i

Facility of Bio-Science Engineering FACULTEIT BIO-INGENIEURSWETENSCHAPPEN

Academiejaar 2005 – 2006

COMPARISON OF PHYSICAL FRACTIONATION METHODS TO SEPARATE FUNCTIONAL SOIL

ORGANIC MATTER POOLS

Md. Abdul Kader

Promotoren : Prof. dr. ir. Stefaan de Neve Dr. ir. Steven Sleutel

Thesis submitted in partial fulfillment of the requirements for the degree of Master of

Science in Physical Land Resources

ii

ACKNOWLEDGEMENTS

With a great pleasure, I want to expresses my gratitude, deepest sense of respect and

profound regard to my promoter, Prof. Stefaan De Neve, Department of Soil management

and Soil Care, Ghent University, Ghent, Belgium for his cordial support, encouragement,

meaningful suggestion during my thesis work.

I am especially grateful to Dr. ir. Steven Sleutel, my co-promoter, for his availability,

constant and untiring supervision, valuable suggestions and friendly co-operation during my

whole period of research and preparation of thesis.

I am also grateful to the Flemish Interuniversity Council (V.L.I.R) and Belgian State

Secretary for development Co-operation for providing me with financial assistance through a

full scholarship during the entire period of my studies in Belgium.

I want to express my sincere thanks to Mathieu, Sophie, Luc, Tina and Olle for their

technical assistance. I also want to extend my sincere thanks to Karoline to allow me to use

some soils of her experiment. I wish to acknowledge all the staff members of Physical Land

Resources program for providing me valuable knowledge and necessary facilities to

accomplish this study successfully. My best regards to Dominique, Anita and Mieke at

Physical Land Resources Secretariat.

Very especial thanks to my wife, Shamim Ara Begum for all the care, sacrifice,

encouragement during my studies in Belgium. Also, I would like to thank all of my

colleagues of Physical land Resources program, who supported me in various aspects.

Ghent, 21 August, 2006

Md. Abdul Kader

iii

SUMMARY

A comparison of the two most widely used soil organic matter (SOM) physical fractionation

methods: the Six et al. (2002) micro-aggregate isolation method and an ultrasonication-

sedimentation method was made by fractionating soil samples from a wide range of

combinations of soil texture and SOM content. In a second part of this thesis, both

methodologies were compared for their ability to assess the impact of soil tillage

management on soil dry matter and organic C and N distribution over isolated fractions. The

Six et al. (2002) method isolates SOM into 4 different fractions namely coarse free

particulate organic matter (fPOM > 250 μm), the fine fPOM, the intra-aggregate POM

(iPOM, 53-250 μm) and the silt and clay associated (63 µm), silt sized (2-63 µm) and clay sized (

grassland > reduced till cropland > conventional till cropland. This OM fraction was further

found to be correlated with soil silt and clay percentages, which demonstrates the iPOM and

the fPOM to be part of two distinctly different SOM pools. The silt and clay associated OM

of the Six et al. (2002) method constituted the largest SOM fraction in all soils and was

found to be strongly related to the soil clay percentage but not to the silt percentage. The silt

sized OM fraction obtained by ultrasonication-sedimentation method showed a poor relation

with the silt percentages indicating that this OM fraction is a composite SOM pool containing

both free as well as mineral associated OM whereas the clay sized OM fraction demonstrated

a strong relation with the clay percentages of soil.

iv

Both methods were found to be effective to compare the tillage treatments on OM

distribution as different isolated SOM fractions responded differently to the tillage treatment.

A relative enrichment of labile (fPOM) and physically protected OM was measured in all

reduced till fields. Between the two investigated physical fractionation methods, the Six et al.

(2002) method was found to be promising for a meaningful separation of labile OM fractions

and has merit in the fact that it considers different SOM stabilization mechanisms, and in that

it’s SOM fractions are equivalent with conceptual model pools. The ultrasonication-

sedimentation method has merit in its simplicity and its ability to isolate SOM fractions,

which show distinct differences in their relation to the soil mineral phase. Based on these

above findings, a new physical fractionation procedure may be proposed by combining both

methods. The POM fractions should be isolated by a procedure similar to the Six et al.

(2002) method with a lower sieving cut-off for the isolation of POM. The individual clay and

silt fractions should be further separated by an ultrasonication-sedimentation method.

v

SAMENVATTING

Een vergelijking van de twee meest gebruikte bodem organisch stof (BOS) fysische

fractionaerings methoden (1° de microaggregaat isolatie methode naar Six et al. (2002) en 2°

een ultrasonicatie-sedimentatie methode) werd uitgevoerd door fractionering van een selectie

bodems met uiteenlopende combinaties van textuur en BOS gehalte. In een tweede deel van

deze thesis werden beide methoden vergeleken naar hun geschiktheid om de invloed van

bodembewerking op de verdeling van organische C en N in verschillende bodemfracties te

meten. De methode naar Six et al. (2002) scheidt de BOS in vier fracties, nl. grof (> 250 µm)

vrij particulair organisch materiaal (coarse fPOM), fijn fPOM (53-250 µm), intra-

microaggregaat POM (iPOM) en klei en leem geassocieerde OS. De ultrasonicatie-

sedimentatie methode scheidt de BOS in drie grootte fracties: zand (>63 µm), leem (2-63

µm) en klei (63 µm fractie, die zowel fPOM als het “fysisch beschermde” iPOM bevat, bleken alle

onafhankelijk te zijn van bodemtextuur, maar werden sterk beïnvloed door landgebruik. De

grootste hoeveelheden fPOM werden gevonden in bosbodems gevolgd door grasland en

akkerland. Het iPOM bleek eveneens sterk bepaald te worden door landgebruik en nam af in

de volgorde bos>grasland>akkerland onder beperkte bodembewerking>conventioneel

bewerkt akkerland. De hoeveelheid iPOM was gecorreleerd met het %leem en %klei van de

bodems, wat aantoont dat het iPOM en fPOM deel uitmaken van afzonderlijke BOS pools.

De klei en leem geassocieerde BOS fractie (

vi

Beide methoden bleken doeltreffend te zijn voor het meten van verschillen in de OS

verdeling in verschillende BOS fracties resulterende uit een contrasterend bodembewerking

beheer. Een relatieve aanrijking van labiele OS (fPOM) en fysisch beschermde OS werd

gemeten in de bodems met beperkte bodembewerking. De methode van Six et al. (2002)

bleek veelbelovend te zijn voor de scheiding van deze labiele OS fracties. Het feit dat deze

methode gekoppeld is aan een conceptueel BOS model dat verschillende BOS

stabiliseringmechanismen beschouwd is een verdere verdienste van deze methode. De

ultrasonicatie-sedimentatie methode is echter eenvoudiger uit te voeren en is in staat om BOS

fracties te isoleren die in zekere mate van elkaar verschillen in hun associatie met de

minerale bodemfase. Op basis van de bovenvermelde bevindingen kan een nieuwe

fractioneringprocedure worden voorgesteld die een combinatie behelst van beide

beschouwde methoden. POM fracties zouden moeten worden gescheiden volgens de

methode van Six et al. (2002), echter met een fijnere zeefgrootte dan de huidige 53 µm. De

klei en leem geassocieerde OS fractie moet verder worden gefractioneerd aan de hand van

een ultrasonicatie-sedimentatie methode.

vii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS II

SUMMARY III

SAMENVATTING V

TABLE OF CONTENTS VII

LIST OF ABBREVIATIONS X

CHAPTER 1. INTRODUCTION 1

CHAPTER 2. LITERATURE REVIEW 4

2.1 Soil Organic Matter 4

2.1.1 Role of Soil Organic matter and Carbon Cycle 4

2.2 Soil Organic Matter Fractions 6

2.2.1 Commonly Described SOM Pools and Related Fractions 6

2.2.2 Litter 7

2.2.3 Microbial biomass 7

2.2.4 The Light fraction 8

2.2.5 Inter-microaggregate organic matter 9

2.2.6 Particulate Organic Matter 9

2.2.6.1 Free POM 10 2.2.6.2 Intra-microaggregate POM 10

2.2.7 Silt and Clay sized SOM 11

2.2.8 Humus 12

2.2.9 SOM pools and SOM fractions 13

2.3 Protection Mechanisms of OM in soils 16

2.3.1 Physical protection 17

2.3.2 Chemical stabilization 18

2.3.3 Biochemical stabilization 20

2.3.4 Unprotected SOM 20

2.4 Physical fractionation methodologies 21

2.4.1 Different methodologies 22

2.4.1.1 Density fractionation 22

viii

2.4.1.2 Size based fractionation 23 2.4.1.3 Dispersion of soil fractions 25

2.4.2 Limitations of physical fractionation methods 26

2.4.2.1 Limitations of density based fractionation methods 28 2.4.2.2 Limitations of size based fractionation methods 30 2.4.2.3 Limitations of dispersion techniques 31

