Enzymes in the Environment: Activity, Ecology and Applications - Chapter 9 potx

9
Enzyme and Microbial Dynamics of
Litter Decomposition
Robert L. Sinsabaugh
University of Toledo, Toledo, Ohio
Margaret M. Carreiro
University of Louisville, Louisville, Kentucky
Sergio Alvarez
Universidad Auto
´
noma de Madrid, Madrid, Spain
I. INTRODUCTION
The decomposition of plant litter may be the biosphere’s most complex ecological process
in that it involves the interactions of a large number of taxa, spanning much of the range
of biotic diversity. Because of the complexity, nearly all efforts to model plant litter de-
composition have approached the problem from an ecosystem perspective: predicting mass
loss (the emergent biotic process) from litter composition and physical conditions—the
abiotic template (26,53). Technological developments of the past two decades have re-
moved many impediments to the study of microbial dynamics in natural systems; however,
many of these tools have not been applied extensively to the study of microdecomposer
communities; this is beginning to change (22,75). Consequently, fundamental information
on structural and functional patterns across systems, a requisite for development of general
models, is lacking. The significance of this gap is apparent in the context of global change.
The effects of atmospheric carbon enrichment, nitrogen deposition, climate alteration, and
other anthropogenic processes on ecosystems cannot be predicted without decomposition
models grounded in biotic process (51,52).
The biotic process of decomposition spans three levels of organization: biochemical,
organismal, and community. At the biochemical level, the topics of interest are the struc-
ture of plant fiber and the enzymological characteristics of degradation. The enzymological
features are complex. For the polysaccharides at least one enzyme is required for every
combination of monomer, linkage, and secondary structure (69). For lignin and other aro-

matic molecules, the process is principally oxidative; the enzymes have lower specificity,
but the full range of enzymes and oxidants involved has not been defined (2,25). At the
organismal level, the questions focus on the regulation of enzyme expression and the
kinetics of growth. Studies of model organisms suggest a common pattern for the regula-
Copyright © 2002 Marcel Dekker, Inc.
Figure 1 The decomposition process presented as a successional loop. The diagram emphasizes
the dynamic interactions among microdecomposers, extracellular enzymes, and substrate and high-
lights the role of extracellular enzymes as the rate-controlling agents of decomposition.
tion of extracellular enzyme expression that proceeds from environmental induction, to
derepression of transcription, translational expression, and then transcriptional repression,
if enzymatic reaction products exceed metabolic needs (70). This system optimizes the
allocation of resources to the production of enzymes that once deployed outside the cell
are subject only to environmental regulation (5). At the community level, the subjects
include metabolism, structure, competition, succession, and diversity. Aside from patterns
of biomass and respiration, and in some cases fungal succession (19), there is probably
less known at this level than the others.
The first step toward integrating microbial decomposition with traditional ecosystem
perspectives is to integrate these three levels into a useful representation. This can be done
by considering the decomposition process as a successional loop (Fig. 1). The substrate
selects the microbial community, which produces extracellular enzymes that degrade and
modify the substrate, which in turn, drives community succession. In this model, extracel-
lular enzymes link substrate composition and microbial community metabolism. This cen-
tral role, plus substrate specificity and ease of assay, make enzyme kinetics a powerful
tool for investigating the functional diversity of decomposers and the mechanisms that
link environmental disturbance to ecosystem responses (60,61,72).
In this chapter, we review the literature on enzyme activities and decomposition,
propose some metrics for comparative analyses, and present conceptual models for future
research. Our review is limited to studies of natural systems that include quantitative data
on the activities of one or more extracellular enzymes in relation to mass loss from a
cohort of particulate plant detritus. Studies in which enzyme activities are measured in

