCl, with phenolphthalein as an indicator.
Because the amount of HCl consumed in the titration is known, the amount of
KOH left in the solution can be determined, and the amount of CO2 generated can
then be calculated.

Equipment and Instrumentation

• Anaerobic glove box (if anaerobic incubation conditions are desired)
• Incubator
• Balance with precision to 0.01 g
• Drying oven
 Organic Matter Mineralization and Decomposition 395

Materials and Reagents

• 20-mL glass vials (scintillation vials work well)
• 50-mL beakers
• Burette and stand
• Compressed O2–free N2 gas if anaerobic conditions are desired
• Cu tubing and gas-tight fittings can be used to construct a manifold, but in-
expensive plastic manifolds can be purchased also (part no. EW-30600-43,
Cole-Palmer). Cut the top off of plastic 1-mL syringes with a razor blade and
connect to the manifold with appropriate sized tubing to allow flushing of
incubation vessels.
• Soil incubation vessels of appropriate size that can form a gas-tight seal.
Canning jars (a.k.a. Mason jars) are an inexpensive choice that works well.
The jars must be drilled to accept a rubber septum. The septum must fit tight-
ly into the lid. Vacuum grease is useful to ensure a tight seal of the septum
to the lid and also to smear over the septum after puncturing with a needle.
• Rubber septa
• 1.0 mol L−1 HCl: to a 1-L volumetric flask containing 700 mL of deionized wa-
ter, slowly add 83 mL of concentrated HCl and bring to volume with deion-
ized water in a fume hood.
• 1.0 mol L−1 KOH: in a 1-L volumetric flask containing 700 mL of deionized wa-
ter, dissolve 56.1 g of KOH and bring to volume with deionized water. Make
sure that the bottle is tightly closed.
• 1 mol L−1 BaCl2: in a 0.5-L volumetric flask containing 400 mL of deionized
water, dissolve 183.2 g BaCl2×H2O and bring to volume with deionized water.
• 1% phenolphthalein indicator solution: dissolve 1.0 g of phenolphthalein in
100 mL of ethyl alcohol (95%).
• Vessel for weighing and oven drying
• Deionized water

Sample Preparation
Collect soil cores from the field to the desired depth (often the zone of maximum
plant root density) and place on ice. Wetland soils often have a water-table level
close to the soil surface, below which heterotrophic respiration is anaerobic. To
estimate in situ microbial respiration rates, intact cores should be taken with poly-
vinyl chloride tubes with rubber caps with metal compression bands to ensure a
tight seal. Use pore water from the site to maintain the water-table level in the core
at the same level as it would be in situ. Unsaturated portions of the soil column
should be incubated under aerobic conditions in the laboratory, and saturated por-
tions of the soil column should be incubated under anaerobic conditions. If an
anaerobic glove box is available, waterlogged samples should be processed in it
to avoid exposure to room air. If a glove box is not available, process waterlogged
samples in the air as rapidly as possible to minimize excessive exposure to O2.
Upon arrival in the laboratory, roots and other live plant parts are removed
to avoid plant respiration during the experiment. Pebbles >2-mm diameter and
large pieces of wood are also typically removed by hand, and then soil samples are
396 Bridgham & Ye

thoroughly homogenized. Keep storage of samples to a minimum, but if samples

must be stored for a short time do so at 4°C.