2.4.3 Combined use of size, density and ultrasonic fractionation 33

2.5 Reduced tillage management and Soil Organic Carbon 36

CHAPTER 3. MATERIALS AND METHODS 40

3.1 Site description and soils 40

3.1.1 Comparison of physical fractionation methods 40

3.1.2 Fractionation of conventional and reduced tilled soils 42

3.2 Physical fractionation according to the method by Six et al. (2002) 44

3.2.1 Introduction 44

3.2.2 Soil prewetting 45

3.2.3 Wet sieving 45

3.2.4 Density fractionation 46

3.2.5 Dispersion and sieving 47

3.3 Separation of particle size fractions by ultrasonication-sedimentation 48

3.3.1 Introduction 48

3.3.2 Dispersion by ultrasonication 49

3.3.3 Sedimentation 51

3.4 Carbon and nitrogen analysis of the soil fractions 52

3.5 Texture 53

3.6 pH 53

3.7 Calculation 53

CHAPTER 4. RESULTS AND DISCUSSION 55

4.1 Comparison of physical fractionation results for all the soils 55

4.1.1 Physical fractionation according to the method of Six et al. (2002) 55

4.1.1.1 Soil dry matter distribution of the isolated soil fractions 55 4.1.1.2 OC and ON distribution and the C:N ratios of the isolated fractions 57

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4.1.2 Separation of particle size fractions by the ultra.-sedimentation method 63

4.1.2.1 Soil DM distribution over the isolated soil fractions 63 4.1.2.2 OC and ON distribution and C:N ratios over the isolated fractions 65

4.1.3 Comparison of the results obtained from the two fractionation methods 71

4.1.3.1 Recovery percentage of OC and ON 71 4.1.3.2 Relation between texture and distribution of SOC and SON fractions 72 4.1.3.3 Carbon and nitrogen enrichment in different SOM fractions 80

4.2 Ability of the fraction methods to differentiate between the RT and the CT fields 85

4.2.1 Physical fractionation of the RT and the CT samples (Six et al., 2002) 85

4.2.1.1 Dry matter distribution over the isolated size and density fractions 85 4.2.1.2 OC and ON distribution and C:N ratios of the isolated SOC fractions 85

4.2.2 Separation of particle size fractions by the ultra-sedimentation method 89

4.2.2.1 Soil DM distribution over the isolated soil fractions 89 4.2.2.2 OC and ON distribution and the C:N ratios of the isolated fractions 89

4.2.3 Comparison of the results obtained from the two fractionation methods 92

CHAPTER 5. CONCLUSION 95

REFERENCES 99

x

LIST OF ABBREVIATIONS

CMI

coarse fPOM

CPI

CT

DM

fine fPOM

free POM

iPOM

L

LF

LI

OC

OM

ON

POM

RT

SOC

SOM

SON

SPT

Stdev

Crop Management Index

coarse (>250 µm) inter-microaggregate POM

Carbon Pool Index

Conventional Tillage

Dry Matter

fine (

1

CHAPTER 1. INTRODUCTION

Soil organic matter (SOM) influences all soil functions and represents one of the largest

reservoirs of carbon on the global scale (Kögel-Knabner et al., 2005). Approximately 81% of

the organic carbon (OC) that is active in the terrestrial carbon cycle is stored in soils

(Paustian et al., 2000; Wattel-Koekkoek et al., 2001). Consequently, any change in the size

and the turnover rate of soil C pools may potentially alter the atmospheric CO2 concentration

and the global climate. Thus SOM is a central element in the global carbon cycle and the

subject has come to the focal point since the Kyoto Protocol on climate change in 1992

which demands for fundamental understanding of mechanisms of SOM stabilization and

their regulating factors. The mechanisms for C stabilization in soils are still not well

understood and the ultimate potential for C stabilization in soils is unknown (Lützow et al.,

2006). Current SOM turnover models are not fully process-orientated and thus the simulation

of ecosystem response to environmental changes such as management and the changing

climate is still difficult (Patron, 1996). The lack of understanding of the processes that

maintain SOM pools makes SOM management difficult. This also causes great uncertainty

when simulation models of carbon turnover are used to calculate the development of C pools

in soils under changing environmental conditions and different land use management

(Christensen, 1992). A key element to reliably assessing SOM dynamics is the experimental

identification of SOM pools linked to the mechanisms of stabilization.

Many techniques have already been established to measure the size and turnover of SOM

pools based on chemical, physical or biological separation. Classical chemistry has

historically had the most apparent impact on the methodology applied in SOM research.

Traditionally, wet chemical methods, which are based on sequential extraction of organic

matter (OM) with acids and bases, have been widely applied, but have not proven to be

particularly useful in modeling the dynamics of SOM. Alternatively, physical fractionation

methods which separate the soil on the basis of size and density have recently been

anticipated to relate better to the structure and function of SOM in situ than classical wet

chemical methods (Golchin et al., 1994b). This technique emphasizes the function of soil

2

minerals and structure in SOM turnover (Christensen, 2001). Therefore, to determine the

association of SOM with primary particles and to quantify the amount of particulate organic

matter between and within soil aggregates, physical fractionation of SOM has been widely

used (Beare et al., 1994; Six et al., 1998; Aoyama et al., 1999; Puget et al., 2000). Moreover,

it is envisaged that physical fractionation of SOM may contribute in reducing the

discrepancies between the capabilities of SOM fractionation techniques and the requirement

of biologically founded and mathematically formulated SOM turnover models (Christensen,

1992). Consequently, the last decade there has been a mushrooming of studies on separating

soil aggregate fractions. Accordingly, assortments of physical fractionation methods have

been developed for the separation of the SOM using a multitude of combinations of the

existing density, size and ultrasonic methods for physical fractionation of SOM, which

severely limits standardization and the ability to interpret results by comparison to other

studies. Therefore, the goal of this thesis research was to make a comparison of the two most

widely used physical fractionation methods for the isolation of particle size and density

fractions of SOM to reveal whether the results of both methods can be related. The fact that

to date, little or no effort has been put into making such comparison renders the study unique.

Moreover, another objective was to identify fractionation steps that are relevant for

investigating the impact of management or other soil properties or SOM dynamics. The two

selected physical fractionation methods were physical fractionation of SOM based on a

combination of wet sieving with density separation as outlined by Six et al. (2002) and the

other one was based on ultrasonication and sedimentation techniques. For the purpose of this

comparison, soil samples from a wide range of combinations of soil texture (sandy to clay)

and OM content were fractionated according to both methods. In addition, both

methodologies were also compared for their ability to assess the impact of soil tillage

management on soil dry matter and organic C and N distribution over isolated fractions on

three conservation tilled sites located at Heestert, Court–St- Etienne and Villers-le- Bouillet.

Chapter 2 presents a review of the available literature on SOM and its importance for the

global carbon cycle, different fractions of SOM and their composition and the most recent

insights into different stabilization mechanisms of SOM. Existing physical fractionation

methods of SOM are reviewed including discussion on their limitations. The review

3

concludes with an overview of conservation tillage and its applicability in SOM

management.

In Chapter 3 the materials and methodologies used in this study are described in details.

Chapter 4 presents and discusses the dry matter, OC, and OM distribution of the selected

soils obtained by the two selected physical fractionation methods. A first part covers the

comparison of two methods based on the results of 18 soils where different isolated fractions

of SOM were mainly correlated with the different soil organic matter (SOM) pools and soil

texture. A second part assesses the ability of these two methods to measure the impact of

tillage management on OC and organic nitrogen (ON) distribution based on the SOM

fraction of three conservation tillage sites in the loess region of Belgium.

In chapter 5 an overall conclusions are made together with on outlook for further

experimental work.

4

CHAPTER 2. LITERATURE REVIEW

2.1 Soil Organic Matter

2.1.1 Role of Soil Organic matter and Carbon Cycle

Soil Organic Matter (SOM) is both a source and a sink of plant nutrients (Duxbury et al.,

1989); it is an ion exchange material; it promotes the formation of soil aggregates and

thereby influences soil physical properties and soil moisture; and it is an energy substrate for

soil microbes and macrofauna (Allison, 1973). Soil aggregation and soil organic matter

(SOM) dynamics are closely linked. Well-aggregated soils possess a larger pore space, a

higher infiltration rate and better gaseous exchange between soil and atmosphere than poorly

aggregated soils, leading to enhanced microbial activity (Lynch and Bragg, 1985). Even in

well-fertilized soils, soil productivity is reduced by loss of SOM (Aref and Wander, 1997).

Accompanying these losses in productive potential are losses in agroecosystem efficiency.

Crop response to mineral inputs is increased in soils where biological and physical properties

influenced by OM are enhanced (Cassman, 1999; Avnimelech, 1986). Important SOM-

mediated processes include mineralization and nutrient supply (N, P, S), enhancement of the

soil water retention capacity and hydraulic permeability, reductions in energy required for

tillage, enhanced soil tilth, pH buffering and, disease suppression. Collectively these

influence crop production and environmental outcomes.

