Priming Effects of Dissolved Organic Substrates on the Mineralisation of Lignin, Peat, Soil Organic Matter and Black Carbon Dete

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Priming effects of dissolved organic substrates on the mineralisation of lignin, peat, soil organic matter and black carbon determined with 14C and 13C isotope techniques

Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Geowissenschaften der Ruhr-Universität Bochum

vorgelegt von Dipl.-Geographin Ute Hamer geboren 01.05.76 in Wickede (Ruhr)

2004 Contents 2

Contents

Abbreviations ...... 3

Chapter 1 ...... 5 Introduction

Chapter 2 ...... 29 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat Journal of Plant Nutrition and Soil Science (2002), 165: 261-268

Chapter 3 ...... 53 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions Soil Biology & Biochemistry (2004), in press

Chapter 4 ...... 78 Priming effects in soils after combined and repeated substrate additions Geoderma (2004), in press

Chapter 5 ...... 110 Interactive priming of black carbon and glucose mineralisation Organic Geochemistry (2004), 35: 823-830

Chapter 6 ...... 129 Isotopic 13C fractionation during the mineralisation of organic substrates Rapid Communications in Mass Spectrometry (2004), in review

Chapter 7 ...... 151 Epilogue

Summary...... 167

Zusammenfassung...... 170

Acknowledgements...... 174

Curriculum vitae ...... 175 Abbreviations 3

Abbreviations

BC black carbon

Bq bequerel

C carbon

C3 plant plant with the C3 pathway of photosynthesis

C4 plant plant with the C4 pathway of photosynthesis

CaCl2 calcium chloride

CFE chloroform fumigation extraction

CHCl3 chloroform

Cmic microbial biomass C

CO2 carbon dioxide

Corg organic carbon

/13C 13C/12C ratio expressed relative to the PDB standard

DGGE denaturing gradient gel electrophoresis

DOC dissolved organic carbon

DOM dissolved organic matter dw dry weight

KOH potassium hydroxide

N nitrogen n number of replicates

ND not determined

NMR nuclear magnetic resonance spectroscopy

NPK fertilisation with nitrogen, phosphorus and potassium n. s. not significant at p < 0.05 Abbreviations 4

PDB PeeDeeBelemnite, standard for 13C-analysis

PE priming effect

Py-GC/MS-IRMS pyrolysis - gas chromatography/mass spectrometry - isotope ratio mass spectrometry r correlation coefficient

SD standard deviation

SOC soil organic carbon

SOM soil organic matter

TOC total organic carbon

WHC water holding capacity

Chapter 1

Introduction

Chapter 1 Introduction 6

Global carbon cycle

On a global basis, terrestrial soil carbon exceeds that within aboveground biomass by far. It is estimated that about 1580 Gt carbon have been accumulated over centuries and millennia in our soils while the global pools of living biomass and atmospheric carbon amount to 620 and 720 Gt C, respectively (Gleixner et al., 2001). About two- thirds of soil carbon worldwide is stored in forest soils (Sedjo, 1993; Solomon et al.,

1993). On the background of increasing atmospheric CO2 levels there is much interest whether soils act as net carbon sink or source in the global carbon cycle (Kätterer and Andrén, 1999; Ehleringer et al., 2000; Gleixner et al., 2001; IPCC, 2001; Krull et al., 2003). Therefore, it is important to know the processes resulting in a sequestration of CO2 into soil organic carbon (SOC) and those leading to the mineralisation of

SOC resulting in a release of CO2 to the atmosphere. Atmospheric CO2 is trans- formed into plant organic matter via photosynthesis. The amount and the composition of plant litter inputs into soils are essential controlling factors for the formation of SOC (Kögel-Knabner, 2002). In addition to the above-ground input of litter, a considerable proportion of organic material is incorporated into soils below-ground.

Cereals, for example, translocate between 20 and 30 % of assimilated CO2-C into the below-ground. However, only a portion of this carbon is incorporated in soil microorganisms and in SOC (Kuzyakov and Domanski, 2000). The stability of SOC is influenced by many different factors. In addition to the physical and chemical environment (e.g. moisture, temperature, pH, mineralogy), the chemical structure of organic matter as well as the location of organic matter within the soil matrix determine their susceptibility to degradation by microorganisms and enzymes (Sollins et al., 1996; Baldock and Skjemstad, 2000). Although conceptual models about stabilisation mechanisms of SOC exist, quantitative knowledge about the processes is scarce. There is considerable disagreement over carbon pool sizes and fluxes for various ecosystems. It is estimated that on a global scale between 68 and 100 Gt carbon are released to the atmosphere per year due to soil respiration (Rustad et al., 2000).

Although soils contain considerable amounts of SOC, most of it is not available for microorganisms. However, substrates may be available locally, e.g. in decaying ma- Chapter 1 Introduction 7 terial of plant and animal origin. Due to this inhomogenity there are “hot spots” of microbial activity and growth in soils (van Elsas and van Overbeek, 1993).

Priming effect

As early as 1926 Löhnis observed an increase in SOC mineralisation after the addition of fresh organic residues to soil. This phenomenon was termed “priming effect” by Bingemann et al. (1953). The term priming effect is used until today although this sometimes causes confusion, since it was observed that the addition of organic substrates to soil may also retard the mineralisation of SOC. Therefore, the term “positive priming effect” is used when the mineralisation of SOC is accelerated by the substrate addition compared to the control (Figure 1). The term “negative priming effect” is used when the mineralisation of SOC is retarded (Kuzyakov et al., 2000). Within this thesis, the amount of SOC which is mineralised more than in the control is designated as additional SOC mineralised (Figure 1).

Positive priming effect Substrate SOC additional SOC Negative n

o priming effect i t a s li a r e n i M

Soil without Soil with Soil with substrate substrate substrate

Figure 1: Schematic diagram of the influence of organic substrate additions on the mineralisation of soil organic carbon (SOC). The acceleration of SOC mineralisation is termed as positive priming effect and the retardation as negative priming effect (changed after Kuzyakov et al., 2000).

The research on priming effects between 1950 and 1970 mainly focused on arable soils. In agriculture, the main goal was the maintenance of the soil organic matter content. Therefore, the carbon balance of the soils after incorporation of plant resi- Chapter 1 Introduction 8 dues was investigated experimentally. As reviewed by Sauerbeck (1966), most studies of this period showed that more carbon of the plant residues remained in soil than additional SOC was lost by priming. Sauerbeck (1966) concluded that priming effects in arable soils are not important for their carbon stocks.

Recent research on priming effects has shown that especially in experiments with planted and unplanted soils a stimulation of SOC mineralisation in the planted soils of up to 300 % occurred (Cheng et al., 2003). These so-called rhizosphere priming effects are discussed in detail by Kuzyakov (2002). Such high positive priming effects have also been reported after the incorporation of fresh plant shoot residues into the soil (Kuzyakov et al., 1997). It has been shown that not only plant residues induce positive priming effects (Liang et al., 1999; Stemmer et al., 1999; Bell et al., 2003; Malosso et al., 2004), but also simple easily available substrates such as glucose or different amino acids (Dalenberg and Jager, 1989; Vasconcellos, 1994; De- gens and Sparling, 1996; Shen and Bartha, 1997; Aoyama et al., 2000; De Nobili et al., 2001; Falchini et al., 2003). In some cases, positive priming effects due to sew- age sludge or compost addition were reported (Luna-Guido et al., 2001; Leifeld et al., 2002). The addition of model root exudates, root mucilage or roots to soil can also enhance SOC mineralisation (Mary et al., 1993; Traoré et al., 2000; De Nobili et al., 2001). However, most of the above mentioned substrates have also induced negative priming effects in several studies. For example, Szolnoki et al. (1963) and De- gens and Sparling (1996) observed negative priming effects after glucose addition. Roots and dry foliage of vegetable residues (Kuzyakov et al., 1997) and high molecular tannin fractions (Fierer et al., 2001) can cause negative priming effects. Rea- sons for this different behaviour of the same substrate are still uncertain. It is suggested that priming effects are higher in soils with high contents of organic matter (Kuzyakov et al., 2000). Kuzyakov (2002) reports that the rhizosphere priming effect in a soil with 4.7 % organic carbon (Corg) was 30 to 100 times higher than in a soil with 0.7 % Corg. However, these two experiments were not comparable since different plant species were used. In almost all studies only a few arable soils under different management systems were examined. Priming effects in forest soils and in subsoils were not considered. On this basis statements on relationships between soil organic matter content and priming effects are only speculative. It is not only unknown which conditions favour the occurrence of priming effects, but also how long they Chapter 1 Introduction 9 persist and whether they are repeatedly inducible when substrates become available in soils. Furthermore, there is no insight into the pools of soil organic matter (SOM) which are affected by priming effects.

Our knowledge about occurrence, magnitude and mechanisms of this interdependent decomposition of different types of organic matter is still small. In recent research it becomes evident that the mechanisms of priming effects are indispensable to better understand the carbon cycle in soils. Up to now this interdependent decomposition of different organic materials in soils is not considered when calculating the mean residence time of organic matter in soils. Factors involved in many models simulating SOM dynamics are soil physical properties, meteorological data and management information (Franko et al., 1997; Smith et al., 1997).

Mechanisms of priming effects

The activation of microorganisms through easily available substrates is mostly considered to be the main reason for the occurrence of positive priming effects in soils. One possible mechanism, as reviewed by Kuzyakov et al. (2000), is co-metabolism, i.e. the enhanced degradation of SOM is due to microbial growth and the accompanying increased enzyme production. Additionally, the abolishment of energy limitation of microorganisms may enable them to produce energetically expensive enzymes capable of degrading SOM. De Nobili et al. (2001) suggested that some microorganisms invest low amounts of energy to maintain the cell in a state of "metabolic alertness" thus being able to react more rapidly to substrates than dormant cells. They showed that even minute amounts of easily available substrates trigger microorganisms into activity. In contrast, the theory developed by Fontaine et al. (2003) assumes that the addition of easily available, energy rich substrates only promotes the growth of r-strategist microorganisms which are characterised by their ability to respond to substrate additions by rapid growth, but are not able to utilise the more complex organic compounds typical of SOM. Furthermore, several authors suggest that additional CO2 evolution from soils after substrate addition is only an apparent priming effect assuming that the additional CO2 originates from the turnover of native microbial biomass instead of SOC mineralisation (Chander and Joergensen, Chapter 1 Introduction 10

2001; De Nobili et al., 2001). In contrast, Vanlauwe et al. (1994) reported on priming effects after the addition of maize residues which could not be related to enhanced microbial turnover. The results from Chotte et al. (1998) clearly indicated that the additional CO2 evolution after glucose, starch, wheat or legume residue addition to soil could not be explained exclusively by the decrease of native biomass after 66 days of incubation. In both studies, the chloroform-fumigation-extraction method was used to determine the amount of native and new microbial biomass during the incubation.

Possible mechanisms of negative priming are toxicity of the substrate to microorganisms and inhibition of enzyme activities or structural change of organic matter by binding (Gianfreda et al., 1993; Fierer et al., 2001). A preferential utilisation of the easily available substrate compared to SOC is a further explanation (Kuzyakov et al., 2000).

Priming effects induced by dissolved organic matter

Many organic substrates which induce priming effects in soils may be dissolved in the soil solution. Dissolved organic matter (DOM) may be the most important C- source in soils since all microbial uptake mechanisms require an aqueous environment (Metting, 1993). Therefore, the amount and composition of DOM in the soil solution strongly influences microbial activity (Nelson et al., 1994). In turn, degradation changes the properties of the remaining DOM and consequently is expected to affect the stabilisation and decomposition of organic matter (Marschner and Kalbitz, 2003). DOM in soil solutions is operationally defined as the fraction of organic compounds thaWSDVVDPPHPEUDQHILOWHU'20LVDYHU\KHWHURJHQHRXVPL[WXUH of organic substances. It contains complex high molecular weight compounds of unknown structure as well as organic compounds of known structure such as carbohydrates, carboxylic acids, amino acids, and hydrocarbons (Stevenson, 1994). The latter group can constitute about 20 % of DOM in soil solutions (Herbert and Bertsch, 1993). These substrates are continuously entering the soil solution, e.g. by canopy leaching, root exudation or microbial decay. For forest ecosystems it has been reported that about 10 to 40 g dissolved organic carbon (DOC) m-2 year-1 is translo- cated from the organic surface layer into the mineral soil horizons (Guggenberger Chapter 1 Introduction 11 and Kaiser, 2003). During one vegetation period cereals and grasses allocate between 150 and 220 g C m-2 into the below-ground (Kuzyakov and Domanski, 2000). The rhizodeposition, the total amount of C allocated by plants into the soil, comprises several compound groups depending on their mode of release. It is estimated that the water soluble root exudates constitute between 1.7 to 10 % of the rhizodeposition (Grayston et al., 1996).

Maize root exudates, for example, consist of 52.7 % sugars, 1.7 % amino acids and 24 % organic acids (Traoré et al., 2000). The sugar fraction of maize root exudates is dominated by glucose and fructose (Matsumoto et al., 1979; Gransee, 1997). Glycine and alanine are common amino acids in root exudates (Matsumoto et al., 1979) and in soils (Kuzyakov, 1997; Beavis and Mott, 1999). According to Jones and Kielland (2002) the amount of amino acids added to the soil in the form of exudates from liv- -3 -1 LQJURRWVFDQEHHVWLPDWHGWR0FP soil d based upon a root density of 0.9 cm root cm-3 soil. Organic acids were found in concentrations between 0.5 and 10 µM in soil solutions (Jones, 1998). Oxalic acid is one of the most common low molecular weight organic acids in forest soils (Certini et al., 2000). Fox and Cromerford (1990) observed oxalic acid concentrations between 25 and 1000 µM in soil solutions. According to Küsel and Drake (1999), acetate can make up to 25 % of DOC. Phenolic compounds, such as catechol, represent only a minor part of 3 to 10 % of total DOC (Gallet and Keller, 1999).

Only a few studies on priming effects of defined dissolved organic substrates on soil organic matter mineralisation have been conducted (Shen and Bartha, 1996; Shen and Bartha, 1997), although experiments on rhizosphere priming effects (Fu and Cheng, 2002; Kuzyakov, 2002) and with model root exudates (Traoré et al., 2000) indicate that dissolved organic substrates are important for inducing priming effects. Only glucose is a substrate often used in biodegradation experiments (Sauerbeck, 1966; Vasconcellos, 1994; Degens and Sparling, 1996; Aoyama et al., 2000).

Chapter 1 Introduction 12

Soil organic matter affected by priming

According to Baldock and Skjemstad (2000) SOM is defined as the total of all biologically derived organic matter residing within the soil matrix and directly on the soil surface including thermally altered materials. Up to now it is unknown which types of organic matter are affected by priming. There are only few studies considering priming effects on different types of organic matter (Szolnoki et al., 1963). Gen- erally, it is expected that only the labile pool of organic matter is concerned by priming and not the stable one (Jenkinson, 1971; Kuzyakov et al., 2000). In almost all SOM turnover models, SOM is considered to consist of pools with different turnover times: a small C pool which has a rapid turnover time (called as labile, active or dynamic pool) containing relatively young SOM with a mean age of less than a few decades and a large C pool that turns over slowly (called as stable, passive or refractory pool) characterised by old SOM (Rühlmann, 1999). Some models include an inert organic carbon pool which is defined as biologically not decomposable. In some models this C pool is estimated by the content of black carbon in the soil (Krull et al., 2003).

Black carbon (BC) is formed by incomplete combustion of fossil fuels and vegetation and occurs ubiquitously in soils and terrestrial sediments. BC is a continuum from partly charred plant materials to charcoal and soot particles recondensed from the gas phase. It contains over 60 % carbon with the major accessory elements hy- drogen, oxygen, nitrogen and sulphur (Goldberg, 1985; Schmidt and Noack, 2000). Up to 60 % of SOC has been attributed to BC in Canadian Chernozems (Ponoma- renko and Anderson, 2001). In German Chernozems up to 45 % of SOC (Schmidt et al., 1999), in Australian soils up to 30 % (Skjemstad et al., 1996) and in U.S. agricultural soils up to 35 % of SOC (Skjemstad et al., 2002) have been identified as charcoal. These data indicate that BC can constitute a significant part of the soil carbon pool. Hence, charred plant materials may play an important role in carbon sequestration. However, only little is known about its degradation rates and mechanisms. Pho- tochemical or microbial co-metabolic breakdown are proposed to be responsible for BC degradation (Gleixner et al., 2001). Oxidative enzymes (lignin and manganese peroxidase, laccase), hydrolytic enzymes (esterases), alkaline metabolites as well as Chapter 1 Introduction 13 natural chelators are considered to be important for coal degradation or liquefaction (Fakoussa and Hofrichter, 1999).

Lignin and its breakdown products are further common organic compounds in soils. Plant residues, e.g. straw, litter, leaves, roots and wood, consist of 5-30 % of lignin depending on plant species and age (Haider, 1996). Lignin is a random polymer of sinapyl, coniferyl and coumaryl alcohols containing a variety of complex organic linkages (Tate, 2000). Due to its aromatic structures, lignin is not easily degradable and therefore can accumulate during the initial phases of litter decomposition (Sol- lins et al., 1996).

Peat is an organic soil developing in consequence of incomplete decomposition of wetland vegetation under conditions of high moisture level at a deficiency of oxygen. The peatland area worldwide is estimated to 422 Mio. ha (Kivinen and Pakarinen, 1981). Peat is composed to more than 30 % of organic matter. In contrast to peat from groundwater dependent mires, peat from ombrotrophic mires receives water solely from precipitation and thus its nutrient content is low (Scheffer and Schachtschabel, 2002). Peat from ombrotrophic mires contains relative high amounts of polysaccharides and only low amounts of lignin (Wilson, 1987). Due to its nutrient deficiency it is relatively resistant to biodegradation.

Measurement of priming effects

The mineralisation of organic matter is usually determined by measuring the CO2 evolution. For determining priming effects, it is necessary to differentiate between the CO2 originating from the soil organic carbon and the CO2 originating from the amended organic substrate (Figure 1). The development of analytical methods using the isotopic label of organic matter first enabled us to differentiate between the sources of CO2. Elements exist as both stable and nonstable (radioactive) isotopes. Due to different numbers of neutrons in the atomic nucleus the isotopes differ in their masses (Ehleringer and Rundel, 1989). It is possible to determine the turnover of organic carbon in soils using the radioactive 14C isotope or the ratio of the stable 13C/12C isotopes. Labelling organic matter with the stable 15N isotope would also allow to track the fate of nitrogen in soils as discussed by Kuzyakov et al. (2000). Chapter 1 Introduction 14

Since this study focuses on the turnover of organic carbon in soils, the methods using 14C and 13C/12C will be discussed.

