Concept
CSIRO PUBLISHING
M. Kleber, Environ. Chem. 2010, 7, 320–332. doi:10.1071/EN10006
www.publish.csiro.au/journals/env
What is recalcitrant soil organic matter?
Markus Kleber
Department of Crop and Soil Science, Oregon State University, 3017 Agriculture and Life Sciences
Building, Corvallis, OR 97331, USA. Email: markus.kleber@oregonstate.edu
Environmental context. On a global scale, soils store more carbon than plants or the atmosphere. The cycling
of this vast reservoir of reduced carbon is closely tied to variations in environmental conditions, but robust
predictions of climate–carbon cycle feedbacks are hampered by a lack of mechanistic knowledge regarding the
sensitivity of organic matter decomposition to rising temperatures. This text provides a critical discussion of the
practice to conceptualise parts of soil organic matter as intrinsically resistant to decomposition or ‘recalcitrant’.
Abstract. The understanding that some natural organic molecules can resist microbial decomposition because of certain
molecular properties forms the basis of the biogeochemical paradigm of ‘intrinsic recalcitrance’. In this concept paper
I argue that recalcitrance is an indeterminate abstraction whose semantic vagueness encumbers research on terrestrial
carbon cycling. Consequently, it appears to be advantageous to view the perceived ‘inherent resistance’ to decomposition
of some forms of organic matter not as a material property, but as a logistical problem constrained by (i) microbial ecology;
(ii) enzyme kinetics; (iii) environmental drivers; and (iv) matrix protection. A consequence of this view would be that the
frequently observed temperature sensitivity of the decomposition of organic matter must result from factors other than
intrinsic molecular recalcitrance.
Introduction
It is widely accepted within the scientific community that
there is an easy logic that ties these three examples of recalcitrance together in a convincing causal relationship. This logic
assumes that (i) if a compound had a certain molecular property
like very low aqueous solubility or a high proportion of polycondensed aromatic moieties, then the compound would not be
decomposed during the early stages of a hypothetical incubation
experiment (ii) and would as a consequence achieve a long
residence time (iii) in the system.
But there are issues with this easy logic. For example, age is a
property that is physically independent of molecular composition. This can be illustrated by putting a perishable food item like
ice cream in a freezer. Freezing does not change the molecular
structure of ice cream, so when the ice cream is taken out of the
freezer on day zero þ X, it has aged but it still has the same
disposition of becoming decomposed as on day zero. Similarly,
the fact that OM persists in the presence of decomposer organisms does not automatically mean that it has resisted decomposition. It can, at least theoretically, have been left behind
because the decomposers deliberately choose not to decompose
it. A potato left on the plate after a family dinner is not left
uneaten because it has been able to resist the onslaught of the
hungry dinner participants, its persistence is the result of a
decision triggered by food-saturation, which was independent of
the molecular structure of the potato.
The scientific community is aware of the existence of
mechanisms other than recalcitrance that can protect organic
matter against decomposition.[25] Mechanisms of organic matter
protection like sorptive interactions with minerals and physical
separation from enzymes and decomposers[26] are treated as
independent of the intrinsic chemical properties of organic
carbon and are not considered here. Within the context of this
paper, the term ‘recalcitrance’ and its many synonyms such as
‘resistant carbon’ or ‘refractory organic matter’ are understood
Estimates for the amount of global soil carbon (0–300 cm) have
recently been raised from a previous 2344 pg[1] to a new datum of
43300 pg.[2] It is not well understood how this vast amount of
carbon will respond to environmental change. Models that have
coupled climate and carbon cycles[3] show a large divergence
in the size of the predicted feedback[4–6] between enhanced
decomposition of biospheric carbon pools and the heat content
of the atmosphere. One of the issues that remains to be resolved
for a more mechanistic understanding of carbon-cycle climate
feedbacks is the intensely disputed[7–12] temperature sensitivity of
soil organic matter decomposition. Of particular concern is the
sensitivity of the resistant pools of organic matter,[13] which are
often synonymously referred to as stable, recalcitrant or ‘old’.[9]
Compounds that are classified as recalcitrant or ‘stable’ are
assumed to comprise a much greater proportion of the total soil
carbon pool than those that are classified as labile.[14] Theoretical models predict greater sensitivity of recalcitrant compounds to rising temperatures,[14,15] but empirical studies do
not equivocally support this prediction.[7,16] Laboratory incubations tend to show a strong temperature response of recalcitrant
organic matter, whereas field studies show either a minor
temperature response or one that quickly re-equilibrates at a
low level.[17] Part of the problem may be that a large variety of
mechanistically independent interpretations of the terms ‘recalcitrance’ and ‘inherent stability’ can be encountered in the
literature (Table 1). Some authors, for example, understand
recalcitrance as a material property[15,18–21] based on molecular
structure (Table 1, item I1); another approach (Table 1, item I2)
considers ‘old’ organic compounds that have achieved long
mean residence time as recalcitrant[14,16,22] and a third view
defines recalcitrant organic matter operationally as such
that is decomposed during the later stages of an incubation
experiment[23,24] (Table 1, item III1).
Ó CSIRO 2010
320
1448-2517/10/040320
What is recalcitrant soil organic matter?
Table 1.