Nowadays, study of SOM is not limited to agricultural crop production but also concerns

environmental stress such as global warming and climate change by the potential of

sequestration of atmospheric CO2 as SOM. The concentration of CO2 has increased from 270

ppm (mid 1800) to 370 ppm at present and between 15% and 17% of this CO2 is believed to

be derived from the decomposition of SOM (Houghton et al., 1991). So, the retention of

organic carbon (OC) in soils is becoming more important since the rise in atmospheric CO2

and global warming are recent concerns. Terrestrial vegetation and soils are significant

reservoirs of OC containing about three and a half times as much carbon as the atmosphere

amounting to 2500 Gt C (Fig 2.1). Soils and vegetation always actively exchange CO2 with

5

the atmosphere (Fig. 2.1). Carbon is accumulated in the soil, mainly in an organic form. This

OM undergoes a series of biotransformations, including decomposition and finally

mineralization by microorganisms, with the release of CO2. SOM in the surface soil contains

1550 Pg C (Batjes, 1996) and the Intergovernmental Panel on Climate Change (IPCC)

reports a total of 128 Pg C till a depth of 1 m in cropland soils (IPCC, 2001). Small

alterations of this large amount of C can increase the atmospheric CO2 concentration

dramatically, which ultimately can change the global climate system. Agricultural production

is accelerating the decomposition of SOM, resulting in a loss of C to the atmosphere, which

contributes to the greenhouse effect and global warming. The total cumulative SOC losses

from cultivated soils due to land-use changes were estimated at 40-60 Pg by Paustian et al.

(1997). SOC dominates the terrestrial carbon cycle in terms of total quantity, yet the long-

term sequestration of soil organic carbon is relatively low (only 0.7% of net primary

production) (Schlesinger, 1990).

Figure 2.1 Global Carbon Stocks (Gt C) and Carbon Flows (in Gt C yr-1) from 1989 to 1998 (Schimel et al., 1996)

6

2.2 Soil Organic Matter Fractions

2.2.1 Commonly Described SOM Pools and Related Fractions

The term fraction is generally used to describe measurable organic matter components

whereas the term pool is used to refer to theoretically separated, kinetically delineated

components of SOM (Wander, 2004). It is very difficult to summarize the general

relationships between kinetically conceived SOM pools and related OM fractions because

overlapping terminology is often applied to fractions and pools that are not closely related

and this gives rise to confusion (Wander 2004). Commonly isolated and measured SOM

fractions will be discussed below (2.2.2 to 2.2.8) in a general ascending order of their

resistance against microbial decomposition (Table 2.1).

Table 2.1 Estimated ranges in the amount and turnover times of various types of organic matter stored in agricultural soils (according to Christensen, 1996; Jastrow & Miller, 1997; Sleutel, 2005)

Organic Matter fraction Proportion of whole soil OM (%) Turnover time (y)

Litter - 1-3

Unprotected OM

Microbial biomass 2-5 0.1-0.4

Free POM 18-40 5-20

Light fraction 10-30 1-15

Inter-microaggregate OM 20-35 5-50

Intra-microaggregate OM 5-40 20-50

Silt and clay sized OM 50-90 1000-3000

It should be understood that these fractions are often strongly overlapping and may have

slightly different meaning depending on the author and fractionation procedures. For

instance, the light fraction is the part of the free POM which is separated by density

fractionation. It may contain inter- or intra-microaggregate or both OM depending on the

disruption of macro and microaggregates before density fractionation. On the other hand,

inter and intra-microaggregate OM cover the free POM, light fraction and part of the

microbial biomass outside and inside microaggregates, respectively. Free POM also overlaps

with unprotected POM, the light fraction, and the microbial biomass. The term humus is

7

more relevant in terms of chemical fractionation instead of physical fractionation and

comprises a part of silt and clay associated OM.

2.2.2 Litter

Fresh plant residues are considered as the litter fraction and many definitions of SOM

exclude fresh plant residues. Litter can be an important component of the active fraction.

Residues play a significant biological and physical role in soils and represent a principal

means by which SOM can be managed. Studies of the factors controlling microbial decay of

litter provide the basis for the understanding of how residue quality influences SOM

dynamics. Litter quality is equated with the rate at, or ease with, which organic substrates

are, decomposed (Paustian et al., 1997). Litters are mostly abundant in unmanaged or

minimally managed systems, such as in forest soils. The physical activity of litter- and plant-

derived carbohydrates is important. Surface litter also provides protection against erosion.

2.2.3 Microbial biomass

The microbial biomass is a fraction of the soil organic matter (SOM) that is actively involved

in the transformation of organic residues in the soil and in the dynamics of N, P and S. Soil

microbial biomass and its activity, especially its sensitivity to human activity, are suitable

predictors of soil biological status in terms of soil fertility (Elliot et al., 1996). Carbon and

nutrient turnover are mediated by the soil microbial biomass, which responds to crop residue

or tillage management (Dalal et al., 1991). Microbial biomass is usually related to the carbon

in the soil light fraction and to the in vitro carbon mineralization (Bremer et al., 1994;

Alvarez et al., 1998).

The microbial biomass fraction has been very often related to chloroform-labile C and N

(Brookes et al., 1985) and is one of the few measurable SOM fractions included in several

multipool models of SOM dynamics (Hansen et al., 1991). According to Franzluebbers et al.

(1999), the microbial biomass, estimated by fumigation extraction, is a good general measure

8

of active SOM if the C recovered from control soils is not subtracted from treatment soils.

They found that subtraction of control obscured resolution of differences. Phospholipid-P, a

more direct measure of the living biomass has been used effectively to reflect the biomass

component of active SOM (Kerek et al., 2002). Amino sugars, which occur in soils as macro-

polysaccharides, including chitin (Stevenson, 1994), have been related to bacterial and fungal

biomass and can be used to estimate contributions to the biologically active pool. Newly

immobilized N also contains microbially derived amino compounds (Kelly & Stevenson,

1985; He et al., 1988).

2.2.4 The Light fraction

The so called "light fraction" consists of mineral-free OM composed of partly decomposed

plant and animal residues, which turn-over rapidly and have a specific density that is

considerably, lower than that of soil minerals (Alvarez & Alvarez, 2000). The light fraction

is occasionally also composed of biologically inert SOM (e.g. charcoal; Skjemstad et al.,

1990). Dalal & Mayer (1986) found for Australian clay soils that the rate of loss of OC from

the light fraction (density < 2 g ml-1) was 2 to 11 times greater than that of the heavy fraction.

Six et al. (2002) suggested that the light fraction and the free Particulate Organic Matter

(POM) (see 2.2.6) can be combined into a conceptual similar pool. The carbohydrate

concentration of both fractions is low compared to the smaller sized or heavier fractions

(Solomon et al., 2000). The mannose + galactose /arabinose + xylose ratio of POM and LF is

lower than of the smaller sized and heavier soil fractions. The net N mineralization potential

of both the POM and the light fraction is low due to the high C:N ratio of these fractions.

Lastly, these two SOM fractions are labile, easily decomposable and greatly depleted by

cultivation (Camberdella & Elliot, 1992; Six et al., 1999; Solomon et al., 2000). Therefore,

they play an important role in OM turnover and decomposition.

9

2.2.5 Inter-microaggregate organic matter

Inter-microaggregate OM comprises free POM, the light fraction and microbial biomass. It is

also known as unprotected or uncomplexed OM. This fraction of OM is neither present as

readily recognizable litter components (which are typically > 2 mm) nor is it incorporated

into primary organo-mineral complexes. It is recovered by density and size fractionation

procedures or combinations thereof. Separation by density in heavy liquids (1.2-2.0 g ml-1)

has been widely used. The inter-microaggregate OM consists mainly of particulate, partly

decomposed plant and animal residues but can also encompass fungal hyphae, spores, faecal

pellets, faunal skeletons, root fragments, and seeds (Gregorich & Janzen, 1996). For soils on

which the vegetation is frequently burned, charcoal can account for a significant proportion

of the uncomplexed OM (Skjemstad et al., 1990; Cadisch et al., 1996). It is a transitory pool

between litter and mineral-associated OM. Its turnover is also slower than that of recently

shed litter but faster than that of OM associated with clay and silt.

Accumulation of inter-aggregate OM is favoured in cold and dry climates, in acid soils, and

in continuously vegetated soils with a large return of plant litter such as those under forest

and grass. In soils with permanent vegetation it can account for 15-40% of the OM in surface

horizons, whereas in long cultivated arable soils it can be very low (less than 10% of the OM

in the tilled layer) (Sextone et al., 1985). Uncomplexed OM tends to be readily depleted

when soils under permanent native vegetation are brought into cultivation (Besnard et al.,

1996; Balesdent et al., 1998; Six et al., 1998), and it increases when arable soils are reverted

to native grassland (Jastrow, 1996). Often the decrease accounts for a major part of the initial

loss of OM in the soil when it is first cultivated.

2.2.6 Particulate Organic Matter

Particulate organic matter (POM) is an active SOM fraction, which supplies nutrients to the

growing plant. It is more responsive to changes in agricultural management than total SOM.

As such, POM was considered as an indicator of soil quality (Gregorich & Ellert, 1993).

Koutika et al. (2001) found an improvement of soil quality by an increase in N content of

10

POM fractions under two legumes compared to natural regrowth. POM is enriched in

phenolic CuO oxidation products vanillyl, syringyl and cinnamyl with a low acid aldehyde

ratio of the vanillyl units and had high syringyl-to-vanillyl ratio, indicating a high lignin

content which is only little altered by microbes (Six et al., 2001; Solomon et al., 2000).