In general, laboratory incubation experiments with differently labelled organic matter are carried out. Thus it is possible to work under controlled environmental conditions. Besides, the use of radioactive substrates in field studies is forbidden. One approach to measure the influence of sucrose on soil respiration of a forest soil in situ was conducted by Högberg and Ekblad (1996). They used the natural abundance of 13C and 12C isotopes of organic substances to differentiate between the sources of

CO2. However, here problems due to isotopic fractionation during mineralisation arise, as discussed below. Furthermore, Högberg and Ekblad (1996) were not able to differentiate between soil and root respiration.

During the incubation experiments of this thesis, samples were incubated in 250 ml incubation vessels (Nalgene) in an Respicond-apparatus at 20 °C (Nordgren Innova- tions, Sweden). The CO2 evolved from the sample was trapped in 10 ml of 0.6 M KOH solution placed inside the incubation vessels. By measuring the changes in conductivity of the KOH solution with platin electrodes, the Respicond determined the total amount of CO2 hourly (Nordgren, 1988). The isotopic composition of the 14 13 CO2 trapped in the KOH solution was recorded with C or C isotope techniques at different time intervals, as described in the following sections. Without using differently labelled organic substrates, the only evidence of positive priming effects is given when the difference of CO2-C evolved between the substrate amended sample and the control is higher than the amount of added substrate-C.

14C

The radioactive 14C isotope is continuously produced in the atmosphere due to neutrons from the cosmic radiation hitting on nitrogen leading to the release of a proton. 14 - The C isotope decays by emitWLQJQHJDWLYHO\FKDUJHGEHWDSDUWLFOHV ) called nega- - WURQV,QWKLVVWXG\WKH emission is measured using liquid scintillation counting, because the CO2 evolved during incubation was trapped in KOH solution. For counting an aliquot of the KOH solution is mixed with a scintillation cocktail containing a - VROYHQWDQGIOXRUV7KH particles emitted from the sample interact with the fluor Chapter 1 Introduction 15 molecules, creating flashes of light, which are detected by paired photomultiplier tubes, positioned 180° apart, in the liquid scintillation counter. Quenching may interfere the measurement leading to an underestimation of decay. Therefore, the counting efficiency has to be taken into account for converting counts per minute into disintegrations per minute, the actually occurring decay in the sample. Most instruments KDYHD-emitting external standard built into the counter enabling them to calculate counting efficiencies automatically. Otherwise, internal standardisation can be used. Here, the sample is counted, removed from the counter and measured again after the addition of a standard material of known disintegrations per minute. Chemilumines- cence and phospholuminescence may also interfere the measurement, but are generally prevented when storing the samples in the dark. Bequerel (Bq) is the unit of ra- dioactivity and is defined as one disintegration per second (Coleman and Corbin, 1991; Wilson and Walker, 2000).

For studies on the turnover of organic matter, it is most appropriate to use uniformly 14C-labelled organic substrates, i.e. substrates in which every carbon atom is 14C. Many chemical compounds are commercially available as uniformly 14C-labelled compounds. When a substrate is not uniformly labelled and decomposition rates of the labelled part differ from those of the unlabelled part, this could lead to erroneous calculations of its turnover. This is mainly problematic in studies with 14C-labelled 14 plant residues, which can be produced by growing plants in a CO2 atmosphere (Jenkinson, 1971; Voroney et al., 1991; Wolf et al., 1994). A further source of error is carbonate in soil (Jenkinson, 1971). Sauerbeck (1966) suggests that some of the early experiments were erroneous.

13C

The natural variations of stable carbon isotopes (13C/12C) between different plant species is a useful tool to determine the turnover of organic matter in soil. Approxi- mately 98.89 % of all C in nature is 12C, and 1.11 % is 13C. To determine the influence of "real" DOC in soil solutions on the mineralisation of SOM it is necessary to use the natural differences in the 13C/12C isotopic ratio of organic matter. Chapter 1 Introduction 16

Stable isotope ratios are measured by mass spectrometry and are expressed relative 13 WR WKHL QWHUQDWLRQDO3 '%OL PHVWRQHV WDQGDUG DV / C, because the natural absolute variation in the ratio 13C/12C is small:

13 / C [‰] = [Rsample-RPDB] / RPDB x 1000,

13 12 where R is the isotopic ratio of C/ C. RPDB is the defined isotope ratio of the lime- stone fossil Belemnitella americana from the Cretaceous PeeDee Formation in South 13 13 12 &DUROLQDZKLFKLVVHWWR/ C = 0 ‰ as basis of the scale. It has an absolute C/ C UDWLR RI &UDLJ $ QHJDWLYH /YDOXHL QGLFDWHVW KDW WKHV Dmple is “lighter”, i.e. contains less 13C than the standard.

The 13C/12C ratio of organic carbon in terrestrial ecosystems is determined largely by the C isotope fractionation occurring during photosynthesis (Wolf et al., 1994).

Plants with the C3 photosynthetic pathway reduce CO2 to phosphoglycerate, a 3-C compound, via the enzyme ribulose bisphosphate (RuBP) carboxylase. This enzyme 13 13 discriminates against CO2UHVXOWLQJLQUHODWLYHO\ORZ/ C values between -20 to -

32 ‰. The enzyme phosphoenol pyruvate (PEP) carboxylase of C4 plants reduces

CO2 to aspartic or malic acid, both 4-C compounds, without discriminating against 13 13 &DVPXFKDV5X%3FDUER[\ODVH7KXV/ C values of C4 plants range from -9 to - 17 ‰. Some plants with the Crassulacean acid metabolism (CAM) are able to switch 13 between those photosynthetic pathways. C3SODQWVSHFLHVZLWKDQDYHUDJH/ C value of -27 ‰ dominate in most temperate zone and all forest communities. C4 plant spe- 13 FLHVZLWKDQDYHUDJH/ C value of -13 ‰ are as well as CAM plants more common in warm, arid, or semiarid environments (Boutton, 1991; Ehleringer, 1991). Maize is a typical widespread C4 crop species.

13 :LWKLQSODQWVWKH/ C values of different compounds vary. It has been observed that lignins and lipids are usually 13C depleted compared to the bulk plant material while sugars, amino acids and hemicelluloses are 13C enriched (Boutton, 1996).

13 In several studies it was observed, that the / C value of CO2 evolved during the mineralisation of organic substrates differed significantly from the /13C value of the substrate (Mary et al., 1992; Schweizer et al., 1999; Santrucková et al., 2000; Fer- nandez et al., 2003; Kristiansen et al., 2004). However, there are also studies in which this isotopic fractionation did not occur or was considered to be negligible Chapter 1 Introduction 17

(Cheng, 1996; Ekblad and Högberg, 2000; Nyberg et al., 2000). Hence, it is still uncertain which factors control the magnitude of isotopic 13C fractionation. According to Fernandez and Cadisch (2003), carbon isotope discrimination by heterotrophic microorganisms seems to depend on temperature, molecule isotopic distribution, chemical nature of the substrate, metabolic pathways of carbon, and physiological conditions of microbial growth.

It is important to know this isotopic fractionation for determining the turnover of organic substrates in soils and the influence of these substrates on the mineralisation of SOC, i.e. for determining priming effects. Bol et al. (2003) tried to account for isotopic C fractionation during slurry mineralisation in soils by comparing experiments with C3- and C4-slurry and assuming that isotopic fractionation will be the same for C3- and C4-slurry. However, they were only able to calculate priming effects for the first 9 days of incubation, because thereafter mineralisation between C3- and C4-slurry differed significantly.

Objectives and outline of this thesis

Although priming effects are reported in many different studies since the beginning of the 20th century and research was repeatedly concentrated on it, knowledge about occurrence and mechanisms of priming effects is still uncertain, as presented above. This thesis focuses on the role of dissolved organic substrates on the mineralisation of different organic materials typically occurring in soils and should provide a broad data base concerning priming effects in soils.

The main objectives can be summarised beneath the following three points which sometimes can not be strictly separated:

1. Occurrence of priming effects

• To determine the importance of soluble organic substrates for inducing positive and negative priming effects.

In a first step, the influence of seven uniformly 14C-labelled soluble organic substrates (glucose, fructose, alanine, glycine, oxalic acid, acetic acid and cate- Chapter 1 Introduction 18

chol) on the mineralisation of lignin and peat was examined in a model system, as presented in Chapter 2.

• To investigate whether priming effects are ubiquitously occurring in soils and whether their occurrence and magnitude is related to physical or chemical soil properties or to the composition of SOM.

Those substrates causing strong positive or negative priming effects on lignin or peat were tested in a second step in 11 soil samples originating from two contrasting forest soils and one arable soil, as presented in Chapter 3.

• To examine the types of organic matter in soils which are affected by priming effects (lignin, peat, stable or labile pools of SOM, black carbon). A broad range of organic materials occurring in soils was incubated with several of the 14C-labelled soluble organic substrates. Results on lignin and peat are described in Chapter 2, those on SOM in Chapter 3. Furthermore, priming effects on a soil sample with extended pre-incubation were examined to elucidate whether stable and labile pools of SOM are affected, as demonstrated in Chapter 4. Chapter 5 deals with priming effects on black carbon mineralisation.

• To elucidate how long priming effects persist and whether they are repeatedly inducible. The effects of repeated substrate additions to soil samples during 1 to 4 month incubations and those to black carbon are presented in Chapter 4 and 5, respectively.

2. Mechanisms of priming effects

• To investigate the contribution of the turnover of the microbial biomass to the

additional CO2 evolution after substrate additions.

As shown in Chapter 4, during incubation the amount of the native and new microbial biomass was determined with the chloroform-fumigation-extraction method.

• To estimate if positive priming effects are only due to co-metabolism.

Indications of co-metabolism are only indirect as discussed in Chapter 3, 4 and 5. Chapter 1 Introduction 19

3. Suitability of natural 13C abundance for studying priming effects

• To examine whether the natural differences in the isotopic label of organic matter are suitable for determining priming effects.

Isotopic 13C fractionation during the mineralisation of DOC extracted from maize and forest floor material as well as during the mineralisation of the respective solid materials was investigated. Results are presented in Chapter 6.

Structure of this thesis

The following chapters (Chapter 2 to 6) are self-containing papers, which have been published, accepted or submitted to reviewed journals. An overall discussion and recommendations for future research are presented in the final chapter (Chapter 7). The thesis is written in British English, except Chapter 2 which is written in Ameri- can English.

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Chapter 2

Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat

Co-author: Bernd Marschner

Journal of Plant Nutrition and Soil Science (2002), 165: 261-268.

Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 30

Abstract

The 14C-labeled substrates glucose, fructose, alanine, glycine, oxalic acid, acetic acid and catechol were incubated at 20 °C in a model system that consisted of sand mixed with lignin or peat (3 % Corg (DFKVXEVWUDWHZDVDGGHGDWHLWKHURUJ& J sand)-1. During 26 days of incubation with an inoculum extracted from forest soil, the 14 amount of CO2 evolved was measured hourly. The amount of CO2 was determined after 4, 6, 12, 19 and 26 days. After 26 days of incubation, each substrate showed priming effects, but not in all examined treatments. Most substrates stimulated the degradation of the model substances (positive priming effects). Negative priming effects only were found in the lignin system with oxalic acid and catechol addition at both concentrations. The strongest positive priming occurred in the peat system with -1 WKHR[DOLFDFLGDGGLWLRQRIJ&J where 1.8 % of the peat were mineralized after -1 GD\V FRPSDUHG WR L Q WKH FRQWURO 7KHD GGLWLRQ RI J DODQLQH-C g caused the strongest increase in lignin mineralization, amounting to 3.9 % compared to 2.8 % in the control. During the incubation the extent of priming changed with time. Most substrates caused the strongest effects during the first 4 to 10 days of incubation. The extent of priming depended on substrate type, substrate concentration, and organic model substance. Possibly this is due to the activation of different microorganisms. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 31

Introduction

The addition of organic substrates to soil may accelerate soil organic matter (SOM) mineralization (positive priming effect) or retard it (negative priming effect) (Kuzya- kov et al., 2000). Priming effects first were investigated by Löhnis (1926), who observed an acceleration of soil organic matter (SOM) turnover after the addition of fresh organic residues to soil. In the last years, priming effects were investigated in several studies (e.g. Dalenberg and Jager, 1989; Shen and Bartha, 1996; Kuzyakov et al., 1997; Shen and Bartha, 1997; Sørensen, 1998). They generally showed that the addition of different substrates to soil can accelerate or retard the decomposition of soil organic matter to different extents. In some cases, no effect was observed. Up to now it is not clear which substances cause priming effects and what the mechanisms are. Furthermore, the importance of priming effects for SOM turnover is in discussion. According to Jenkinson (1971) priming effects are only temporary and small in comparison to the amount of SOM and therefore not of practical interest. On the other hand, Kuzyakov et al. (1997) measured positive priming effects of up to 336 % of basal respiration after the addition of fresh shoot residues to soil, indicating that SOM mineralization can be greatly stimulated. Lignin and its breakdown products are common organic compounds in soils. Plant residues, e.g. straw, litter, leaves, roots and wood, consist of 5-30 % of lignin depending on plant species and age (Haider, 1996). Due to its aromatic structures, lignin is not easily degradable and therefore can accumulate during the initial phases of litter decomposition (Sollins et al., 1996). Peat from ombrotrophic mires generally contains only low amounts of lignin, but can still be relatively resistant to biodegradation due to its low nutrient content. Dissolved organic matter (DOM) may be the most important C-source in soils since soil microorganisms are aquatic and all microbial uptake mechanisms require a water environment (Metting, 1993). Therefore, the amounts and composition of DOM in the soil solution strongly influence microbial activity (Nelson et al., 1994) and consequently are expected to affect the stabilisation and decomposition of organic matter (Marschner and Kalbitz, 2003). DOM is a very heterogeneous mixture of organic Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 32 substances. It contains simpler organic compounds of known structure, e.g. sugars, polysaccharides, proteins and low molecular weight organic acids (Guggenberger et al., 1994), and complex high molecular weight compounds of unknown structure such as the so called fulvic and humic acids (Sposito, 1989; Stevenson, 1994). Im- portant sources of DOM in soils are litter, root exudates and the solid organic matter (Zsolnay, 1996). Matsumoto et al. (1979) and Gransee (1997) found in the sugar fraction of maize root exudates glucose and fructose as dominant species. Glycine and alanine are common amino acids in soils (Kuzyakov, 1997; Beavis and Mott, 1999) and root exudates (Matsumoto et al., 1979). Organic acids were found in concentrations between 0.5 and 10 µM in soil solutions (Jones, 1998). Oxalic acid is one of the most common low molecular weight organic acid in forest soils (Certini et al., 2000). Fox and Cromerford (1990) observed that oxalic acid was the dominant low molecular weight organic acid in all examined soil solutions with concentrations between 25 and 1000 µM. According to Küsel and Drake (1999), acetate can make up to 25 % of DOC. Phenolic compounds, such as catechol, represent only a minor part of 3 to 10 % of total DOC (Gallet and Keller, 1999). Guggenberger and Zech (1993) observed free phenol concentrations in the DOC of acid forest soils from 0.11 g kg-1 DOC (sum of 6 phenols). There have been only few studies on priming effects of defined water soluble organic substrates on soil organic matter mineralization (Shen and Bartha, 1996; Shen and Bartha, 1997). Only glucose is a substrate often used in biodegradation experiments (e. g. Szolnoki et al., 1963; Vasconcellos, 1994; Degens and Sparling, 1996; Aoyama et al., 2000).

The objective of this study was to identify DOM-typical substances that cause positive or negative priming effects. Incubation experiments were used to examine the effects of the 14C-labeled substrates glucose, fructose, glycine, alanine, oxalic acid, acetic acid and catechol on the decomposition of the model substances lignin and peat. All these compounds were detected in soil solutions and root exudates and may therefore play an important role in the stabilization process of soil organic matter. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 33

Materials and methods

Model system

A model system that consisted of sand (acid washed and ignited, Baker, Griesheim,

Germany) mixed with lignin or peat (3 % Corg) was used, instead of soil samples. Hence, adsorption of organic materials on minerals that could lead to their stabilization is avoided (Saggar et al., 1996; Miltner and Zech, 1998). Lignin was an 'organosolv-lignin' extracted from spruce wood (Federal Research Centre for Forestry and Forest Products, Hamburg, Germany). According to Schweers and Meier (1979) organosolv-lignins derived from ethanol-water delignification proved to be largely unchanged. The peat used in this study was a commercial horticultural product, presumably collected in the high fens of Northwestern Germany. Lignin contained 63.4 % C and N < 0.01%, peat 51.4 % C and 1.07 % N (C/N-elemental-analyser, Carlo Erba). Solid-state 13C NMR spectra were obtained on a Bruker DSX 200 spectrometer (Bruker, Karlsruhe, Germany) applying the cross polarization magic angle spinning technique with magic angle spinning at 6.8 kHz, a contact time of 1 ms and pulse delay of 400 ms. The 13C chemical shifts, according to Schmidt et al. (2000), were relative to tetramethylsilane (0 ppm). Aromatic-C was the dominant lignin carbon type (56.4 %) followed by alkyl-C (15.5 %). The main constituent of peat was alkyl-O-C (30.7 %), followed by alkyl-C (23.6 %) and aromatic-C (21.6 %).

The model systems consisting of 50 g samples of the sand/peat or sand/lignin mixtures were adjusted to pH 6.5 with Ca(OH)2. Furthermore, we added 1 ml nutrient -1 solution (11.72 g NH4NO3 + 9.4 g K2HPO4 + 12.34 g KH2PO4 l ) and 1 ml inoculum. The inoculum was obtained from the Of- and Oh-horizons of a Podzolic Cambi- sol from the Fichtelgebirge, Germany (Manderscheid and Göttlein, 1995). After sampling in October 2000, the forest floor sample was air-dried. Two weeks prior to use, the material was rewetted to 60 % water holding capacity and preincubated at

15°C. Then the inoculum was obtained by shaking with 4 mM CaCl2-solution (at 1:5 soil:solution ratio) for 30 minutes and subsequent 5 µm filtration. To test the viability of the inoculum, we incubated 60 µl of the soil extract over a period of 5 days with inorganic nutrients and glucose as the only C-source according to Marschner and Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 34

Bredow (2002). In all experiments, between 87 and 90 % of the glucose was mineralized by the inoculum.