Procedure
Weigh out an ?10-g soil sample (dry weight equivalent) and place in an incu-
bation vessel. Add 10 mL of 1.0 mol L−1 KOH to a 50-mL beaker, fill a 20-mL
scintillation vial with deionized water (to maintain air humidity), and place both
into the incubation vessel. Cap the vessel and flush with O2–free N2 gas for 10
min by inserting a needle through the septum, with a second needle as an escape
valve. Record the time. For aerobic incubations, the vessels are flushed with room
air (CO2–free air and reagents may also be used for greater precision by running
an air stream through a 4 mol L−1 NaOH trap and bubbling reagents with N2 gas).
Blanks without soil samples are prepared in the same way. Because many samples
are typically handled during a short time, it useful to make a gas manifold so that
five to 10 samples can be flushed at a time.
Take two subsamples from each soil type and dry at 60°C for determination of
the initial dry mass equivalent of the samples.
Place the incubation vessels into an incubator at in situ temperatures, or at
some other temperature if in situ rates are not desired. Incubation intervals vary
depending on the study design, but 1 d to approximately 1 wk are common. At
the end of the incubation interval, quickly remove the beaker with KOH, pour the
KOH into a 20-mL vial, and tightly cap the vial for subsequent titration. Record
the time. If longer incubations are desired, place fresh KOH solutions into the
incubation vessels every 2 to 3 d. The vessels are then flushed and incubated as
described above.
For titration, add an excess of 1 mol L−1 BaCl2 and three drops of phenolphtha-
lein indicator into the solution and then titrate with 1.0 mol L−1 HCl. The HCl must
be added to the solution drop by drop to the endpoint where the solution color
just changes from pink to clear. Record the initial and final volumes of 1.0 mol L−1
HCl in the burette.

Calculations
Calculate the dry mass equivalent of soil (DM) in each sample:

SDM
DM = wet weight of sample ´ [10]
SWM

where SDM is the dry mass of the subsample that was oven dried and SWM is the
wet weight of that sample before drying.
Calculate CO2 production (g CO2–C kg−1 soil d−1) as

1.0 mmol HCl 1 mmol CO 2 12 mg CO 2 1 [11]

CO 2-C = ( HCl B - HCl S ) ´ ´ ´
mL 2 mmol HCl mmol CO 2 DM ´ t
 Organic Matter Mineralization and Decomposition 397

where HClB and HClS (mL) are the amounts of 1.0 mol L−1 HCl used to neutralize
the KOH solution in the blank and sample vessels, respectively, and t (d) is the
time of incubation.

Gas Chromatography Method

This GC method is similar to the AA method except for how gases in the head-
space are measured. Small volumes of headspace are periodically removed with
a syringe and injected into a gas chromatograph, which consists minimally of
an injection port, an inert carrier gas (usually He or N2), a separation column in
a heated oven, and one or more detectors. Different gases are separated in the
column, giving them unique retention times. Known gas standards are used to
determine the retention times and the concentration/peak area relationship of
the gases of interest. Thermal conductivity (TCD) and flame ionization (FID)
detectors are most commonly used for CO2 and CH4. A TCD can measure both
gases but is less sensitive than a FID, particularly for CH4. A FID can only directly
measure reduced gaseous C compounds, which includes CH4 but not CO2; how-
ever, a methanizer placed after the column can effectively reduce CO2 to CH4 so
the FID can measure both gases. Gas chromatographs can also be configured to
run the gas stream first through a TCD, which is nondestructive for samples, and
then through a FID. A gas chromatograph equipped with a FID with a metha-
nizer is our preferred choice. Relatively inexpensive gas chromatographs with
packed columns work fine for routine soil gas measurements and, in fact, may
be preferred over more expensive models because of their relative simplicity of
operation.
Calculations are given both for short-term incubations that more closely
approximate in situ rates and for long-term soil incubation potentials. In the lat-
ter method, CO2 for aerobic incubations or CO2 + CH4 for anaerobic incubations
is measured until the cumulative production begins to reach an asymptote that is
interpreted as the total amount of labile C in the sample (Updegraff et al., 1994).
The labile soil N pool also can be measured simultaneously with this method.
The only equipment and materials that are different than the AA method
above are mentioned here to avoid redundancy.