relation to microbial biomass, production, or respiration are also included if they are di-
rectly related to plant litter decomposition. Information on the enzymatic capabilities of
individual taxa is not presented. Assay methodologies are not described except when rele-
vant for cross-study comparisons.
II. ENZYMES OF INTEREST
The enzymes of interest in decomposition studies are generally those that break down the
principal components of plant fiber (cellulose, hemicellulose, pectin, lignin) into soluble
units that either are directly assimilated by microorganisms or enter the dissolved organic
Copyright © 2002 Marcel Dekker, Inc.
matter pool. Because the main constituents of plant fiber do not contain nitrogen (N) or
phosphorus (P), extracellular enzyme systems involved in the acquisition and recycling
of N and P for microbial growth are also of interest. The most commonly measured classes
of enzymes are cellulases, hemicellulases (xylanases, mannanases), pectinases, (poly)
phenol oxidases, peroxidases, chitinases, peptidases, ureases, and phosphatases. Each of
these functional classes includes multiple forms of the enzymes, whose structure, kinetics,
and deployment may vary considerably across taxa. In general, a different enzyme is
required for each type of linkage and each type of monomer; also enzymes that act on
the interior linkages of polymers (endoenzymes) are usually distinct from those that attack
free ends (exoenzymes). This complexity creates a hierarchy of synergistic interactions:
individual enzymes are components of multienzyme systems that collectively degrade
specific polymers, multiple systems of enzymes degrade the matrix of polymers that con-
stitute plant cell walls, and diverse microbial taxa deploy enzyme systems that interact to
effect decomposition. The biochemical characteristics of these enzyme systems have been
reviewed extensively (3–5,9,14,20,21,25,28,34,41,47,56,58,69,83,84).
A. Aquatic Systems
The literature on enzyme dynamics in relation to litter decomposition is not extensive.
The first study, by Sinsabaugh and associates (64), reported patterns of cellulase activity
(β-1,4-exoglucanase, β-1,4-endoglucanase, β-glucosidase) for senescent Cornus florida
(flowering dogwood), Acer rubrum (red maple), and Quercus prinus (chestnut oak) leaves
decomposing in a woodland stream. They observed that each enzyme showed a distinct

temporal pattern and that the ratio of endoglucanase to exoglucanase activities increased
through time and with initial lignin content. Chamier and Dixon (8) found that pectinolytic
enzymes produced by hyphomycetes, the principal fungal decomposers of plant litter in
aquatic systems, were important components of the decomposition process. Tanaka
(76,77) studied decomposition of senescent leaves from the reed Phragmites communis
in a coastal saline lake. The microbial community, initially dominated by bacteria, became
fungus-dominant after a few months. Both groups produced cellulolytic and xylanolytic
enzymes whose activities were correlated with mass loss.
Sinsabaugh and Linkins (68) collected particulate organic matter (POM) from depo-
sitional areas of a boreal river and examined the distribution of enzyme activities in rela-
tion to particle size and composition. This descriptive study was followed by two others
(74,85) that included analyses of structural similarity in POM-associated microdecom-
poser communities. These studies showed that POM generally becomes more recalcitrant
with decreasing particle size and that carbohydrase activities tend to decrease while oxida-
tive activities increase. Fungi become scarce as POM size decreases below 1 mm and
microbial community diversity steadily increases as size approaches 0.1 mm.
To determine how particle comminution and the shift from a fungus-dominated to
a bacteria-dominated decomposer community affect decomposition, POM was collected
from a woodland stream and sorted into three ranges (1–4, 0.25–1, 0.063–0.25 mm),
which were dried, placed in litter bags, and returned to the stream (73). Mass loss and
the activities of seven enzymes involved in lignocellulose and chitin degradation were
followed. Natural POM accumulations were also collected, size-sorted, and assayed. When
the enzyme activities were integrated over time and regressed against mass loss, it ap-
peared that the decomposition of particles Ͻ1 mm-was less efficient (i.e., lower mass loss
Copyright © 2002 Marcel Dekker, Inc.
increment per unit of enzyme activity) than the decomposition of POM Ͼ 1 mm by factors
of 1.5 to 7. In addition, enzyme activities associated with the POM confined in litter bags
were generally lower than those associated with in situ POM, suggesting that the litter
bag technique was underestimating in situ turnover rates. Estimates of in situ turnover
rates, generated from regression models relating enzyme activities and mass loss, were

