Developments in Fractionation and Measurement of Soil Organic Carbon: A Review

Soil organic carbon (SOC) is the percentage measure of carbon (C) derived from living organisms in soil. Stability of soil organic matter (SOM) can be defined in terms of how easily C and nitrogen in the SOM can be decomposed. Due to the implications in the permanence of SOC during sequestration there is scientific interest in fractionation of SOM into different fractions. A large number of SOM fractionation procedures have been developed to distinguish between SOM to study whether it is liable or recalcitrant to activities of soil microbes. There are physical and chemical fractionation techniques. The former is based on particle size and density of soil samples or combination of the two, and the latter on the reaction of chemical on SOM for the separation of stable SOC. Each fraction of SOC in the laboratory can be commonly determined using wet oxidation by Walkley-Black method and dry combustion by LECO CN Analyzer. With the advancement in chemometric statistical techniques; faster, robust, cheaper and non-destructive methods are emerging. The chemometric statistical techniques do not require any reagents for analysis compared with the wet oxidation or dry combustion methods. Thus, these emerging techniques are highly attractive for studies where a large number of analyses are required. For in situ measurement of SOC, spectral reflectance technology is developed to facilitate instant measurement in the field using the sensors or by remote sensing.


Introduction
Soil organic matter (SOM) is the vast array of carbon (C) compounds in soil and the soil organic carbon (SOC) is the C component of the SOM.Therefore, the SOC is the percentage measure of C deriving from living organisms and it is commonly represented as SOM = SOC 1.72, with 1.72 as the most commonly used conversion factor [1].This is based on the assumption that SOM has 58% C content, on average.It is advantageous to report SOC rather SOM for making consistent and reliable comparisons between studies.Recently, a conversion factor of 2.0 was proposed to be more accurate and representative based on the fact that most of the published literature averaged content of 50% C rather than 58% [2].Slight change in the SOC stock is reported to influence atmospheric C concentration and the fluxes of SOC differ in response to environmental conditions and land manage-ment factors [3].Stability of SOC can be studied using fractionation techniques.This paper summarises the methods of SOC fractionation which helps us understand the characteristic of SOC specially its stability, and provides the summary of main methods used for measurement of SOC in the laboratory and field.

Need for SOC Fractionation
Stability of SOM can be defined in terms of how easily C and nitrogen in the SOM can be decomposed.The identification, isolation and characterisation of SOM fractions have received a great deal of scientific interest because of their implications in the permanence of SOC during sequestration [4,5].The estimates of different pools are used in mechanistic models (e.g.Roth-C and Century) that predict changes in SOM storage [6,7].A large number of SOM fractionation procedures have been devel-oped that seek to distinguish between SOM that is more easily decomposed (low stability) and less easily decomposed (high stability) by the soil microbes.The procedures have been recently reviewed and include physical fractionation by size or density, and various chemical fractionation methods that separate SOM by solubility, hydrolysability, or resistance to oxidation [3,8].

Physical Fraction by Particle Size
Physical fractionation procedures by size is based on the idea that the association of soil particles and their spatial arrangement play a key role in SOM dynamics as bioaccessibility is a prerequisite for decomposition [9,10].Physical fractionation involves the application of various degrees of disaggregating treatments by dry and wet sieving [11][12][13], dispersion [14], and density separation and sedimentation [11,15].The extraction method developed by Cambardella and Elliott [14] is commonly used to isolate particulate (>53 µm) and mineral-associated organic C fractions (<53 µm) after dispersion of soil samples in potassium hexa-meta phosphate solution (5 g L −1 ) and tumbled overnight.Based on the extraction component of Cambardella and Elliott [14], an automated wet sieving technique was developed in Australia using 50 µm sieve so that >50 µm fraction consists of particulate organic matter (POM) plus sand and <50 µm fraction of mineral-associated organic matter [16].

Combination of Size and Density Fraction
The general wet sieving process uses sieves of 250 μm and 53 μm to isolate fractions into three aggregate groups.The >250 μm is macroaggregate, 250 -53 μm is microaggragate and <53 μm is silt + clay size fraction [12,13].Density fractionation is applied to isolate SOM that is not firmly associated with soil minerals from organo-mineral complexes [11,15].Associations of SOM to mineral surfaces are most often characterised by a density >1.6 -2 g cm −3 .The fraction of SOM that floats in the >1.6 -2 g cm −3 medium is the light fraction and that settles down is the heavy fraction.Lighter fraction or POM with a density of <1.6 -2 g cm −3 consists mostly of pieces of plant residues and heavier fraction consists of mineral-associated organic C [11,15].POM also has been separated by a combination of size and density fractionation [17][18][19] in several steps for each particle size group.