Concept
I. General and indirect
1. Molecular recalcitrance
2. Turnover time and age
II. Mechanistic
1. Quality concept
2. Energy content
3. Carbon oxidation state
4. Molecular size
III. Operational
1. Persistence in incubation experiment
2. Resistance to hydrolyses
3. Resistance to alkaline extraction
4. Resistance to chemical oxidation
List of definitions to characterise the recalcitrance of organic matter
Definition
Unit of measurement
Molecular-level characteristics of organic substances that
influence their degradation by microbes and
enzymes.[18,19,118]
Refractory soil organic matter is defined as a compartment that
is either considered inert, or has a very slow turnover time[119]
No generally accepted single value parameter
Quality ¼ number of enzymatic steps required to release
a carbon atom from an organic molecule as carbon
dioxide,[15,68] large number of enzymatic steps ¼ low
quality ¼ high requirement of activation energy
Amount of energy that is stored in bonds and can be released
upon oxidation and combustion, compounds are considered
as being labile when they are ‘energy-rich’[120]
Degree of oxidation of organic matter,[109,121] with lower
(more negative) oxidation state indicating higher
vulnerability to microbial decomposition
A molecule that cannot penetrate the microbial cell and is not
modified by an extracellular enzyme is recalcitrant[18]
Arrhenius energy: Ea (kJ mol 1)
Labile C is defined as C that is respired first. Arbitrary time
or mass criterion (C respired after X% of total have
been respired) separates labile from stable C[23]
Organic matter left after acid hydrolyses[78,122]
Organic matter left after an alkaline extraction
has been performed (humin)[123]
Labile fraction is defined as the organic matter that is
oxidised, stable fraction is defined as organic matter
that resists oxidation[124]
in a material property sense and do not refer to carbon that has
achieved long turnover time for reasons that are not related to its
chemical composition, that is, because it was protected against
decomposition by physical isolation from or through interactions with its environmental matrix.
There appear to be serious concerns that a large proportion of
organic matter in soils may have chemical properties, operationally termed recalcitrance, which render it particularly sensitive to feedbacks initiated by the expected rise of heat content
within the atmosphere. The carbon quality-temperature (CQT)
theory of the temperature sensitivity of organic matter decomposition links the temperature sensitivity of ‘recalcitrant’
organic carbon to the increase in activation energy that can
theoretically be expected when molecular structures become
more complex.[14,15] The classical Humic Polymer Model of soil
organic matter[27] explains recalcitrance as the consequence of
humification processes, which are thought to create large,
covalently bonded humic polymers.[28,29] But if soil organic
matter fragments are not large, complex, polymeric humic
macromolecules[30,31] and are therefore not intrinsically recalcitrant, there is no longer a molecular justifcation for the
assumption of complexity needed by the CQT theory to explain
higher activation energies and the resulting greater temperature
sensitivity of recalcitrant materials.
Attempts to clarify the issue and to predict the response of
recalcitrant organic matter to climate change are confounded
by a large uncertainty about the precise physical and chemical
determinants for the phenomenon of recalcitrance and by the
Time: t (years)
Bond energy (kJ mol 1)
Mean oxidation state Cox (dimensionless)
Moleculer weight (Dalton)
Carbon respired (mg CO2-C g soil
1
day 1)
Residual C (nonhydrolysable C) (percentage
of total)
Residual C (humin) (percentage of original
carbon concentration)
Residual C (oxidation resistant)(percentage
of original carbon concentration)
semantic indeterminacy of the category ‘recalcitrance’. It
recently has been suggested that contradictory evidence may
result, in part, from a large ambiguity and a lack of conceptual
rigidity within the complex problem space of organic matter
decomposition.[17] It appears therefore that continuing the practice of classifying organic matter as recalcitrant may adversely
affect progress in many areas, including the temperature sensitivity of decomposition, carbon-cycle climate feedbacks, carbon
turnover models and agricultural soil management. For example,
it has been suggested to sequester atmospheric CO2 in soils by
increasing the proportion of recalcitrant aliphatic compounds in
soil organic matter.[32] This would be a questionable strategy
if aliphatic compounds cannot be classified as recalcitrant, as
some recent experimental work suggests.[33,34] Consequently, the
objective of this manuscript is to highlight the lack of mechanistic
depth of the concept of recalcitrance, invite the reader to step
outside the conceptual box of recalcitrance and to point at
alternative ways of incorporating information about the molecular structure of organic matter into representations of soil carbon
turnover dynamics.
Background and relevance
Terminology
The multitude of non-numerical expressions used to convey
the presumed ability of organic materials to resist microbial
decomposition illustrates the difficulty to constrain a characteristic, which traditionally is thought to be of great consequence
321
M. Kleber
Recalcitrance as a mechanism of carbon stabilisation in soils
Recalcitrance has long been accepted as one of the major
mechanisms involved in the persistence of organic matter in soils,
next to interactions,[19] accessibility,[19] climatic stabilisation[25]
and facultative non-utilisation by the decomposer community.[47,48] Some authors even determined that chemical recalcitrance of largely pyrogenic carbon was the only mechanism in
their soils that could account for long turnover times of organic
matter.[36] This understanding is reflected in current soil carbon
turnover models[38,49] in which organic matter has been conceptualised as consisting of several functionally homogeneous
compartments decomposing at different rates following firstorder kinetics. Decomposition rates decrease continuously with
time, with ,10% of organic matter remaining after a decade
while some organic matter remains in soil for greater than millennia.[50] The supposition that the more carbon ages in a soil, the
more resistant against decomposition it becomes (‘new humus
decomposes faster than old humus’[51]) is an important characteristic of carbon turnover modelling concepts. A decrease in
decomposition rate with time is accounted for in some models by
the depletion of fast-decomposing pools and transfer of organic
matter into pools with lower rate constants.[38,49] Other modelling
approaches allow the rate of decomposition to decrease with time
as a function of decreasing substrate quality[52] or as an empirical
relationship.[53]
The decrease in rate of decomposition over time is frequently
viewed as resulting from either (i) the combined effects of the
gradual incorporation of carbon into the protective structural
fabric of soils and sediments (interactions and accessibility
in the model of Sollins et al.[19]); from (ii) biotic and abiotic
syntheses processes that fuse decomposition products into more
recalcitrant organic materials (humification or biochemical
protection in the model of Six et al.[54]) or as being caused by
(iii) preferential decomposition of more palatable compounds
(‘selective degradation’ in the model of Sollins et al.[19]).
