Mineralization of dissolved organic carbon (DOC) and nitrogen (DON) in the soil solution of different forested stands in Flanders

 iv
The observed DOC degradation rate was quite higher in the soil solution collected from the 
site with background N-deposition than for the plot affected by high N-deposition. Whereas 
no DON removal was detected in the case of elevated N-deposition, a relevant DON increase 
was observed in the solution from the site with background N-deposition. 
 
After all, DOC and DON biodegradability was low (2-24 %) for all the solutions, meaning 
that mineralization is likely to be a minor process in removal of DOC and DON from mineral 
soil compared to adsorption interactions.  
 v
Samenvatting 
 
Opgeloste organische koolstof (DOC) en stikstof (DON) spelen een belangrijke rol in de C en 
N cycli in natuurlijke ecosystemen. Het uitlogen van deze componenten naar grond- en 
oppervlaktewaters resulteert in het verlies van nutriënten uit het ecosysteem en in eutrofiëring 
van aquatische ecosystemen. Hoewel sorptie aan minerale bodembestanddelen beschouwd 
wordt het belangrijkste regulerende mechanisme te zijn bij de uitspoeling van DOC en DON 
(Qualls en Haines, 1992), blijkt mineralisatie een belangrijk verwijderingsmechanisme te zijn 
voor DOC en DON in percolerend bodemwater. Het doel van deze scriptie is te bepalen in 
hoeverre mineralisatie betrokken is bij de verwijdering van DOC en DON uit percolerend 
bodemwater in bosecosystemen in Vlaanderen. 
 
Monsters van humuspercolatiewater, bodemwater op verschillende dieptes in de minerale 
bodem, ongestoorde bodemmonsters en inoculum monsters werden verzameld op drie 
verschillende sites in het bosreservaat van Ravels, Kempen.   
Humuspercolatiewater en bodemwater verzameld op het veld en in het labo, werden over een 
0.45 µm filter gebracht en de initiële DOC concentratie werd op ongeveer 60 ppm gebracht. 
Om het merendeel van de opgeloste anorganische stikstof (NO
3
-
 + NO
2
-
 + NH
3
/NH
4
+
)uit de 
verschillende bodemoplossing te verwijderen, werden de monsters gedialyseerd. Daarna werd 
het inoculum toegediend en werden de monsters gedurende 84 dagen geïncubeerd bij 15 °C. 
Totale organische koolstof (TOC) en totale stikstof (TN) concentraties werden bepaald op 
gefilterde en ongefilterde submonsters en opgeloste anorganische stikstof (DIN) werd bepaald 
op de gefilterde monsters.  
 
Er werd gevonden dat zowel de mineralisatiesnelheid van DOC als van DON afhankelijk zijn 
van de diepte in het profiel, het vegetatietype en het N-depositie niveau.  
In de minerale bodem bleek de mineralisatiesnelheid van DON af te nemen met de diepte 
onder de A-horizont, terwijl er voor DOC eerder een trend aanwezig was dat de 
mineralisatiesnelheid toenam met de diepte. Daarnaast werd de hoogste mineralisatiesnelheid 
voor DOC in het humuspercolatiewater gevonden, terwijl de DON concentratie toenam wat 
erop wijst dat DON eerder werd vrijgesteld dan gemineraliseerd.  
 vi
De afname van de DOC concentratie in het bodemwater van het Corsicaanse dennenbestand 
was groter in het bodemwater bemonsterd in het Berkenbestand.  
Daarnaast was de afbraaksnelheid van DOC hoger in het bodemwater gewonnen onder het 
Corsicaans dennenbestand met achtergrond N-depositie en in het bodemwater gewonnen 
onder het Corsicaans dennenbestand met een verhoogde N-depositie. Terwijl er geen DON 
verwijdering werd gedetecteerd in het geval van verhoogde N-depositie, werd een 
aanzienlijke verhoging in DON concentratie waargenomen in de oplossing van de site met 
achtergrond N-depositie.  
 