14C-labeled substrates

The commercially available uniformly 14C-labeled substances D-glucose, D-fructose, glycine, L-alanine (Amersham Pharmacia Biotech, Freiburg, Germany), oxalic acid, acetic acid and catechol (Sigma-Aldrich, Taufkirchen, Germany), with a radiochemical purity between 98 and 99 %, where used as DOM-typical substances. The effects of these DOM-typical substances on the decomposition of the model substances lignin and peat were tested at two concentrations, 80 µg C (g sand)-1 and 400 µg C (g sand)-1. To obtain these C-concentrations, it was necessary to dilute the radioactive chemicals with unlabeled substrates. The activity per incubation vessel was approximately 3000 Bq. The carbon amounts added are realistic for soils as Bremer and Kuikman (1994) reported DOC-concentrations of up to 750 µg g-1 in rhizosphere soil.

Incubation

For the incubation experiment, the pH-adjusted and nutrient supplemented sand/peat or sand/lignin mixtures (n = 3 for peat, and n = 4 for lignin) were placed into 250 ml incubation vessels and wetted to 60 % of the previously determined water holding capacity with 10 ml (for peat) or 6 ml (for lignin) of aqueous solutions containing the inoculum, the nutrients and the respective DOM-typical substrate. The model systems were incubated for 26 days at a temperature of 20 °C in an Respicond-apparatus

(Nordgren Innovations, Bygdeå, Sweden), which measures the CO2-evolution hourly by determining the changes in electrical conductivity in 10 ml of 0.6 m KOH solu- 14 tion placed inside the incubation vessels (Nordgren, 1988). The amount of CO2 evolved was determined after 4, 6, 12, 19, and 26 days. From the exposed KOH solution, subsamples of 1 ml were taken for counting radiocarbon using 8 ml of a scintillation cocktail (Rotiszint 22, Roth, Karlsruhe, Germany). A Beckmann LS 6000 TA (Fullerton, USA) was used for liquid scintillation counting with correction for back- Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 35 ground and efficiency by the external standard ratio method. Chemiluminescence was predominantly below 1 %. Therefore correction was not necessary.

Statistical analysis

A t-test for unpaired groups was used for statistical analysis to compare means of

CO2-evolution. Prior to calculating priming effects, we tested whether the lignin- or 14 peat-derived CO2-C [mg] from the C-treated samples differed significantly

(p < 0.05) from the CO2-C from the untreated control. To quantify CO2-C [mg] 14 evolved from lignin or peat in the C-treated samples,CO2 − Ctreatment , it was neces- 14 sary to subtract the CO2-C [mg] from the C-labeled substrates of the total CO2-C 14 [mg] evolved. Substrate-derived CO2-C was calculated from the amount of CO2-C evolved, assuming that the 14C- and 12C-substrates were mineralized equally. The dynamics of the priming effect intensity [PI] during the incubation was calculated for HDFKWLPHLQWHUYDOûWDQGH[SUHVVHGLQSHUFHQWRIWKHFRQWUROUHVSLUDWLRQSHUGD\

∆CO2−Ctreatment − ∆CO2−Ccontrol PI t []% = 100⋅ , ∆CO2−Ccontrol ⋅ ∆t where PIt is the priming effect intensity, and ∆CO2-Ctreatment and ∆CO2-Ccontrol are the 12 cumulative amounts of CO2-C during each time interval, respectively. It must be considered that it is not possible to sum up the priming effect intensities to yield the priming effect after 26 days of incubation, because the calculation is based on different amounts of ∆CO2-Ccontrol. The priming effect [PE] after 26 days of incubation was calculated by

CO2 −Ctreatment − CO2 −Ccontrol PE []% = 100 ⋅ 26 26 . 26 CO −C 2 control26

Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 36

Results

Substrate mineralization

After 26 days of incubation with high (400 µg C g-1) substrate addition, the amounts of substrate mineralized in the lignin system differed significantly from those in the peat system (p < 0.05) except for the alanine addition (Table 1). The greatest differences between the two systems were found for oxalic acid and catechol mineralization. In the lignin system only 26.9 % of oxalic acid was decomposed. In contrast, in the peat system oxalic acid was the substrate mineralized to the largest extent (81.7 %). Catechol was decomposed very well in the lignin system to 56.8 % and in the peat system to only 20.1 %. The decomposition of the other substrates was between 54 and 75 %. Between high and low substrate addition, the percentage of substrate mineralized was similar. Only in the lignin system, 6.8 % of oxalic acid were mineralized (low substrate addition), whereas 26.9 % were mineralized at high substrate addition. In the peat system 12.6 % of catechol was mineralized in contrast to 20.1 % by the high C-addition. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 37

Table 1: Mineralization of different 14C-labeled substrates in the model systems and change of lignin and peat mineralization after substrate addition compared to control without substrate addition after 26 days of incubation.

Substrate Substrate addition Lignin Peat Lignin Peat

-1 14 14 a b [µg C g ] [% CO2-C of added C] [% of control]

Glucose 400 67.6 (0.61) 58.3 (0.73) n. s. +23

Fructose 400 54.2 (0.22) 60.9 (0.62) +22 +31

Glycine 400 74.6 (1.91) 63.4 (1.82) +25 n. s.

Alanine 400 61.1 (0.73) 66.0 (1.82) +40 +129

Oxalic acid 400 26.9 (0.56) 81.7 (1.73) -13 +53

Acetic acid 400 62.4 (0.82) 70.7 (1.53) +7 +130

Catechol 400 56.8 (0.98) 20.1 (1.66) -10 +23

Glucose 80 65.2 (0.84) 60.8 (0.83) n. s. n. s.

Fructose 80 53.0 (1.26) 64.1 (1.17) +18 n. s.

Glycine 80 68.2 (0.85) 66.1 (0.83) +15 +149

Alanine 80 61.2 (1.85) 65.4 (0.77) +18 +33

Oxalic acid 80 6.8 (4.92) 85.4 (1.05) -9 +157

Acetic acid 80 65.8 (0.40) 69.6 (0.98) n. s. +28

Catechol 80 58.5 (1.04) 12.6 (0.55) -6 n. s. a Standard error in parenthesis. b < 0: negative priming, > 0: positive priming, n. s.: not significant, no priming.

Nearly in all cases the lag phase was longer in the lignin system than in the peat system which is shown exemplary in Figure 1 for fructose mineralization. This might be due to the impact of peat-derived microbial biomass since control experiments showed that peat mineralization also occurred without an inoculum. We observed only a delay of one day in comparison to the sample with inoculum addition. In contrast, no lignin mineralization occurred without inoculum during a seven-day incubation (data not shown). Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 38

] 60 % [

2 50 O C

4 40 1 -

se 30 o

t Lignin

c 20 u r

F 10 Peat 0 0 5 10 15 20 25 30 Time [d]

Figure 1: Cumulative fructose mineralization in % of added fructose-14C in the lignin and peat system after high substrate addition over the incubation period (bars represent standard error of means and were omitted when smaller than the symbols).

Effect of substrate addition on lignin and peat mineralization

Figure 2 shows the CO2-evolution of the unamended controls. After an initial delay of CO2-evolution of nearly 5 days, lignin was mineralized to a greater extent than peat. At the end of the incubation, the amount of CO2 evolved from lignin reached 42 mg CO2-C, and thus was nearly fourfold higher than that from peat.

45 Lignin 40

] 30 g m

[ 25 C -

2 20 O

C 15 Peat 10

0 0 5 10 15 20 25 Time [d]

Figure 2: Cumulative CO2-C evolution from the unamended peat and lignin system (control with an initial carbon content of 1500 mg C). Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 39

After 26 days of incubation, nearly all substrates caused priming effects (Table 1). -1 7KH DGGLWLRQ RIIU XFWRVH DODQLQH DQG DFHWLFDF LG DW J &J accelerated peat mineralization to a larger extent than lignin mineralization. For example, the mineralization of peat was more than doubled by the addition of alanine and acetic acid, while lignin mineralization only increased by 40 and 7 %, respectively. Oxalic acid and catechol caused priming effects of different directions. They accelerated the decomposition of peat and retarded that of lignin. Glycine had a positive effect on lignin mineralization and no effect on peat mineralization. In contrast, the addition of glucose caused a positive priming effect for peat and had no effect on lignin decomposition.

-1 The priming effects resulting from the low substUDWHDGGLWLRQV J&J ) differed from those caused by the high substrate additions (Table 1). In the lignin system, the direction of priming was generally the same for low and high substrate additions. -1 2QO\WKHDGGLWLRQRIJDFHWLFDFLG-C g did not affect lignin mineralization significantly. The other substrates caused smaller priming effects when added at low concentration. However, this decrease in additional CO2-evolution was not proportional to the decrease in substrate addition. For example, the addition of 400 µg fructose-C g-1 caused an increase in lignin mineralization of 22 %, while the addition of 80 µg fructose-C g-1, had almost the same effect (+18 %) and thus was much more effective in causing priming effects. In the peat system, the reduced substrate addition of alanine and acetic acid led to approximately proportional smaller priming effects. The high acetic acid addition caused an increase in peat mineralization of 130 %, the low addition one of 28 % only. In contrast, the low addition of glycine and oxalic acid accelerated the decomposition of peat much more than the high addition. The peat mineralization with 80 µg oxalic acid-C g-1 was threefold higher than with 400 µg oxalic acid-C g-1. Glucose, fructose and catechol in the low concentration had no significant effect on peat mineralization. The results showed that both direction and intensity of priming depended on substrate type, substrate concentration, and organic model substance.

During the incubation, the priming effect intensity changed with time. For the peat system it was possible to distinguish three curve types as shown in Figure 3 for the high substrate addition. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 40

150

] Glucose 1 - d

l Fructose

o 100 r t

n Glycine o c f

o Alanine 50 % [ C - 2 O

C 0 t- ea P

-50 0 5 10 15 20 25 3 Time [d]

300

] Acetic acid

1 250 - d l

o Oxalic acid r 200 t n o

c Catechol

f 150 o % [ 100 C - 2

O 50 C t- ea

P 0

-50 0 5 10 15 20 25 3 Time [d]

Figure 3: Dynamic of priming effect intensity after high substrate addition (400 J&J-1) to the peat system in percent of control.

The first type showed the maximal (positive) priming during the first four days of incubation and then diminished slowly towards the end of incubation. This was the case when glucose and fructose were added to the system (Figure 3). Alanine caused a slight increase up to day 6. Although glucose and fructose caused negative priming Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 41 after day 12, at the end of incubation the total priming effect (Table 1) was still positive. The second curve type was that of glycine which showed the most negative priming during the first four days of incubation. Thereafter, no clear effect was observed. The third type was characterised by a maximum effect between day 4 and 6 which decreased with increasing incubation time and was caused by the addition of acetic acid, oxalic acid and catechol. In all these cases, the first days of incubation were the most important ones for the magnitude of the priming effects. This was very obvious for the addition of acetic acid when peat mineralization between day 4 and 6 was about 3.5 times larger than that in the control. Apart from the intensity, the curve types were nearly the same for the low substrate addition as mentioned above except for acetic acid and glycine which belong to the first curve type.

For the lignin system, the changes of priming efficiency were different from those mentioned above and are shown in Figure 4. The curves of fructose and glycine were similar for the high substrate additions. They showed a first maximum between day 4 and 6 and a smaller second one between day 12 and 19. The shapes of the curves of low substrate addition were very similar differing in their maximum CO2-C- evolution and the subsequent decrease in CO2-C-evolution (not shown). In contrast, the curve of high catechol addition showed that the lignin mineralization decreased between day 4 and 6 to about -39 % of the control per day and thereafter increased up to 15 % per day between day 12 and 19, and then again decreased to -8 % per day between day 19 and 26. This was not the case for low catechol addition where the priming was positive at the beginning of incubation, thereafter declined, and was negative after day 12 (not shown).

Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 42

200 Glucose ] 1 - d

l 150 Fructose o r t

n Glycine o c

f 100 Alanine o % [ C

- 50 2 O C - n i

n 0 g i L

-50 0 5 10 15 20 25 3 Time [d]

100

] Acetic acid 1 - d l

o Oxalic acid r t n

o 50

c Catechol f o % [ C - 2

O 0 C - n i n g i L

-50 0 5 10 15 20 25 3 Time [d]

Figure 4: Dynamic of priming effect intensity after high substraWHDGGLWLRQ J&J-1) to the lignin system in percent of control.

Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 43

Discussion

Substrate mineralization

The amount of glucose, fructose, glycine, alanine and acetic acid mineralized after 26 days of incubation was between 54 and 75 % in both model systems. This is in the range of mineralization often reported in the literature. Saggar et al. (1999) for ex- 14 ample reported, that the amount of CO2 respired after 35 days of incubation ac- counted for 51 to 66 % of the glucose14C input to soils with different clay contents. Martin and Haider (1986) showed a glucose mineralization of 82 % after four weeks. Verma et al. (1975) observed after four weeks a glycine decomposition of 86 % and an alanine decomposition of 79 %. Differences are due to different experimental de- signs, e. g. incubation temperature, water content and soil type. Values reported in the literature for catechol mineralization are in the range of 11 % (Martin et al., 1979) to 18 % (Martin and Haider, 1986). This is in agreement with our findings for the peat system. In contrast, the catechol mineralization observed in the lignin system is very high with 57-59 %. Catechol is formed during degradation of many aromatic substances like lignin (Haider, 1996). This indicates, that inoculum-derived microorganisms capable of degrading aromatic structures were selectively promoted in the lignin system. The 13C NMR-spectra have shown more aromatic-C in lignin than in peat. The low oxalic acid mineralization in the lignin system compared to the peat system is probably due to a larger amount of binding sites for oxalic acid in lignin. Piccolo et al. (1996) showed that oxalic acid causes a rearrangement of humic substances. The part of oxalic acid that paticipates in rearrangement may thus become less available for decomposition. The higher oxalic acid mineralization in the peat system can also be related to peat-borne microorganisms adapted to oxalic acid utilization due to their history of exposure under field conditions.

Effect of substrate addition on lignin and peat mineralization

Glucose is a common substance used in biodegradation tests. The observed effects of glucose on the mineralization of organic matter vary strongly. Mary et al. (1993) Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 44 found a positive priming effect after 6 months of incubation. Shen and Bartha (1996; 1997), who added 400 µg glucose-C (g soil)-1, observed a positive priming effect during a 30 d incubation. Vasconcellos (1994) measured positive priming effects after the addition of 1000 µg glucose-C (g soil)-1. The intensity of priming depended on incubation temperature and soil type. Szolnoki et al. (1963) showed that the influence of glucose on the decomposition of different alfalfa fractions depended on composition and degree of decomposition of the plant material. They reported a negative priming effect for the cellulose-lignin alfalfa fraction. For the water soluble fraction, no effect was observed and the decomposition of the whole plant material was accelerated. Degens and Sparling (1996) incubated different aggregate size classes with 615 or 2457 ng glucose-C (g soil)-1. For the 0.25-0.5 mm size class, they found a retardation of soil organic matter mineralization after 56 days of incubation. Simi- larly to Baldock et al. (1989), we found no priming effects by glucose addition in most cases. Only the high glucose addition to peat caused a positive priming effect. This may be due to the higher carbohydrate contents in peat than in lignin as shown 13 by C NMR. Schutter and Dick UHSRUWHGWKDWWKHDGGLWLRQRIJ JOu- cose-C (g soil)-1 led to a larger utilization of carbohydrates in Biolog®-Microplates compared to the control. Some studies indicated that especially high glucose addition to soil favours the development of fast growing microorganisms which are less effective than indigenous biomass in utilizing more resistant C pools (Bremer and van Kessel, 1990; Wu et al., 1993).

In contrast, fructose, which is structurally related to glucose, caused positive priming effects in most cases. Only the high addition to peat showed no significant effect. Shen and Bartha (1997) did not observe a priming effect by fructose addition. Fur- ther studies dealing with priming effects caused by substrates tested in this study were not found.

When priming was observed, the direction of priming caused by substrate addition was the same for most substrates tested in both the lignin and peat system. Only the additions of oxalic acid and catechol at both concentrations caused negative priming effects in the lignin system and positive ones in the peat system. In the peat system, negative priming effects after 26 days of incubation were never detected. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 45

The priming efficiency, i.e. the amount of model substance-C mineralized additionally per amount of added substrate-C, between high and low substrate addition was different. We found an increase in lignin mineralization by high substrate addition which was not proportional to the increased amount of added C. Similar results are shown by Degens and Sparling (1996), who incubated different aggregate size classes with 615 or 2457 ng glucose-C (g soil)-1 for the size class < 0.25 mm. In contrast, for the 1-2 mm size class the mineralization of native organic matter was greater by low substrate addition. This effect was observed in our study for the addition of glycine and oxalic acid to the peat system. On the other hand, Mary et al. (1993) reported a priming increase proportional to the substrate-C addition similar to our study for alanine and acetic acid addition to the peat system. These varying effects may be due to different microbial communities active at different substrate concentrations. Griffiths et al. (1999) observed structural changes in the microbial community after adding different amounts of synthetic root exudate to soil. The higher was the root exudate addition, the more fungi became dominant over bacteria.

Most substrates caused the strongest effects during the first 4 to 10 days of incubation. This is in agreement with De Nobili et al. (2001) who observed a decreasing additional C mineralization after 8 days. In soils, sugars, amino acids, organic acids and phenols are continuously supplied by root exudation, leaching and microbial acitivity. De Nobili et al. (2001) showed that the addition of 1/3 of the full substrate -1 UDWH J&J soil) at three intervals during a 24 d incubation increased the additional CO2-C evolution, depending on substrate type, up to five-fold in comparison to a single addition. Therefore, it is of ecological relevance to examine the effects of repeated substrate additions to soil. When considering the relevance of priming effects, it must be kept in mind that the carbon loss through priming is not always greater than the carbon input with non-mineralized substrate. After 26 days of decomposition some substrates left more carbon in the system than was lost due to priming.

Mechanisms of priming are yet not well understood. Some authors suggest that the observed additional CO2-C evolution is due to an increased turnover of native microbial biomass instead of an increased SOM mineralization (Dalenberg and Jager, 1989; De Nobili et al., 2001). Wu et al. (1993) and Luna-Guido et al. (2001) could Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 46 not relate all observed priming effects to changes of microbial biomass, because the additional CO2-C evolution was greater than the decrease of native biomass-C. We did not measure biomass-C during the incubation, but estimated that the maximum input of microbial biomass with the inoculum amounts to 3.5 mg C. In the lignin system, where no autochthonous microbial activity was observed, the excess C released after substrate additions generally greatly exceeded this amount, indicating that the observed positive priming effects were due to an acceleration of lignin mineralization. For the peat system this was not that clear since the peat-borne biomass- C could not be estimated. It must be concluded that turnover of biomass-C may contribute to measured additional CO2-C evolution after substrate addition, but the solid organic matter seemed to be the main CO2-C source.