up to twice that of confined POM. This approach was applied by Jackson et al. (29) to
study the spatial and temporal dynamics of POM turnover in a Typha sp. (cattail) marsh.
Three size ranges of POM were collected and placed in litter bags at two sites. In situ
enzyme activities were monitored along transects across the marsh. Size-specific relation-
ships between enzyme activity and mass loss generated from the litter bag data were used
to estimate instantaneous mass loss rates across the spatial grid.
Sinsabaugh and Findlay (65) collected four size ranges of POM from a Typha sp.
wetland, a Trapa sp. (water chestnut) wetland, and two channel sites along the Hudson
River estuary and assayed for lignocellulose and chitin-degrading enzyme activities; bacte-
rial and fungal biomass and productivity were estimated. Bacterial biomass and productiv-
ity increased as particle size declined, whereas fungal biomass decreased. Using estimates
of POM turnover rate based on enzyme activities, they calculated that production effi-
ciency, i.e., production rate/decomposition rate, ranged from 1% to 30%; thus most of
the soluble products of decomposition were exported as dissolved organic matter (DOM)
rather than metabolized in situ.
Denward et al. (15) used microcosms containing Phragmites australis to assess the
effects of solar radiation on decomposition. Compared to that of shaded controls, bacterial
abundance increased relative to that of fungi. β-Glucosidase activity also increased, shift-
ing the α-glucosidase/β-glucosidase ratio from Ͼ1toϽ1.
Other studies in aquatic systems have taken a comparative ecosystem approach. Kok
and Van der Velde (36) placed litter bags containing fragments of senescent water lily
leaves (Nymphaea alba) in alkaline (pH ca. 8) and acidic (pH ca. 5) freshwater ponds.
The contents of each bag were analyzed for mass loss, cellulase activity, and xylanase
activity. In a parallel study, they followed the decomposition of Nymphaea alba leaf disks
in six freshwater microcosms with pH values from 4.0 to 8.0. Litter from each microcosm
was analyzed for mass loss and the activities of cellulase, xylanase, polygalacturonase
(pectinase), and pectin lyase. Corroborating the work of Chamier and Dixon (8), they
concluded that the pH dependence of pectinolytic activity was a critical factor underlying
differences in mass loss rates with system pH.
The decomposition of Liriodendron tulipifera (tulip poplar) wood was studied in a

small mountain stream from which new litter inputs were excluded (78). The activities of
phosphatase and five lignocellulose-degrading enzymes were followed along with fungal
biomass and breakdown rates. Compared to those in a reference stream, fungal biomass,
enzyme activities, and breakdown rates were higher in the litter-excluded stream. Differ-
ences in the ratios of phosphatase to carbohydrase and phenol oxidase to carbohydrase
between the systems suggested that the increased decomposition activity was the result
of higher nitrogen and phosphorus availability, a finding confirmed by water chemical
analyses.
Raviraja et al. (54) looked at hyphomycete diversity in relation to enzyme activities
and mass loss at organically polluted river sites, using two litter types: Ficus benghalensis
and Eucalyptus globulus. Although diversity was strongly depressed compared to that of
unpolluted sites, mass loss rates and enzyme (cellulase, amylase, xylanase, pectinase)
activities did not differ.
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Alvarez et al. (2) examined POM turnover in two ephemeral wetlands. Toro pond
was surrounded by a Pinus pinea forest and had a littoral belt of Juncus and Scirpus spp.
Oro pond was surrounded by a eucalyptus plantation and had a disturbed littoral zone.
Both ponds dried completely during the summer. Two size ranges of POM (Ͼ1 mm and
0.063–0.5 mm) were collected from each site and placed in litter bags. In using the ap-
proach of Sinsabaugh et al. (73) and Jackson et al. (29), confined and in situ POM samples
were assayed monthly for β-glucosidase, β-N-acetylglucosaminidase, β-xylosidase,
phenol oxidase, and alkaline phosphatase. When regressions of integrated enzyme ac-
tivity and mass loss were compared, it appeared that decomposition of coarse particles
was about 20 times more efficient than that of fine particles at the Toro site and about
10 times more efficient at the Oro site. Differences between sites were attributed to dif-
ferences in organic matter quality and to the lower pH of Oro pond. At both sites, enzyme
activities measured on material confined in litter bags were lower than those measured
in situ.
B. Terrestrial Systems
Like those in aquatic systems, the terrestrial studies can be roughly classified into those

dealing with fine-scale questions, such as the relationship between enzyme activities and
litter composition or microbial dynamics and those that take a larger-scale comparative
ecosystem approach. Linkins et al. (42) followed the decomposition of senescent Cornus
florida (flowering dogwood), Quercus prinus (white oak), and Acer rubrum (red maple)
leaves in a deciduous woodland in southwest Virginia. This step was followed by a micro-
cosm study using the same litter types (43). They found that activity levels varied with
litter type, that cellulose disappearance and mass loss were correlated with cellulase activi-
ties, and that cellulolytic activity declined sharply as the lignocellulose index (LCI) ap-
proached 0.7. (LCI is the fraction of acid-insoluble material in the residual plant fiber:
[lignin ϩ humus]/[lignocellulose ϩ humus]).
Zak et al. (88) examined fungal diversity, lignocellulase activities, and mass loss
rates on mesquite sticks incorporated into the middens of desert wood rats. The dominant
fungal taxa and fungal diversity varied with moisture availability, but these structural
changes did not correlate with enzyme activity patterns or mass loss in these arid systems.
Litter decomposition in a suburban forest in relation to N deposition has been studied
(6). Senescent leaves of Quercus rubra (red oak), Acer rubrum (red maple), and Cornus
florida (flowering dogwood) were placed on forest floor plots that were sprayed monthly
with distilled water or with NH
4
NO
3
solution at dose rates equivalent to 2 or 8 g N m
Ϫ2
y
Ϫ1
.
Mass loss responses to N amendment varied with the lignin content of the litter. Dogwood,
a fast-decomposing, low-lignin litter, decomposed up to 25% faster than did the control
plots. Maple, intermediate in lignin content, decomposed slightly faster at the lower N
deposition rate and slightly slower at the higher rate. Mass loss rates for heavily lignified