2.2.6.1 Free POM

Free Particulate Organic Matter (free POM) includes loose organic particles in the soil i.e.

not included in microaggregates, and therefore, free POM is also termed inter-aggregate

POM. Golchin et al. (1997) concluded that free POM consists mainly of partly decomposed

litter residues. However, Six et al. (1998) suggested that free POM represented a mixture of

recently deposited crop residues and older uncomplexed OM previously occluded within

aggregates but released from degraded aggregates following the depletion of available

substrates in the occluded OM. Free POM is more decomposable than occluded POM as

reflected by differences in 13C signatures of fractions from soils on which C3 vegetation has

been replaced by a vegetation with C4-type photosynthetic pathway (Besnard et al., 1996;

Gregorich et al., 1997).

2.2.6.2 Intra-microaggregate POM

Intra-microaggregate POM is the POM that is contained within microaggregates with a size

limit of 53-250 μm (Six et al., 2002). The distinction between free and occluded fractions of

uncomplexed POM is based experimentally on a stepwise dispersion of soil combined with

separations according to particle size or density or both. Free POM is recovered from

minimally dispersed samples in which microaggregates remain intact, while occluded OM

subsequently is isolated after dispersion of microaggregates (see also 2.3.1). Microaggregates

are made up of primary organomineral complexes and uncomplexed OM particles held

together by transient and temporary binding agents. SOM present in free microaggregates or

in microaggregates within macroaggregates is protected from decomposition (Balesdent et

al., 2000; Besnard et al.,1996; Denef et al., 2001; Six et al., 2000).

11

C mineralization of crushed free microaggregates was 3 to 4 times higher than from crushed

macroaggregates (Bossuyt et al., 2002), which demonstrates that microaggregate protected

SOM is decomposed very quickly if it is exposed by disturbing soil management practices.

Based on the differences in the OC turnover time, the stabilization of OM is greater within

free microaggregates (turnover time 412 y) than within macroaggregates (turnover time

140y) (Jastrow et al., 1996). Further corroborating evidence for the crucial role which

microaggregates play in C sequestration were reported by Besnard et al. (1996), Gale et al.

(2000) and Six et al. (2000). These studies clearly indicate that OM stabilization is greater

within microaggregates than within macroaggregates (see 2.3.1).

2.2.7 Silt and Clay sized SOM

Most of the SOM may be observed in silt and clay sized primary organo-mineral separates.

Clay generally accounts for over 50% of the SOM, clay and silt ( clay > silt fraction. Following addition of simple substrate, new SOM is found in all

size separates although clay sized SOM shows a higher accumulation. In the long term

however the silt sized SOM dominates over the clay sized SOM (Christensen, 1996).

Refractory SOM in arable soils is primarily stored in fine-particle-size fractions (Kiem &

Kögel-Knabner, 2002). According to Kiem & Kögel-Knabner (2002), organic structures that

are chemically recalcitrant by nature do not contribute to recalcitrant pools unless they are

affiliated with fine-particle-size separates; exceptions include charcoal, which is highly

resistant to degradation and which is recovered in POM fractions. Measurement of SOM

fractions associated with fine-silt and coarse-clay sized separates (Six et al., 2000;

Christensen, 2001; Guggenberger & Haider, 2002) is often used to estimate size of the stable

SOM pool.

12

Stable SOM constituents are primarily related to the proportion and characteristics of fine

particles in soils (Carter et al., 2003). Hassink (1997) and Six et al., (2002) established

relationships between the silt and clay associated OM and the soil texture (Fig. 2.2).

Figure 2.2 The relationship between the silt + clay content and silt + clay associated C for grassland, forest and cultivated ecosystems (from: Six et al., 2002). Size boundaries for silt+ clay that were used were 0-20 μm (A) and 0-50 μm (B)

Here, the relationships indicate a maximum of C associated with silt and clay which differs

between forest and grassland ecosystems and between clay types. The intercept variation of

these two figures may be the result of the presence of larger sized (20-50 μm) silt particle in

0-50 μm which have more C per unit material. Particle surface area and the abundance of Fe

and Al oxides as well appear to play a key role in SOM stabilization in the fine fraction

(Curtin, 2002). The upper limit of carbon content associated with primary particles

13

The average properties of Fulvic Acids (FAs) and Humic Acids (HAs) are distinctly and

remarkably uniform across soils (Mahieu et al., 1999). The abundance of C in FAs is lower

(40–50%) than that in HAs (53–60%), and the abundance of O in FAs higher (40–50%) than

that in HAs (32–38%). This is consistent with the higher exchange capacity of FAs, which is

640–1420 cmol (+) kg–1 FA, compared with 560–890 cmol (+) kg–1 HA (Stevenson, 1994).

Reported molecular weight ranges are 3000 Da for HA, 1000 to 3000 Da for FA, and lower

(

14

heterotrophs, and those that are likely to be mineralized are followed by the letter B in

parentheses. Fractions produced by methods used to separate physically active from

protected organic matter or that isolate material associated with physical function are

followed by the letter P in parentheses. The SOM fractions produced by methods designed to

isolate chemically labile from persistent OM or separate OM that usefully describes soil's

exchange and sorption characteristics are followed by the letter C in parentheses.

Table 2.2 Soil Organic matter fractions (Wander, 2004)

Organic Matter Pools, Theorized Kinetics and Function

Procedurally Defined Fractions of Organic Matter

Labile or Active SOM Half- life days to a few years Equated with material of recent origin or embodied living components of SOM Material of high nutrient or energy value Physical status (not physically protected) makes soil incorporated matter likely to participate in biologically or chemically based reactions Physical role of materials located at the soil surface and of compounds that promote macroaggregation is transient.

Chloroform-labile SOM (B) Microwave- irradiation-labile SOM (B) Amino compounds (B,P) Phospholipids (B) Labile Substrates Mineralzable C or N, estimated by incubation (B) Substrate-induced activity (B) Soluble, extractable by hot water or dilute salts (C,B) Residues for which chemical formula can be described, inherited from living organisms Litter vegetative fragments or residues (B,P) POM not protected within aggregates (B,P) Pollysaccharides, carbohydrates (C,P)

Slow or Intermediate SOM Half- life of a few years to decades Physical protection, physical status, or location help separate this fraction from the other two fractions

Partially decomposed residues and decay products Amino compounds, glycolproteins (B,P) Aggregate protected POM (B,P) Some humic materials Acid / base hydrolysable (B,C) Mobile humic acids (B,C)

Recalcitrant, Passive, Stable, and Inert SOM Half life of decades to centuries Recalcitrance because of biochemical characteristics and/ or mineral association

Refractory compounds of known origin Aliphatic macromolecules (lipids, cutans, algaenans, suberans) (C) Charcoal (C) Sporopollenins (C) Lignins (C) Some humic substances High molecular weight, condensed SOM (C,P) Humin (C) Nonhydrolyzable SOM (C) Fine-silt, coarse-clay associated SOM (C,P)

15

The functional importance of SOM of different ages varies systematically, with the youngest

materials being most biologically active and materials of recent origin and intermediate age

contributing notably to the physical status of soils. Materials with longer residence times

exert more influence on the physicochemical reactivity of soils.

Six et al. (2002) conceptualized a model of SOM dynamics based on four measurable pools

defining soil C-saturation capacity: (1) a biochemically protected C pool, (2) a silt and clay

protected C pool, (3) a microaggregate protected C pool and (4) an unprotected C pool (Fig.

2.3). Each pool has its own dynamics and stabilization mechanisms, which determine a level

at which soil C become saturated.

Figure 2.3 The maximum OC content for the soil as defined by the dynamics of different measurable soil C pools (Six et al., 2002)

16

2.3 Protection Mechanisms of OM in soils

Mechanisms for C stabilization in soils have received much interest recently due to their

relevance in the global C cycle. Lützow et al. (2006) reviewed the C stabilization

mechanisms. Several author (Sollins et al., 1996; Badlock et al., 2004; Mayer, 2004)

considered three broad headings that are currently, but often contradictorily or inconsistently,

considered to contribute to organic matter (OM) protection against decomposition in

temperate soils were:

(1) Selective preservation due to recalcitrance of OM, including plant litter, rhizodeposits,

microbial products, humic polymers, and charred OM. Krull et al. (2003) reviewed

mechanisms and processes of the stabilization of soil OM and concluded that in active

surface soils, adsorption and aggregation can retard decomposition processes but 'molecular

recalcitrance' appears to be the only mechanism by which soil OM can be stabilized for

longer periods of time.

(2) Spatial inaccessibility of OM against decomposer organisms due to occlusion,

intercalation, hydrophobicity and encapsulation; and

(3) Stabilization by interaction with mineral surfaces (Fe-, Al-, Mn-oxides, phyllosilicates)

and metal ions.

Other researchers (Stevenson, 1994; Christensen, 1996; Six et al., 2002) proposed four main

mechanisms of SOM stabilization which are described below in detail. Those are (1) physical

protection, (2) stabilization by organo-mineral bonding (3) biochemical stabilization and (4)

not protected by any mechanisms. Basically the first three mechanisms involve the

accessibility of OM to microbes and enzymes, interactions between the organic and mineral

compounds and chemical resistance of organic molecules against microbial attack,

respectively. If SOM is not protected by one of these mechanisms it is considered as (4)

unprotected.