Another explanation for positive priming effects often suggested is an increase in microorganism activity after addition of easiliy available organic substrates leading to an acceleration of SOM mineralization by means of cometabolism (Kuzyakov et al., 2000). Negative priming effects are due to a direct inhibition of activity of microorganisms or their enzymes. Gianfreda et al. (1995) for example observed an inhibition of enzyme activity through bonding of phenolic compounds. Another explanation is the switch of microbial biomass from SOM decomposition to the utilization of the easily available C source (Kuzyakov et al., 2000). The negative priming effects of oxalic acid in the lignin system coincide with a strongly reduced oxalic acid mineralization (see above). This could be due to an oxalic acid induced rearrangement of lignin components, as observed by Piccolo et al. (1996) for humic materials. It is conceivable that the very polar oxalic acid molecule binds to polar lignin structures, thus making them less accessible for microorganisms and enzymes.

The results show that the observed priming effects depend on substrate type, substrate concentration and organic matter in the system. The direction, intensity and course of priming is very different and the causes for this are not fully understood. Some results indicate that this might be due to different microbial communities activated by different substrates and substrate concentrations.

Further research is needed to identify mechanisms of priming and to identify the relevance of priming effects for the carbon cycle in soils. Chapter 2 Priming effects of sugars, amino acids, organic acids and catechol on the mineralization of lignin and peat 47

Acknowledgments

This project is financial supported by the German Foundation of Research (DFG). It is part of the Schwerpunktprogramm 1090 “Soils as source and sink of CO2 – mechanisms and regulation of organic matter stabilization in soils”. Thanks also to Dr. Heike Knicker, München, for 13C NMR analysis and Dr. Thilo Rennert, Bochum, for critically reading and improving the text.

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Chapter 3

Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions

Co-author: Bernd Marschner

Soil Biology & Biochemistry (2004), in press.

Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 54

Abstract

It is well established that certain substrate additions to soils may accelerate or retard the mineralisation of soil organic matter. But up to now, research on these so called "priming effects" was almost exclusively conducted with arable soils and with plant residues or glucose as additives. In this study, the effects of the uniformly 14C- labelled substrates fructose, alanine, oxalic acid and catechol on the mineralisation of soil organic carbon (SOC) from different horizons of two forest soils (Haplic Podzol and Dystric Cambisol) and one arable soil (Haplic Phaeozem) under maize and rye cultivation were investigated in incubation experiments for 26 days. Apart from the -1 controls, alOVDPSOHVUHFHLYHGVXEVWUDWH DGGLWLRQVRI J VXEVWUDWH-C mg Corg. 14 During the incubation, CO2-evolution was measured hourly and the amount of CO2 was determined at various time intervals. In almost all soils, priming effects were induced by one or several of the added substrates. The strongest positive priming effects were induced by fructose and alanine and occurred in the Bs horizon of the Haplic Podzol, where SOC mineralisation was nearly doubled. In the other soil samples, these substrates enhanced SOC mineralisation by +10 to +63%. Catechol additions generally reduced SOC mineralisation by -12 to -43% except in the EA horizon of the Haplic Podzol where SOC-borne CO2-evolution increased by +46%. Oxalic acid also induced negative as well as positive priming effects ranging from -24 to +82%. The data indicate that priming effects are ubiquitously occurring in surface and subsoil horizons of forest soils as well as in arable soils. Although a broad variety of soils was used within this study, relationships between soil properties and priming effects could not be ascertained. Therefore, a prediction on occurrence and magnitude of priming effects based on relatively easily measurable chemical and physical soil properties was not possible. Nevertheless, the data suggest that positive priming effects are most pronounced in forest soils that contain SOC of low biodegradability, where the added substrates may act as an important energy source for microbial metabolism.

Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 55

Introduction

The mineralisation of soil organic matter (SOM) can be accelerated or retarded by the addition of organic substrates to soil thus causing positive or negative priming effects (Kuzyakov et al., 2000).

Several hypotheses about the mechanisms of priming effects have been stated in the past. The activation of microorganisms through easily available substrates is considered to be the main reason for the occurrence of positive priming effects in soils. One possible mechanism, as reviewed by Kuzyakov et al. (2000), is co-metabolism, i.e. an enhanced SOM degradation due to microbial growth and the accompanying increased enzyme production. Additionally, the abolishment of energy limitation of microorganisms may enable them to produce energetically expensive enzymes able to degrade SOM for nutrient acquisition. De Nobili et al. (2001) suggest that some microorganisms invest low amounts of energy to maintain the cell in a state of "metabolic alertness", thus being able to react more rapidly to substrates than dormant cells. They showed that even trace amounts of easily available substrates trigger microorganisms into activity. In contrast, the theory developed by Fontaine et al. (2003) assumes that the addition of easily available, energy rich substrates only promotes the growth of r-strategist microorganisms which are characterised by their ability to respond to substrate additions by rapid growth, but are not able to utilise the more complex organic compounds typical for SOM. Several authors suggest that additional CO2 evolution from soils after substrate addition is only an apparent priming effect, assuming that the additional CO2 originates from the turnover of native microbial biomass instead of SOM mineralisation (Chander and Joergensen, 2001; De Nobili et al., 2001).

Possible mechanisms of negative priming are toxicity of the substrate to microorganisms and inhibition of enzyme activities or structural change of organic matter by binding (Gianfreda et al., 1993; Fierer et al., 2001). A preferential utilisation of the easily available substrate compared to SOM is a further explanation (Kuzyakov et al., 2000). Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 56

Until now, priming effects were mainly studied in arable soils after the addition of plant residues (Sauerbeck, 1966; Vanlauwe et al., 1994; Kuzyakov et al., 1997; Bell et al., 2003) or glucose (Szolnoki et al., 1963; Vasconcellos, 1994; Aoyama et al., 2000). Only few studies have investigated priming effects induced by other defined water soluble organic substrates (Shen and Bartha, 1996; Shen and Bartha, 1997; Falchini et al., 2003), although experiments on rhizosphere priming effects (Fu and Cheng, 2002; Kuzyakov, 2002) and model root exudates (Mary et al., 1993; Traoré et al., 2000) indicate that such mixtures of dissolved organic substrates can effectively induce priming effects. Most of the studies were conducted with arable topsoils. Priming effects in forest soils and in subsoils have so far not been considered. How- ever, forest soils contain approximately 66% of the terrestrial soil C (Smith et al., 1993) and for forest soils it has been reported that up to 52% of SOC is stored in the subsoil (Rumpel et al., 2002). Little attention has also been paid to negative priming effects, although positive as well as negative priming effects are reported for most of the above mentioned substrates (Szolnoki et al., 1963; Degens and Sparling, 1996; Kuzyakov et al., 1997). Vanlauwe et al. (1994) concluded that especially in models for short term organic matter dynamics, the interdependent decomposition of different fractions of organic matter makes simulation very complicated. Altogether, the cited studies show that priming effects are quite common, but that it is not possible to predict how a certain substrate will act in a certain soil.

Our previous work has shown that many dissolved organic substrates (glucose, fructose, alanine, glycine, acetic acid, oxalic acid, catechol) cause priming effects on lignin and peat (Hamer and Marschner, 2002). Among these substrates, fructose and alanine generally induced strong positive priming effects while oxalic acid and catechol induced positive as well as negative priming effects. Therefore in this study, these four substrates were chosen to examine (i) if these dissolved substrates influence the decomposition of organic matter in a variety of soils and (ii) how different chemical and structural properties of SOM (e.g. C/N, degradability, structural composition as determined by 13C NMR) and different soil properties influence priming effects. A set of 11 soil samples originating from three contrasting soil types under different land use (2 forest sites, 1 arable site) was chosen to investigate the effects of the 14C-labelled substrates fructose, alanine, oxalic acid and catechol on SOC mineralisation. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 57

Materials and methods

Soils

Soil samples were collected in October 2001 at three sites in Germany, air-dried and sieved (2 mm). All three sites are part of a major German research programme on SOM stabilisation mechanisms. Thus, extensive soil data exists for statistical data analysis. The first site is located in the Steigerwald in Northern Bavaria. The soil is a Dystric Cambisol under a mixed stand of European beech (Fagus sylvatica L.) and European oak (Quercus robur L.). The second site is located in the Fichtelgebirge in North Eastern Bavaria. It is a Norway spruce (Picea abies (Karst.) L.) forest with Haplic Podzol as soil type. Samples at these two forest sites were taken from different horizons of the reference profiles described in Kaiser et al. (2002). The abbrevia- tion Cambisol or Podzol with the respective horizon symbol are used for sample identification. The third site is the long-term field experiment “Ewiger Roggenbau” in Halle (Saxony-Anhalt), which is described in detail in Merbach et al. (1999). This field experiment was established in 1878. It was continuously cropped with rye under different fertiliser treatments. In 1961, a continuous maize cropping system was started on a part of the continuous rye field. Samples were taken from the Ap horizon (0-25 cm) from four plots. The treatments maize and rye cultivation without fertilisation and with NPK fertilisation were chosen (abbreviations: maize, maize NPK, rye, rye NPK). The soil type is a Haplic Phaeozem.

Soil characteristics for all soils are given in Table 1. The soil pH was determined potentiometrically in 0.01 M CaCl2 at a soil:solution ratio of 1:2.5 in mineral soil and 1:5 in organic horizons. Total C and N contents were determined with a CN-analyser (Vario max). Particle size distribution was analysed by sieving and sedimentation (Schlichting et al., 1995).

C P r h i m a p i n t e g r

Table 1: Physical and chemical characteristics of the soil samples (ND: not determined). e 3 f f

ec t a a b b b b s i

Corg C/N DOC (K2SO4) pH (CaCl2) Sand Clay Alkyl C O-Alkyl C Aryl C Carboxyl C n d i f f

0-45 ppm 45-110 ppm 110-160 ppm 160-220 ppm e r e n

-1 -1 -1 t [g kg ] [mg kg ] [g kg ] [% of signal intensity] s o i l t y

Dystric Cambisol p e s i

Oa 122 18.7 887 3.9 ND ND 30 43 17 10 n d u A 44 18.8 424 3.4 472 140 33 42 14 11 ce d b

B 6 17.4 172 3.7 547 100 44 31 16 10 y f r u c t

Haplic Podzol o s e ,

Oa 340 23.6 1157 2.7 ND ND 32 49 15 4 a l a n i

EA 32 26.1 243 3.1 516 104 46 30 13 10 n e , o

Bs 53 26.8 422 3.7 340 164 34 37 14 14 x a li

Bw 24 22.4 337 4.1 447 104 41 34 11 14 c ac i d

Haplic Phaeozem a nd

Maize 10 14.7 32 5.5 662 125 ND ND ND ND ca t ec

Maize NPK 12 15.3 34 5.6 662 104 24 24 32 18 ho l a

Rye 11 14.5 41 5.5 686 102 24 30 25 20 d d iti

Rye NPK 12 14.6 44 5.6 690 100 24 28 29 17 o n s a Particle size distributions of the Dystric Cambisol and the Haplic Podzol are obtained from Kalbitz (unpublished). b 13C NMR spectra of the Dystric Cambisol and the Haplic Podzol are obtained from Rumpel et al. (2002) and of the Haplic Phaeozem from Rumpel and

Wiesenberg (unpublished). 58

Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 59

Microbial biomass C (Cmic) was estimated by a slightly modified fumigation- extraction method (Vance et al., 1987). Prior to fumigation soil samples were rewetted to 60% water holding capacity (WHC) and pre-incubated for 10 days at 15°C.

Then the samples were fumigated with ethanol-free CHCl3 (Merck) for 24 h. After fumigant removal the soils were extracted by shaking for 30 min with 0.5 M K2SO4 (soil:solution ratio: 1:4 for mineral horizon samples, 1:20 for organic horizon samples) and then filtered through a Whatman GF/A filter. Control soils were extracted immediately after pre-incubation. The content of organic C in the extracts was measured with a TOC-analyser (Shimadzu 5050). Cmic was calculated as EC/0.45, where

EC is organic C extracted from fumigated soils minus organic C extracted from non- fumigated soils (Jörgensen, 1995).

Solid-state 13C NMR spectra were obtained on a Bruker DSX-200 NMR spectrometer. Cross polarisation with magic angle spinning was applied using a spin speed of 6.8 kHz and a contact time of 1ms as described in detail in Rumpel et al. (2002). The NMR-analyses of mineral soil samples were carried out after treatment with 10% HF to remove paramagnetic compounds.

14C-labelled substrates

The uniformly 14C-labelled substances D-fructose, L-alanine (Amersham Pharmacia Biotech), oxalic acid and catechol (Sigma-Aldrich) have a radiochemical purity between 98 and 99%. They were mixed with the respective unlabelled substrate and added to the soils. Every soil received a substrate addition of 13.3 µg substrate-C -1 mg Corg with an activity between 3000 and 5000 Bq.

Incubation

For the incubation, 50 g of the air dried soil samples from mineral horizons, 10 g from Podzol Oa and 20 g from Cambisol Oa were placed in 250 ml incubation vessels, rewetted to 40% WHC 10 days prior to use and stored at 15°C. Then the samples were wetted to 60% WHC with the respective 14C-labelled substrate solution or in the case of the controls with deionised water, thoroughly mixed and incubated Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 60 with 3 replicates for 26 days at a temperature of 20°C in a Respicond-apparatus

(Nordgren Innovations). In the Respicond, CO2-evolution is recorded hourly by determining the changes in electrical conductivity in a 0.6 M KOH solution placed in- 14 side the incubation vessels. After 4, 6, 12, 19 and 26 days, the amount of CO2 trapped in the KOH solution was quantified using liquid scintillation counting (Beckmann LS 6000 TA). A more detailed description of analytical methods is given by Hamer and Marschner (2002).

Calculations

All statistical calculations were performed with SPSS 11.0 (SPSS Inc., Chicago, IL).

A t-test for unpaired groups was used to compare means of CO2-evolution. Priming effects during the considered time interval [t] were calculated according to the following equation

CO2−Ctreatment − CO2−Ccontrol PE []% = 100 ⋅ t t , t CO −C 2 controlt where CO2-Ctreatment is the accumulated amount of total evolved CO2 minus the ac- 14 cumulated amount of CO2 derived from the C-labelled substrate, assuming that the 14C- and 12C-substrates were mineralised equally. The total priming effect after 26 days of incubation only was calculated when the amount of CO2-Ctreatment differed significantly from CO2-Ccontrol (p < 0.05, t-test). The absolute amount of additionally derived CO2-C from SOC is obtained by subtracting CO2-Ccontrol from CO2-Ctreatment.

The calculated priming effects after 26 days of incubation were correlated with physical, chemical and biological characteristics of the soil samples. Since many variables were not distributed normally, the Spearman correlation coefficient was chosen. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 61

Results

Substrate mineralisation

After 26 days of incubation, the mineralisation of fructose, alanine, oxalic acid and catechol was very similar among the four treatments from the field experiment “Ewiger Roggenbau” (data not shown). About 58% fructose, 66% alanine and 92% oxalic acid were mineralised. Catechol mineralisation was very low (9 to 14%). In contrast, substrate mineralisation in the forest soils differed between soil types and horizons. For the Dystric Cambisol the mineralisation of fructose, alanine and catechol was highest in the Oa horizon and declined with soil depth while the mineralisation of oxalic acid increased from 74% in the Oa horizon to 97% in the B horizon. A similar increase in oxalic acid mineralisation with soil depth was observed for the Haplic Podzol with a minimum value of 56% for the Oa horizon and a maximum of 90% in the Bs horizon. In samples from this profile the mineralisation of fructose was highest in the EA horizon and lowest in the Bw horizon. Alanine mineralisation declined with soil depth. The mineralisation of catechol in the forest soils was generally also very low (6 to 16%). However, in the Oa and EA horizon of the Haplic Podzol, this phenolic compound was mineralised to a much higher degree (42 and 28% respectively).

Priming effects after 26 days of incubation

The addition of fructose, alanine, oxalic acid and catechol to the soil samples influenced SOC mineralisation to different extents (Fig. 1). For the Dystric Cambisol samples, the highest positive priming effect (+37%) was observed after the addition of alanine to the Oa horizon, where SOM-borne C-mineralisation increased from 2.2% in the control to 3.0%. In the A horizon, alanine caused a priming effect of +22% and in the B horizon the mineralisation in the amended sample did not differ significantly from the control. Fructose only had a significant effect on the SOC mineralisation in the Oa horizon (+10%) and oxalic acid only showed a significant negative priming effect in the B horizon. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 62

control fructose alanine oxalic acid catechol 3.5 +37% a) Dystric Cambisol 3.0 ] %

[ +10% n.s. +22% C 2.5 O

S n.s.

f n.s. n.s.

o 2.0 -19% n.s. -12% n o i -17% t

a 1.5 -21% s i l a r

e 1.0 n i

M 0.5

0.0 Oa A B

2.0 b) Haplic Podzol ] % [ 1.5 C O

S +82% f o +49% n 1.0 +46% o i

t +30% a +17% s +63% i l +91%

a +14%

r +50% n.s. +85% e

n 0.5 -24% n.s. i +49% -23% M n.s.

0.0 Oa EA Bs Bw

2.0 c) Haplic Phaeozem n.s.

] n.s. +19% % [ 1.5 C

O +22% +40% S n.s. f n.s.

o +10% n.s. +19% n.s.

n n.s. 1.0 n.s.

o +16% i

t -43%

a n.s. s i l a r e

n 0.5 i M

0.0 Maize Maize NPK Rye Rye NPK

Figure 1: Mineralisation of SOC after fructose, alanine, oxalic acid or catechol addition compared to the control in samples from a) the Dystric Cambisol, b) the Haplic Podzol and c) the Haplic Phaeozem after 26 days of incubation (means and standard deviation, n = 3). When the difference between SOC mineralisation of the control and the substrate amended sample was significant (p < 0.05), the magnitude of the priming effect is shown in percent (n.s.: not significant). Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 63

The addition of catechol caused negative priming effects in all horizons of the Dystric Cambisol with the strongest retardation of SOC mineralisation in the B horizon (-21 %).

In the Haplic Podzol, mineralisation rates were much lower than in both the Dystric Cambisol and the Haplic Phaeozem. The strongest enhancement of SOC mineralisation was induced by the addition of fructose to the Bs horizon where the amount of SOC mineralised was nearly doubled from 0.20% in the control to 0.37% (Fig.1). Fructose also enhanced mineralisation in the Oa and EA horizons, whereas mineralisation in the Bw horizon was not affected. Similar to fructose, alanine caused the strongest acceleration of SOC mineralisation in the Bs horizon (+85%). Oxalic acid caused negative priming in the Oa horizon of -24% and positive priming in the other three horizons with the strongest stimulation of SOC mineralisation in the EA horizon (+82%). The addition of catechol did not significantly affect SOC mineralisation in the Oa and Bs horizon and retarded the mineralisation in the Bw horizon (-23%). In contrast, in the EA horizon catechol caused a positive priming effect of +46% thus being the only sample that showed an acceleration of SOC mineralisation after catechol addition.