Whereas mechanism (i) is independent of the molecular properties of organic matter, mechanisms (ii) and (iii) rely on the
supposition that some forms of organic matter are able to
withstand decomposition based on their chemical structure.
for the fate of organic carbon in the environment. Historically,
categories like ‘molecular recalcitrance’,[35] ‘intrinsic chemical
recalcitrance’,[19] or simply ‘resistant’ v. ‘labile’ carbon[8] have
been used to suggest that some organic materials in the biosphere
may have a greater ability than do others to resist microbial
attack.[21] The widespread use of these terms indicates a broad
scientific consensus that one or more material properties of natural organic carbon compounds can prevent the compound from
being decomposed. This assumption, the basis of general theories
to explain the turnover of soil organic matter,[25,27,36] is used as a
control on carbon turnover time in models of soil carbon turnover,[37,38] has been used to describe certain kinds of marine
dissolved organic matter[39,40] and has informed research on
mobile forms of refractory organic carbon in the terrestrial
environment.[41]
When scanning the large amount of literature on ‘recalcitrance’, the first issue is the absence of standardised use of the
concept. For example, the terms ‘recalcitrant organic matter’
and ‘refractory organic matter’ are often used synonymously in
biogeochemistry. However, whereas there is semantic overlap
between the two, the dictionary indicates an important difference in the conventions of use. The term recalcitrance has a more
behavioural connotation (resistant in the sense of active opposition), whereas the term refractory implies an acquired material
property (resistant in the sense of being immune, especially,
being resistant to heat, see ‘recalcitrant’ at www.merriamwebster.com/dictionary/recalcitrant and ‘refractory’ at www.
merriam-webster.com/dictionary/refractory, both accessed 28
June 2010). The latter convention has made its way into the
Standard Terminology chosen by the American Society for
Testing and Materials (ASTM). The ASTM C71 standard[42]
defines refractory materials as ‘non-metallic materials having
those chemical and physical properties that made them applicable for structures, or as components of systems, that are
exposed to environments above 10008F (811 K; 5388C)’.
The term ‘refractory’ appears to be favoured by that part of
the community of biogeochemists who work with thermally
altered forms of organic matter like charcoal, but it also is
encountered in situations where plain resistance to microbial
decomposition is discussed.[43] Recalcitrance has been made
popular as a concept in soil microbiology in the early 1960s
by Alexander,[35,44,45] who defined the term as ‘a stubbornness
on the part of specific molecules to succumb to microbiological
attack’.[35] This early view has been refined to ‘Recalcitrance
comprises molecular-level characteristics of organic substances
including elemental composition, presence of functional groups,
and molecular conformation, that influence their degradation
by microbes and enzymes’,[19] implying that there are certain
material properties of organic molecules that allow them to
actively resist microbial degradation. More recently it has been
posited that recalcitrance mainly accounts for protection on
shorter time scales[46] and that it may be necessary to distinguish
between ‘primary recalcitrance of plant litter and rhizodeposits
as a function of their indigenous molecular characteristics, and
the secondary recalcitrance of microbial products, humic polymers and charred materials’.[26] Besides revealing the struggle
to find an adequate denominator for an enigmatic phenomenon,
the non-numerical character of this latter definition also illustrates the strong contrast between the ‘hard’ formalisms (known
algorithms) of physical chemistry and related science disciplines[17] and the ‘soft’ semantic category of recalcitrance,
which lacks specifity and cannot easily be expressed using
numerical categories.
Recalcitrance and litter quality
The plant litter available to the decomposer community
encompasses a broad range of tissues that differ in chemical and
physical properties.[55] Decay rates of different plant organs
reflect this diversity: fruits decompose faster than leaves, which
in turn decompose much faster than woody plant parts.[55,56] The
reader is referred to the review of Sanderman and Amundson[57]
for references to a wealth of studies illustrating the point that
the chemical composition of plants plays a role in determining
the decomposition rate. This observation is often articulated by
calling woody plant parts more recalcitrant, following the notion
that materials with slow turnover behave in this way because
they are able to resist decomposition.
Generally, tissues with higher lignin, polyphenol and wax
contents and higher lignin : N and C : N ratios decompose more
slowly. Aber et al.[58] found that the ratio of lignin : cellulose
(lignin–cellulose index, LCI) was a good predictor of the
decomposition rate k of plant litter, and substrate quality
measured as lignin content was the primary control on the
decomposition rate of plant litter in coniferous forests.[59] But
lignin content was not a major factor in a study of global patterns
of root decomposition, where Silver and Miya[60] found that
322
What is recalcitrant soil organic matter?
100
14
C remaining (percentage of 14C added)
calcium concentrations and C : N ratios of root materials
together explained 89% of the variability of decomposition
rates in a multiple linear regression. It is not plausible that lignin
should be a control on the aboveground decomposition of plant
litters but have no influence on the belowground decomposition
of roots. If methodological difficulties of lignin analysis[61] are
ruled out as an explanation for contradictions of this kind, then
there must be other system properties with the ability to override
lignin content as a ‘quality control’ on decomposition. It can be
summarised that the chemical composition of litter unquestionably influences its decomposition rate; it does not do so because
it renders the substrate chemically invulnerable, it merely determines the complexity of the decomposition operation. To an
extent this was recognised in a recent analyses of the relevance
of recalcitrance for carbon turnover in soils, which posited that
‘molecular recalcitrance of natural OM is not absolute, but
relative’.[26] The same study proposed to differentiate between
primary and secondary recalcitrance – the former refering to the
influence of indigeneous molecular characteristics on variations
in turnover time of plant litter and rhizodeposits, the latter
comprises products of secondary microbial syntheses, humification processes and thermally altered materials.[26]
90
80
70
60
50
7 ⫽ Ring C of caffeic acid in polymer
6 ⫽ 14COOH of caffeic acid in polymer
5 ⫽ Ring C in vanillic acid
4 ⫽ Ring C in caffeic acid
3 ⫽ Ring C in benzoic acid
2 ⫽ Amino acids, glucose
1 ⫽ 14COOH in p-hydroxybenzoic acid
40
30
20
10
0
1
2
3
4
5
6
7
Compound
Fig. 1. Polymerisation stabilises organic matter against decomposition.