Equipment and Instrumentation

• Digital pressure meter
• Gas chromatograph with TCD or FID
• pH meter

Materials and Reagents

• Soil incubation vessels. Canning jars work well, but smaller gas-tight glass
containers with appropriate septa for headspace sampling may be used.
Many laboratories use serum bottles, but the small openings make washing
difficult. Wide-mouth I-CHEM jars (Thermo Fisher Scientific) also work well
and come in a variety of sizes.
• 1- and 5-mL syringes with Luer-Lock tips. Standard plastic syringes work fine
398 Bridgham & Ye

if headspace samples are immediately injected into the gas chromatograph

from the incubation vessel or storage container.
• 2-mL (if samples are to be stored) and 120-mL serum bottles, septa, aluminum
caps, and crimping tool
• Appropriate gas standards

Sample Preparation
Sample preparation is the same as for the AA method above. If only CH4 produc-
tion is to be measured, intact soil cores can be used, which reduces the disturbance
effect of removing roots.

Procedure
Weigh out an appropriate amount of the soil sample into the incubation vessel that
is being used. Typically 60 to 90% of the incubation vessel volume is headspace.
Prepare samples aerobically or anaerobically as for the AA method. Because no
procedural control typically is used in the GC method, make CO2–free headspaces
for aerobic incubation by running an airstream through a 4 mol L−1 NaOH solution.
Record the start time of the incubation.
Take two subsamples from each soil type and dry at 60°C for determination of
the initial dry mass equivalent of the samples (DM0), as in Eq. [10].
Determine the pH in a subsample of each soil type. For longer incubations,
the pH may have to be determined at multiple time points.
Place incubation vessels into an incubator at the desired temperature.
Sampling times vary depending on the goals of the study. In situ rates of soil
heterotrophic respiration are typically measured across time intervals <24 to 48
h. It is best to take multiple headspace samples during the incubation period to
ensure linearity of the flux, but researchers often take only a final time point for
short incubations after establishing the linearity of response in a pilot experiment.
If only a final sample is taken, it is essential to establish that the initial flushing
protocol removes all CO2 and CH4 from the headspace and pore water.
For determination of soil mineralization potentials (see calculations below),
incubations go for much longer (e.g., >1 yr; Bridgham et al., 1998). Aerobic incu-
bations should remain open to the air to avoid excessive buildup of CO2 except
for periodic closed incubations to determine 24-h rates. Placing glass wool in the
incubation vessel neck allows adequate air exchange but reduces moisture loss.
Nevertheless, the sample and container should be weighed and any water loss
compensated for by regularly adding deionized water.
In anaerobic incubations, the sample containers are left closed between sam-
pling times, and the cumulative amount of CO2 and CH4 produced during the
sampling interval is determined. Inhibitory concentrations of gases can build up,
however, so the samples should be sparged of all gases with an O2–free N2 stream
as detailed above after each gas measurement.
At each sampling interval, measure and record the pressure inside the incubation
vessel with a pressure meter, then withdraw 1 mL of gas sample from the head-
space with a 1-mL syringe and inject into the gas chromatograph. Record the time of
 Organic Matter Mineralization and Decomposition 399

injection. The baseline of the gas chromatograph should be stable before sample injec-
tion. Typical operating conditions are a 2-m Hayesep-D packed column, 20 to 30 mL
min−1 carrier flow, flame ionization detector at 380°C, and column at 60°C.
It is always best to inject headspace samples directly into the gas chromato-
graph, but headspace samples can be stored if necessary for 1 to 2 wk. Two-milliliter
serum bottles work well for gas storage after they have been previously flushed
with N2 and evacuated. A gas manifold makes bottle preparation much more
efficient. Inject at least 3 mL of headspace sample into the serum bottles to over-
pressurize. The vacuum will not be complete, so standards must also be made up
in serum bottles to account for gas dilution.
Make a standard curve for CO2 and CH4 using at least five points in 120-mL
serum bottles. Standards must incorporate the full range of the samples. Different
soil samples often have very different CH4 production rates, so sometimes it is nec-
essary to make low and high CH4 standard curves, with the high standard curves
including the full range of samples. Even with an excellent correlation coefficient,
a wide CH4 standard curve will often predict low-CH4 samples poorly.
Pure CO2 and CH4 can be used to make standards in serum bottles with N2
as a background gas, but this is laborious, error prone, and often requires a serial
dilution to make the low-concentration standards. Commercially made standards
(e.g., Air Liquide America Specialty Gases) are much quicker and easier to use,
and at least one such standard should be used to ensure that homemade standards
are correctly made. Gas chromatographs measure absolute amounts of a gas, so
different volumes of standards can be injected to make standard curves. Deter-
mine the actual volume of the serum bottles because the advertised volume is only
approximate. It is useful to overpressurize stand bottles so that multiple samples
can be taken, as standards remain good for several days. (For example, a 120-mL
bottle would be overpressurized by 10 mL, and the effective volume for dilution
calculations would be treated as 130 mL—see Ideal Gas Law below. This gives a
concentration corrected to the atmospheric pressure of the laboratory.)