The priming effects in the samples from the arable soil were often not significant because of the high variability among the three replicates. In the unfertilised rye soil, none of the four substrates caused significant effects. In the other samples, fructose caused only weak positive priming effects ranging between +10 and +19%. Alanine only showed a positive effect in the maize NPK soil. Among all substrates, oxalic acid caused the highest positive priming effects, but only in the maize soils. Catechol only affected the SOC mineralisation of the rye NPK soil significantly where it caused the strongest inhibition of all samples (-43%). In general, the arable soils with NPK treatment showed more pronounced priming effects than the unfertilised treatments. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 64

The statistical analysis of possible soil parameters and the observed priming effects showed very few significant correlations (Tab. 2). Only the priming effect induced by alanine and the C/N ratio showed a significant positive correlation (r = 0.64*). The priming effects were not significantly correlated to either SOC degradability in the controls nor to the degree of substrate mineralisation in the treatments (Tab. 2). Nevertheless, the strongest priming effects occurred in the horizons of the Haplic Podzol where SOC mineralisation in the controls was lower than in the horizons of the two other soil types.

Table 2: Spearman correlation coefficients between priming effects (PE) by the different substrates after 26 days of incubation and soil organic matter properties as well as activity parameters of the microbial community (*significant at the 0.05 probability level, n = 11).

a a a a Corg N C/N DOC Alkyl C O-Alkyl C Aryl C Carboxyl C PE [%] [mg kg-1] [mg kg-1] [% of signal intensity]

Fructose 0.44 0.39 0.34 0.15 0.15 -0.05 -0.14 -0.18

Alanine 0.60 0.57 0.64* 0.50 0.57 0.36 -0.60 -0.22

Oxalic acid -0.10 -0.15 0.18 -0.36 0.23 -0.48 -0.33 0.43

Catechol 0.53 0.53 0.55 0.35 0.42 0.31 -0.38 -0.54

a a a degradability [%] Cmic Cmic/Corg qCO2

-1 -1 -1 PE [%] of SOC of substrate [mg kg ] [mg CO2-C h g Cmic] Fructose -0.53 0.32 0.20 -0.39 -0.29

Alanine -0.48 0.30 0.37 -0.13 -0.16

Oxalic acid -0.58 -0.16 -0.32 -0.19 -0.29

Catechol -0.44 0.36 0.18 -0.53 0.04 a n = 10. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 65

Temporal dynamics of priming effects

In almost all samples with positive priming effects, the period of highest additional SOC mineralisation coincided with the period of highest absolute substrate mineralisation. In all samples, the highest absolute mineralisation occurred during the first four days of incubation. Only in Podzol EA horizon with oxalic acid addition mineralisation was highest between days 6 and 12 (data not shown). In general, the observed positive priming effects in the samples from the Dystric Cambisol persisted during the whole incubation, whereas positive priming in the arable Haplic Phaeozem ceased after the first six days at the latest. All arable samples showed a positive priming effect of more than 100% between days 0 and 4 and after day 6 negative ones. Positive priming effects in the samples from the Haplic Podzol lasted longer than in the arable soils but sometimes also became negative during incubation. In the samples showing negative priming effects no relationship between substrate mineralisation and retardation of SOC mineralisation was observed.

Carbon balance

In order to evaluate the impacts of the observed priming effects on the carbon pools in the soil samples, C balances were calculated for each treatment by taking into account the amount of substrate-C remaining in the samples at the end of the incubation period. As shown in Table 3, the C balance was generally positive even in treatments with positive priming effects, because the additionally released SOM-borne

CO2-C did not surpass the amount of residual substrate-C in the samples. Only the addition of alanine to the Cambisol Oa and A horizons and the addition of oxalic acid to the Podzol EA and to the two Phaeozem maize samples caused a net loss of carbon. This loss was highest in the Cambisol Oa where alanine caused a decrease of -1 0.51 mg C g soil which is equivalent to 0.4% of Corg. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 66

Table 3: Substrate effects on the net carbon balance of the soil samples relative to the controls after 26 days of incubation [(+), (-) or (n.s.) indicate positive, negative or non-significant priming effects].

Fructose Alanine Oxalic acid Catechol

[% of Corg]

Dystric Cambisol

Oa 0.24 (+) -0.42 (+) 0.23 (n.s.) 1.58 (-) A 0.55 (n.s.) -0.02 (+) 0.21 (n.s.) 1.40 (-) B 0.73 (n.s.) 0.42 (n.s.) 0.30 (-) 1.58 (-)

Haplic Podzol

Oa 0.38 (+) 0.21 (+) 0.58 (-) 0.63 (n.s.) EA 0.32 (+) 0.09 (+) -0.18 (+) 0.70 (+) Bs 0.37 (+) 0.22 (+) 0.02 (+) 1.04 (n.s.) Bw 0.68 (n.s.) 0.40 (+) 0.04 (+) 1.19 (-)

Haplic Phaeozem

Maize 0.48 (+) 0.39 (n.s.) -0.09 (+) 1.40 (n.s.) Maize NPK 0.42 (+) 0.29 (+) -0.19 (+) 1.23 (n.s.) Rye 0.87 (n.s.) 0.49 (n.s.) 0.33 (n.s.) 1.54 (n.s.) Rye NPK 0.32 (+) 0.45 (n.s.) -0.04 (n.s.) 1.71 (-)

Apparent or real positive priming effects?

Positive priming effects may be real or apparent. During the incubation, a certain portion of the 14C-substrate is incorporated into the microbial biomass and may thus 12 12 substitute native C biomass. This substituted Cmic can contribute to the observed 12 additional CO2-C evolution, which is termed apparent priming. Only in the case of real priming the mineralisation of SOC is stimulated. For the Podzol EA horizon, apparent priming can be excluded, because the amount of additionally evolved 12 CO2-C is about 1.5 to 4 times higher than the amount of Cmic present at the beginning of incubation (Tab. 4). For the other soil samples, such apparent priming effects can not be fully excluded, since the amount of initial Cmic was higher than that of 14 additional CO2-C. In these cases it is helpful to consider the amount of residual C in the samples after incubation. The amount of residual 14C gives the maximal Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 67

14 12 amount of C which could be incorporated in Cmic and substitute native Cmic. Based on data from other experiments (Hamer and Marschner, 2004) we can assume that Cmic did not change very much during incubation. Therefore, if the amount of 12 14 additionally evolved CO2-C is higher than the amount of residual C the observed additional CO2 evolution can be related to SOC mineralisation. This is the case for Cambisol Oa and A with alanine, for Podzol EA, maize and maize NPK with oxalic 12 acid (Tab. 3). In a third step it can be assumed that not all native Cmic is substituted 14 by new Cmic, since Chotte et al. (1998) show that after 38 days of incubation the new 14C biomass only constituted between 13 and 32% of the total biomass. Assum- ing a maximal value of 30% exchange, most positive priming effects would be real, i.e. are due to an enhanced turnover of SOC. Only for fructose addition to Podzol Oa, maize and maize NPK and for alanine addition to maize NPK apparent priming can not be excluded under these assumptions.

Table 4: Amount of microbial biomass (Cmic) in the samples before incubation compared to the a- mount of additional CO2-C released from the substrate treated samples due to positive priming after 26 days of incubation (mean values, n = 3, ND: not determined, -: negative priming, n.s.: non- significant priming).

-1 Cmic Additional CO2-C [mg kg ]

[mg kg-1] Fructose Alanine Oxalic acid Catechol

Dystric Cambisol

Oa 1095 268 984 n.s. - A 428 n.s. 184 n.s. - B 16 n.s. n.s. - -

Haplic Podzol

Oa 628 272 217 - n.s. EA 34 51 89 148 84 Bs 161 95 89 51 n.s. Bw ND n.s. 47 37 -

Haplic Phaeozem

Maize 33 10 n.s. 20 n.s. Maize NPK 72 14 18 37 n.s. Rye 49 n.s. n.s. n.s. n.s. Rye NPK 76 27 n.s. n.s. - Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 68

Discussion

Substrate mineralisation

The mineralisation of the four substrates differed between the horizons and showed no common pattern. Oxalic acid mineralisation in the forest soils was lowest in the Oa horizons (56 and 74%) and increased to values of about 90% in the mineral horizons. These results seem to be contradictory to those of van Hees et al. (2002). They observed a decrease of oxalic acid mineralisation with increasing soil depth in three podzolic forest soils. This could be due to our differential substrate additions which were normalised to Corg content, thus leading to an absolute higher oxalic acid addi- -1 -1 WLRQWRWKH2DKRUL]RQV 3RG]RO2D0J GU\VRLO&DPELVRO2D0J dry -1 soil) than to the subsoil horizons (3 –0J dry soil), whereas van Hees et al. -1 D GGHG0J field moist soil to each horizon. Since Corg and Cmic are correlated in our samples (r = 0.64*), this differential substrate addition also resulted in a fairly narrow range of substrate-C:Cmic ratios (1.4 – 5.7, except 12.5 in Podzol EA). Consequently, the higher oxalic acid mineralisation in the subsoil horizons indicates a strongly substrate limited microbial activity while microorganisms in the organic forest floor are probably more limited by other factors such as nutrient availability. Since no significant increase of microbial biomass was observed in other experiments (unpublished data) it is unlikely that lower substrate mineralisation in the Oa samples is due to microbial incorporation of the substrate.

The observed catechol mineralisation rates of 6 to 16% are in the same range as those reported by Martin et al. (1979) and Martin and Haider (1986). In contrast to this, catechol mineralisation in the Oa and EA samples from the Haplic Podzol were two- to threefold higher and similar to values obtained when catechol was incubated with lignin (Hamer and Marschner, 2002). It is quite likely that lignin degrading fungi also metabolise the phenolic compound catechol and that these fungi are more abun- dant in the topsoil of the very acidic Haplic Podzol.

Alanine mineralisation was 70 to 74% in the O and A horizons of the forest sites and decreased to 55 - 66% in the B horizons. While Verma et al. (1975) also report an alanine mineralisation of 79% after 4 weeks of incubation in a sandy loam with pH 7, Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 69 the decreasing mineralisation with soil depth may be due to increased sorption to soil minerals in the subsoil, as suggested by Vinolas et al. (2001). Fructose mineralisation followed the same pattern as alanine, but was generally lower, thus indicating that microbial activity in these soils may not only be limited by energy substrates but also by nitrogen.

Priming mechanisms

We have shown that all examined dissolved organic substrates were able to induce strong priming effects in at least one of the soil samples. The period of strongest priming during the first four days of incubation coincided with the period of strongest substrate mineralisation. This has also been reported by Chotte et al. (1998), Hamer and Marschner (2002) and Luna-Guido et al. (2003). Since we have not ob- 12 14 served a significant relationship between additional CO2- and CO2-evolution during incubation, the observed priming effects cannot easily be explained by co- metabolism.

In the case of oxalic acid, positive priming effects may be facilitated by an additional mechanism. According to Dutton and Evans (1996) oxalic acid initialises the de- polymerisation of cellulose and thus plays an important role in lignocellulose degradation by wood-rotting basidiomycetes. The data of Piccolo et al. (1996) suggests, that oxalic acid can also cause a structural rearrangement of humic materials, which of course can also make them less accessible for degradation and therefore could explain the observed negative priming effects in two of our soil samples. Similar observations were made during lignin degradation (Hamer and Marschner, 2002).

Negative as well as positive priming effects were also observed after catechol addition. In Podzol EA the strong positive priming effect (+46%) was accompanied by high catechol mineralisation (+28%). However, a better adaptation of the microorganisms on catechol mineralisation seems not to be the only reason for this positive priming effect, since in Podzol Oa 42% catechol were mineralised while priming was negative. The same was reported for lignin (Hamer and Marschner, 2002). Further- more, some samples showed no significant change in SOC mineralisation after catechol addition similar to results from Inderjit and Mallik (1997). Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 70

These inconsistent results indicate, that the direction and magnitude of priming effects is not only due to substrate quality and soil properties but is also strongly influenced by the composition of the soil microflora as hypothesized by Bell et al. (2003). They observed that positive priming effects were higher in arable soils where the fungal:bacterial ratio was also higher. Besides, Falchini et al. (2003) showed that glutamic acid and glucose induced different changes in the microbial community composition, accompanied by an additional SOC mineralisation. Therefore, a controlling factor of priming effects most likely is the composition of the indigenous microbial community of the respective soil and the activation of different groups of microorganisms with different substrates.

Priming effects and soil properties

The priming effects we have observed, are highly variable in direction and intensity between substrates and soil samples. But this variability is not related to chemical and physical soil characteristics or to SOM properties. It was only ascertained that alanine induced higher priming effects in soils with higher C:N ratios. This indicates that SOC mineralisation in the N-poor forest soils was limited by N-availability and thus increased strongly after the addition of this N-containing compound. This would also explain the low or lacking alanine effects in the arable soils where the low C:N- ratio indicates that N is not a limiting factor for microbial activity. Similarly, Kuzya- kov et al. (2000) stated that the direction of priming often depends on the C:N ratio and the nutrient content in soils. Fontaine et al. (2003) also suggest that nutrient-poor soils are more often affected by priming effects than nutrient-rich soils.

In an earlier study with the soils from the long-term field experiment “Ewiger Rog- genbau” in Halle, Freytag and Igel (1968) also observed stronger positive priming effects induced by glucose in the NPK treatment (+26%) than in the control (+12%). Other factors such as low P-availability (Merbach et al., 1999) and regular inputs of fresh crop residues may also explain the low response of microbial activity to the substrate additions.

Jenkinson (1971) suggested that only the labile pool of SOC will be susceptible to priming effects and not the stable one. Therefore, soils with SOC of higher degrad- Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 71 ability should show higher positive priming effects. However, we observed the opposite trend that priming effects were most pronounced in the subsoil horizons of the Haplic Podzol where SOC degradability in the controls was extremely low. These findings are interesting in two respects. Firstly, they show that microorganisms in these horizons apparently are more limited by the availability of energy substrates than those in the other soil samples. This is probably related to the amounts and composition of above- and below-ground plant residue inputs at this spruce site. Sec- ondly, these results indicate that the low SOC degradability in the subsoil horizons is not primarily caused by some structural recalcitrance or by stabilisation through sorption to mineral soil components. This would explain the lacking correlation between priming effects and the structural composition of SOM as determined by 13C NMR analysis. On the other hand, Baldock and Skjemstad (2000) point out that SOM in subsoils may have the same structural composition as in surface soils and still be stabilised against microbial breakdown through sorption to soil minerals. In this profile too, Kaiser et al. (2002) have found an increased association of SOM with clay sized particles with depth and Rumpel et al. (2004) determined a relatively high O-alkyl and carboxyl content of SOM in this size fraction. Both interpret their data in accordance with other studies as evidence for the sorptive stabilisation of SOM in the subsoil. However, our data suggest that the low in-situ SOM degradability is at least partly due to the low input of energy substrates for microbial activity. Qualls and Haines (1992) have shown that dissolved organic matter extracted from subsoils is highly recalcitrant and therefore unavailable as an energy source for microorganisms. Root exudates and root litter as other sources of easily degradable inputs to the subsoil also only play a minor role at this site since the spruce trees have a very shallow rooting system.

Since no statistical relationships between priming effects and any of the available soil properties were found, a different composition of the soil microbial community in the soil samples may be an important factor controlling the direction and magnitude of priming effects, as already discussed in section 4.2. This is supported by the findings that catechol was mineralised to very different degrees in the samples. In addition, it is quite conceivable, that the observed priming effects are due to different mechanisms in the different soil samples and therefore are not associated with a specific set of soil properties. For example, in the forest top soils, alanine may have Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 72 acted primarily as a N-source, while in subsoils, the priming effects of alanine are mainly due to its utilisation as an energy source.

Conclusions

Priming effects occur in almost all examined soils, in organic and subsoil horizons of forest soils as well as in the plough horizon of arable soils. All organic substrates (fructose, alanine, oxalic acid, catechol) induced priming effects. The occurrence of the most pronounced positive priming effects in the mineral horizons of the Haplic Podzol where the mineralisation of SOC in the controls was lowest may indicate that especially in such forest soils the stability or recalcitrance of SOC depends strongly on the input of available substrates to the soil solution. Although a variety of soils was used within this study, relationships between soil properties and priming effects were not ascertained, thus indicating that the mechanisms of priming effects are quite diverse. A prediction on occurrence and magnitude of priming effects based on relatively easily measurable chemical and physical soil properties may therefore not be possible. Further research should focus on activity and dynamics of the soil microbial community and on the specific limiting factors for their mineralisation activity.

Acknowledgements

This project was financially supported by the German Research Foundation (DFG).

It is part of the priority programme “Soils as source and sink of CO2 – mechanisms and regulation of organic matter stabilisation in soils”. Thanks also go to Cornelia Rumpel (Paris) and Guido L. Wiesenberg (Köln) for 13C NMR spectra, to Karsten Kalbitz (Bayreuth) for particle size distribution data and to Thilo Rennert (Bochum) for helpful comments on the manuscript. Chapter 3 Priming effects in different soil types induced by fructose, alanine, oxalic acid and catechol additions 73

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Chapter 4

Priming effects in soils after combined and repeated substrate additions

Co-author: Bernd Marschner

Geoderma (2004), in press.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 79

Abstract

In incubation experiments with soil samples from two forest soil profiles (Dystric Cambisol, Haplic Podzol) we investigated the influence of the addition of easily available 14C-labelled organic substrates on the mineralisation of soil organic carbon (SOC). Substrates were added in combination (fructose, alanine) or repeatedly at different time intervals (fructose, alanine, oxalic acid, catechol). During the 1-4 14 months incubation CO2 evolution was monitored hourly while CO2, and microbial biomass 12C and 14C were determined at different time intervals. The combined addition of fructose and alanine to the Bs horizon of the Haplic Podzol induced a higher positive priming effect (+127 %) than the single substrate additions (+91 and +85 %, respectively). During the first 4 days, seven times more SOC was mineralised than in the control while microbial biomass only increased by a factor of 1.4. These results and the C-balance at the end of the incubation indicate that the observed priming effects can not be solely attributed to co-metabolism or to the turnover of microbial biomass. When added repeatedly, most substrates induced higher positive priming effects, than single additions, thus indicating that the SOC pool affected by priming was not depleted. Since priming effects were not depressed after extended pre- incubation of a soil sample, it seems unlikely that only the labile SOC pool is susceptible. With repeated substrate additions, substrate mineralisation increased with time, indicating changes in the microbial community structure. In the case of catechol, the increased substrate utilisation was accompanied by subsiding negative priming effects, which in one case even became positive. Although many factors controlling priming effects still remain unclear, the study shows that some of the priming mechanisms discussed by other authors such as co-metabolism and microbial biomass turnover are insufficient to explain the observed data.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 80

Introduction

Although soils contain considerable amounts of soil organic carbon (SOC), most of it is not available for microorganisms. However, substrates may be available locally, e.g. in decaying material of plant and animal origin. Due to this inhomogenity there are “hot spots” of microbial activity and growth in soils (van Elsas and van Over- beek, 1993). As early as 1926 Löhnis observed an increase in SOC mineralisation after the addition of fresh organic residues to soil. This phenomenon was termed as “priming effect” by Bingemann et al. (1953). Since the incorporation of substrates into soils may not only accelerate SOC mineralisation but also retard it, Kuzyakov et al. (2000) differentiated between positive and negative priming effects. It has been shown that not only plant residues induce priming effects (Sauerbeck, 1966; Stem- mer et al., 1999; Bell et al., 2003), but also easily available substrates such as sugars or amino acids, which are present in soil solutions and root exudates (Vasconcellos, 1994; Shen and Bartha, 1997; Hamer and Marschner, 2002). Until now it is not clear how long priming effects persist in soils and if they are induced every time when a substrate becomes available in soils. Since there is a continuous supply of easily available substrates in the soil solution this is important for assessing the role of priming effects for the carbon turnover in soils. For example, De Nobili et al. (2001) showed that repeated substrate additions induced a higher additional CO2-C evolution than a single full addition at the beginning of incubation.