Decomposition of specifically 14C labelled benzoic and caffeic acids, caffeic
acid linked into phenolic polymers and some other simple organic compounds in a Greenfield sandy loam. Bars indicate amount of 14C that remains
in the soil after 12 weeks of incubation. Grey bars indicate monomers, black
bars indicate polymers. Redrawn using data from Haider and Martin.[65]
Recalcitrance and molecular complexity
The idea that some molecular characteristics of organic matter,
especially its molecular complexity and degree of polymerisation, should be linked to its susceptibility to decomposition
can be traced back to the early decades of the 20th century[62]
and received strong support from the work of Martin et al.[63–66]
These scientists introduced 14C into the molecular structure of
phenolic acids and artificially prepared phenolic polymers. This
allowed them to identify the molecular origin of the carbon in
the 14CO2 that evolved during decomposition of these molecules. In a seminal publication, Haider and Martin[65] showed
how linkage into polymeric structures was able to prolong the
residence time of organic carbon (Fig. 1) in 12-week incubation
experiments.
The evidence obtained by Martin and co-workers can be
interpreted to indicate that labile carbon may become stabilised
through abiotic or biotic syntheses into polymeric molecules
that are necessarily more complex than the monomers from
which they originate. The syntheses of polymeric molecules –
assuming this to be a quantitatively significant process in soils
and sediments – would necessarily take some time. This in turn
would suggest that the longer an organic matter fragment
persists in soil, the more likely it is to become resistant to
decomposition as a consequence of being involved in some
polymerisation reaction incorporating it into a more complex
molecule. This idea was paraphrased by Stott and Martin[51] as
the paradigm of ‘old humus is more stable than young humus’.
Classical humification theories[67] can be seen as attempts to
propose potential chemical pathways that would create such
complex polymeric structures in soils.
number of enzymatic steps required to release as carbon dioxide a
carbon from an organic compound. The larger the number of steps
the lower is the quality of the carbon atom’. It can be shown that
decomposing a complex molecule requires more enzymatic steps.
Since each of these enzymatic steps has a characteristic, reactionspecific, temperature dependent activation energy (Ea), the total
activation energy required to decompose a complex molecule is
higher than that for one of its monomers. Thus the carbon-qualitytemperature hypothesis assumes that the enzymatic reactions
required to metabolise structurally complex, low-quality C substrates should have a higher net activation energy than reactions
metabolising C substrates that are structurally simpler.[68] As the
net activation energy increases, the temperature sensitivity as
expressed by the Q10 value should also increase (Eqn 3).
There are three important corollaries of this approach: it
(a) allows the general term ‘quality’ to be expressed in a numerical
fashion by connecting it to the thermodynamics of an enzyme
catalysed reaction, it (b) provides a mechanistic explanation for
the observed decrease in decomposition rate (Fig. 1) with
decreasing substrate quality, and most importantly, it (c) suggests
that the decomposition rate of low quality substrates (¼ complex
polymeric macromolecules) should have a stronger temperature
dependence than that of high-quality substrates (¼ simple monomers) – always assuming quality to be a function of complexity,
and complexity to result from the polymerisation of simple, labile
compounds into more complex and more stable compounds.
According to the Arrhenius-theory,[69] the relation between
the sensitivity of a reaction to a temperature increase (Q10) and
the activation energy for this reaction (Ea) can be obtained by
combining Eqns 1
The carbon quality-temperature (CQT) theory
Because complex geopolymers decompose at a slower rate than
the monomers from which they originate, they can be perceived
as having a lower quality as a substrate for microbial decomposition than the monomers. The term ‘quality’ is rather general
and not easily parameterised, which is an obstacle to its incorporation into numerical models. To alleviate this disadvantage,
Bosatta and Ågren[15] proposed to represent quality as ‘the
Q10
323
T 10T
2
1
k2
¼
k1
ð1Þ
M. Kleber
forever[25,50] – unless it is protected against decomposition.
Such protection can be very powerful, as illustrated by reports of
charcoal in Silurian sediments.[77]
and 2
Ea ¼
to define
T1 T2 R
k2
ln
T2
T1 k1
Q10 ¼ e
10Ea
RT1 T2
ð2Þ
Activation energy as a measure for recalcitrance
Todays atmosphere contains ,21 wt% of oxygen, which is the
second most powerful electron attractor in the periodic table.
This fact brings up the question: why is all the electron-rich
reduced carbon in the biosphere not spontaneously reacting with
this abundance of oxygen to satisfy the 2nd law of thermodynamics and achieve greater entropy by converting few highly
organised, energy rich, condensed organic biomolecules into
many energy-poor, gaseous and thus more disordered CO2
molecules? The transformation of organic macromolecules into
carbon dioxide requires that the chemical bonds of the former be
broken first, a process that requires an initial input of activation
energy (Ea). As long as this energy investment is not made, no
reaction will take place and the compound will appear stable or
‘recalcitrant’. Activation energies are specific for given combinations of reactants and catalysts, but they can be modified by
the response of the catalyst to environmental conditions such
as substrate availability[71] and variations in pH. Experiments
carried out on the trypsin-catalysed hydrolysis of amino acid
esters illustrate this point. A histidine in the basic form is an
essential part of the catalytic site of this enzyme. The activation
energy at pH 6.0 is 76 kJ mol 1 and at pH 7.5 (when the histidine
is predominantly in the basic form) it is 46 kJ mol 1.[69] Activation energy requirements for enzyme-catalysed decomposition reactions will therefore change as a function of the
susceptibility of the catalyst towards variations in environmental conditions like pH.
