[Advances in Ecological Research] Litter Decomposition: A Guide to Carbon and Nutrient Turnover Volume 38 || Methods in Studies of Organic Matter Decay

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  • Methods in Studies of OrganicMatter Decay

    I. Introductory Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291ADVAN

    # 2006CES IN ECOLOGICAL RESEARCH VOL. 38 0065-250

    Elsevier Ltd. All rights reserved DOI: 10.1016/S0065-25044/06


    8009-II. Incubation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

    A. In Situ (Field) Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

    B. Decomposition RateLaboratory Methods . . . . . . . . . . . . . . . . . 309III. Studying Chemical Changes During Decomposition. . . . . . . . . . . . . . . 313

    A. Introductory Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

    B. Preparation of Samples for Chemical Analysis and

    Some Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

    IV. Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319A. Regression Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

    B. Analysis of Variance (ANOVA) . . . . . . . . . . . . . . . . . . . . . . . . . . 324

    C. Multivariate Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325V. Presentation of the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327I. INTRODUCTORY COMMENTS

    Although the book has been devoted so far solely to litter de-

    composition processes, described mostly using case studies from boreal

    forests, we recognize that the reader may require some insight into methods

    used more broadly in soil biology. Thus, in this last chapter, we present

    an overview of a range of field and laboratory methods to study decompo-

    sition. Since the book is addressed mainly to students and younger scientists,

    we also discuss briefly some methods of data analysis and presentation in the

    latter part of the chapter. They all represent more general techniques and

    conventions used in data handling and we discuss advantages and disadvan-

    tages of using particular models and methods rather than giving detailed

    formulas for calculating statistics, which may be found in relevant text-

    books. Our impression from many years of teaching at the university level

    is, however, that it is easy to get lost in the plethora of statistical methods

    and ways to present data graphically, and we hope that this short guide is


    As decomposition of organic matter is a set of most complex biological,

    physical, and chemical processes, a broad range of research techniques and

    tools is required to study it. Depending on the research problem, techniques0


  • 292 BJORN BERG AND RYSZARD LASKOWSKImay be needed to expose plant litter in the field or to apply atomic absorp-

    tion spectrometry (AAS), nuclear magnetic resonance (NMR), chromatog-

    raphy, or isotopic analyses. Some of these methods are fields of studies in

    themselves and it would be impossible to cover them all in detail in this

    book. Our intention is to present in detail those methods that are used solely

    in decomposition studies, and to mention briefly some more general techni-

    ques to make the reader familiar with other possibilities and what to look for

    when more detailed studies are required. We also try to pinpoint the pitfalls

    and indicate some solutions that pertain especially to the studies of organic

    matter decomposition. Thus, this chapter can be used as a reference for

    specific litter decomposition techniques; however, for more general methods,

    specialized handbooks will be indispensable.

    Generally, research techniques might be divided between in situ and

    laboratory methods. However, a number of methods can be used equally

    well, with only minor modifications, both in the field and in a laboratory,

    although the interpretation of results may be diVerent between laboratoryand field techniques. From the point of view of research questions, we dis-

    tinguish between studies on decomposition rates and patterns and studies

    on chemical changes, although they are frequently performed in parallel.

    One might also diVerentiate between direct and indirect methods. An exam-ple of the first group would be studies with litter bags, while the latter could

    be represented by, say, calculation of decomposition rates from litter fall/

    organic matter accumulation balance. There is probably no single good

    classification of the research techniques used in decomposition studies. For

    the purpose of this book, we decided to describe the methods grouped into

    two major classes, with studies on decomposition rates, patterns, and chemical

    changes in one group and analytical techniques in another. The first category

    contains detailed descriptions of a number of in situ (field) and laboratory

    methods. Analytical techniques will be presented in a general guideline to

    assist the researcher in choosing the most appropriate tools for specific

    studies and avoiding common problems. Finally, we present a brief overview

    of mathematical decomposition models and some useful statistical methods.II. INCUBATION TECHNIQUESA. In Situ (Field) Methods