Furthermore, we do not know which pools of SOC are affected by priming effects and if one pool will become exhausted. Jenkinson (1971) suggests that only the labile SOC pool is concerned and not the stable one. Neff et al. (2002) report that long-term nitrogen fertilisation accelerated the decomposition of SOC in the light fractions with decadal turnover times while SOC in heavier, mineral-associated fractions was stabilised. Results from Hagedorn et al. (2003) indicate that N fertilisation reduced the mineralisation of old and humified SOC.

Also, little is known about the interactions between substrates when these are added to soils in combination instead alone. Results from De Nobili et al. (2001) and Cheng et al. (2003) suggest that root exudates, containing many different organic substances, induce especially high positive priming effects. Chapter 4 Priming effects in soils after combined and repeated substrate additions 81

We concentrated on forest soil samples, since our previous work showed that the turnover of SOC from forest soils was more strongly affected by substrate additions than SOC from arable soils (Hamer and Marschner, in press). In addition, little is known about priming effects in forest soils. The objectives of this study were (i) to examine the influence of a combined substrate addition on SOC turnover and (ii) to determine the effects of repeated substrate additions on SOC turnover. The experiments with repeated substrate additions were conducted to determine if the pool of SOC that is susceptible to priming effects is limited, if microorganisms adapt to substrates and if other limiting factors restrict microbial activity.

Materials and methods

Soils and substrates

Soil samples were taken in October 2001 from the different horizons of to forest soil profiles in Germany, air-dried and sieved (< 2 mm). The first soil was classified as Dystric Cambisol under a mixed stand of Fagus sylvatica L. and Quercus robur L. (Steigerwald, Northern Bavaria). The second soil was a Haplic Podzol under Picea abies (Karst.) L. (Fichtelgebirge, North Eastern Bavaria). More detailed site and soil information is presented in Kaiser et al. (2002) and Hamer and Marschner (in press). Some important characteristics of the soil samples are shown in Table 1. The abbreviations Cambisol and Podzol with the respective horizon symbol are used for sample identification.

The uniformly 14C-labelled substances D-fructose, L-alanine (Amersham Pharmacia Biotech), oxalic acid and catechol (Sigma-Aldrich) with a radiochemical purity between 98 and 99 % were used as substrates. It was necessary to mix these radioactive chemicals with the respective unlabelled substance to obtain the required carbon concentrations for the incubation experiments.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 82

Table 1: Chemical and physical characteristics of the soil samples (ND: not determined).

SOC C/N Biomass C pH Sand Clay

-1 -1 -1 [g kg ] [mg kg ] (CaCl2) [g kg ]

Dystric Cambisol

Oa 122 18.7 1095 3.9 ND ND

B 6 17.4 16 3.7 547 100

Haplic Podzol

EA 32 26.1 34 3.1 516 104

Bs 53 26.8 162 3.7 340 164

Bw 24 22.4 ND 4.1 447 104

Incubation

Before incubation the air-dried soil samples (n = 3) were placed in 250 ml incubation vessels, rewetted to approximately 40 % of the water holding capacity (WHC) and stored at 15 °C. If not otherwise stated, after 10 days the water content was adjusted to 60 % WHC with the respective 14C-labelled substrate solution or in the case of the controls with deionised water. All samples were thoroughly mixed and incubated at 20 °C in a Respicond-apparatus (Nordgren Innovations). During the incubation the

CO2 evolved was trapped in 0.6 M KOH solution placed inside the incubation vessels. The Respicond automatically records the total amount of CO2 evolved every hour by measuring the changes in electrical conductivity of the KOH solution. The 14 amount of CO2 trapped in the KOH solution was quantified several times at different time intervals during the incubation using liquid scintillation counting (Beck- mann LS 6000 TA). Analytical methods are described in more detail in Hamer and Marschner (2002).

Microbial biomass

The amounts of total microbial biomass and substrate derived 14C-biomass were determined several times during incubation by the chloroform-fumigation-extraction Chapter 4 Priming effects in soils after combined and repeated substrate additions 83 method (Vance et al., 1987). All experiments were done in triplicate. The samples were fumigated with ethanol-free CHCl3 (Merck) for 24 h. After fumigant removal the soils were extracted by shaking for 30 min with 0.5 M K2SO4 (soil:solution ratio: 1:4 for mineral horizon samples, 1:20 for organic horizon samples) and then filtered through a Whatman GF/A filter. Control soils were extracted immediately. The content of organic C in the extracts (DOC) was measured with a TOC-analyser (Shima- 14 dzu 5050). The amount of C in the 0.5 M K2SO4 extracts was determined by liquid scintillation counting. For counting, an aliquot of 1 ml extract was mixed with 8 ml scintillation cocktail (Rotiszint 22, Roth). Total microbial biomass and 14C-biomass were calculated using a kEC factor of 0.45 (Joergensen, 1996). Since it was not possible to measure DOC in radioactive samples, additional samples were incubated receiving the same amounts of non-labelled substrate. In the following the content of organic C extractable from the non-fumigated soils is termed as DOC (K2SO4).

Combined and repeated substrate additions

An overview of all 4 experiments is given in Table 2.

Experiment 1

Soil samples from the Bs horizon of the Podzol were incubated 26 days with combined addition of fructose and alanine. At the beginning of incubation a substrate solution containing the same amounts of fructose- and alanine-C was added to 40 g -1 dry weight (dw) soil. The samples received in total 13.3 g substrate-C mg SOC. The activity per vessel was 15300 Bq. Additional samples were incubated for determining the amount of total microbial biomass and 14C-biomass after 4, 6 and 26 days as described above.

Experiment 2

This experiment with repeated additions of individual substrates was carried out with soil samples from the Oa and B horizon of the Cambisol and the EA and Bw horizon of the Podzol and all 14C-labelled substrates (fructose, alanine, oxalic acid, catechol). Chapter 4 Priming effects in soils after combined and repeated substrate additions 84

These samples were incubated 52 days and received two substrate additions. At the -1 beginQLQJDQGDWGD\RIWKHLQFXEDWLRQJVXEVWUDWH-C mg SOC was mixed into the soil. All vessels with mineral soil contained 50 g dw soil, those with samples from Cambisol Oa 20 g. The different samples received an activity between 3000 and 5000 Bq with each substrate addition.

Table 2: Design of the different experiments showing the used soil samples, added substrates (fru: fructose, ala: alanine, ox: oxalic acid, cat: catechol), substrate amounts and time of substrate addition.

Substrate Substrate amount Time of addition

>JVXEVWUDWH-C >JVXEVWUDWH-C [day] g-1 soil] mg-1 SOC]

Experiment 1

Podzol Bs fru + ala each 354 each 6.65 0

Experiment 2

Cambisol Oa fru, ala, ox, cat 1617 13.3 0 and 26

Cambisol B fru, ala, ox, cat 80 13.3 0 and 26

Podzol EA fru, ala, ox, cat 426 13.3 0 and 26

Podzol Bw fru, ala, ox, cat 314 13.3 0 and 26

Experiment 3

Cambisol Oa ala 808 6.65 0, 28, 56 and 84

Cambisol Oa ala 808 6.65 56 and 84

Experiment 4

Cambisol B ox 13 2.2 0, 6, 12 and 18

Podzol EA ox 106 3.3 0, 6, 12 and 18

Experiment 3

This experiment with repeated substrate additions over 111 days was carried out with samples from the Cambisol Oa horizon (20 g dw soil). At days 0, 28, 56 and 84 of -1 the incubation 6.65 g alanine-C mg SOC with an activity of 8600 Bq was mixed in -1 the soil. In total the samples received 26.6 g alanine-C mg SOC. This is the same substrate amount as in experiment 2, but distributed over a longer time period. One Chapter 4 Priming effects in soils after combined and repeated substrate additions 85 part of the samples received only the last two alanine additions. After the last substrate addition at day 84 a water content of approximately 60 % of the WHC was reached. Total microbial biomass and 14C-biomass were determined after 28, 56, 84 and 111 days of incubation.

Experiment 4

For the fourth experiment, soil samples from Cambisol B (50 g dw) and Podzol EA (40 g dw) were incubated 24 days with repeated additions of 14C-labelled oxalic acid every six days. After each addition the samples were thoroughly mixed. In total sam- -1 SOHVIURP&DPELVRO%UHFHLYHGJR[DOLFDFLG-C mg 62& JR[DOLFDFLG-C -1 mg 62&Z LWK %T HYHU\ GD\V D QG WKRVHIU RP3 RG]RO( $ J R[DOLF -1 -1 acid-C mg SOC JR[DOLFDFLG-C mg SOC with 9000 Bq every 6 days). At day 18 a water content of approximately 60 % of the WHC was reached.

Calculations

A t-test for unpaired groups was used to compare means between control and substrate treated samples. Changes in microbial biomass and DOC during incubation were tested with an analysis of variance for significance using the Tamhane’s T2 test. All statistical calculations were performed with SPSS 11.0 (SPSS Inc., Chicago, 14 IL). Substrate derived CO2-C was calculated from the amount of CO2-C evolved.

The amount of SOC mineralised in the substrate amended samples (CO2-Ctreatment) was obtained by subtracting the amount of substrate derived CO2-C from the total amount of evolved CO2-C during the regarded time interval t. A significant difference between CO2-Ctreatment and CO2-Ccontrol (p < 0.05, t-test) during a time interval t was the prerequisite for the following calculation of the priming effect [PE]:

CO2 −Ctreatment − CO2 −Ccontrol PE []% =100 ⋅ t t , t CO −C 2 controlt where CO2-Ccontrol is the amount of SOC mineralised in the control without substrate addition. In the following, we will use the term additional (SOC-derived) CO2-C (the Chapter 4 Priming effects in soils after combined and repeated substrate additions 86 difference between CO2-Ctreatment and CO2-Ccontrol), when referring to the absolute amount of the priming effect.

Results

Priming effect after combined fructose and alanine addition

Experiment 1

The combined addition of fructose and alanine to Podzol Bs caused a positive prim- -1 ing effect of +127 % after 26 days of incubation (Table 3). About J62&J soil were additionally mineralised in these samples. The possible contribution of the mineralisation of died microbial biomass to this value can be estimated from the difference in biomass 12C at day 0 and 26 (Table 3). Microbial biomass 12C decreased -1 E\DERXWJJ soil. However, a statistical evaluation of this value was not possible, because the biomass value at day 0 represents a difference from two means. 12 Thus, at the most 34 % of the observed additional CO2 evolution can be explained by the turnover of native microbial biomass.

The strongest acceleration of SOC mineralisation occurred during the first four days, when the mineralisation of the substrate was highest. During this period SOC mineralisation was nearly sevenfold higher than in the control (Table 3). This was accompanied by a significant increase in the total amount of microbial biomass from 187 -1 -1 JJ VRLOLQWKHFRQWUROWRJJ soil in the substrate treatment. In the substrate -1 14 WUHDWPHQWJJ of the total biomass was new C-labelled biomass. After day 4, the total amount of biomass tended to decrease in the substrate treated samples. In the control biomass significantly decreased between day 12 and 26.

C P r h i m a p i n t e

14 14 g

Table 3: Cumulative mineralisation of SOC and C-labelled substrate (fructose and alanine), cumulative priming effect (PE), total and new ( C) micro- r e 4 f f

bial biomass and K2SO4-extractable organic carbon during 26 days of incubation of samples from Podzol Bs with combined fructose and alanine addition ec t

(mean values, n = 3, SD in parenthesis, ND: not determined). s

i n s o il s

-1 -1 14 -1 14

Substrate a

SOC mineralisation [CO2-C µg g ] PE [%] Total biomass C [µg g ] Biomass C DOC (K2SO4) [µg g ] DO C (K2SO4) f t mineralisation e r

14 c o

[ CO2-C in % m

14 -1 -1 b [d] Control + Substrate Control + Substrate Control + Substrate i of added C] [µg g ] [µg g ] n e d a n d

0 0 0 0 0 162 (25) ND ND 426 (9) ND ND r e p ea t e 4 18 (4.9) 128 (6.6)*** +596 45.6 (0.9) 187 (18) 264 (31)* 40 (22) 311 (9) 296 (4) 23 (1) d s u b s t r a t

12 52 (8.8) 174 (13.7)*** +237 57.4 (0.7) 213 (22) 221 (48) 25 (10) 308 (1) 304 (11) 21 (1) e a dd i t i 26 95 (11.9) 216 (20.9)** +127 61.8 (0.7) 101 (35) 150 (18) 28 (3) 308 (1) 286 (9)* 8 (2) on s

*, **, *** indicate significant differences between control and substrate treated soil at p < 0.05, p < 0.01 and p < 0.001, respectively. 87

Chapter 4 Priming effects in soils after combined and repeated substrate additions 88

During the first four days of incubation the amount of K2SO4-extractable DOC decreased in the control as well as in the substrate amended samples to the same extent -1 (Table 3). After four days, extractable DOC decreased by abRXWJ &J soil -1 ZKLOHRQO\J&J soil were mineralised in the control, indicating that soluble C was immobilised through sorption or microbial incorporation. Only 3 % of the added substrate-C was extractable as DO14C after 4 and 12 days of incubation and at the end of incubation the amount decreased to 1 %.

A comparison of respiration rates during the first 10 days of incubation between combined and single fructose and alanine additions (adapted from Hamer and Mar- schner, in press) is shown in Figure 1. Highest respiration rates were observed when alanine was added as single substrate. After 2.4 days, maximum respiration occurred -1 -1 ZLWKJ &22-C g soil h . In the fructose treated samples the lag phase was -1 shorter and maximum respiration wasREVHUYHGDIWHUGD\ZLWKJ&22-C g soil h-1. With the combined substrate addition, two respiration peaks appeared. The first -1 -1 RQHDIWHUGD\VZLWKJ&22-C g soil h and the second one after 2.2 days -1 -1 ZLWKJ&22-C g soil h (Figure 1).

20 Control

with Fructose and Alanine ] 1

- 15 h with Fructose l i o s

1 with Alanine - 10 µg g [ C - 2

O 5 C

0 0 2 4 6 8 10 Time [d]

Figure 1:0HDQUHVSLUDWLRQUDWHVRI3RG]RO%VDIWHUVLQJOHDGGLWLRQRIJIUXctose- or alanine-C mg-162& +DPHUDQG0DUVFKQHULQSUHVV DQGWKHFRPELQHGDGGLWLRQRIJIUXFWRVH- and 6.65 JDODQLQH-C mg-1 SOC during the first 10 days of incubation. Chapter 4 Priming effects in soils after combined and repeated substrate additions 89

Priming effects after repeated substrate additions

Experiment 2

During the first 26 days of incubation, the mineralisation of SOC in the controls was not significantly different (p < 0.05) from mineralisation during the second period, except for Cambisol Oa (Table 4). In this sample, 2.19 % SOC were mineralised during the first 26 days of incubation, whereas only 1.55 % SOC were mineralised between day 26 and 52. In nearly all soil samples, alanine caused the highest positive priming effects after the first substrate addition (Table 4). Only in Podzol EA the first oxalic acid addition induced a higher priming effect than alanine (+82 vs. +49 %). In contrast, oxalic acid addition caused negative priming in Cambisol B. The first catechol addition caused negative priming effects in most soils, except in Podzol EA where it stimulated high positive priming (+46 %). Fructose showed positive (+10 to +30 %) or non significant priming effects.

The second addition of alanine caused much higher positive priming effects than the first one in all samples, except for Cambisol B (Table 4). In Cambisol Oa and Podzol EA the mineralisation of SOC was more than doubled after the second alanine addition (+115 and +129 %, respectively). The influence of the second fructose addition on mineralisation of SOC was similar to that of the first addition in most samples. Only in Podzol Bw a much higher positive priming effect was observed (+70 %) after the second substrate addition. The second addition of oxalic acid to the samples from the Podzol did not enhance SOC mineralisation more strongly than the first addition. In Podzol EA no more significant effects were observed. In Cambisol B, oxalic acid did not further retard SOC mineralisation and in Cambisol Oa the second oxalic acid addition accelerated the SOC mineralisation by +11 %. With the second catechol addition, the negative priming effects generally subsided and in Podzol Bw even became positive (Table 4).