Uiteindelijk was de DOC en DON afbreekbaarheid relatief laag en afbreeksnelheid traag, wat 
erop neer komt dat mineralisatie een eerder inferieure rol speelt in de verwijdering van DOC 
en DON uit de bodemoplossing vergeleken met de verwijdering dmv adsorptie. 
 1
1. LITERATURE REVIEW 
 
 
 
 
1.1  General aspects of  dissolved organic matter (DOM) 
 
 
1.1.1  Definition of DOM, DOC, DON and DOP 
 
Organic matter in terrestrial and aquatic environments consists of two operationally defined 
phases, particulate organic matter (POM) and dissolved organic matter (DOM). 
DOM is a complex mixture of many organic compounds. DOC, DON and DOP are then 
defined as the amount of carbon, nitrogen and phosphorous included in the dissolved organic 
matter. It is composed of both humic materials and other organic compounds, such as amino 
acids, aliphatic and aromatic acids, carbohydrates and hydrocarbons.  
Soil solution DOM is usually defined as a continuum of organic molecules that can pass a 
0.45 μm filter. In fact the word “dissolved” means all particles smaller than 0.45 µm; so this 
is not only the fact for soil solution (and not only for organic molecules). 
Some workers have used finer filter paper (i.e., 0.20 μm) to separate ‘true’ DOM from 
colloidal materials which are not retained in 0.45 μm filters (Bolan et al., 2004). 
In soils dissolved organic matter is believed to be the most bioavailable / reactive fraction of 
soil organic matter, since all microbial uptake mechanisms need a water environment.  
 
 
1.1.2 Functions of DOM 
  
Dissolved organic matter (DOM) comprises only a small part of soil organic matter; 
nevertheless, it affects many processes in soil and water including the most serious 
environmental problems like soil and water pollution and global warming (Kalbitz and 
Kaiser, 2003). 
Dissolved organic matter plays an important role in the cycling of nutrients, such as nitrogen 
(N), carbon (C), phosphorous (P) and sulphur (S), within soil, and the transport of these 
nutrients from terrestrial to aquatic ecosystems.  
 2
Dissolved organic matter mediates the transport of inorganics, for instance Al and other 
metals, within soils, and moreover is important in the translocation of oxides, humus and clay 
in soils. It is also a substrate for microbial growth (Zsolnay and Steindl, 1991; Qualls and 
Haines, 1992).  
 
The presence of DOM can influence the fate and transport of organic compounds, including 
polycyclic aromatic hydrocarbons (PAH), chlorinated hydrocarbons, pesticides and herbicides 
in the environment, contributing to pollutant transport or to micronutrient availability. 
In fact it has often been shown that organic pesticides added to soil and aquifers are 
partitioned preferentially onto DOM, which can act as a facilitator for the movement of 
pesticide residues to groundwater. Similarly, the organic acids present in the DOM can act as 
a chelating agent, thereby enhancing the mobilisation of toxic heavy metals. 
Dissolved organic matter increases the solubility of organic contaminants, such as DDT 
[1,1,1-trichloro-2,2 bis(p-chlorophenyl)ethane], and may decrease the volatilisation as well. It 
may facilitate the transport of these compounds through soils and groundwaters. Dissolved 
organic matter can reduce the abiotic hydrolysis of pesticides and other organic contaminants 
photosensitize transformations of toxic organic compounds (Zepp et al., 1981; Mills and 
Schwind, 1990).  
 
TABLE 1.1 
Summary of Physical, Chemical, and Biological Contributions of DOM to Soil Functions 
(Source: M. Wander, 2004) 
 
Physical Functions 
Color, water retention, helps prevent shrinking and drying, combines with clay minerals, 
improves moisture-retaining properties, stabilises structure, permits gas exchange. 
 
Chemical Functions 
Formation of chelates improves micronutrient availability; buffer action maintains uniform 
reaction in soil and increases cation exchange. 
 
Biological Functions 
Mineralization provides source of nutrients; combines with xenobiotics, influencing 
bioavailability and pesticide effectiveness. 
 3
1.1.3 The biogeochemistry of DOM 
 
One of the main sources of DOM is decomposition of soil organic matter, defined as organic 
material that has undergone decomposition and humification. It derives principally from the 
vegetation, biological waste materials, the release of root exudates and the lysis of 
microorganisms. Most of the DOM in soil is the end product of microbial metabolism of 
organic residues. 
However, the most important processes which cause the increase in DOM concentration are 
microbial degradation and assimilation, lateral flow, sorption and leaching.  
In cultivated and pastoral soils, crop residues provide the major source of DOM while in 
forest soils, litter, plant residues and throughfall serve as the major source. In forest soils, 
DOM represents a significant proportion of the total carbon budget. 
 