ð3Þ
with Q10 ¼ a dimensionless number representing temperature
sensitivity (¼ the change in decomposition rate k for a temperature difference of 10 K under otherwise constant conditions[70]) and k1 ¼ reaction rate (often expressed as mol CO2
evolved per time unit) at temperature T1 (K), k2 ¼ reaction
rate at temperature T2 ¼ T1 þ 10 K; R ¼ the gas constant
(8.314 J mol 1 K 1) and Ea ¼ activation energy.
Varying Ea in Eqn 3 illustrates that a compound with low
Ea will necessarily exhibit a lesser response to a temperature
increase than will a compound with a high Ea. With more heat in
the atmosphere, more energy will be available to overcome
activation energy barriers for decomposition reactions with high
activation energies. Since the increase in reaction rate (¼ the Q10
value) is larger for reactions with high activation energies
(that is, for reactions that require a large energy investment
before they can proceed), materials requiring high Ea will show
a more pronounced response to rising temperatures (¼ will
decompose disproportionately faster) than materials requiring
low Ea.[14]
Factors like substrate availability,[71] interactions of organic
compounds with the abiotic matrix,[7] and freezing, drought, or
flooding[14] have been shown to affect apparent temperature
sensitivities of organic compounds. Yet it has become quite
common to equate high Ea values of organic materials with
‘chemical recalcitrance’,[14,72,73] and to divide soil organic
matter into labile and ‘more resistant’ fractions based on
observed Q10 values.[23]
Recalcitrance an inherent property?
If recalcitrance is defined as inherent biochemical stability,[54]
that is if it is seen as a molecular property that operates independent from interactions with the abiotic environment,[19] then
the ability of an organic compound to resist microbial decomposition is conceptualised as a characteristic that rests completely with the organic compound in question (¼ an ‘intrinsic’
or ‘material’ property). The implication is that the persistence
of an intrinsically recalcitrant compound would not be affected
by changes in environmental conditions, whereas a protected
compound becomes a ready substrate as soon as the protective
mechanism ceases to operate. By definition, an inherently stable
compound must exhibit resistance to decomposition regardless
of the environment in which it is placed. However, abundant
observational data for the biodegradation of pesticides in soils
have long shown that degradation rates of the same chemical can
vary greatly depending on extrinsic factors like clay content, pH
and microbial community composition that are not at all related
to the molecular composition of the compound in question.
Recently, Feng and Simpson[72] showed that activation energies
and Q10 values of lignin Vanillyl, Syringyl and Cinnamyl units
varied when they were incubated in two different grassland soils
from the Canadian prairie. The same lignin unit qualified as relatively recalcitrant in one soil (Ea ¼ 88.3 kJ mol 1 and corresponding temperature sensitivity of Q10 ¼ 3.5 at 158C) but showed
a decidedly more labile character in another (Ea ¼ 49.4 kJ mol 1
and Q10 ¼ 2.0, also at 158C). Significantly, the magnitude of
change in activation energy was not constant for the three
compounds, but amounted to a factor of 1.8, 2.3 and 1.4 for
lignin Vanillyl, Syringyl and Cinnamyl units respectively.
Challenging the concept of recalcitrance
Basic thermodynamics
The second law of thermodynamics commands that energy
spontaneously flows only from being concentrated in one place
to becoming diffused or spread out, not vice versa. Consequently, reviews[74] and textbook chapters[75] addressing the
cycling of organic matter in the environment typically introduce
natural organic matter as a thermodynamic anomaly dependent
on life processes. Life is recognised as a situation where matter
is in a state of particularly high order and thus, low entropy.
The 2nd law prevents this state from becoming permanent; it
forces organisms to continuously process energy to escape
disintegration into disordered systems.[75] To counteract their
inevitable energy losses, living organisms are forced to procure
highly ordered molecules that have low entropy, high energy
content and high free energy and to convert them into disordered
molecules with high entropy, low energy content and low free
energy.[76] Once life terminates, the 2nd law demands that
energy stored in the reduced carbon constituents of dead
organisms be dissipated throughout the system. Dead reduced
carbon is forced to react with suitable electron acceptors (like
O2, NO3 , Feþ
3 and others) to form molecules with higher
entropy and lower free energy. The 2nd law of thermodynamics
thus creates a boundary condition for the assumption of
inherent stability by determining that no carbon will stay in soil
324
What is recalcitrant soil organic matter?
Table 2. Relation between refractory nature and stability in soils as assessed by Derenne and Largeau[78]
Compound
Refractory nature based onyA
Stability in soilsA
Lignin
Sporopollenins
Exhibits a higher resistance to microbial degradation than cellulose
Survive drastic nonoxidative treatment
Algaenans
Conspicuous resistance to degradation observed for both drastic
laboratory hydrolyses and microbial attack
Fossil remains of suberised tissues with well preserved morphologies are
observed commonly in sedimentary materials of terrestrial origin
Proanthocyanidin polymers are resistant to acid and base hydrolyses,
phlorotannins also exhibit a high resistance to nonoxidative
chemical degradation
BC particles can survive harsh thermal and chemical oxidations
as well as photooxidations
Protein-N is usually considered labile
No significant preservation occurred in arable soils
Some of the tested spores and pollen disappeared
within years in various types of soils
No clear-cut evidence of contribution to soil
organic matter
No clear-cut evidence for preservation in soils
Cutans and Suberans
Tannins
Black carbon
Protein
A
Not assessed (decomposition in litter reported to be
near complete within weeks[125])
Charcoal retrieved from a soil depth of 2 m reported
to be 8800 years old
Preservation as cross-linked melanoidin-like
materials[126] or through association with
mineral surfaces[127]
Quoted from or as assessed by Derenne and Largeau[78] unless otherwise indicated.