Chemical Fractionation of SOC
Of the five SOC pools in the Roth-C model, the inert pool [6] is considered to be totally unaffected by microbial attack and thus to undergo no decomposition with time [20].Several chemical fractionation methods have been used for the isolation of stable SOC; hydrolysis using trifluoroacetic acid (TFA) and hydrochloric acid (HCl) [21,22], oxidative treatments using several oxidants like hydrogen peroxide (H 2 O 2 ) [23], disodium peroxodisulphate (Na 2 S 2 O 8 ) [24], or sodium hypochlorite (NaOCl) [25].Hydrolysis removes compounds that are supposed to be potentially biodegradable [21,22], whereas it has been suggested that the treatments using oxidizing reagents to mimic biodegradation to the extent that treatments preferentially remove less protected SOC [23,25].Helfrich et al. [26] compared five chemical fractionation methods and found that all chemical treatments caused a preferential removal of young, maize-derived SOC, with Na 2 S 2 O 8 and H 2 O 2 being most efficient.However, none of the methods were reported to be generally suited for the determination of the inert organic matter pool of the Roth-C model [26].

Wet Oxidation and Dry Combustion Methods
The most commonly used methods for determination of SOC are wet oxidation by Walkley-Black method [27] and dry combustion by LECO CN Analyzer [28].The latter method estimates SOC more accurately.Due to incomplete oxidation of SOC in a dichromate-sulphuric acid mixture, the Walkley-Black method underestimates SOC concentration when no heating is applied.To overcome the problem of incomplete C recovery, which leads to the underestimation of C concentrations by Walkley-Black method, the analytical results of this technique are adjusted by 1.32 as a correction factor assuming the recovery rate of 76% [29].However, modified dichromate oxidation techniques that involve extensive heating do not require a correction factor because most of the organic C in the soil is oxidized to CO 2 [27].Some authors reported that the recovery of 76% is generally too large [30,31] and that it may vary according to land use, soil texture and sampling depth [30][31][32].

Chemometric Statistical Methods
With the development and emergence of reflectance spectroscopy techniques, near infrared (NIR) and mid-infrared (MIR) are also used to determine SOC in the laboratory.Using chemometric statistical methods, both NIR and MIR spectroscopy techniques have been used in the past two decades to determine soil properties, including soil organic C and total nitrogen [33][34][35].Compared with wet and dry combustion methods, the NIR and MIR spectroscopy techniques are faster, robust, cheaper and non-destructive, and they do not require any reagents for analysis [36,37].These emerging techniques are, therefore, likely to replace the tedious job of wet and dry combustions in future, and are highly attractive for studies where a large number of analyses are required [34].

In Situ Methods
The techniques used for in situ measurement of SOC in the field are by sampling, or by on-the-go detection with sensors mounted on a tractor [38,39] that facilitate instant measurement of SOC.These methods are also non-destructive and relatively cheaper compared to the destructive wet laboratory [27] or dry combustion [28] methods.Remote sensing is another tool for in situ measurement of soil properties in the field [40,41] based on spectral laboratory data and this method captures large spatial area unlike the other previously mentioned techniques.
The other in situ methods include Laser-Induced Breakdown Spectroscopy and Inelastic Neutron Scattering techniques [41].A recent review covers more detail on SOC dynamics with fractionation and measurement techniques [3].

Conclusion
It is advantageous to report SOC rather SOM to consistently and reliably compare between studies.Due to the implications in the permanence of SOC during sequestration, there is a need to fractionate of SOM into different fractions.Further, mechanistic models that predict changes in SOM storage require the estimates of different SOC pools.Thus, a large number of SOM fractionation procedures have been developed to distinguish between SOM to study whether it is liable or recalcitrant.Once fractionated, the most common methods for determination of SOC in laboratory are wet oxidation and dry combustion.More advanced; chemometric statistical techniques are faster, robust, cheaper and non-destructive than wet oxidation and dry combustion methods.With the advancement in spectral reflectance technology, the in situ measurement of SOC is done using with sensors or by remote sensing to facilitate instant measurement of SOC in the field.