oak litter declined by up to 25%. Fungal biomass increased for all litter types (40% maple,
32% dogwood, 15% oak). Cellulolytic activity, measured by assays for β-glucosidase,
cellobiohydrolase, and endoglucanase, increased with N deposition for all litter types;
ligninolytic activity, measured by assays for phenol oxidase and peroxidase, varied with
the lignin content of the litter. With added N, oxidative activity increased on dogwood
litter, decreased on oak litter, and stayed about the same on maple litter. For all litter
types, phosphatase activity increased with N deposition, indicating higher P demand. For
dogwood, the activities of peptidase and chitinase, enzymes involved in N acquisition,
Copyright © 2002 Marcel Dekker, Inc.
wererepressedbyaddedN;formapleandoak,theseactivitiesincreased.Theresults
suggestedthatwhiterotfungi,whichproduceligninasesinresponsetolowNavailability,
weredisplacedbysupplementalN,slowingthedecompositionofrecalcitrantlitter.
HenriksenandBreland(27)alsofocusedontheroleofNinthedecomposition
process.Usingamicrocosmsystemofwheatstrawandsoil,theyfoundthatcarbonminer-
alization,fungalbiomass,andactivitiesofcellulolyticandhemicellulolyticenzymesde-
creasedwithNavailability.
Intheareaofcomparativeecosystemstudies,Sinsabaughetal.(62,63)followed
massloss,NandPimmobilization,andactivityof11typesofextracellularenzymesfor
birchsticks(Betulapapyfera)decomposingateightupland,riparian,andloticsitesover
afirst-orderwatershed.Masslossratesamongsitesvariedbyafactorof5andwere
correlatedwithlignocellulaseactivities.Incontrast,relationshipsbetweenmasslossand
activitiesofacidphosphataseandβ-1,4-N-acetylglucosaminidasevariedwidelyamong
sites.TheserelationshipsalongwithanalysesoftheNandPcontentofthestickssuggested
thatdifferencesinmasslossratesamongsitesweretiedtodifferencesinnutrientavail-
ability.
Inanotherexperiment,litterbagscontainingsenescentleavesofAgeratumconi-
zoidesandMallotusphilippinensiswereplacedonthefloorofayoungtropicalforestsite
innortheastIndia(38).OtherlitterbagscontainingleavesofHolarrhenaantidysenterica
andVitexglabratawereplacedatamaturetropicalforestsite.Athigher-elevationsubtrop-
icalsites,litterbagscontainingPinuskesiyaandMyricaesculentaleaveswereplacedin

ayoungforestandbagscontainingPinuskesiyaandAlnusnepalensisleaveswereplaced
inamatureforest.Sampleswereanalyzedformassloss,bacterialandfungalnumbers,
cellulosecontent,Ncontent,solublesugarcontent,andactivitiesofcellulase,amylase,
andinvertase.Cellulaseandamylaseactivitieswerecorrelatedwithmicrobialnumbers.
Invertaseactivitycorrelatedwithsolublesugarcontent.Enzymeactivitiesandmassloss
rateswerehigheratthelowerelevationsitesbutwerenotrelatedtostandage.Inasimilar
study,thedecompositionofPinuskesiyaandAlnusnepalensisatadisturbedroadside
forestsitewascomparedwiththatatanundisturbedsite(30).Againcellulaseandamylase
activitieswerecorrelatedwithmicrobialnumbers,whereasinvertaseactivitywaslinked
tosolublesugars.
DillyandMunch(18)studiedenzymeactivitiesandmicrobialrespirationforAlnus
glutinosa(blackalder)leavesdecomposingatwetanddrysiteswithinafenforest.Mass
lossratesweremorethantwiceasfastatthewetsite.Microbialbiomassandrespiration
decreasedovertime(16to2.3µmolg
Ϫ1
h
Ϫ1
),buttheefficiencyofCutilizationincreased.
Thesetrendswereparalleledbydecreasingβ-glucosidaseactivityandincreasingprotease
activity.
III.COMPARATIVEANALYSES
Inthecontextofthesuccessionalloopmodel(Fig.1),therearethreedimensionsfor
comparing studies of enzymatic decomposition: enzyme activity and litter composition,
enzyme activity and mass loss rate, enzyme activity and community composition. A fourth
dimension is large-scale patterns in relation to ecosystem type or disturbance type. These
comparisons are external to the model but integrate enzymatic decomposition into large-
scale perspectives. At present, there are too few studies in any of these areas to support
Copyright © 2002 Marcel Dekker, Inc.
much beyond inference, and in any case, comparisons are generally complicated by meth-
odological diversity (72).