The mineralisation of the added substrates increased in the order catechol << fructose < alanine < oxalic acid in all examined soils and was significantly higher after the second substrate addition than after the first one in almost all treatments (Table 4). Especially the mineralisation of catechol was nearly doubled during the second incubation period in all soil samples. The highest catechol mineralisation was observed in

C P r h i m a p i n t e g r e 4 f f

ec t

Table 4: Mineralisation of SOC in the controls, priming effects and mineralisation of the added substrate during the 52 days of incubation with two sub- s

-1 i VWUDWHDGGLWLRQVRIJVXEVWUDWH-C mg SOC at day 0 and 26 of incubation (mean values, n = 3, SD in parenthesis, n.s.: no significant difference n s o

between control and substrate treated soil at p < 0.05). il s a f t e SOC mineralisation Substrate mineralisation r c o m b i n

Incubation period Control Fructose Alanine Oxalic acid Catechol Fructose Alanine Oxalic acid Catechol e d a

14 14 n [d] [%] Priming effect [%] [ CO2-C in % of the respective C addition] d r e p ea

Cambisol Oa 0-26 2.19 (0.04) +10 +37 n.s. -19 65.5 (3.1) 70.7 (1.1) 74.1 (3.1) 12.7 (0.2) t e d s u

26-52 1.55 (0.02) +6 +115 +11 -7 71.8 (2.0) 87.2 (1.9) 92.2 (5.6) 22.9 (1.4) b s t r a t Cambisol B 0-26 1.61 (0.13) n.s. n.s. -17 -21 44.1 (2.1) 59.9 (1.7) 97.2 (2.4) 5.9 (1.6) e a dd i t i 26-52 1.84 (0.35) n.s. n.s. n.s. n.s. 58.2 (4.7) 71.9 (3.3) 95.0 (4.4) 12.5 (1.8) on s

Podzol EA 0-26 0.57 (0.10) +30 +49 +82 +46 64.1 (1.3) 72.5 (0.5) 79.0 (1.2) 27.8 (0.6)

26-52 0.57 (0.00) +49 +129 n.s. +33 74.8 (0.4) 78.7 (2.2) 106.4 (0.7) 40.0 (0.8)

Podzol Bw 0-26 0.32 (0.00) n.s. +63 +50 -23 47.7 (2.7) 54.9 (0.3) 85.4 (3.6) 15.9 (0.4)

26-52 0.31 (0.01) +70 +80 +15 +15 59.7 (1.9) 67.1 (0.6) 105.4 (4.9) 40.8 (1.3)

Chapter 4 Priming effects in soils after combined and repeated substrate additions 91 both horizons of the Podzol. Here, about 40 % of the added catechol were mineralised between days 26 and 52. Oxalic acid in Cambisol B was mineralised at equal rates during the two incubation periods. In Podzol EA and Bw a strong increase in oxalic acid mineralisation after the second addition was observed. During this period more than 100 % of the second oxalic acid addition were mineralised, which shows that residual oxalic acid or its metabolites from the first addition were also mineralised during this period.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 92

Experiment 3

In this experiment with repeated alanine additions to Cambisol Oa, the mineralisation of SOC in the control declined from 2.2 % during the first 28 days to 1.2 % between days 84 and 111 of incubation (Table 5). Each alanine addition increased SOC mineralisation during the respective incubation period by 16 to 29 % with a slightly higher positive priming effect after the second and fourth alanine addition (Table 5). When alanine was added after a 2 month pre-incubation, the priming effects were not significantly different from the first two additions in the treatment with four alanine additions. In contrast, during this later period, alanine mineralisation was significantly higher in the samples which had received alanine since the beginning of incubation than in those with alanine addition starting day 56 (Table 5).

Table 5: Mineralisation of SOC in the controls and alanine treated samples, priming effect and mineralisation of alanine during incubation of samples from Cambisol Oa with the addition of 6.65 g -1 alanine-C mg SOC at days 0, 28, 56 and 84 (alanine 4 times) or in the case of alanine (2 times) at days 56 and 84 of incubation (mean values, SD in parenthesis).

Alanine mineralisation 14 SOC mineralisation [%] Priming effect [%] [ CO2-C in % of the respective 14C addition]

+ Alanine + Alanine + Alanine + Alanine + Alanine + Alanine [d] Control (4 times) (2 times) (4 times) (2 times) (4 times) (2 times)

0-28 2.18 (0.08) 2.59 (0.16) 2.26 (0.17) +19 - 73.5 (2.2) -

28-56 1.65 (0.07) 2.09 (0.09) 1.72 (0.17) +26 - 79.0 (1.7) -

56-84 1.49 (0.07) 1.73 (0.09) 1.81 (0.13) +16 +22 80.2 (3.2) 72.8 (3.0)

84-111 1.19 (0.03) 1.53 (0.06) 1.50 (0.08) +29 +26 81.5 (2.0) 77.2 (0.5)

-1 In total, the priming effect induced by the addition of 13.3 g alanine-C mg SOC during the last two months of incubation was half of that induced by the addition of -1 26.6 g alanine-C mg SOC during the whole incubation period (+12 and +25 %, respectively). These priming effects were much lower than in experiment 2, where Chapter 4 Priming effects in soils after combined and repeated substrate additions 93

-1 the addition of 13.3 g alanine-C mg SOC at day 0 and 26 of incubation caused a positive priming effect of +69 % after 52 days (Table 4).

The time course of additional (SOC-derived) CO2 evolution shows that the strongest enhancement of SOC mineralisation always occurred during the first 4 to 6 days after alanine addition (Figure 2). Respiration rates in the alanine amended samples also peaked during this time interval. Immediately after the subsequent alanine additions, -1 -1 UHVSLUDWLRQ UDWHVL QFUHDVHG WR YDOXHVD URXQG J CO2-C g soil h and declined after 4 days to values slightly above the control respiration rates. Samples receiving the first alanine additions after 2 month of pre-incubation showed the same pattern (not presented).

40 2.5 Control with Alanine additional C C -

2.0 2 ] O 1

- 30 C h l l i a ] l o i s

1.5 on

o i 1 t s - i

1 g d 20 - d g g µ g [

1.0 e a m v C [ i - t 2 a l O

10 u C

0.5 m u c

0 0.0 0 20 40 60 80 100 120 Time [d]

Figure 2: Mean respiration rates of the control and alanine treated soil as well as cumulative release -1 of additional (SOC-derived) CO2-&IRU&DPELVRO2DZLWKWKHDGGLWLRQRIJDODQLQH-C mg SOC at days 0, 28, 56 and 84 of incubation.

The amount of total microbial biomass in the control and the samples treated with four alanine additions changed strongly during incubation, but followed the same pattern (Table 6). During the first month, total biomass C decreased from 1095 to -1 DQGJJ , respectively, and then increased at day 56, decreased at day 84 and again increased at the end of incubation. The amount of new 14C-labelled microbial biomass significantly increased during the last incubation period (Table 6). At the end of incubation, the 14C-labelled microbial biomass amounted to about 32 % of Chapter 4 Priming effects in soils after combined and repeated substrate additions 94 total biomass. In the samples with two alanine additions only 9 % of total biomass was 14C-labelled (not shown). During the whole incubation period, the microbial biomass in the samples with four alanine additions tended to be lower than in the control (Table 6). The samples which received the first alanine addition after 2 months of pre-incubation contained significantly more biomass C than the control at the end of incubation and compared to day 0 biomass C only slightly declined (not shown).

14 Table 6: Development of total and new ( C) microbial biomass and K2SO4-extractable DOC and DO14C during 111 days of incubation in samples from Cambisol Oa with the addition of 6.65 g -1 alanine-C mg SOC at days 0, 28, 56 and 84 (mean values, n = 3, SD in parenthesis, ND: not determined).

14 14 Total biomass C Biomass C DOC (K2SO4) DO C (K2SO4)

>JJ-1] >JJ-1] >JJ-1] >JJ-1] day Control + Alanine + Alanine Control + Alanine + Alanine

0 1095 (83) ND ND 887 (40) ND ND

28 516 (128) 446 (33) 35 (12) 305 (6) 460 (17)** 11 (5)

56 884 (61) 672 (107) 86 (13) 501 (20) 896 (43)** 11 (3)

84 397 (5) 133 (30)** 59 (13) 447 (16) 973 (11)*** 16 (4)

111 667 (15) 589 (134) 191 (39) 485 (13) 1076 (39)*** 13 (1)

*, **, *** indicate significant differences between control and substrate treated soil at p < 0.05, p < 0.01 and p < 0.001, respectively.

Assuming that the disappearance of biomass 12C during incubation is entirely due to its mineralisation, this would explain about 35 % of the additionally released CO2 in the treatment with four alanine additions and 16 % in the treatment with two alanine additions. During the incubation, periods with higher priming effects correlated with increased microbial biomass (r = 0.76, p < 0.01, n = 12).

The amount of K2SO4-extractable DOC in the control decreased more than half during the first month of incubation (Table 6). Thereafter, changes were similar to the development of microbial biomass. In the treatment with four alanine additions DOC decreased by about 50 % during the first month, but then continuously increased to -1 JJ until the end of incubation and was thus 2-fold higher than in the control. Chapter 4 Priming effects in soils after combined and repeated substrate additions 95

At the same time, the specific UV-absorbance also increased to 2-fold higher values than in the control. Also the pH was higher (5.7) than in the control (4.6) (not shown). The amount of 14C-labelled DOC did not change significantly during incubation (Table 6).

Experiment 4

This experiment was set up to study in more detail the strong but opposing effects of oxalic acid additions to the Cambisol B and the Podzol EA as observed in experiment 2 (Table 4). The addition of four low doses of oxalic acid in 6-day intervals caused very different effects than a single higher dosage (Figure 3a). In the Cambisol B, each substrate addition caused an increase in respiration rates that generally subsided within less than two days. Only the response after the first oxalic acid addition was retarded and lasted for almost three days. Therefore, the basic response pattern was similar to the single dose treatment where the much higher oxalic acid addition caused a slightly retarded large increase in respiration rates that lasted for about four days. In the Podzol EA, the initial respiration response to the low oxalic acid addition was almost identical to the single high dosage (Figure 3b). The following substrate additions also caused an immediate response in CO2-release, but levels remained elevated compared to the control throughout the incubation and even increased after the last oxalic acid addition. Chapter 4 Priming effects in soils after combined and repeated substrate additions 96

3 with 1 x Oxalic acid Control with 4 x Oxalic acid

a) Cambisol B ] 1 - h l i 2 o s

1 - µg g [ C -

2 1 O C

0 0 7 14 21 28 Time [d] 4 b) Podzol EA ] 1 - 3 h l i o s

1 - 2 µg g [ C - 2 O

C 1

0 0 7 14 21 28 Time [d]

Figure 3:0HDQUHVSLUDWLRQUDWHVDIWHUVLQJOHDGGLWLRQRIJR[DOLFDFLG-C mg-1 SOC and four UHSHDWHGDGGLWLRQVRIR[DOLFDFLGDWGD\DQGRILQFXEDWLRQWRD &DPELVRO% [J oxalic acid-C mg-162& DQGE 3RG]RO($ [JR[DOLFDFLG-C mg-1 SOC). Chapter 4 Priming effects in soils after combined and repeated substrate additions 97

In the Podzol EA, elevated respiration rates can largely be attributed to the mineralisation of the added oxalic acid (Table 7). Priming effects only occurred after the first two substrate additions, thereafter oxalic acid mineralisation increased with each dosage. In the Cambisol B, total oxalic acid mineralisation was similar to the Podzol EA but more evenly distributed over the incubation period (Table 7). Here, priming effects were highest after the second substrate addition (+81 %) but also almost reached 50 % after the first and third addition. Here too, no significant priming effects occurred after the fourth substrate addition. Considering the whole incubation period, the four low oxalic acid dosages increased SOC mineralisation in the Cambi- sol B by 38 %, while the effects in the Podzol EA were not significant. The respective priming effects after the single high dosage in experiment 2 of -17 % and +82 % are clearly not easily related to these results. It must be noted that the control of Cambisol B showed lower respiration rates during the first days of incubation than in experiment 2 (not shown).

In almost all four experiments the carbon balance was positive, because the amount of residual substrate-C in the samples surpassed the amount of additionally mineralised SOC. Only alanine often caused a net loss of carbon.

C P r h i m a p i n t e g

Table 7: Mineralisation of SOC in the controls and oxalic acid treated samples, priming effect (PE) and mineralisation of oxalic acid during incubation r e 4 -1 -1 f f of samples from Cambisol B with the addition of 2.2 g oxalic acid-C mg SOC and Podzol EA with the addition of 3.3 g oxalic acid-C mg SOC at ec t days 0, 6, 12 and 18 of incubation (mean values, SD in parenthesis, n.s.: no significant difference between control and substrate treated soil at p < 0.05). s i n s o il s

Cambisol B Podzol EA a f t e r c o m

SOC mineralisation [%] PE SOC mineralisation [%] PE b i

Oxalic acid mineralisation Oxalic acid mineralisation n e

14 14 d

[ CO2-C in % of the [ CO2-C in % of the a n

14 14 d

respective C addition] respective C addition] r [d] n Control + Oxalic acid [%] Control + Oxalic acid [%] e p ea t e d s u

0-6 12 0.26 (0.03) 0.39 (0.08) +49 61.5 (9.5) 0.19 (0.02) 0.25 (0.03) +30 78.0 (3.4) b s t r a t e a

6-12 9 0.27 (0.03) 0.49 (0.10) +81 72.2 (4.8) 0.12 (0.02) 0.14 (0.01) +13 44.7 (4.4) dd i t i on s

12-18 6 0.33 (0.02) 0.48 (0.10) +46 57.2 (7.2) 0.16 (0.01) 0.17 (0.01) n.s. 61.0 (13.8)

18-24 3 0.36 (0.02) 0.32 (0.03) n.s. 72.5 (0.8) 0.15 (0.03) 0.19 (0.04) n.s. 95.4 (37.5)

0-24 3 1.22 (0.11) 1.68 (0.24) +38 68.7 (3.5) 0.63 (0.08) 0.75 (0.09) n.s. 67.7 (13.7)

Chapter 4 Priming effects in soils after combined and repeated substrate additions 99

Discussion

Priming effect after combined fructose and alanine addition

The positive priming effect due to the combined addition of fructose and alanine was much higher (+127 %) than the priming effects due to the single addition of these two substrates (+91 and +85 % respectively, as shown in Hamer and Marschner, in press). However, substrate mineralisation apparently was not affected by the form of application, since 61.8 % of the added combined 14C-substrates were mineralised after 26 days (Table 3) compared to an expected 59.5 % calculated from the single substrate additions (Hamer and Marschner, in press). The strongly increased priming effect from the combined addition of fructose and alanine compared to the expected additive effects of single additions indicates some form of synergism. Above all, Hamer and Marschner (2002) always observed lower priming effects with lower fructose and alanine additions. Since mixtures of many different substrates are per- manently released into the soil solution, this outlines the importance of priming effects in soils. Therefore, the high positive priming effects of over 200 % observed in planted soils (Cheng et al., 2003) may also be the result of such synergistic effects from different substrates present in the root exudates. De Nobili et al. (2001) also report higher positive priming effects after the addition of a root extract compared to glucose or amino acids.

The priming effect due to the combined fructose and alanine addition not only was higher but also occurred more rapidly than after the single additions, since 91 % of the total additional CO2-C evolved during the first four days of incubation compared to 54 % and 70 % after single fructose and alanine addition, respectively.

The seven-fold increase of SOC mineralisation during the first four days of incubation was only accompanied by an increase of total microbial biomass by a factor of 1.4 compared to the control. Therefore, it seems unlikely that the priming effect was only due to co-metabolism, i.e. due to increased metabolic activity from an increased microbial biomass as suggested as possible mechanism (Kuzyakov et al., 2000). It therefore seems more likely, that the added substrates provided the microorganisms Chapter 4 Priming effects in soils after combined and repeated substrate additions 100 with energy, to produce more "expensive" enzymes that are capable of breaking down more complex components of SOC.

The lag phase after alanine addition was twice as long as that of fructose which is consistent with results from Marstorp (1996a). But in contrast to Marstorp (1996b), who reported that the combined addition of glucose and alanine reduced the lag phase of alanine mineralisation, we observed a longer lag phase when fructose and alanine were added simultaneously. However, the total amount of mineralised substrate was similar between single and combined addition, which is in accordance to Marstorp (1996b). According to Harder and Dijkhuizen (1982) the simultaneous utilisation of the substrates in a mixture appears to be the general response of the microorganisms when the substrate concentrations are growth-limiting. It is likely that only the combination of fructose and alanine makes optimal microbial activity possible. If fructose and alanine are added individually to soils, the microorganisms must satisfy their energy- and C-demand from the respective substrate. However, amino acid uptake by microorganisms is typically an energy-dependent process (Vi- nolas et al., 2001) and sugars are used by the microbial biomass preferentially for energy production (Coody et al., 1986). Thus presumably the presence of fructose facilitated the alanine uptake by the microorganisms satisfying their N-demand for growth and enzyme production. This is in accordance with Dilly (1997), who observed that the addition of glucose decreased the ammonification rate from arginine in different soils. Furthermore, some bacteria depend on the availability of amino acids for growth since they are not able to synthesize them (Gottschalk, 1986).

Our results also show, that the turnover of native microbial biomass could have contributed only up to 34 % to the observed additional CO2-C release. Similar low con- tributions of microbial biomass turnover to the calculated priming effects occurred with repeated alanine additions in experiment 3. In contrast, Chotte et al. (1998) de- 12 termined a contribution of respired biomass C to the additional CO2-C release of 53 to 85 %. Assuming that 50 % of dead biomass will be mineralised during 20 days of incubation, Wu et al. (1993) were able to relate the total positive priming effect induced by glucose addition to the decrease in biomass 12C, but not that induced by ryegrass. Chander and Joergensen (2001) explained the total additional CO2-C evolution after glucose addition by the decrease in biomass 12C. Chapter 4 Priming effects in soils after combined and repeated substrate additions 101

A further important factor contributing to the observed high priming effect may be a qualitative change of the microbial community structure as reported by Falchini et al. (2003). They observed positive priming effects after glucose and glutamic acid addition to a grassland soil accompanied by changes in the DGGE profiles of soil bacterial communities after 3 and 7 days of incubation. Baudoin et al. (2003) showed, that the addition of artificial root exudates to soil caused changes in substrate utilisation patterns and the genetic structure of the microbial communities. According to Koz- drój and van Elsas (2000) the addition of artificial root exudates to heavy metal con- taminated soils reduced the bacterial diversity towards domination of r-strategist microorganisms while the unamended soils were dominated by K-strategists. The rapid increase in SOC mineralisation after substrate additions observed here and in numer- ous other studies (Chotte et al., 1998; De Nobili et al., 2001; Hamer and Marschner, 2002; Hamer and Marschner, in press; Hamer et al., 2004; Luna-Guido et al., 2003) indicates that these priming effects are probably due to the activity of r-strategists rather than slow growing K-strategists although Fontaine et al. (2003) assumed that r-strategists are not able to use SOC and therefore should not induce a priming effect.

Is there only a limited (labile) pool of SOC available for priming effects?

As formerly shown in section “Priming effect after combined fructose and alanine addition”, the turnover of microbial biomass can not explain the additional CO2 release, even if a complete mineralisation of dead microbial biomass is assumed. Therefore, the observed positive priming effects are definitely not apparent ones and the turnover of the SOC pool is indeed affected.