    1. General CommentsStandard field methods include incubation of plant litter using the litterbag technique and microcosms. The rate of organic matter degradation can

    also be measured as the amount of organic carbon mineralized and evolved

  • METHODS IN STUDIES OF ORGANIC MATTER DECAY 293from soil as carbon dioxide (respirometry). Other methods may also include

    the use of isotopes such as 13C, 14C, and 15N, often labeling specific mole-

    cules. Depending on the problems to be studied, diVerent methods arepreferred. For example, the classical litterbag technique is the method ofchoice when the decomposition rates and patterns of diVerent plant speciesare to be compared and when chemical changes are studied. To measure

    the maximal extent of litter decomposition or the potential accumulation of

    resistant material, an important point is to follow the decomposition for as

    long as possible.

    The litter bag technique does not allow for estimating total release of carbon

    from organic matter or humus of the forest floor. Thus, if this is the study

    subject, the respirometric techniques would be preferred. Regular respirome-

    try, in turn, does not allow us to distinguish between the CO2 originating

    from dead organic matter and that evolved by roots and mycorrhizain this

    case, isotope labeling, for example, using 14C, may be of use.

    The aim of this section is to provide help in choosing the most suitable

    methods for field studies of particular decomposition processes.2. LitterBagsThis is one of the most commonly used field techniques. Despite its relative

    simplicity, it is a very powerful method indeed, allowing us to address a wide

    range of problems connected with plant litter decomposition. It is also

    frequently used as a first, indispensable step in more detailed studiesfor

    example, on dynamics of organic compounds and chemical elements during

    litter decomposition (see Chapters 4 and 5). Because of the abundance of

    directly in situ measured data which may be gathered using this method, it

    has become a sort of standard in decomposition studies: a quick search

    through the database of the Institute of Scientific Information for litterbag resulted in 198 articles published in the 9 years from 1996 through

    2004, and these include only the articles in which the term occurred in the

    title, in the abstract, or as a keyword.

    Essentially, a litterbag is exactly what the word saysa bag contain-ing some plant litter. Such a bag is filled with weighed dry litter, exposed

    to field conditions for a specific time period, brought back to the labora-

    tory andafter cleaning from contamination with ingrown roots, small

    soil invertebrates, or mineral particlesthe remaining contents are dried

    and weighed. This allows us to calculate the rate and follow the pattern

    of one of the most crucial ecosystem processesthe decay of dead organic

    matter. Thus, important information about an ecosystem can be obtained

    with that simple method. This determination of mass loss is a first step

  • 294 BJORN BERG AND RYSZARD LASKOWSKIin a study but the basic one since it allows us also to quantify the dynamics

    of the litter chemical components.

    Although incubation of litter in litterbags is a simple method, it stillrequires good and detailed planning for each single study. There are no gen-

    eral rules regarding the litterbag size, mesh type, or material from whichit is made. In practice, a typical litterbag measures from 10 10 cm to20 20 cm and is made of flexible but biologically resistant polyester net.Nylon is an alternative, but since nylon contains nitrogen, we cannot

    exclude that this material is suboptimal in many studies, for example, if

    litter nitrogen should be studied. The mesh size should be adjusted depend-

    ing on type of litter and the aim of studies; for example, by using diVerentmesh sizes, one can exclude particular groups of soil invertebrates from

    degradation processes. However, the size of the litter is the main factor

    that determines the mesh size. For needles of spruce or larch, a fine mesh

    size is required, 0.5 mm or less. With leaf litter of broadleaf species, larger

    mesh sizes can be used. Still, litter of several deciduous species is fragmented

    in the late decomposition stage and, in order to prevent such fragments being

    lost, a fine mesh size may be needed. Most frequently, a mesh size of approxi-

    mately 0.5 to 1 mm is used. This allows a number of small invertebrates that

    are active in organic matter degradation (micro and mesofauna) to partici-pate in the process, at the same time excluding most of the macrofauna, such

    as worms, which might drag large parts of litter from the bag.