formed mainly by enzymatic depolymerisation and oxidation of
plant biopolymers. These reactions are thought to transform the
originally nonpolar aromatic and lipid plant components into
amphiphilic molecules. These amphiphiles form membrane-like
aggregates on mineral surfaces and micelle-like aggregates in
solution. Individual molecules within supramolecular aggregates are pushed in place by entropic interactions with the polar
solvent water and are not covalently bonded. Since the Molecular Aggregate Model does not provide a structural reason for
inherent stability against decomposition, the fundamental difference between the ‘Humic Polymer’ concept and the ‘Molecular Aggregate’ model is that the latter does not invoke the
creation of refractory phases with extended turnover time.[81]
The experimental evidence from the recent past fails to identify
distinct humic molecules in soils[84] and in alkali extracted
humic substances.[85] In addition, alkaline extracts recently have
been found to decompose on an annual time scale.[86] In combination with the Molecular Aggregate Model, these indications
challenge the scientific bases of the CQT theory, where the
temperature sensitivity of ‘recalcitrant’ organic carbon is
explained through an increase in molecular complexity with
progressing decomposition state.[14,15]
Clearly, the decomposition of these lignin fragments was, to a
large extent, controlled by other factors than their ‘intrinsic
molecular properties’.
Resistance to chemical degradation and decomposability
The issue of the decomposability of seemingly refractory
materials was discussed in a review by Derenne and Largeau,[78]
who defined the adjective ‘refractory’ to mean being ‘insoluble
and nonhydrolysable in a laboratory procedure’. This approach
assumes that an organic compound that can resist rather violent
chemical treatment (involving, for example, boiling in 6 M HCl
as in acid hydrolyses) should be expected to be able to resist the
supposedly much less aggressive natural decomposition process
as well. Derenne and Largeau[78] found that of the seven major
classes of supposedly refractory biopolymers investigated
(Table 2), only thermally altered carbon showed the presumed
relationship between being refractory (¼ insoluble and nonhydrolysable) in the laboratory and being able to persist in a soil
environment.
Complexity and recalcitrance as the result of humification
Classical carbon turnover models assume that the component
molecules of soil organic matter are produced from degradation
products by secondary syntheses reactions or so-called humification processes.[79] This assumption has led to the Humic
Polymer Model of soil organic matter,[80] in which the component molecules are depicted as large, covalently bonded
(‘humic’) polymers with unique chemical structures that are
different from those of the starting materials. Consequently, the
Humic Polymer Model implies inherent resistance of so-called
humic substances to decomposition.[81] This model forms the
basis of a huge literature (see reviews by Haider et al.,[82] Stott
and Martin,[51] Huang and Hardie[79]) on methods to synthesise
model humic substances like the ones that Haider and Martin[65]
used to demonstrate how carbon in polymers decomposes more
slowly than carbon in monomers (Fig. 1). The fact that scientists
managed to reproduce some of the characteristics that they
observed in alkaline humic extracts in laboratory settings was
taken as evidence that they had been successful in reproducing
the mechanisms operating in nature. The competing Molecular
Aggregate Model[83] assumes that organic materials in soils are
Slow turnover without recalcitrance
Old organic matter may contain microbial polysaccharides and
proteins,[87–89] indicating that chemically labile organic matter
can persist for long times in soils.[26] This observation is often
explained by matrix protection through sorptive interactions and
reduced accessibility,[25] but recent investigations have revealed
that significant quantities of organic material in soil may persist
in spite of being chemically labile, unprotected, accessible and
decomposable.[48] Using modified Michaelis–Menten kinetics,
Schimel and Weintraub[90] showed that the reactivity constant of
a substrate (¼ its decomposability) cannot by itself induce limitation of the decomposition rate as long as enzyme concentration
is a term in the reaction rate equation. Above a given enzyme
concentration, enzymatic products from substrate decomposition
are insufficient to meet the cell energy demands associated with
enzyme syntheses. This means that microbial substrate decomposition can be strongly limited even if substrate availability is
unlimited. In such a case the decomposability of a substrate does
325
M. Kleber
(a)
Unaltered plant
material
Transition
char
Amorphous
char
Composite
char
Turbostratic
char
1.0
Relative phase distribution
Amorphous
lignin
Crystalline
cellulose
Turbostratic
crystallites
Amorphous
hemicellulose
Pore space
0
100
Ash
Volatile matter
wt %
(b)
Pyrogenic
amorphous carbon
Non-volatile matter (fixed-C)
Char yield
0
100°C
Charring intensity
700°C
Fig. 2. Dynamic molecular structure of charcoal across a charring gradient and schematic representation of four char categories and their individual phases as
proposed by Keiluweit et al.[128] (a) Physical and chemical characteristics of organic phases. Exact temperature ranges for each category are controlled by both
charring conditions (i.e. temperature, duration and atmosphere) and relative contents of plant biomass components (i.e. hemicellulose, cellulose and lignin).
(b) Char composition as inferred from gravimetric analysis. Yields, VM, fixed-C and ash contents are averaged across wood and grass chars. Relative
contributions above 7008C are estimates. Source: Keiluweit et al.[128]
not determine its fate: labile organic matter may well be left
behind. This is only one of many mechanisms with the potential to
preserve organic carbon without recalcitrance, more possibilities
are discussed in mechanistic detail elsewhere.[47,91,92]
other words, the likelihood that the decomposer community has
the full suite of required enzymes available to decompose the
multitude of thermally altered phases produced by any fire event
is rather low, a fact that has been experimentally verified by
demonstrating decreasing decomposition rates with increasing
charring temperatures.[98] On the other hand, there can be
no doubt that charcoal has been around long enough in earth
history[77] to give the decomposer community ample chance
to develop enzymatic pathways that can decompose even
highly altered pyrogenic carbon. Observational evidence for
the decomposability of charcoal has accumulated over the last
years[99–104] and found its most recent confirmation in a study
from Scandinavia showing a much lesser mean age of charcoal
in forest soils than would have been expected based on fire
frequency since the onset of the Holocene.[105]
Thermally altered organic matter
Pyrogenic carbon differs from other natural organic matter in
that it has gone through a thermal transformation process after
its precursor materials were assembled by enzymatic processes.