A. Enzyme Activity and Litter Composition
It is clear that enzyme activities vary with litter composition. Patterns are most easily seen
when different types of litter decompose in the same environment. The patterns arise
from both biotic and abiotic processes. The biotic processes are substrate selection of
microdecomposer populations and physiological regulation of enzyme secretion. In addi-
tion, litter-specific activity patterns to some degree reflect physicochemical processes of
adsorption and stabilization (5,66), which are functions of the architecture and composition
of the litter. The relative contribution of biotic and abiotic processes to enzyme activity
patterns across litter types is probably a function of the structural resistance of the enzyme
to inhibition, denaturation, and proteolysis. If the turnover time for a particular enzyme
activity is longer than that for microbial populations, sorption processes may contribute
to litter-specific patterns; if it is lower, then organismal processes predominate.
Enzymes like α-glucosidase and invertase that process soluble saccharides appear
to be in the latter category. Their activities are correlated with the soluble saccharide
content of the litter (38,30). Activities generally peak early in the decomposition process,
then decline markedly; activities tend to be higher on fast-decomposing litter (Fig. 2). β-
Figure 2 Hypothetical distribution of relative enzyme activities with time for a decomposing
cohort of herbaceous plant litter. The patterns reflect general trends reported in the literature. (A),
invertase, α-glucosidase; (B), β-1,4-exoglucanase (exocellulase), (C), β-1,4-endoglucanase (endo-
cellulase), (D), (poly)phenol oxidase; (E), peroxidase.
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Glucosidaseactivityalsotendstobehighestduringtheearlystagesofdecomposition,
butbecauseofitsroleincellulolysis,activityremainssignificantevenathighmassloss
values(18,64).Theactivitiesoftheothercellulases,β-1,4-endoglucanaseandβ-1,4,-exog-
lucanase,increasemoreslowlyandgenerallypeakaboutmidway(40–80%massloss)
throughdecomposition;earlypeaksareassociatedwithheavilylignifiedlitterandlater
peakswithlabilelitter(42,62).Asaccessiblecellulosedisappears,theratioofendogluca-
nasetoexoglucanasetendstoincrease,atleastpartlyasaresultofdifferentialsorption
(66).(Poly)phenoloxidaseactivitytendstoincreasewithlignin-humuscontent(68),but
activitiescanalsoberelativelyhighearlyindecompositionforlittersthathavehightannin

contents(6).Inheavilyhumifiedmaterialperoxidaseactivitiespredominate.Thesetrends
appeartobegeneral:theyoccurinbothaquaticandterrestrialsystems;theyapplyto
individuallittertypesdecomposingthroughtime,aswellastodifferencesamonglitters
ofvaryinginitialcomposition;andtheycanbeobservedalonggradientsofdecreasing
particlesize.Thegeneralitiessuggestthatenzymeactivitiesmaybeusedtomakeinfer-
encesaboutorganicmatterqualityacrossenvironmentaltemplates.Ratiosofβ-1,4-endog-
lucanasetoβ-1,4-exoglucanaseactivity(64)orcellulolytic:ligninolyticactivity(65)have
beensuggestedforthispurpose.However,environmentalfluctuationsthataltertempera-
ture,moisture,andnutrientavailabilityalsoalterenzymeactivitiesandmayobscureor
overwhelmpatternslinkedtolitterquality.
B.EnzymeActivitiesandMassLoss
Correlatingenzymeactivitieswithlittermasslossprovidesinformationonthemechanics
ofdecomposition.Theactivitiesofseveralenzymeshavebeencorrelatedwiththerate
ofdisappearanceofspecificlitterconstituentsorwithmasslossingeneral.Thisinforma-
tioncanbeusedinvariousways.Oneistomodelenzymaticdecompositioninrelation
totemperatureandwaterpotential(48,72).Anotheristousestatisticalmodelstoestimate
instantaneousmasslossratesfromenzymeactivities.Thissecondapproachhasbeenap-
pliedtoprovideestimatesoforganicmatterturnoverinheterogeneoussystems(65)and
toestimatetheturnoverrateoffineparticulateorganicmatter,whichappearstobeunder-
estimatedbythetraditionallitterbagmethod(2,29).Thethirduseistodescribetheeffi-
ciencyandfunctionaldiversityofenzymaticdecomposition.Forthesecomparisons,turn-
overactivitiesareausefulmeasurement.
Turnoveractivitiesarecalculatedfrommodelsofmasslossasafunctionofcumula-
tiveenzymeactivity,analogoustotraditionalmodelsthatdescribelitterdecayovertime
asafirst-orderfunctionofresidualmass(Fig.3).Cumulativeenzymeactivityisexpressed
in units of activity-days, which are calculated by integrating the area under a curve of
enzyme activity vs. time. A linear regression, LN (cumulative activity-days) vs. time,
generates a first-order rate constant called the apparent enzymatic efficiency with units of
activity-day
Ϫ1