17

2.3.1 Physical protection

SOM can be physically protected against microbial decomposition by soil aggregation.

Several studies have elucidated the relationship between aggregate dynamics and associated

SOM dynamics (Jastrow, 1996; Six et al., 1998; 2000). Aggregates protect SOM by forming

physical barriers between microbes and enzymes and their substrates and controlling food

web interactions (Elliot & Coleman, 1988). The current hypothesis of the aggregate hierarchy

concept (Tisdall & Oades, 1982) is that free primary particles are bound together into

microaggregates (50-250 µm) by persistent binding agents (e.g. humified OM). These stable

microaggregates are bound together into macroaggregates (>250 µm) by temporary (i.e.

fungal hyphae and roots) and transient (i.e. microbial- and plant-derived polysaccharides)

binding agents, and in turn, new microaggregates are predominantly formed within

macroaggregates (Fig. 2.4).

Figure 2.4 The chronology of the formation of hierarchical aggregate orders according to Tisdall & Oades (1982), Oades (1984) (Six et al., 2004)

Fungally derived amino sugars and glomalin and POM contribute to aggregate formation and

stabilization (Waters & Oades, 1991). Polysaccharides exudated by roots and micro

organisms, which include sugar and non-sugar forms, adsorb strongly to negatively charged

soil particles through cation bridging (Chenu, 1995) and contribute notably to aggregate

stabilization (Martens & Frankenberger, 1992). This physical protection by aggregates is

further indicated by the positive influence of aggregation on the accumulation of SOM (Six

et al., 2002). Among the mineral particles silt has been considered to be the key player for

aggregate formation. Therefore, the positive correlation between silt content and intra-

18

aggregate POM also again indicate the physical protection of SOM by micro-aggregation

(Sleutel et al., 2006a). Microaggregates have a higher stability than macroaggregates and

particularly POM inside these microaggregates constitutes an OM pool with intermediate

turnover rate. Long-term (decades to millennia) SOC sequestration mechanisms are rather

thought to be mainly due to the physical protection of chemically recalcitrant organic matter

within organomineral complexes and also to charcoal formation (Skjemstad et al., 1996).

2.3.2 Chemical stabilization

Chemical stabilization of SOM is the chemical or physico-chemical binding between SOM

and soil minerals (silt and clay particles) (Six et al., 2002). Under identical annual OM input,

a slower SOM turnover, a larger microbial biomass and more OM are expected in soils with

a high clay content within the same climatic area (Müller & Höper, 2004). Tisdall & Oades

(1982) reported that silt sized aggregates bind more carbon. The mineral fraction has a

profound effect on the quantity and quality of OM in soils due to the adsorption of OM on

mineral surfaces (Fig 2.5).

Figure 2.5 Overview of the different bindings in a clay-humate complex (Stevenson, 1994)

Different criteria of organic mineral particles, such as types of layer silicates, intercalation of

OM and contents of pedogenic oxides, are important for organic-mineral bonds (Schulten &

19

Leinweber, 2000). Particularly smectites and oxides have been shown to have greater

adsorptive potential. Therefore the specific surface area (SSA) has been suggested to be a

greater predictor for the adsorptive capacity of the soil minerals. A number of studies have

indeed established positive relationships between the SSA and SOC contents, and the mean

residence time of SOC has also been shown to increase with SSA (Wiseman & Püttman,

2005). Tiessen & Stewart (1983) observed that SOM in large-sized particle-size classes

mineralized more rapidly than finer components. Moreover, the highest amount of OC and

ON is present either in the finest fraction (fine clay, clay) or coarse clay or fine silt which

was described by enrichment factors EFc and EFn (EFc= %OC in fraction/ %OC in bulk soil,

EFn analogous; Christensen, 1992). EFc and EFn of clay and silt sized fractions decrease

with the increasing clay and silt contents which is represented by an inverse function (Fig

2.6) (Schulten & Leinweber, 2000).

Figure 2.6 Relationship between fraction size and carbon and nitrogen enrichment factors for clay (

20

2.3.3 Biochemical stabilization

Biochemical stabilization is the stabilization of SOM due to its chemical composition (e.g.

recalcitrant compounds such as lignin and polyphenols) and through chemical complexing

processes (e.g. condensation reactions) in soil (Six et al., 2002). Humified OM, i.e. humic

acids and humin in particular, represents the most persistent pool of SOM with mean

residence times of several hundreds of years (Piccolo, 1996). With humification, plant

residues are transformed via chemical, biological and physical processes into more stable

forms (humus). Therefore, humification and degradation processes result in the loss of

structurally identifiable materials (Chefetz et al., 2002). During humification the amount of

aromatic and alkyl C increases whereas the level of O-alkyl C decreases. A commonly

suggested hypothesis is that the O-alkyl C (i.e., carbohydrates) are utilized by the microbial

population in the soil, resulting in a relative increase of the more refractory components of

the SOM (i.e. aromatic and alkyl structures). Proteins are also readily degraded in soils,

whereas the polyphenols decompose more slowly (Schulten et al., 1996).

2.3.4 Unprotected SOM

Recently derived, partially decomposed plant residues that are not closely associated with

soil minerals constitute the unprotected SOM pool. Six et al. (2002) suggest that this

unprotected SOM pool is measurable as either the light fraction (LF) or free POM fraction.

They defined unprotected POM as the 53-2000 μm sized POM not contained with

microaggregates. Since LF and free POM are labile organic matter pools, they are sensitive

to management practices (Solomon et al., 2000) and consequently highly influenced by the

cultivation history of the soil.

The size of the unprotected SOM pool is a function of inputs and specific decomposition rate

of the various components. The decomposition rate of the unprotected C pool is, by

definition, independent of the level of chemical and physical protection. This explains why

the amount of free POM is not dependent on the soil clay content (Kölbl & Kögel-Knabner,

2004), but is related to soil moisture, temperature, intrinsic biodegradability and N

21

availability as principal controls on microbial activity. There are indications that the

unprotected C pool also become saturated (Fig 2.3), but more research is required to prove

the existence of a saturation level and to find out by which mechanisms it is caused (Six et

al., 2002).

2.4 Physical fractionation methodologies

There are many techniques which try to measure the size and turnover of SOM pools, and

they have been used to separate SOM into labile and recalcitrant pools. These methods rely

on chemical, physical, or biological separation (Doran et al., 1999). As chemical

fractionation methods have not been proven particularly useful in following the dynamics of

organic material in soils (Duxbury et al., 1989) soil scientists turned to the physical

fractionation of SOM. Physical fractionation is considered less destructive than chemical

methods and results obtained with physical fractionation methods are anticipated to relate

better to the structure and function of SOM in situ (Golchin et al., 1994b). Physical

fractionation of soil emphasizes the function of soil minerals and structure in SOM turnover

(Christensen, 2001). These techniques have been applied to determine the association of

SOM with primary particles and to quantify the amount of particulate organic matter between

and within soil aggregates (Beare et al., 1994; Six et al., 1998; Aoyama et al., 1999; Puget et

al., 2000). Physical fractionation covers a range of different methods, each designed for

specific purposes, including combinations of ultrasonic, mechanical and chemical dispersion

with size separation using wet or dry sieving and density separation using heavy liquids.

Most fractionation schemes attempt to avoid chemical changes in SOM during the

fractionation step and distinguish between SOM that is not firmly associated with soil

minerals, SOM that is incorporated into primary organo-mineral complexes, and SOM that is

trapped within aggregates (secondary organo-mineral complexes) (Christensen, 1996).

Recovery of soil material and SOM after physical fractionation is never total due to losses of

soil material, solution of dissolved organic matter, measuring errors on the weight of carbon

fractions and measuring errors on the SOC content of individual OM fractions. The recovery

22

may logically depend on the complexity of SOM fractionation: the more complex the

fractionation procedure, the more possible losses of SOM and accumulation of measuring

errors.

2.4.1 Different methodologies

2.4.1.1 Density fractionation

Fractionation by density is based on submersion of soil samples into inorganic salt solutions

with a specific density of typically 1.6 to 2.2 g cm–3 and limited sample dispersion

(Christensen, 1992). Commonly applied heavy liquids are sodium-poly tungstate, silica gel

(Ludox) and NaI. A light fraction can be separated from a heavy fraction by flotation with a

dense liquid (Gregorich & Janzen, 1996). The light fraction is considered to be more labile

with a density lower than the soil minerals whereas the heavy fraction is assumed to be more

processed decomposition products stabilized onto the surface of clay or silt particles

(McLauchlan & Hobbie, 2004) or within soil microaggregates, making it more resistant to

microbial degradation and with a higher specific density due to it intimate association with

soil minerals. Various degrees of dispersion can be used prior to density fractionation for the

breakdown of organo-mineral bonds or the breakdown of soil aggregates which allows the

separation of uncomplexed OM and of various sized organo-mineral complexes. Sieving and

floatation in water has also been used for density separation with dispersion.