In uncontrolled natural fires, the outcome is a stochastic mix of
products with properties that are novel to the environment. Thus
the decompository challenge associated with charcoal originates
from the fact that it does not represent a well defined chemical
compound or structure; rather, it represents a dynamic molecular property space within which molecular structure and
crystallinity vary with the conditions under which the char was
created. Depending on charring intensity, charcoal may contain
individual polycondensate aromatic elements of increasing
size, which eventually grow into graphene sheets that are then
arranged to form increasingly complex turbostratic crystallites
(Fig. 2).
Since natural fires differ in intensity, duration, precursor
materials and many other factors,[93–96] the resulting palette
of often highly alkaline chars and ashes will typically be rather
diverse as well.[97] These considerations suggest that the eventspecific combination of charring products is likely to be
somewhat distant from the mainstream of catabolic pathways
for common microorganisms even in fire-prone ecosystems. In
How molecular characteristics affect decomposition
of organic compounds
Several molecular features have been proposed as having the
ability to render natural organic carbon compounds refractory,
including molecular size,[18] aqueous solubility or polarity,[26]
aromaticity,[106] aliphaticity expressed as alkyl : O-alkyl
ratio,[107] molecular complexity,[15] certain N-containing substituents and functional groups[26] and many others. Fierer
et al.[68] published the results of an incubation experiment that
allows testing the effect of variations in some of these features
(Table 3). To do so, respiration rate constants (B-values) from
Fierer et al.[68] were plotted as a function of aqueous solubility
326
What is recalcitrant soil organic matter?
Table 3. Molecular characteristics of the carbon compounds incubated by Fierer et al.[68]
Compound
Formula
Citric acid
C6H8O7
BondsA
Structure
OH
O
O
C
CH2
HO
C
CH2
C
OH
Molecular
Aqueous
O/C C oxidation
mass
solubility
ratio
stateB
(g mol 1) (g 100 mL 1)
C–C
C–OH
C¼O
192
133
1.17
1
C–C
C–OH
C–O–C
180
91
1
0
C–C
C–OH
C–O–C
342
21.6
0.92
0
C–C
C–OH
C¼O
C–C
C–OH
C¼O
Arom. C
116
1
1
E0.8
0.43
0
C–C
C–OH
C¼O
Arom. C
1701
280
0.61
0.52
C–OH
Arom. C
110
0.33
0.33
C
Glucose
O
HO
OH
C6H12O6
O
HO
HO
Lactose
OH
OH
OH
C12H22O11
O
OH
OH
OH
O
OH
O
OH
OH
OH
OH
Succinate
C4H4O4
⫺
OOC
COO⫺
p-Hydroxybenzoic acid C7H6O3
Tannic acid
C76H52O46
OH
HO
HO
OH
OH O
OH
HO
O
OH
O
O
O
OH
O
O
O
HO
OH
O
O
HO
138
E10
O
O
O
O
O
O
HO
O
OH
O
OH
HO
O HO
HO
OH
OH
O
HO
OH
O
HO
Catechol
C6H6O2
OH
HO
OH
OH
0.043
A
Other than C–H (occurs in every molecule).
Oxidation state calculated according to Hockaday et al.[109] as Cox ¼
B
ð2z
yÞ
x
with x, y, z corresponding to CxHyOz.
(Fig. 3a), molecular mass (Fig. 3b), carbon oxidation state
(Fig. 3c) and atomic oxygen:carbon ratio (Fig. 3d).
Aqueous solubility and molecular mass did not explain any
variability of decomposition rate-constants. In addition, they
varied independent of each other: in the case of tannic acid,
solubility was high (this should, in theory, facilitate decomposition) when molecular size was large (this should, in theory,
make a molecule more recalcitrant). Thus neither parameter
appeared to be suitable as a predictor for the decomposability of
the organic compounds in this experimental setup. The molecular complexity of the compounds, approximated as the number of different bonds within the molecule (Table 3), varies
within the limited range of 3 and 5, with the compound that had
the highest Q10/Ea (catechol) being the least complex in terms of
diversity of bonds. Catechol also has the lowest molecular mass
of the seven compounds. Thus, an examination of what can be
327
M. Kleber
(a)
(b)
0.12
0.12
0.10
Citric acid
B (µg C-CO2 g⫺1 soil h⫺1)
B (µg C-CO2 g⫺1 soil h⫺1)
0.10
0.08
Lactose
0.06
0.04
Glucose
Succinate
p-Hydroxybenzoic acid
0.02
Citric acid
0.08
Lactose
0.06
0.04
Glucose
Succinate
p-Hydroxybenzoic acid
0.02
Tannic acid
Catechol
0.00
0.00
0
50
100
150
200
250
300
350
500
Aqueous solubility (g 100 mL⫺1)
1000
1500
2000
Molecular mass (g mol⫺1)
(c)
(d)
0.12
0.12
0.10
0.10
Citric acid
B (µg C-CO2 g⫺1 soil h⫺1)
B (µg C-CO2 g⫺1 soil h⫺1)
Tannic acid
Catechol
0.08
Lactose
0.06
0.04
0.02
0.00
⫺1
Glucose
Succinate
p-Hydroxybenzoic acid
Tannic acid
Catechol
Citric acid
0.08
Lactose
0.06
0.04
p-Hydroxybenzoic acid
Glucose
Succinate
Tannic acid
0.02
Catechol
0.00
0
1
Oxidation state
2
0.2
0.4
0.6
0.8
1.0
Oxygen/carbon ratio
1.2
Fig. 3. Decomposition rate B as a function of molecular properties for seven organic C compounds added to a sandy, N-poor grassland soil. B values are
related to temperature sensitivity following Q10 ¼ a Bk and were originally published in fig. 2 of Fierer et al.[68] (a) aqueous solubility; (b) molecular mass;
(c) oxidation state; and (d) O/C ratio.
considered as classic molecular-level characteristics for recalcitrance reveals that they are not predictors for the decomposability of the substances examined here.