. Inverting this rate constant produces an estimate of litter turnover expressed
in units of activity-days. Implicit in this model is the premise, supported by field data
(72), that mass loss per unit of enzyme activity decreases through time because of the
increasing recalcitrance of the residual material.
Like the traditional mass loss constant, and its inverse turnover time, these turnover
activities provide a basis for comparison across sites, treatments, and litter types. These
comparisons convey a sense of the quantity and type of ‘‘work’’ a microbial community
has to do to decompose a cohort of litter and how this work is linked to substrate heteroge-
neity and enzyme synergisms. They also provide a way to ‘‘map’’ the functional diversity
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Figure 3 Comparison between turnover time and turnover activity for a litter cohort. In this exam-
ple, β-glucosidase activity was assayed during the decomposition of Acer rubrum litter at a forest
site. The upper graph shows a traditional first-order exponential decay curve with cumulative mass
loss plotted as a function of time. The slope of the linear regression is a rate constant (k) with units
of day
Ϫ1
;1/k is the turnover time for the litter in days. The lower graph shows mass loss as a
function of cumulative β-glucosidase activity. Cumulative activity is calculated by integrating the
area under the curve of β-glucosidase activity vs. time and is expressed in units of activity-days.
The slope of the regression is a first-order rate constant (k) with units of activity-day
Ϫ1
;1/k is the
β-glucosidase turnover activity for the litter.
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Figure 4 Functional profiles of Cornus florida (flowering dogwood), Acer rubrum (red maple),
and Quercus borealis (red oak) leaf litter decomposition based on turnover activities. A comparison
of relative turnover activities shows that the enzymatic decomposition of dogwood leaves was more
efficient than that of maple and oak. Oak leaves required the most phenol oxidase activity, whereas
maple required the most peroxidase activity. Phosphorus acquisition activity was highest for oak;
nitrogen acquisition activity was highest for maple. G, β-glucosidase; CBH, cellobiohydrolase; EG,

β-1,4-endoglucanase; PhOx, phenol oxidase; Perox, peroxidase; NAG, β-1,4-N-acetylglucosamini-
dase; GAP, glycine amino peptidase; AP, acid phosphatase. (Data from Ref. 6b).
of decomposition. One example (6) shows that the decomposition of flowering dogwood
leaves was accomplished with much less enzyme activity than that needed to turn over
red maple and red oak litter and that extensive phenol oxidase activity was needed to
decompose oak leaves, which have a lot of lignin, whereas a lot of peroxidase activity
was required for decomposing maple leaves, which have a lot of nonlignin phenols (Fig. 4).
Other studies suggest that the apparent enzymatic efficiency of decomposition declines
with particle size (2,29), coinciding with the transition from fungal to bacterial dominance
but also with increasing humification.
Turnover activities can also be calculated for enzymes that are not directly involved
in the decomposition of major litter components. For enzymes such as phosphatase, urease,
peptidase, and chitinase, turnover activities are measures of relative effort directed toward
obtaining N and P from organic sources. Such comparisons have proved useful in under-
standing differences in decomposition rates among systems (63,78). Even within the same
system, the enzymatic effort directed toward the acquisition of organic N and P varies among
litter types (Fig. 4). A major constraint on the value of turnover activities is that direct
comparisons across studies cannot be made unless the same assay methodology was used.
C. Enzyme Activity and Community Composition
In some decomposition studies, microbial numbers, biomass, or respiration has been linked
with extracellular enzyme activities, but there have been few attempts to link community
Copyright © 2002 Marcel Dekker, Inc.
composition or biodiversity with enzymatic process. Maire and associates (46) examined
the relationships among soil respiration, microbial diversity (using phospholipid fatty acid
analysis), and activities of xylanase, laminarinase, phosphatase, urease, and chitinase.
They found a correspondence between functional diversity and structural diversity, both
peaking in spring. Others (44,45) found that differences in breakdown rates and cellulolytic
activity between permanent and temporary stream sites were associated with differ-
ences in fungal diversity and bacterial biomass. However, Raviraja et al. (54) and Zak
et al. (88) found no relationships among fungal diversity, enzyme activities, and mass