Calculations
Example Standard Curve: The following standard curves were calculated
for nominal 120-mL serum bottles with an actual volume of 118.9 mL (Tables
20-1 and 20-2). The CH4 and CO2 tanks to make the standards were 99 and

Table 20-1. Dilutions from the 99% CH4 and 100% CO2. Note that all concentrations are corrected to
atmospheric pressure.

Stock CH4 or CO2 N2

—–—–—–—–—–—–—–—–—–—– mL —–—–—–—–—–—–—–—–—–—–
CH4
 1 (71,274 nL mL−1) 10 10
 2 (7127 nL mL−1) 1 19
CO2
 3 (71,994 nL mL−1) 10 10
400 Bridgham & Ye

Table 20-2. Dilutions of stocks to make working standards. Note that all concentrations are corrected to
atmospheric pressure.

CH4 stock CO2 stock N2 stock

to add to CH4 in working to add to CO2 in working to add to
Standard serum bottle standard serum bottle standard serum bottle
mL nL mL−1 mL nL mL−1 mL
1 0.1, Stock 2 5.1 0.75, Stock 3 388.7 19.15
2 0.25, Stock 2 12.8 1.0, Stock 3 518.3 18.75
3 0.5, Stock 2 25.7 1.25, Stock 3 647.9 18.25
4 1, Stock 2 51.3 1.5, Stock 3 777.5 17.5
5 1.5, Stock 2 77.0 2.0, Stock 3 1036.6 16.5
6 0.25, Stock 1 128.3 2.5, Stock 3 1296 17.25
7 0.65, Stock 1 333.5 3.0, Stock 3 1554 16.35
8 1.0, Stock 1 513.1 4.0, Stock 3 2073 15
9 commercially 100
prepared
10 commercially 504
prepared

100% pure, respectively. Dinitrogen gas was used to bring the serum bottles
to 20-mL overpressurization.

Sample calculation—Stock 1 CH4:

10 mL CH 4 ´ 0.99 ( 99% pure )´ 10 6

= 71,274 nL CH 4 mL- 1 headspace [12]
138.9 mL effective volume

Sample calculation—Working Standard 6 CH4:

0.5 mL of 71,274 nL CH 4 mL- 1 stock
= 77.0 nL mL- 1 [13]
138.9 mL effective volume

Rate Calculations: Corrections need to be made for the solubility of CO2 and
CH4 in water. Additionally, dissolved CO2 converts to H2CO3, HCO3−, and CO32− in
a proportion that is dependent on the pH. Total gas production is the sum of all
gaseous and dissolved phases.

Headspace Calculations for Carbon Dioxide and Methane:

The standard curves are used to convert reported peak areas from the GC into
volumetric concentrations. The examples below are given for CO2, but it is the
same for CH4.
Calculate the total volume of CO2 in the headspace:

nL CO 2 mL- 1 headspace ´ headspace volume ( mL ) = nL CO 2 [14]

To convert measurements to millimoles or micromoles, use the Ideal Gas Law:

 Organic Matter Mineralization and Decomposition 401

PV
PV = nRT ® n = [15]
RT

where n is moles of gas, P is pressure (kPa) inside the incubation vessel, V is the
volume of gas (e.g., from Eq. [14] above), R is the gas law constant = 8.31446 L kPa
mol−1 K−1, and T is the temperature (K).