There are two contrasting approaches used in the density fractionation of SOM. The

sequential separation used by Dalal & Mayer (1986) involves successive extractions with

heavy liquids of increasing density. The parallel separation used by Cambardella & Elliott

(1992) separates SOM from replicate samples with heavy solutions of different densities. The

mass and C content of the intermediate fractions are calculated indirectly by taking the

difference between two neighbouring (heavier or lighter) fractions (Cambardella & Elliott,

1992). A general procedure of sequential separation is described for density fractionation in

Fig. 2.7. In this example water is used as a dispersing agent and Ludox solutions of varying

density were used for density separation.

23

Figure 2.7 A general procedure of sequential separation used in density fractionation (Magid et al., 1996)

2.4.1.2 Size based fractionation

In size based fractionation methods different fractions of SOM are separated by a series of

sequential sieving. Dry and wet sieving are both applied depending on the purpose of

fractionation. Sieving soil into different size classes separates small aggregates or particles

from larger particles (e.g. Six et al., 1998; DeGryze et al., 2004) which contain SOC that is

partially protected from microbial degradation, although not necessarily chemically

recalcitrant. Normally, a size based fractionation method separates soil into sand, silt and

clay sized fractions which relating better with the texture of soil (Fig 2.8).

Dispersion in water

Discarded 150

P1.4

P

24

Figure 2.8 Distribution of the whole soil carbon between clay (

25

Figure 2.9 Scheme of the fractionation procedure (Puget et al., 2000)

2.4.1.3 Dispersion of soil fractions

Physical fractionation of SOM depends largely on the preceding dispersion of the soil

samples. For dispersion of SOM physical procedures (e.g., shaking and ultrasonic vibration),

and chemical procedure are used frequently. A dispersion procedure based on ultrasonic

energy has proven to be an attractive method in SOM research (e.g. Schulten et al., 1993;

Amelung et al., 1998), as it generally attains a good level of dispersion without introducing

chemicals or altering the pH (Christensen, 1992). Therefore, the properties of the isolated

fractions are thought to remain unaffected after such a dispersion and fractionation

procedure. However, a standard method for ultrasonic dispersion does not exist. Christensen

(1992) mentioned treatment periods between 3 and 30 minutes at a power output varying

from 60 to 600 W, resulting in applied energies ranging from 480 to 28 800 J g-1 soil. As the

ultrasonic energy varies from soil to soil it is necessary to determine the ultrasonic energy for

complete dispersion for every soil type studied. Furthermore, distribution of SOM between

size separates change with the change of ultrasonication energy level (Fig 2.10). Gregorich et

26

al. (1988) found a decline of sand and silt sized SOM content from 1-0.5% and 2.3-1.4%,

respectively, while clay SOM content increased from 3.9-6.3% with a raise in ultrasonic

energy from 100-1500 Jml-1.

Figure 2.10 Distribution of different sized fractions of SOC after dispersion of soil at different ultrasonic energy level (Gregorich et al., 1988)

2.4.2 Limitations of physical fractionation methods

Methods used to separate SOM fractions from soils rely on a variety of size- and density-

based techniques that are ideally tailored to meet specific objectives (Table 2.3). This means

methods are developed purpose specific. One method is good for separation of a specific

fraction of SOM and another is more suitable for separation of other SOM fractions. For

example, material size, shape, and density influence partitioning when separation methods

rely on sedimentation (Elliott & Cambardella, 1991). This specific character of custom

designed fractionation methods makes comparisons between results throughout literature

often very difficult. Table 2.3 gives an overview of the multitude of specific aims for

27

physical fractionations that exists and a short description of fractionation methodologies

applied to reach these objectives.

Table 2.3 Objectives of POM fractionation methods and associated studies (Wander, 2004)

Method Objectives and References Size-Based Methods

Macro organic matter: Typically emphasizes large residues, clearly identifiable as plant residues. Upper boundary of subdivision is variable, e.g., 100–250, 250–2000, 8000-200 µm

Concentrate recent inputs of plant and organic residues and biologically active SOM, e.g. Magid & Kjaergaard, 2001

POM or coarse fraction: Typically refers to SOM that is sand sized or larger. Common subdivisions include separation of >53-µm material into >53–250 µm and > 250 µm

Concentrate labile SOM influenced by management, e.g. Cambardella & Elliott, 1992; Wander et al., 1998; Nissen & Wander, 2003

Sand-sized class as a constituent of particle-size separates: Methods separate organo-mineral associations into a range of sand-, silt-, and clay-sized components

Characterize dynamics of organic matter and the (a) influence of management or amendment, e.g. Christensen, 1986; Lehmann et al., 1998 and (b) decomposability of SOM or constituents associated with separates, e.g. Christensen, 1987; Cheshire et al., 1990

Density-Based Methods Light fraction : Common density ranges 1.6–1.75, 1.8–1.95, 2.0–2.6 g cm–3; solutions used to recover LF vary, influence on chemical properties not well characterized; dispersion followed by flotation in liquid, denser fractions not collected; energy used to disperse is source of variability as are methods used to recover suspended matter from heavy fraction.

Influence of management and relationship to biologically available or unavailable C or N, e.g. Gregorich et al., 1996; Alvarez et al., 1998; Carter et al., 1998; Fliessbach & Mader, 2000

Combined Size and Density Techniques Active POM fraction: Separate large-sized fraction and then light fraction; size and densities and fraction labels vary as, e.g., >53 µm < 1.6 g cm–3, 150–3000 µm

28

2.4.2.1 Limitations of density based fractionation methods

Despite the frequent use of density fractionation in SOM studies, a number of fundamental

conceptual and experimental limitations appear to be still unsettled (Christensen, 1992).

Turchenek and Oades (1979) summarized the problems encountered in their density

fractionation study as: (1) removal of water and air from microaggregate surfaces and

cavities, (2) disruption of aggregates or maintenance of dispersion during the fractionation,

(3) removal of adsorbed heavy liquids and surfactant from particles, and (4) evaluation of the

changes in organomineral complexes arising from repeated ultrasonic treatments and from

solution effects of heavy liquids and solvents.

The density of the fluid used to separate particulate from organo-mineral constituents

influences the quantity and chemical character of the fractions obtained. Procedures should

be tailored to suit both the soils and experimental scenarios to which they are applied.

Densities used to float out the light fraction (LF) of SOM vary, with values between 1.85 and

1.40 g cm-3 being common. Light fraction yields are very sensitive to change in the density of

fractionation liquid (Dalal and Mayer, 1986). Wander (2004) showed an increase of yield

with the increase of the density of solution. The use of lower densities favours the recovery

of larger POM constituents (Ladd & Amato, 1980). Minor deviations in liquid density may

produce significant differences in the carbon concentration of the light fractions especially in

the density range of 1.9 to 2.4 g cm-3 (Richter et al., 1975). The high variation of densities

used makes comparison between literature results difficult.

Recovery of charcoal in the light fraction skews fraction characteristics, increasing the

abundance of chemical traits attributed to recalcitrant SOM (Roscoe & Buurman, 2003). This

shows that even small amounts of charcoal present in the light fraction may drastically

disturb conclusions on chemical analysis of light fractions that are isolated by density

fractionation. In this case, density fractionation would fail to yield a bio-chemically relatively

homogeneous SOM fraction.

Golchin et al. (1994a) found that selected POM samples isolated in one study by using a

lower density had 13C-NMR spectral characteristics which indicated that it was more

29

decomposed (had lower O-alkyl and higher alkyl C abundance) than did POM obtained at

higher densities. Along with litter, they might have recovered microaggregates (Six et al.,

2002). As this example suggests, the separation between organic and mineral fractions may

be not complete following density fractionation.

Temperature and actual density of the liquid are difficult to control even though they are

important variables and interact with the amount of energy applied. Small differences in

these properties can significantly influence the proportion of C recovered in this fraction

(Christensen, 1992).

The degree of sample dispersion is also critical to the outcome of density fractionation.

Limited dispersion causes density fractions to include a mixture of noncomplexed SOM,

SOM in organomineral complexes, and SOM in aggregates to finer particles.

Microaggregates become included in light fractions because entrapped air and adsorbed

water lower their effective density (Christensen, 1992).

In the past organic liquids such as tetrabromomethane, bromoform and tetramethane were

used for density fractionation (Turchenek and Oades, 1979). The drawbacks of these liquids

was high potential toxicity as halogenated hydrocarbons. Then scientists used aqueous

solutions of inorganic salts such as sodium iodide (Sollins et al. 1984) and sodium

polytungstate (Camberdella & Elliott, 1992). However, these inorganic salts are expensive

and they may alter the chemical characteristics of SOM fractions as they are strong reducing

agents. Other scientists have moved towards non-toxic organic solvents, but still some

limitations remain, for example as silica gels extract humic substances due to a high pH (pH

8 or more) (Wander 2004). Even though it is reported that NaPT is relatively inert, it is

difficult if not impossible to completely remove from POM (Wander 2004).

Physical entrapment of the LF material by the heavy fraction and adhesion of the LF to

container sides can reduce the efficiency of LF recovery. Maximization of the area rather

than the volume of solution to which soil is exposed can reduce entrapment. Efficient

decantation can be facilitated by adding fresh solution after shaking to rinse the adhered

30

material from container sides and increase the distance between suspended light and heavy

materials that will then be pelleted by centrifugation (Wander et al., 1998).