loss.
Pollution gradients may be good systems for investigating such relationships. Kan-
deler et al. (31) reported that heavy metal contamination decreased microbial biomass and
functional diversity in soils. C-acquiring enzymes (cellulase, xylanase, β-glucosidase)
were the least affected, phosphatase and sulfatase the most affected; N-acquiring enzymes
(urease) were intermediate. Another study of heavy metal contamination in grassland soils
(39) showed that reductions in microbial biomass and substrate-induced respiration paral-
leled 10- to 50-fold reductions in extracellular enzyme activities. β-Glucosidase activity
was the most depressed, phosphatase and endocellulase activities were the least; reduction
in β-N-acetylglucosaminidase activity was intermediate.
From a systematic perspective, many fungal taxa can be classified as sugar fungi
or brown rot, soft rot, and white rot decomposers whose extracellular enzyme complements
have been characterized to varying degrees (57). Fungal succession on major categories
of litter has been well studied. Within a particular habitat, the dominant populations vary
more or less predictably through time, selected by their substrate utilization capabilities,
their tolerance of inhibitory phenolic compounds, and their effective ranges of tempera-
ture, water potential, and nitrogen availability (17,19,23,55). The result is a dynamic com-
munity that at any particular time is dominated by a relatively small number of populations,
whose identity varies with litter type and habitat. Thus the taxonomic diversity of fungal
communities may be viewed as either low or high, depending on the spatial and temporal
scales under consideration (86).
From an ecosystem perspective, it seems likely that the prominence of ligninase-
producing basidiomycetes (35) in terrestrial systems and pectinase-producing hyphomy-
cetes (7,89) in aquatic systems probably affects the functional profile and efficiency of
enzymatic decomposition. Such differences have not been explicitly described but have
the potential to influence the quantity of carbon metabolized in situ, and the quantity
exported, as well as the form. Differences in efficiency probably exist between bacterially
and fungally dominated systems as well. Within bacterial systems, the functional diver-
sity and efficiency of enzymatic decomposition probably vary with the distribution of
oxygen and other electron acceptors. For detritus with a high concentration of aromatic

residues, decomposition may all but shut down in the absence of oxygenases and peroxi-
dases. In experimentally manipulated wetland soils, McLatchey and Reddy (49) found
microbial biomass and mineralization of C, N, and P decreased with redox potential; phos-
phatase, protease, and β-glucosidase activities also declined; phenol oxidase was detect-
able only under aerobic conditions.
Much remains to be learned about the significance of functional and structural diver-
sity in the decomposition process. Understanding these mechanics—the relationships
among microbial community composition, enzyme activity, and litter breakdown—is a
requisite for better understanding of ecosystem function.
Copyright © 2002 Marcel Dekker, Inc.
D. Enzyme Activities and Ecosystems
Outside the context of the successional loop, decomposition studies focus on regulation
by climate and nutrient availability. Freeze–thaw and wet–dry events affect the temporal
pattern of decomposition across systems but also influence enzyme activities and the de-
composability of litter (10,11,59,67,79,81). The role of nutrient availability is generally
assessed by using amendment studies. An alternative approach is to examine the relative
distribution of enzyme activities directed toward the acquisition of C, N, and P.
Sinsabaugh and Moorhead (70,71) proposed a model for the distribution of extracel-
lular enzyme activities in relation to mass loss, litter composition, and nutrient availability.
The model, called Microbial Allocation of Resources among Community Indicator En-
zymes (MARCIE), is based on the observation that the production of extracellular enzymes
is often controlled by induction/repression mechanisms tied to substrate availability. At
the community level, this type of regulation resembles an optimal resource allocation
strategy for maximizing microbial productivity. The model links litter mass loss with the
activities of C-acquiring enzymes (e.g., cellulases, hemicellulases), which are constrained
by effort directed toward the acquisition of N and P. In this model, ratios of N acquisition
activity (e.g., chitinase, urease, peptidase) to C-acquistion activity and of P-acquisition
activity (e.g., phosphatase) to C-acquisition activity become indicators of relative nutrient
availability. The general utility of the model remains to be established, but it has provided
insight into nutrient regulation of decomposition rates in at least two studies (63,78) and