    A litterbag usually contains a small amount of dry litterapproximately1 to 10 g, depending on the studys needs. Larger amounts in a small bag

    are not advisable since they make the bags pillowlike so that they do not

    adhere to soil surfaces correctly. A bag should be stitched firmly with a

    thread made from polyester or nylon but not a natural material such as

    cotton, which would decompose rather quickly. To account for possible

    losses during transportation, etc., it is advisable to pack each litter bag in a

    separate envelope. This allows us to retrieve any small parts of leaves that

    may have fallen from a bag. In some cases, for example, spruce needles, the

    lost parts can be even returned to the litter bag without reopening it.

    Preparing litter for litterbag incubation is a compromise between weigh-ing accuracy and retaining the litter in a natural stage. The accurate estima-

    tion of mass lossthat is, the main aim of the studyis possible only if

    weighing errors are minimized and this is achieved in most studies other

    than on litter decomposition by simply weighing the material that is dried

    to constant mass at 105C. Unfortunately, drying litter at that high temper-ature results in the loss of its microbial communities. In addition, the fiber

    structures change and several volatile compounds, such as terpenes, may be

    lost, leading to a mass loss not due to decomposition. The changed and

    collapsed fiber structures and the loss of some chemical compounds may

    delay and change the colonization of the litter with new microflora and aVect

  • METHODS IN STUDIES OF ORGANIC MATTER DECAY 295the decomposition rate and pattern. As a consequence, litter must never be

    dried at high temperatures before the field incubation.

    In practice, this tradeoV between weighing accuracy and retaining originallitter structure and microflora is usually resolved by drying litter at room

    temperature. Only a few subsamples are dried at higher temperature and

    they are used only to calculate the correction factor for recalculating roomtemperature dried mass to waterfree dry mass. However, as has beenmentioned, at high temperatures, some volatile compounds may evaporate,

    thus underestimating the real litter weight. Consequently, we recommend

    that litter is dried at room temperature to an even moisture level. This is

    usually reached within 2 to 4 weeks. Subsamples should be dried at temper-

    ature in the range of 75 to 85C, a range in which most volatile organiccompounds normally would not disappear. The temperature used for drying

    should be the same both before and after the incubation. Note that the

    concept of a volatile compound is a relative one. Some litter types, such as

    eucalypt leaves, may release volatile compounds at our recommended tem-

    perature or even below, and it is simply impossible to give generally valid


    In litterbag experiments, large numbers of bags need to be handled and,considering the time needed for each study, the basic necessary information

    must be given and stored in a way that makes it still available when a shift in

    personnel takes place. We suggest two alternative ways of organizing the

    litterbags and the information. In a first approach, the litter for each litterbag is weighed individually, the weight is stamped on a piece of plastic tape

    (such as, Dymo tape) together with a simple code for the litter moisture.

    DiVerent tape colors allow for diVerentiation between, for example, littertypes, soil treatment, and ecosystem type. With this approach, each bag

    contains all the essential information needed for identifying the bag and

    calculating the mass loss. The Dymo tape may follow each litter sample

    through the handling process after sampling the incubated bags, for exam-

    ple, during the drying process. The printed numbers are still readable after

    drying at 85C.Another approach is to assign a separate number typed on plastic tape to

    each litterbag or to simply put the tape inside the bag together with thelitter. Due to the numbering, in addition to the exact weight of a bag, other,

    even extensive, information can be recorded for each bag, such as, say, the

    tree species from which the litter originates, site names if litters from diVer-ent ecosystems are incubated at one stand, placement of the litterbag in aforest, or diVerent litter treatments if such are used.

    When brought to a laboratory, each bag is opened and its contents

    carefully cleaned from any ingrown material, such as roots, grass, moss, or

    mineral contamination and invertebrates. The cleaned litter is ovendrieduntil constant mass. Usually 24 hours of drying is suYcient. In a final step,

  • 296 BJORN BERG AND RYSZARD LASKOWSKIthe mass loss for the incubation time is calculated. It should be noted,

    however, that cleaning from finer mineral particles cannot always be done

    using just a visual inspection. Contamination of litter with, for example, clay

    particles may result in serious underestimation of the decomposition rate

    because of the higher measured weight of the incubated litter than the actual

    weight of remaining organic material. Thus, analysis for ash content may be

    necessary (see the following text).

    The number of replicate bags is important for the accuracy of the esti-



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