2.4.2.2 Limitations of size based fractionation methods

Quantity and chemical character of the SOM fractions are highly influenced by the size cut-

off of the sieves used to separate particulate from organo-mineral soil constituents. The

simpler size- or density-based methods are well suited to study the influence of land use and

management practices on SOM characteristics. The common use of 53 µm, the lower

boundary for sand sized material, as the cut-off for POM is operationally convenient but

arbitrary. For example, Christensen (1992) used 63 µm as the size dimension that, after

dispersion in water, separated finer organo-mineral complexes from the course fractions. The

upper boundary of the cut-off is also arbitrary and varies notably with sample handling.

Free and occluded fractions of uncomplexed POM can be separated only by stepwise

dispersion of soil combined with separations according to particle size or density or both.

Free OM is recovered from minimally dispersed samples in which aggregates remain intact,

while occluded OM subsequently is isolated after dispersion of aggregates. If, for instance,

the whole soil is dispersed prior to the wet sieving, both free and occluded SOM will end up

in the same size fraction by means of wet sieving. In this sense, size based fractionation

methods by themselves are unable to separate labile SOM from SOM with intermediate

turnover. The size of different fractions as a property for discrimination between soil

fractions cannot be used solely with the aim to isolate unique and non composite SOM

fractions.

Shang & Tiessan (2001) found that the sequential method of SOM fractionation caused a

greater loss of C (8±10%) than the parallel method (3±5%); however, the sequential method

permits direct measurements on C concentration, complexing cations (Fe and Al), and

mineralogical composition.

31

2.4.2.3 Limitations of dispersion techniques

Ultrasonic dispersion

The effectiveness of ultrasonic dispersion depends on instrument specifications, but also on

actual experimental procedures and soil characteristics (Christensen, 1992). There is a great

risk of redistribution of organic matter among the particle-size fractions if ultrasonic energy

is used for dispersion (Elliott & Cambardella, 1991). Oorts et al. (2005) found that

redistribution of SOM fractions increased with the application of increasing sonication

energy. When sonication is used, the energy output and soil solution ratios need to be

optimized for POM recovery. Diaz-Zorita et al. (2002) have shown that the size of fragments

obtained is inversely related to the mechanical stress applied. Work by Elliott et al. (1996)

and Gregorich et al. (1988) suggests that energies lower (300 to 500 J mL–1) than the 1500 J

mL–1 dispersion energy commonly used to obtain complete dispersion of aggregates should

be used to separate POM.

Optimum dispersion energies vary among soils, and this point should be taken into account

during ultrasonic dispersion. For example, in a study of grassland soils, Amelung & Zech

(1999) found that dispersion of macroaggregates (250 to 2000 µm) was achieved at an

ultrasonic energy of 1 kJ for most of the sites considered. Soils from wet extremes in the

prairie were an exception, for which 3 kJ was needed for dispersion and ≥5 kJ was required

to disperse microaggregates (20 to 250 µm). However, use of energies >5 kJ disrupted POM.

On the other hand, Oorts et al. (2005) found redistribution of native and added particulate

organic matter increased with the application of increasing sonication energy in highly

weathered soils (different ultrasonic energies were 750, 1500 and 2250 Jg–1 soil). The mildest

ultrasonic dispersion treatment (750 Jg–1) did not result in adequate soil dispersion as too

much clay was still recovered in the larger fractions. Ultrasonic dispersion at 1500 J g–1 soil

obtained a nearly complete dispersion down to the clay level (0.002 mm), and it did not have

a significant effect on the total amount of carbon and nitrogen in the POM fractions. The

2250 J g–1 treatment was too destructive for the POM fractions since it redistributed up to 31

and 37%, respectively, of the total amount of carbon and nitrogen in these POM fractions to

smaller particle-size fractions. Balesdent et al. (1991) studied the effect of ultrasonic

32

treatment on the breakdown of isolated particulate organic matter (POM) fractions in the

absence of soil minerals. They found increasing redistribution of POM when it was treated

for a longer period with ultrasonic energy. Also, other observations point to POM as the

SOM fraction most susceptible to redistribution by an ultrasonic treatment (Amelung &

Zech, 1999; Schmidt et al., 1999).

Very few systematic studies have considered the energy of the solution. Efforts to optimize

sonication energy can be tailored to maximize the yield or concentration of selected

constituents, including biological activity recovered from soils or retained within selected

fractions (e.g., De Cesare et al., 2000). Cleanliness (purity) of the fraction is decreased when

excess energy is applied (Kerek et al., 2002). For example, Dalal & Meyer (1986) found that

ultrasonic treatment led to greater recovery of total C in the LF, but the average C contents of

the material recovered were lower than those in the LF obtained by shaking alone. Ultrasonic

treatment caused contamination of the LF with mineral matter.

Chemical dispersion

Chemically assisted dispersion procedures may introduce unintended in-process changes of

SOM structure and distribution. Such changes are labile to conflict with the concept of intact

organomineral complexes. Hexametaphosphate or calgon are frequently used for dispersion

before separations of POM. Essington and Mattigood (1990) dispersed soil by vigorous

shaking in ethanol containing 10% polyvinyl pyrrolidene and found that this dispersant was

adsorbed by soil particles. Edwards and Bremner (1965) conclude that dispersion with resin

extract substantial amount of SOM. Dispersants influence the chemical properties of SOM

(Ahmed & Oades, 1984), but their effect on POM composition has not been investigated in

detail. However, Oorts et al. (2005) found that dispersion with sodium carbonate resulted in

the weakest dispersion and affected the chemical properties of the fractions obtained through

its high pH and the introduction of carbonate. Another limitation with the use of chemical

dispersion liquids is that it is very difficult if not impossible to fully separate the dispersed

soil fraction from the dispersive agent, which limits the further chemical analysis of the

dispersed soil fraction.

33

Mechanical dispersion

Soil dispersion can also be done by physical disruption such as stirring, shaking, high speed

mixing or homogenising. Shaking is commonly used as a dispersion technique. According to

Christensen (1992), long term shaking can alter SOM properties as much as ultrasonic

dispersion can. High speed mixing cause unacceptable abrasion of primary particles

(Thornburn and Shaw, 1987) and simple shaking has been found to provide incomplete

dispersion, which can leave air entrapped in microaggregates, which can then contaminate

the LF in a subsequent density fractionation (Turchenek and Oades, 1979; Gregorich et al.,

1989). It is furthermore very difficult to quantify the amounts of energy used for mechanical

dispersion, which limits the potential for standardization of mechanical dispersion methods.

This limitation is even further corroborated by the fact that the dispersion energy that is

required for the isolation of a certain fraction varies from soil to soil.

2.4.3 Combined use of size, density and ultrasonic fractionation

There are many forms of SOM models that describe the dynamics of SOM, ranging from

simple, single compartment models (Jenny, 1994) to multicompartment ones (e.g. Coleman

& Jenkinson, 1996). Within these models some of the pools, such as microbial biomass, can

be measured (Christensen, 1996), whereas many others are not directly measurable. Due to a

lack of experimental methods to verify the partitioning of SOM over pools, pool sizes have to

be determined indirectly by model calibration.

Recent advances in physical fractionation methodologies may now however, be able to yield

SOM fractions that are usable as directly measurable counterparts for SOM model pools. A

separated SOM fraction can only be equivalent to a distinct usable model pool if it is unique

and non composite (Smith et al., 2002). In this context uniqueness refers to the dynamics of a

SOM fraction. The inputs, outputs, the decomposition rate or the order of the decomposition

reaction of a pool should be unique. Non composite refers to whether or not a model pool is a

composite of unique subpools. In reality, currently all model OM pools and fractions are not

unique or non-composite as a consequence of the very strong heterogeneity of SOM (Smith

et al., 2002).

34

An example of the combination of different physical fractionation techniques for the isolation

of distinct SOM fractions is the fractionation scheme proposed by Six et al. (2002) (Fig.

2.11). Six et al. (2002) developed this fractionation scheme based on a conceptual model of

SOM dynamics (Fig. 2.12) by using size, density and ultrasonic fractionation in combination

for SOM fractionation. SOM is separated into 4 measurable pools: biochemically protected C

pool, a pool protected by adsorption to the silt and clay soil fraction, a microaggregate

protected C pool and an unprotected C pool (LF or POM not occluded within

microaggregates). In the first step, coarse non-protected POM, microaggregates, and silt +

clay associated C are isolated from 2 mm air-dried sieved soil by following the method

developed by Six et al. (2000). The method accomplishes a complete break up of

macroaggregates without breaking up microaggregates, which are then separated by sieving.

In the next step, fine non protected POM that was collected together with the

microaggregates on the sieve is isolated by density flotation (Six et al., 2000). Subsequently,

microaggregates are dispersed to isolate microaggregate protected POM versus silt and clay

associated C (Six et al., 2000). The silt and clay associated C fractions from step 1 and 2 are

then hydrolyzed to differentiate the silt + clay protected C versus biochemically protected

carbon.