has potential application in the area of global change research.
Evidence that decomposer communities respond quickly to global change distur-
bances is accumulating (12,24,50,87,90). Ko
¨
rner and Arnone (37) and Dhillion et al. (16),
working in tropical and mediterannean systems, respectively, reported increased activities
of several soil enzymes in response to atmospheric CO
2
enrichment, presumably an effect
of fine-root C priming. Others (6) found that the effects of N deposition on decomposition
were litter-specific because of the connections among N availability, ligninase production,
and distribution of white rot fungi. The effects of atmospheric CO
2
accumulation, warm-
ing, and N deposition on decomposition have the potential to alter soil carbon storage in
ways that may accelerate or mitigate global climate change. At present, these changes are
both difficult to predict and difficult to measure directly. The pool sizes are large. The
addition of nutrients that alter the composition of the microbial community may reduce
decomposition and increase soil organic matter storage even as soil respiration increases.
For these questions, enzyme responses interpreted in the context of the successional loop
and MARCIE model may be the most sensitive indicators of the magnitude and direction
of change.
IV. CONCLUSIONS
Extracellular enzymes directly mediate organic matter breakdown. They link microbial
community organization, litter composition, and environmental conditions. They are also
the most readily monitored components of the decomposition process. This combination
means that models centered on enzyme dynamics have the potential for wide application
in ecological research.
The simplest models relate mass loss to enzyme activities. The potential applications
are monitoring of organic matter pools in heterogeneous systems (65) and conversion of

Copyright © 2002 Marcel Dekker, Inc.
spatial patterns of enzyme activity (13,32) into estimates of decomposition rate. Because
this is an empirical approach, models must be developed for each system. The models
may incorporate the activities of several enzymes, but it is also possible that one to two
‘‘critical activities’’ may have adequate predictive power in some systems. Because sys-
tems with homogeneous litter quality and stable environmental conditions are likely to
produce the most powerful models, this approach may work better for aquatic systems
than for terrestrial ones.
Resource allocation models like MARCIE are valuable for investigating nutrient con-
trols on decomposition, reducing the need for resource intensive amendment studies. Be-
cause they are constructed from the microbial community perspective, they can resolve fine-
scale patterns with respect to time, space, or litter type. As information on the regulation
of extracellular enzyme expression accumulates, the utility of this approach may increase.
At present no models link functional diversity with decomposition. Functional diver-
sity may affect the efficiency of decomposition, which in turn is likely to influence the
growth of microdecomposers, rate of particle comminution, quantity and form of carbon
exported as dissolved organic matter, and nutrient availability for plant growth. Ligninases
are a clear case: loss of activity as a result of insufficient oxygen or excess nitrogen can
markedly reduce the enzymatic efficiency of decomposition. Studies focusing on microbial
production dynamics in relation to enzyme activities and dissolved organic matter produc-
tion have not been done in terrestrial systems. The subject gets a lot of attention in aquatic
systems (1,33,65,81,82), though not in the context of plant litter decomposition. Research
in this area has the potential to improve understanding of food web organization and
carbon transduction in heterotrophic systems.
Simulation models can address mechanical questions of decomposition and commu-
nity metabolism that are not amenable to direct measurement. Models that recreate patterns
of litter composition, mass loss, and enzyme dynamics observed in field studies identify
critical parameters and conceptual gaps and provide predictive hypotheses (51). Some
fundamental questions such as the relationship between microbial biomass turnover and
extracellular enzyme turnover and the number of C or N atoms assimilated for each C or

N atom deployed in extracellular enzymes may be difficult to address outside the realm
of modeling but clearly affect the movement of detrital carbon into the food web.
Over the past four decades, most of the research on enzymatic decomposition has
focused on assay methodology and comparative description of varied systems. The chal-
lenge for the next decade is to move enzymic analysis toward the realm of application.
To achieve this, more research is needed on functional diversity and the efficiency of
decomposition, and on the regulation of enzyme expression and activity. As this informa-
tion develops, enzyme assays will become increasingly useful tools for monitoring and
understanding ecosystem function.
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