nL CO 2 ´ P
µmol CO 2 = [16]
RT ´ 10 3

Pore Water Concentration for Carbon Dioxide:

Convert CO2 concentration (nL CO2 mL−1 headspace) to partial pressure (pCO2) by
multiplying by 10−6:

nL CO 2 mL CO 2
pCO 2 = ´ [17]
mL headspace 10 6 nL CO
2

The combined concentration of dissolved CO2 + H2CO3 in the soil solution is des-
ignated [H2CO3*] and is calculated with Henry’s law:

é H 2CO 3* ù = pCO 2 ´ K H,CO [18]

ë û 2

and

H 2CO 3* ( m mol ) = K H,CO ´ pCO 2 ´ pore water ( mL )´ 10 3 [19]

2

where KH,CO2 is Henry’s law constant (mol L−1 MPa−1). The pore water volume is
determined by the wet weight of the sample minus the dry mass of the sample
(Eq. [10]).
The amount of HCO3− in the soil solution is calculated as

K1 ´ H 2CO 3* ( m mol )
3 ( m mol ) =
HCO-
é H+ ù
[20]
êë úû

where K1 is the acid dissociation constant for H2CO3* and [H+] is calculated as the
negative log10 of the solution pH.
The amount of CO32− in the soil solution is calculated as

K 2 ´ HCO-3 ( m mol ) [21]

CO 23- ( m mol ) =
é H+ ù
êë úû
402 Bridgham & Ye

where K2 is the acid dissociation constant for HCO3−.

The values of KH, K1, and K2 are temperature dependent and can be calculated
with the following equations in low-salinity systems (Stumm and Morgan, 1981,
p. 204–206):

K H = 3.13 ´ 10- 4 T 2 - 2.50 ´ 10- 2 T + 0.763

K1 = - 6.90 ´ 10- 10 T 2 + 8.93 ´ 10- 8 T + 2.59 ´ 10- 6 [22]
- 12 - 10
K 2 = 9.28 ´ 10 T + 2.30 ´ 10

where T is the temperature (°C).

Pore Water Concentration for Methane:

Convert CH4 concentration (nL CH4 mL−1 headspace) to partial pressure (pCH4)
using Eq. [17]. As for CO2, Henry’s law is used to calculate the amount of CH4
in solution:

CH 4 ( m mol ) = KH,CH ´ pCH 4 ´ pore water ( mL )´ 10 3 [23]

4

where KH,CH4 is the Henry’s law constant (mol L−1 MPa−1) for CH4. The value of
KH,CH4 is temperature dependent and is calculated as (Clesceri et al., 1998, p. 6–31)

KH,CH = 8.18 ´ 10- 6 T 2 - 6.54 ´ 10- 4 T + 2.43 ´ 10- 2 [24]

4

where T is the temperature (°C).

Production Rates of Carbon Dioxide and Methane:

Total CO2 production from soil heterotrophic respiration (mmol kg−1 soil d−1) is
calculated as

Total CO 2 =
HS CO 2 ( m mol ) + H 2CO 3* ( m mol ) + HCO- [25]
3 ( m mol ) + HCO 3 ( m mol )
-

DM ´ t

where HS CO2 is the CO2 in the headspace, DM is the dry mass equivalent of the
soil sample, and t is time (typically in days). If multiple samples were taken dur-
ing the incubation, use a linear regression of the total amount of CO2 produced in
the headspace and solution on the y axis vs. time on the x axis, and the slope is the
above rate.
Total CH4 production (mmol kg−1 soil d−1) is calculated as

HS CH 4 ( m mol ) + dissolved CH 4 ( m mol )

Total CH 4 = [26]
DM ´t

where HS CH4 is the CH4 in the headspace.