The picture changes when parameters are considered that
link the chemical composition of the organic compounds to an
outside resource: oxygen. The oxidation state of organic carbon
(Cox) can vary between 4 (dominance of energy rich C–H
bonds as in CH4) and þ4 (dominance of low energy C¼O bonds
as in CO2). The range for oxidation states in different soil
environments has been estimated between 0.45 and 0.3, with
a mean of 0.26.[108,109] The ability to undergo further oxidation will depend on the availability of oxygen as a resource, and
in this sense the parameter Cox integrates a molecular potential
with a logistical constraint, that is, oxygen availability. Fig. 3c
shows a clear trend of increasing decomposition rate with
decreasing electron richness of the substrate. If universally
applicable, such a trend should be reflected in the relative
proportions of oxygen that is already incorporated in organic
matter, because energy-poor bonds are made when organic
matter is oxidised, and oxidation involves the introduction
of oxygen-containing functional groups. Fig. 3d shows that
B-values are lower (and activation energies higher, since B,
Q10 and Ea are mathematically linked with each other, Eqn 1–3)
for energy-rich compounds that contain less oxygen than do
compounds of comparable size, similar solubility and similar
molecular complexity. It can be deduced that even mild oxygen
deprivation should have greater consequences for the decomposition of energy-rich substrates with low oxygen content and
high activation energies than for substrates that already are
partially oxidised. This inference is supported by observations
of increasing relative proportions of aliphatic compounds with
increasing soil depth and thus increasing distance from the
atmospheric oxygen source.[110,111] Oxygen depleted microenvironments at the centre of aggregates were reported to persist
for extended times after soils have been allowed to drain,[112]
indicating that limitations in O2 supply may be common to many
soil environments[113] and may explain long turnover times that
were previously thought to be the result of ‘recalcitrance’. The
length of time that organic carbon is exposed to molecular
oxygen (oxygen exposure time, OET) at the location of deposition has long been identified as an important control on organic
carbon preservation in continental margin sediments,[114–116]
further corroborating the importance of oxygen limitation for
organic matter preservation.
328
What is recalcitrant soil organic matter?
References
But do these findings not suggest that an energy-rich lipid
with low molecular oxygen content and high activation energies
should be viewed as being able to resist decomposition, and
classified as ‘recalcitrant’?[32] I advise the reader to resist the
temptation to do so. Lipids turned over on decadal time scales in
well aerated topsoils[33] and selective preservation of any kind of
organic material, including lipids, was not found to occur in a
topsoil studied by Bol et al.[34] Thus a classification of a lipid as
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oxygen depleted subsoil microenvironment. But it may not
apply to the overlying topsoil or even the outside of an
aggregate, creating the paradoxon that the same organic compound could be labile and recalcitrant at the same time and in the
same soil, simply as a function of oxygen availability within the
given microenvironment. Dependence of recalcitrance on environmental conditions, however, is logically incompatible with a
definition of recalcitrance as ‘intrinsic molecular property’.
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Conclusions
Classifications determine our attitudes and behaviour towards
the object or phenomenon classified. For example, ‘when
lightning was classified as ‘‘evidence of divine wrath’’, no
courses of action other than prayer were suggested to prevent
one’s being struck by lightning. As soon, however, as it was
classified as ‘‘electricity’’, Benjamin Franklin achieved a measure of control over it by his invention of the lightning rod’.[117]
One can make a similar point for the widely used classification
of soil organic matter as either labile or recalcitrant: research
will have to explore new avenues if the category of intrinsic
recalcitrance is abandoned.
The evidence suggests that the persistence of reduced
organic carbon in the environment is co-determined by the
interaction between substrates, microbial actors and abiotic
conditions. Organic matter turnover should thus be seen as
dependent on microbial ecology and the logistic state of a
specific environment, not on the ill-defined ‘recalcitrance’ of
an organic compound. Varying degrees of structural organisation, microbial readiness and resource limitation within a given
environment (soil aggregate, soil horizon) render it likely that
identical organic compounds may turn over with different
velocities as a result of variations in driving variables. Much
confusion may be due to the fact that ‘recalcitrant carbon’ is
often used as a synonym for ‘old’ carbon, neglecting that old age
can be achieved through many other mechanisms than ‘inherent
chemical recalcitrance’.
What is recalcitrant soil organic matter? In the opinion of this
author, ‘recalcitrance’ is a category of semantic convenience
and not a useful classification of material properties. Conceptual
progress appears to be certain if soil organic matter is reclassified as a reservoir of reduced carbon in different states of
protection against decomposition. If such a conceptual step is
taken, the enigma behind the temperature sensitivity of organic
matter decomposition may be a step closer to a solution, and
robust strategies of soil carbon sequestration can be based on
mechanisms of protection.
Acknowledgements
I am indebted to Noah Fierer for sharing the data for Fig. 3 and to Joan Sandeno
for language edits to the final version of the manuscript. Marco Keiluweit,
Peter S. Nico and Dave D. Myrold provided helpful comments on earlier
versions of the manuscript. Three anonymous reviewers are acknowledged for
their constructive suggestions to improve the manuscript.
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