Besides, there is a temporal variation in DOM concentration which occur on two scales: 
seasonal and by storm event. Significant fluxes of DOM take place mostly after snowmelt, in 
the spring after plant growth is initiated, and in the autumn after leaf fall (Herbauts, 1980; 
Antweiler and Drever, 1983). In general, DOM in soil solution is higher in summer than in 
winter (Cronan and Aiken, 1985). Dissolved organic matter concentrations in soils also vary 
during and after storm events, depending on the water flux through the vadose zone;  the 
variation of DOM concentrations is generally more relevant in the A horizon than in B and C 
horizon. This is probably a function of the strong DOM sorption to the solid phase in the 
lower horizons. Therefore, hydrology has a strong influence on nutrient fluxes.  
DOM fluxes through terrestrial ecosystems are substantial and likely to play important roles 
in internal ecosystem carbon re-translocation and in the movement of carbon from litter to 
soils.  
DOM fluxes from litter and surface organic horizons to lower soil layers also play a role in 
soil heterotrophic activity (Cleveland et al., 2004).  
 4
 
         INPUT FROM PRECIPITATION, 
THROUGHFALL AND LITTER LEACHATE 
 
      
 
 
                                                                                                   
 
 
                                                                                SOLUBILISATION  
 
                                                                       
                                                                                SORPTION 
 
                                                                                LEACHING  
 
 
 
                                                                                MICROBIAL  
                                                                                DEGRADATION 
 
 
 
 
Figure 1.1: The biogeochemistry of DOM in soil. The hydrophobicity as DOM leaches 
through the soil profile decreases (Source: United States Department of 
Agriculture, 2005 ). 
 
 
While dissolved organic matter leached from the surface horizons is attenuated in the soil 
profile, a significant fraction can be transported to ground- or surface waters. DOM mobility 
is one of the major factors affecting cycling of nutrients in the soil system and the export of 
nutrients from soils to ground and surface waters. This is particularly true for N and P, as the 
organic forms of these nutrients can be a significant fraction of total dissolved concentrations 
(Qualls et al., 1991). 
 
Sorption of DOM onto soil particles is an important process which controls its transport and 
persistence in soil. Dissolved organic matter sorbs to many different surfaces; the degree of 
DOM sorption to these surfaces strongly depends on pH, the nature of the surface and the 
average molecular weight of DOM, and is less dependent on ionic strength and the source of 
DOM. Specifically sorption raises with the decrease of pH. Furthermore DOM sorbs to 
D
E
C
R
E
A
S
I
N
G
 
H
Y
D
R
O
P
H
O
B
I
C
I
T
Y
 
 5
natural surfaces through several different mechanisms due to its complex nature, such as 
anion-exchange reactions, which are very important as indicated by the decrease in DOM 
sorption in the presence of SO4
2-
, which binds to mineral surfaces primarily through 
electrostatic interactions. Finally, the importance of physical adsorption is shown by 
preferential sorption of the higher molecular weight fraction of DOM.  Anyway, system’s 
conditions can control which is the dominant adsorption mechanism. 
 
Another important process involved in the transportation of DOM through terrestrial 
ecosystems is leaching, which is considered to be the dominant mechanism of loss of 
numerous mineral  and  nutritive elements  in humid  forest  soils. 
Finally, microbial degradation and immobilization of soil organic matter are an integral part 
of nutrient cycling in soils and soil organic matter dynamics (Mazzarino et al., 1983). 
Biodegradation can mediate DOM transport in soils and influence nutrient cycling; besides, 
microbes can degrade DOM in soil solution and DOM leached from litterfall.  
The biodegradability of DOM is also a function of depth of sampling in the soil (Qualls and 
Haines, 1992).  
 
 
1.1.4  Measurement and characterization of dissolved organic matter 
 
Different methods have been used to measure the concentration of DOM in soils and aquifers.  
These methods are grouped into three categories: spectrophotometry, wet oxidation and dry 
combustion.  
 