 Organic Matter Mineralization and Decomposition 403

Long-Term Production Potentials

Occasionally graph the cumulative CO2 + CH4 production in samples on the y axis
vs. time on the x axis. As the curve begins to come to an asymptote, use a nonlinear
model-fitting protocol that exists in many statistical programs to fit the following
two-pool decomposition model (Bridgham et al., 1998; Updegraff et al., 1995):

X t = X o éê 1 - exp(-kt )ùú + ( TX - X o ) éê 1 - exp(-ht )ùú [27]

ë û ë û

where Xt is the cumulative amount of mineralized C at time t, Xo is the amount

of labile C, k and h are the decay coefficients for the labile and recalcitrant pools,
respectively, TX is the total amount of organic C in the soil, and TX − Xo is the
recalcitrant C pool size. Data sometimes fit better to only a single-pool decomposi-
tion model, in which case only the part of the equation dealing with labile C on the
right side of the equation is used.

PHOTODEGRADATION
During photodegradation, DOM is mineralized to CO2, but there is also a substan-
tial conversion of high-molecular-weight components of DOM to lower molecular
weight components, with corresponding changes in the optical properties of the
DOM (Larson et al., 2007). We present a methodology for the determination of
DOM mineralization to CO2 and the change in optical properties of the DOM due
to photodegradation. The SUVA254 (specific ultraviolet absorbance at the 254-nm
wavelength) is the absorbance of a sample standardized by its dissolved organic C
(DOC) concentration; it is often used as an index of DOM aromaticity (Weishaar et
al., 2003). A decrease in the SUVA254 thus represents photobleaching of the DOM
and a decrease in its aromaticity.

Equipment and Instrumentation

• Muffle furnace
• Spectrophotometer
• Autoclave

Materials and Reagents

• Amber glass bottles
• Pre-ashed Whatman GF/F filters (put in muffle furnace at 450°C for 3 h)
• Polycarbonate 0.22-mm filters
• Filtering apparatus
• Quartz spectrophotometer cells
• 7.5- by 18.5-cm (118-mL) Nasco Whirl-Pak bags
• 10% HCl water bath: dilute full-strength HCl 1:10 in a covered plastic container
404 Bridgham & Ye

Sample Preparation
Take surface water samples in amber glass bottles and store on ice for return to the
laboratory. It is essential that transport time be short between sample collection
and filtering. If this is not possible, consider filtering the samples in the field with
syringe filters.

Procedure
Soak all glassware in a 10% HCl bath followed by rinsing in deionized water.
Filter samples sequentially through the pre-ashed GF/F glass-fiber filter and
polycarbonate 0.22-mm filter (prerinse with 100 mL of deionized water to remove
contamination (Yoro et al., 1999) to filter-sterilize the sample. Autoclave the filter-
ing flasks beforehand.
Pour 100 mL of filtered samples into sterile Whirl-Pak bags, which allow the
entire spectrum of light through while minimizing air pockets. Place the bags in
the appropriate light environment of interest. Because ultraviolet radiation (UVR)
is the primary mechanism for photodegradation and UVR is rapidly attenuated
by DOM and foliage (Frost et al., 2006), this step is of obvious importance. It is
also best if the bags can be placed in an environment that allows passive mixing to
reduce self-shading of the sample water. Artificial light sources can also be used,
but it is essential then to know the spectral distribution of the light source and to
relate it to natural levels of UVR radiation.
Incubate the bags for 1 to 3 d, noting the exact incubation time. Use a control
set of bags that are covered with aluminum foil to prohibit any photodegradation.
Store samples in the dark at 4°C until determination of DOC concentrations and
SUVA254 in the light- and dark-incubated samples (DOCL and DOCD, respectively).
There are a number of automated and manual digestion procedures for determina-
tion of DOC concentrations from which to choose (Clesceri et al., 1998).
Determine the absorbance of the samples at 254-nm wavelength in quartz
cells with a 1-cm path length.

Calculations
Photodegradation of the DOM is calculated as
æ DOC - DOC ÷÷ö
DOM photodegradation ( % ) = ççç L D 100 [28]
çè DOC D ÷÷ø

absorbance at 254 nm
(
SUVA 254 L mg- 1 m - 1 = ) [30]
DOC concentration mg L- 1( )
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