Figure 2.11 Fractionation scheme to isolate SOM fractions used in conceptual model by proposed by Six et al. (2002) by combined use of size, density and ultrasonic energy

35

This conceptual SOM model of Six et al. (2002) differs from most SOM models in that the

model state variables are measurable SOM pools. Here, all three SOM pools (Fig 2.12):

unprotected, physically protected and biochemically protected SOM pools are measurable as

they have real physical well-defined counterparts. Unprotected SOM pools are composed of

free coarse POM (>250 μm), and the free fine POM (53-250 μm) which is not present as

intra-microaggregate POM (Fig 2.11). The physically protected SOM pools are the intra-

microaggregate POM occluded inside microaggregates (53-250 μm) and also contains OM

that is protected by adsorption to clay and silt particles (Fig. 2.11). Six et al. (2002)

suggested that this pool can be measured as the OM present in the silt and clay sized soil

fraction that is lost after acid hydrolysis. The third SOM pool, being the biochemically

protected OM is constituted by OM which is stabilized against microbial decomposition by

its inherent chemical recalcitrance (by its composition). This pool should be measurable as

the OM present in the silt and clay soil fraction that is retained after acid hydrolysis.

Physicochemical characteristics inherent to soils define the maximum protective capacity of

these pools, which limits increases in SOM (i.e. C sequestration) with increased organic

residue inputs. For example, the size of unprotected SOM pool is a function of C inputs and

the specific decomposition rate of the various C components (Six et al., 2002).

Figure 2.12 Conceptual model of soil organic matter (SOM) dynamics with measurable pool (from Six et al., 2002)

36

The model of Six et al. (2002) fully incorporates current understandings of the protection and

dynamics of SOM. Figure 2.12 shows that the behavior of SOM can be explained by

generalizing basic physicochemical soil processes and the model structure relating this

process is proposed to be capable of describing the behaviour of measurable SOM pools. Soil

processes such as soil aggregate turnover, SOM decomposition, and adsorption and

desorption of SOM to clay and silt particles and formation of stable SOM through

complexation and condensation are explicitly defined in this model (Fig. 2.12). In this model

both chemical (litter quality) and physical factors (soil aggregation, soil clay content) are

taken into account. Although other models recognize the existence of some or all of these

processes and protection mechanisms, they do not incorporate them directly into the model’s

structure. Such as incorporation of the processes of desorption and adsorption was used by

Hassink & Whitemore (1997) to model silt and clay protection of SOM, as is also the case in

several other models. However, the physical protection of POM through aggregation has

been neglected in most models.

2.5 Reduced tillage management and Soil Organic Carbon

Tillage affects decomposition processes of SOM through the physical disturbance and

mixing of soil, by exposing soil aggregates to disruptive forces, and through the distribution

of crop residues in the soil (Beare et al., 1994; Paustian et al., 1997). By increasing the

effective soil surface area and continually exposing new soil to wetting/drying and

freeze/thaw cycles at the surface, tillage makes aggregates more susceptible to disruption and

physically-protected intra-aggregate organic material becomes more available for

decomposition (Beare et al., 1994; Paustian et al., 1997). Soil tillage gives rise to a sudden

reinforcement of mineralization, because SOM that was previously protected in inaccessible

pores becomes partly available for decomposition. This can be desirable at the beginning or

during the growing season, but has to be avoided at the end of the growing season to

minimize losses of nutrients. When native ecosystems are converted to agricultural land soil

disturbance from tillage is a major cause of organic matter depletion and reduction in the

number and stability of soil aggregates. No-till cropping systems usually exhibit increased

37

aggregation and SOM in the top layer relative to conventional tillage (Paustian et al., 1997;

Six et al., 2000). Paustian et al. (1997) summarizes the results of 39 paired comparisons of

CT with NT on the effect on SOC and observed 285 g m-2 higher SOC under NT than CT.

On a relative basis, soil C was 8% higher in NT than in CT. Six et al. (2004) also reviewed

several studies on OC sequestration under NT and found that in humid climates the mean net

OC sequestration rate under NT compared to CT is 222 kg C ha-1 yr-1 over 20 years. In dry

climates this mean sequestration rate amounts only to 97 kg C ha-1 yr-1 over 20 years,

whereas after 5 year of NT adoption the net OC sequestration is even negative, -306 kg C ha-

1 yr-1. Bayer et al. (2006) found an increased amount of particulate SOM under NT

compared with CT and the rate of C accumulation was higher in the clay soil (0.62 Mg C ha-1

yr-1) than sandy clay loam soil (0.31 Mg C ha-1 yr-1) whereas the effect of tillage was lower in

the mineral associated SOM (Fig. 2.13).

Figure 2.13 Mean C stocks in particulate and mineral-associated SOM fractions in the top 20-cm layer of two Brazilian Cerrado soils under CT and NT systems

38

Six et al. (2000) explain the higher SOC contents under NT with the conceptual model

postulating that microaggregates are formed within macroaggregates (Fig 2.14). This model

suggests that a slow macroaggregate turnover in NT allows time for the formation of fine

intra-aggregate particulate organic matter (iPOM) from recent crop-derived coarse iPOM and

the subsequent encapsulation of this fine iPOM by mineral particles and microbial

byproducts to form stable microaggregates containing young crop-derived C. In contrast, the

turnover of macroaggregates in CT is faster, providing less opportunity for the formation of

crop-derived fine iPOM and stable microaggregates. The incorporation of new C into free

microaggregates is an important factor contributing to C-sequestration (Skjemstad et al.,

1990) since C contained in free microaggregates has a slower turnover than C in

macroaggregates (Jastrow et al., 1996).

Figure 2.14 Conceptual model showing the "life cycle" of a macroaggregate and the formation of microaggregates as influenced by tillage (Six et al., 2000)

39

To justify the suggestions of the model, Six et al. (2000) studied aggregation in a long-term

tillage experiment. They found that the rate of macroaggregate turnover is approximately

halved in NT compared with CT, which leads to twice as much accumulating fine crop-

derived iPOM, held within twice as many (stable) microaggregates, formed within

macroaggregates.

However, in the study of Del Galdo et al. (2003) long-term cultivation decreased only the

organic C content and not the aggregation capability. They compared the carbon dynamics

under permanent grassland and maize cultivation and found that although tillage strongly

decreased SOC content of all aggregate fractions, it did not affect their proportional weight

distribution. There was no evidence of an acceleration of the disruption of the

macroaggregates under maize cultivation compared to permanent grassland.

Golchin et al. (1995) found that cultivation decreased the content of readily available O-alkyl

in the SOM occluded within aggregates. They suggested that this is the result of the

continuous disruption of aggregates, which leads to a faster mineralization of SOM and a

preferential loss of readily available O-alkyl C. Thus, the enhanced protection of SOM by

aggregates in less disturbed soil results in an accumulation of more labile C than would be

maintained in a disturbed soil. More specifically, cultivation leads to a loss of C-rich

macroaggregates and an increase of C-depleted microaggregates (Elliott, 1986).

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CHAPTER 3. MATERIALS AND METHODS

3.1 Site description and soils

3.1.1 Comparison of physical fractionation methods

A total of 18 soils representing a textural gradient from loamy sand to silty clay with

different organic matter contents have been selected for the comparison of the results of two

physical fractionation methods. Fifteen soil samples from each location were collected with

an auger (Ø 2.5 cm) within a radius of 4 m for the Koekelare, Veurne, Gontrode, Herent and

Bertem sites and in 10x15 m rectangle subplots for the other locations. These samples were

bulked and mixed per location. Sampling sites were roughly located in a central belt from the

eastern to the western parts of Belgium (Fig.3.1) and were selected to cover a wide range in

texture, organic matter content and land use. The annual mean temperature of the sampling

area is 9.3°C near the North Sea (Middelkerke) and slightly increases towards the centre

(10.2°C, Ukkel) and again decreases towards the Ardens (8.8°C, Stavelot). Precipitation of the

sampling areas follows a gradient from 670 mm near the coast to 1057 mm in the Ardens.

Figure 3.1 Geographical location of sampling site in Belgium

41

Bulked (3 field replicates) whole soil samples were physically fractionated into different

fractions according to the different fractionation methodologies. For this purpose, prior to

fractionation, the field moist soil was gently broken apart by hand and was passed through an

8-mm sieve to break down large macroaggregates. The soil was then dried at 50°C.

Table 3.1 Land use and physico-chemical properties of the selected soils

Sample numbera

Location Land use Organic Carbon (% OC)

Total N

(%N)

pHKCl (-)

Sand (%)

Silt (%)

Clay (%)

Bulk densityb (g cm-3)

Conventional tillage (CT)

LS 1 Veurne cropland 0.74 0.08 7.41 77 19 4 1.59

SL 1 Koekelare cropland 1.10 0.08 5.20 72 23 5 1.42

SL 3 Veurne cropland 1.08 0.11 7.29 61 31 8 1.54

SiL 1 Villers-le- Bouillet

cropland 1.09 0.10 5.28 11.4 77.0 11.6 1.35

SiL 4 Court-St- Etienne

cropland 0.91 0.10 5.60 13.5 72.3 14.1 1.41

SiL 5 Heestert cropland 1.06 0.11 6.47 29.5 56.2 14.3 1.47

CL 1 Veurne cropland 2.11 0.22 5.54 30 31