The most frequently used method involves the measurement of absorption of light by DOM 
using a spectrophotometer at a wavelength of 250 nm. The relationship between the 
absorbance and the concentration of DOM is often found to vary amongst DOM sources, 
which have been related to the difference in the nature of organic matter and the presence of 
coloured inorganic compounds, such as soluble iron oxides. 
Moreover, groups of organic compounds are known to have different extinction coefficients 
so the difference in the relative amount of these groups may produce differences in 
absorbance, independent of DOM concentration. 
 6
The second method involves dry oxidation of DOM to CO2 at high temperature in the 
presence of a stream of oxygen.  The amount of CO2 produced is measured either by infra red 
detector or by titration after absorbance in an alkali or by weight gain after absorbance in 
ascarite.  The most commonly used dry combustion techniques include LECO
(1)
 combustion 
and total organic carbon (TOC) analyzer. The method has high oxidation efficiency and a 
precision of 1-2 %.  
 
The third method involves wet oxidation of samples containing DOM and the subsequent 
measurement of either the CO2 released or the amount of oxidant consumed.  
Dichromate or permanganate are the most common oxidising agents used in this method and 
the amount of oxidant consumed in the oxidation of DOM is measured either by titration with 
a reducing agent or by a colorimetric method (Bolan et al., 2004). 
 
The proportion of DOM oxidised by wet oxidation is often found to vary amongst DOM 
sources which may be attributed to the differences in the nature of soluble carbon. DOM with 
low molecular weight fractions are considered to be highly mobile and are liable for both 
chemical and microbial oxidation. Meili (1992) observed that most estimates of organic 
carbon from wet oxidation using chromate were within 80 and 90 % of DOM.   
 
DOM is sometimes characterized by separating into fractions based on molecular weight and 
sorption  chromatography. DOM is often fractionated into three nominal molecular weight 
(Da) fractions (<5000, 5000 - 100000 and >100,000) using Sephadex chromatography gels 
with different exclusion limits.   
The most common technique for the fractionation of DOM is based on the sorption of DOM 
to non-ionic and ion-exchange resins (Leenheer, 1981).  Based on this, DOM is fractionated 
into hydrophobic acids, bases, neutrals and hydrophilic acids, hydrophilic bases and neutral 
fractions.  
 
 
 
 (1)
 An instrument manufacturer whose name has become synonymous with a method for determining carbon (C) 
and sulphur (S). Samples are heated in an induction furnace operating at >1650°C causing the volatilization of 
all C and S bearing minerals and compounds. The vapours are carried through an infrared spectrometric cell 
wherein the concentration of C and S is determined by absorption of specific infrared wavelengths. 
 7
1.1.5 Dissolved Organic Carbon 
 
Dissolved organic carbon is operationally defined as the carbon included in organic molecules 
of different sizes that pass through a 0.45 µm filter. A minor fraction of the DOC in forest 
floor leachate consists of chemically well-defined compounds e.g. low molecular weight 
organic acids such as acetic, citric, lactic and oxalic acids (Hongve et al., 2000). 
 
The major fraction of DOC in soils consists of a polydisperse mixture of complex 
macromolecules with a typical molecular weight of about 1000 (Berdén and Berggren, 1990). 
However, a large proportion of these molecules are not in true solution, but are rather 
colloids, defined as having linear dimensions ranging from 1nm to 1µm. Colloidal molecules 
are larger in size than those found in true solutions, but smaller than those found in 
suspensions.   
 
1.1.5.1  The importance of DOC  
 
Dissolved organic matter represents an important carbon input flux to forested mineral soils. 
Seepage water of mineral subsoils contains only small amounts of DOC because of 
mineralization, sorption or the formation of particulate organic matter. However, the relation 
between these processes is largely unknown. 
Dissolved organic matter plays important roles in nutrient transport through ecosystems. DOC 
provides a potential source of carbon for microbial growth; for this reason his role as an 
energy source for microbial metabolism is of particular interest. 
 
Dissolved organic carbon (DOC) is one of the most actively cycling soil organic carbon pools 
and is of significance for transport of nutrients, such as nitrogen, phosphorus and sulphur 
(Qualls et al., 1991; Kaiser, 2001), metals (Tipping and Hurley, 1992) and pollutants (Chiou 
et al., 1986) in soils. It also plays a key role in soil formation of, for example, podzols 
(Lundström et al., 2000). Furthermore, DOC is also involved in the biogeochemistry of 
carbon and redistributes organic carbon with soil depth. 
 
Several studies have attempted to characterize and measure internal fluxes of DOC through 
forest ecosystems, such as measurements of DOC in precipitation, throughfall and soil water 
 8
(Qualls and Haines, 1991; Currie et al. 1996, Dalva and Moore, 1991; McDowell and Likens, 
1988).  
 
Determination of DOC lability in these components, however, has not been attempted. The 
lability of DOC determines how much carbon can be used to support higher trophic levels. It 
is also necessary to understand the global carbon cycle since marine and terrestrial ecosystems 
are huge DOC reservoirs compared to the atmosphere and it is important to know how fast 
these reservoirs can undergo changes. 
DOC concentrations can influence water acidity, the mobility and toxicity of metals, and 
nutrient availability (Dalva and Moore, 1991). Phosphorus fertilization has also been shown 
to cause a dramatic increase in DOC lability (Schindler et al., 1992). 
 
1.1.5.2  Processes controlling production and transport of DOC in forest soils 
 
DOC leached from the O horizon of forest soils is a very important source of soil organic 
carbon in the mineral soil, where most of the organic carbon in forest ecosystems is located. 
Recent studies have shown that about half or more of the DOC leached from recent litter is 
lost during passage through the O horizon, which can be further divided into three sub-
horizons: the Oi, horizon with recent or slightly decomposed litter, the Oe horizon, which 
contains organic matter under degradation and the Oa horizon with humified organic matter. 
Despite this, both recent litter in the Oi horizon and more humified organic matter in the Oe 
and Oa horizons contribute significantly to the DOC leaving the O horizon, but with the major 
proportion coming from the Oe and Oa horizons. 
Soil moisture had no effect on DOC leaching out of the O horizon (Fröberg, 2004). The DOC 
concentration in the B horizon, which is a sink for DOC, is largely governed by the physical 
and chemical properties of the mineral soil. 
The major proportion of the DOC in the B horizon is derived from the carbon stored in the 
mineral soil itself, rather than in the O horizon, suggesting extensive exchange of DOC by 
sorption/desorption processes in the mineral soil. 
In fact, only 20-30 % of the total amount of organic carbon to a depth of one metre occurs in 
the O horizon of well-drained forest soils, the rest being located in the mineral soil (Fröberg, 
2004). 
 9
A well-known model which is used to describe DOC dynamics is called DyDOC
(2)
 
(Michalzik et al., 2003); this model treats the soil as a three- horizon profile and simulates 
metabolic carbon transformations, sorption reactions and water transport. 
Furthermore, in many field studies (Qualls et al., 1991; Huang and Schoenau, 1996; 
Michalzik and Matzner, 2003) the highest amounts of DOC have been measured under the Oi 
horizon, with no further increase in DOC concentrations and fluxes in the lower O horizon. 
 
The DOC in throughfall consists of a large fraction of carbohydrates, which is consistent with 
the fact that DOC from throughfall in incubation studies has been shown to be a labile pool of 
carbon (Qualls and Haines, 1992; Hongve et al., 2000, Yano et al., 2000). 
This indicates that a large part of the DOC from throughfall is degraded quickly and does not 
reach the bottom of the O horizon. Nevertheless, throughfall may play a role in the carbon 
cycle of the forest floor. 
Positive effects of throughfall on the DOC leaching from the forest floor due to the high 
proportion of easily decomposable carbon in throughfall 
(3)
, which can act as a co-substrate or 
promoter for decomposition, have been proposed by Zech and Guggenberger (1996), based on 
the observation that degradation of lignin is co- metabolic in nature and by Michalzik et al. 
(2001) as a possible explanation for a co-variation between DOC fluxes and forest floor 
leachate.   
 
As a consequence, decomposition of the labelled litter-derived DOC alone could not account 
for the loss of DOC from labelled litter. Sorption is probably also involved in this process. 
Potential sorption mechanisms involve electrostatic attraction or binding, hydrogen bonding 
and van der Waals forces (Qualls, 2000). Consequently, sorption is likely to be an important 
first step in the removal of DOC in the O horizon. In a longer-term perspective however, 
mineralization is of course the ultimate fate of the sorbed DOC. 
 
 
 
(2)
 The model operates on hourly and daily time steps, and can simulate carbon cycling over both long 
(hundreds-to-thousands of years) and short (daily) time scales. 
(3)
 
Rain that falls through and can be collected under the tree canopy of a forest. Throughfall interacts with leaves 
and materials present on leaves (e.g., dust, plant secretions, insect droppings, etc.) and therefore can be 
chemically very different from rain that falls directly to the surface.