In several cases, subsequent substrate additions induced higher priming effects than the first ones, thus indicating that the pool of SOC available for priming effects was not depleted. In other cases, priming effects decreased after repeated substrate additions, especially after oxalic acid additions to Podzol EA and Podzol Bw (experiment 2) as well as after four repeated low oxalic acid additions to Podzol EA and Cambi- sol B (experiment 4). Since the mineralisation of oxalic acid increased with time this indicates that repeated oxalic acid additions promoted the growth of oxalic acid utilising microorganisms, which were less efficient in SOC mineralisation than the initial microbial population. Chapter 4 Priming effects in soils after combined and repeated substrate additions 102

On the other hand, even after the prolonged pre-incubation of Cambisol Oa without substrate, where decreasing mineralisation rates indicated that a more labile pool of SOC became depleted, priming effects were similar as with immediate alanine additions. However, in this case N may have limited degradation (C/N ratio of Cambisol Oa: 18.7), so that even after a two month incubation at 20 °C enough labile SOC was present to be rapidly degraded as soon as an easily available N source was added.

Are changes induced in the microbial community structure?

Although we have not done direct measurements of microbial community structure our data provide some indirect information for this. All tested substrates were generally mineralised to a higher degree after repeated additions. This was most pronounced for oxalic acid and catechol and may be due to an adaptation of microorganisms to the respective substrate. In the course of increasing rates of catechol mineralisation, negative priming effects tended to subside or even become positive. Only in soils with high catechol mineralisation, this potentially inhibitory compound induced positive priming effects. There are two possible mechanisms to explain this. Either samples with high catechol mineralisation are colonized by specialized microorganisms that can utilise this phenolic compound as a C-source for growth and energy demands. The other possible mechanism is that catechol additions selectively favoured the activity of organisms that were able to detoxify this inhibitory compound by releasing unspecific oxidizing exoenzymes like laccases or peroxidases thus also enhancing mineralisation of similar structures in SOM.

The respiration rates in Podzol EA (Figure 3) after different oxalic acid additions suggest that different microorganisms were active in the degradation process. Some seem to be able to react immediately to oxalic acid additions in both treatments, while others need between 5 and 20 days. On the other hand this data may indicate that a certain threshold value of oxalic acid or its metabolites appears to be needed to greatly enhance microbial activity. This is supported by the lower cumulative oxalic acid mineralisation after repeated low addition than after a single high addition. This too may be due to a shift in microbial community structure, since Falchini et al. (2003) observed some extra bands in the DGGE profiles of a grassland soil amended -1 ZLWKJR[DOLFDFLG-C mg SOC. Chapter 4 Priming effects in soils after combined and repeated substrate additions 103

In Cambisol B presumably other microorganisms were active than in Podzol EA. Here, the single high addition induced a negative priming effect. However, during the first four days where more than 80 % of oxalic acid was mineralised positive priming occurred. After the exhaustion of this energy substrate, possibly the oxalic acid utilising microorganisms became energy limited and died, resulting in the observed negative priming effect. In contrast, the repeated low oxalic acid additions may have not provided enough substrate for a rapid growth of these microorganisms. In contrast, Hamer and Marschner (2002) observed a threefold higher positive priming effect in peat when the amount of added oxalic acid was reduced to 20%, although the percentage of mineralised oxalic acid was nearly the same. De Nobili et al. (2001) also report that trace amounts of substrates triggered the microorganisms -1 into aFWLYLW\ 7KH\ DGGHG EHWZHHQ DQG J VXEVWUDWH-C mg SOC to arable soils and observed that the addition of 1/3 of the full substrate rate at three intervals during 24 days of incubation increased the additional CO2-C evolution compared to the single full addition. On the other hand, Morris and Allen (1994) showed that the addition of oxalic acid to soil increases the activity of oxalate mineralising microorganisms and increases the amount of available P, which could thus enable other P- limited organisms to increase their mineralisation activity.

With oxalic acid, direct chemical effects on structural components of SOC have also to be considered. Dutton and Evans (1996) point out that oxalic acid is important in lignocellulose degradation by wood-rotting basidiomycetes and initialises the de- polymerisation of cellulose. Piccolo et al. (1996) observed that oxalic acid caused a structural rearrangement of humic materials. However, this occurred at much higher oxalic acid concentrations than used in the present study.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 104

Are other limiting factors affected?

The repeated alanine additions to Cambisol Oa caused a pH increase, which is known to promote microbial activity (Paul and Clark, 1996) and to increase the release of dissolved organic carbon (Marschner, 1997). But if this was the only cause for the observed priming effects, then the high single alanine addition in experiment 1 should have induced a higher priming effect than the lower addition in combination with fructose. Besides, in Cambisol Oa microbial biomass decreased with alanine addition relative to the control. Possibly alanine selectively promoted the growth of microorganisms not able to use SOC after alanine exhaustion and which were again in starvation 28 days after alanine addition. The more microbial biomass was present 28 days after alanine addition the higher was the priming effect. Presumably, N may be a further limiting factor, since in many soils, alanine caused the most pronounced priming effects. But this whole issue has not been investigated systematically in this study. Therefore, future investigations are necessary.

Conclusions

Our results point out the importance of priming effects in forest soils. First of all, the combined substrate addition induced stronger positive priming than the single additions, indicating synergistic effects due to multiple substrate limitations. Besides, priming effects are repeatedly inducible even during a four month incubation. Since priming effects were not depressed after extended pre-incubation of a soil sample, it seems unlikely that only the labile SOC pool is susceptible. Co-metabolism and the turnover of native microbial biomass are insufficient to explain the observed positive priming effects. Our results suggest that the activation of different microorganisms with different substrates and substrate concentrations as well as the adaptation of microorganisms to the added substrate are important factors controlling priming effects.

Chapter 4 Priming effects in soils after combined and repeated substrate additions 105

Acknowledgements

This project is financially supported by the German Research Foundation (DFG). It is part of the priority program 1090 “Soils as source and sink of CO2 - mechanisms and regulation of organic matter stabilisation in soils”.

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Chander, K. and Joergensen, R.G., 2001. Decomposition of 14C glucose in two soils with different amounts of heavy metal contamination. Soil Biology & Biochemistry, 33: 1811-1816.

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Chapter 5

Interactive priming of black carbon and glucose mineralisation

Co-authors: Bernd Marschner, Sonja Brodowski and Wulf Amelung

Organic Geochemistry (2004), 35: 823-830.

Chapter 5 Interactive priming of black carbon and glucose mineralisation 111

Abstract

Black Carbon (BC) in soil is important in the global C cycle, but only little is known about the mechanisms and rates of BC degradation. We investigated the influence of 14C-glucose additions on the mineralisation of charred maize and rye residues (thermally altered at 350 °C) and oak wood (thermally altered at 800 °C). The different BC materials were mixed with sand and incubated for 60 days at 20 °C. The samples UHFHLYHGJJOXFRVH-C per mg black carbon at the beginning and at day 26 of the experiment. At the beginning, a nutrient solution ((NH4)2SO4 + KH2PO4) and an inoculum extracted from an arable soil were added. In the controls without glucose addition, between 0.3 % (wood) and 0.8 % (maize) of the initial charred materials were mineralised and 0.6-1.2 % when glucose was added. The two glucose additions accelerated BC mineralisation, with the 2nd glucose addition inducing a stronger enhancement of BC mineralisation than the 1st one. A close correlation (r = 0.94, p < 0.001) between glucose mineralisation and additional BC mineralisation suggests that BC degradation may be due to co-metabolism, i.e. to the enhanced growth of microbial biomass and the accompanying increased enzyme production. On the other hand, glucose mineralisation was enhanced over the control by the presence of charred material, i.e., there was an interactive priming of BC and glucose mineralisation. We conclude that BC in soils may also promote growth of microorganisms and the decomposition of labile C compounds rather than stabilising them against degradation.

Chapter 5 Interactive priming of black carbon and glucose mineralisation 112

Introduction

Black carbon (BC) is produced by incomplete combustion of fossil fuels and vegetation and occurs ubiquitously in soils and terrestrial sediments. It is relatively inert and thus contributes to refractory soil organic matter (Goldberg, 1985; Schmidt and Noack, 2000). Up to 60 % of the soil organic carbon (SOC) pool has been attributed to BC in Canadian Chernozems (Ponomarenko and Anderson, 2001). In German Chernozems up to 45 % of the SOC (Schmidt et al., 1999), in Australian soils up to 30 % (Skjemstad et al., 1996) and in US soils up to 18 % (native prairie; Glaser and Amelung, 2003) and 35 % in agricultural soils (Skjemstad et al., 2002) has been identified as charcoal. These data indicate that BC can constitute a significant part of the soil carbon pool. Hence, charred plant materials may play an important role in carbon sequestration. To be able to estimate the resilience of BC in soils, knowledge is required concerning its degradation rates and mechanisms.

Shindo (1991) did not observe a significant degradation of charred plant residues in volcanic soils after 40 weeks of incubation and it has been suggested that BC does not commonly serve as C and energy source for microorganisms (Albrecht et al., 1995). In contrast, Shneour (1966) found that over a 96 days period, 2 % of artificial graphitic carbon was oxidised in non-sterile soils. Hofrichter et al. (1999) discovered wood-decaying fungi that were able to degrade low rank coals, indicating that spe- cialised microorganisms may grow on BC as sole C source. However, the presence of a second easily available C source was required to induce microbial degradation of brown coal (Willmann and Fakoussa, 1997). In soils, a variety of substrates are available to microorganisms, e.g. in the rhizosphere where many easily available substrates are released into the soil; hence, at least locally, co-metabolism might be a major pathway of BC degradation. In general, organic substrates are considered to be degraded co-metabolically if another C-source is required by microorganisms for their degradation (Paul and Clark, 1996). We hypothesized that an additional C source accelerates BC mineralisation (positive priming effect), since microorganisms are then no longer C and energy limited.

Positive priming effects of different organic substrate additions on SOC mineralisation frequently have been observed (Kuzyakov et al., 2000; Hamer and Marschner, Chapter 5 Interactive priming of black carbon and glucose mineralisation 113

2002). The reverse effect, i.e., the effect of refractory SOC on the degradation of the added substrate has received only limited attention. It may be assumed that the presence of BC in soils might retard the decomposition of other C sources, since BC of- fers surface areas for the adsorption and, hence, the protection of other compounds from degradation (e.g., Cooney, 1998; Jonker and Koelmans, 2002). On the other hand, it is also conceivable that the high surface area of BC facilitates the growth of microorganisms, leading to a faster decomposition of other C sources (Andrews and Tien, 1981).

The objectives of this study were to: (i) examine the influence of glucose as easily available substrate on the mineralisation of black carbon materials; (ii) test the hypothesis that the presence of charred materials retards glucose degradation. We used 14C-labelled glucose in order to differentiate between glucose and BC mineralisation. The BC materials were obtained from the charred residues of maize, rye and oak wood.

Materials and methods

Black carbon

To produce BC from maize (Zea mays L.) and rye (Secale cereale L.) straw, we heated 75 - 140 g ground maize/rye straw (particle size about 5 - 10 mm) in stainless steel containers (15 cm x 26 cm x 5 cm) at 350 °C for two hours in a muffle furnace. The containers were closed with a cap to reduce the oxygen entry. It took one hour to reach the final temperature. After cooling, the charred residues from 12-32 replicates were pooled. The mass loss of the material after heating averaged 67.6 % ± 0.5 (SE; n = 12) of the initial mass for maize and 68.0 % ± 0.3 (SE; n = 32) of the initial mass for rye. The charred wood was prepared by heating oak wood for 20-24 h at 800 °C (Unique Forst, Freiburg, Germany).

The elemental composition of the charred materials was determined by microanalysis (Ilse Beetz laboratory, Kronach, Germany). The charred materials did not contain bicarbonates. We checked this by HCl pretreatment that did neither result in visible Chapter 5 Interactive priming of black carbon and glucose mineralisation 114 bubbling from CO2 release nor in any change of the total C content. To ascertain that the charred materials mainly contained aryl C, solid-state 13C NMR spectra were obtained on a Bruker DSX 200 spectrometer (Bruker, Karlsruhe, Germany) applying the cross polarization magic angle spinning technique with a spin speed of 6.8 kHz (acquisition parameters reported by Rumpel et al., 2004). A contact time of 1 ms and a pulse delay of 500 ms were used. The 13C chemical shifts were referenced to tetramethylsilane (0 ppm). According to Schmid et al. (2002) the spectra were inte- grated across four major chemical shift regions.

Incubation

The different BC materials were finely ground with a ball mill, mixed with sand (grain size: 61 % 200-PDQG-PDFLGZDVKHGDQGLJQLWHG%DNHU Germany) at a BC:sand ratio of 1:10 (w/w); 30 g of these mixtures were placed in a 250 ml incubation vessel (n = 3) with the addition of 1 ml inoculum and 0.5 ml nutri- -1 ent solution [60 g (NH4)2SO4 + 6 g KH2PO4 l ] at the beginning of the experiment. The water content was adjusted to approximately 60 % water holding capacity. Thereafter, the samples were incubated for 60 days at 20 °C in a Respicond- apparatus (Nordgren Innovations, Sweden).

The inoculum was obtained from the Ap horizon of an Haplic Phaeozem under continuous rye cultivation since 1878 from the long-term field experiment “Ewiger Rog- genbau” of the Halle University (Germany). More detailed information concerning this field experiment is presented in Merbach et al. (1999). The sample of the Ap horizon had been stored air-dried and sieved (2 mm) after sampling in October 2001. Two weeks prior to use, it was re-wetted to 60 % water holding capacity and preincubated at 15 °C. The inoculum was obtained by shaking with 4 mM CaCl2 solution (at 1:2 soil:solution ratio w/v) for 30 min and subsequent 5 m filtration. At the beginning and at day 26, a 14C-labelled glucose solution was added. With each addition the samples received 20 g glucose-C per mg BC. The solution had a radio- activity of 3300 Bq. It was produced by diluting UL 14C D-glucose (Amersham Pharmacia Biotech, Germany) with unlabelled glucose to obtain the required C concentration. Sand samples without glucose or BC addition served as controls. In the control without BC, the pH of the sand was adjusted to 6.5 with a Ca(OH)2 solution Chapter 5 Interactive priming of black carbon and glucose mineralisation 115 to assure comparable environmental conditions. The pH values (0.01 M CaCl2) of the charred maize/sand, rye/sand and wood/sand mixtures were 8.0, 6.7 and 6.5, respectively. During the incubation, the CO2-evolution was measured hourly by determining the changes in electrical conductivity of a 0.6 M KOH solution placed inside the 14 incubation vessels (Nordgren, 1988). The amount of CO2 evolved was determined in 13 time intervals using liquid scintillation counting (Beckmann LS 6000 TA, USA). A more detailed description of the analytical methods is given in Hamer and Marschner (2002). All experiments were performed in triplicate.

Calculations

14 Glucose derived CO2-C was calculated from the amount of CO2-C evolved, assuming that the 14C- and 12C-glucose was mineralised at equal rate. The amount of BC mineralised in the glucose amended samples (CO2-Ctreatment) was obtained by subtracting the amount of glucose derived CO2-C from the total amount of evolved CO2- C during the regarded time interval t. Priming effects [PE] were calculated after 26 and 34 days of the respective glucose additions, using the following equation

CO2−Ctreatment − CO2−Ccontrol PE []% = 100 ⋅ t t , t CO −C 2 controlt where CO2-Ccontrol is the amount of BC mineralised in the control without glucose addition. A significant difference between CO2-Ctreatment and CO2-Ccontrol (p < 0.05, t- test) was the prerequisite for the calculation of a priming effect. In the following, we use the term additional (BC-derived) CO2-C (the difference between CO2-Ctreatment and CO2-Ccontrol), when referring to the absolute amount of the priming effect. Chapter 5 Interactive priming of black carbon and glucose mineralisation 116

Results

Characteristics of charred materials

Elemental and structural characterisation of the BC materials showed the expected typical properties of coal particles, such as high C and aryl-C content. There were no pronounced differences in elemental and structural composition between the charred maize and rye residues. Both contained 66 % C. About 70 % of the total signal intensity of the 13C NMR spectra was attributed to aryl C, followed by alkyl C (13 % and 14 % of total area intensity, respectively; Table 1). The N content in the maize residues was twice that in the rye ones, resulting in a C/N ratio of 27 for maize char and 52 for rye char. The O content was higher in the rye than in the maize residues (Ta- ble 1). The charred wood had the highest C and lowest N, S and P contents of all charred residues, with a C/N ratio of 392 (Table 1). Aryl C was also the dominant C type in the charred wood. It contributed to 77 % of the total signal intensity, followed by alkyl C with 9 %. We conclude that both charring procedures were successful and that N-content discriminated among the different BC materials. For plant materials it has been shown that a higher C/N ratio reflects lower “decomposability” (Paul and Clark, 1996). If the same holds also true for BC, the biodegradability of our BC materials should increase in the order charred wood << charred rye < charred maize.

Table 1: Bulk chemical features of the charred materials.

C C/Na O H S P Alkyl C O-alkyl C Aryl C Carboxyl C

(0-45) b (45-110)b (110-160 )b (160-240 )b

[g kg-1] [g kg-1] [g kg-1] [g kg-1] [g kg-1] [% of total signal intensity]

Maize 664 26.5 160 34 5 4 13 7 73 7

Rye 663 52.2 210 31 3 3 14 6 71 8

Wood 785 392.3 172 34 1 0.01 9 5 77 8 a Weight ratios. b The values in parenthesis correspond to resonance range (ppm = Hz MHz-1) notation for the chemical shift in nuclear magnetic resonance spectroscopy.

Chapter 5 Interactive priming of black carbon and glucose mineralisation 117

Glucose mineralisation

The lowest extent of glucose mineralisation was detected in the control sand without BC addition. After a lag phase of 6 days, 47 % of added glucose-C was lost during the first part of the experiment (Figure 1a). In contrast, no lag phase was observed when BC had been added to the samples and more glucose-C was lost. In total, after 26 days, 64 % of the added substrate had been mineralised in the samples containing charred rye residues and wood. In the samples with charred maize, up to 77 % of the glucose was mineralised.

Maize Rye Wood Sand

] % [

2 40 O C 14 20 st a) 1 addition 0

0 5 10 15 20 25 30 Time [d]

] % [

2 40 O C 14 20 b) 2nd addition

0 26 31 36 41 46 51 56 61 Time [d]

Figure 1: Cumulative glucose mineralisation (% of added glucose-14C) after a) the first glucose addition at day 0 and b) the second glucose addition at day 26 (bars represent standard deviation, n = 3). Chapter 5 Interactive priming of black carbon and glucose mineralisation 118

During the second incubation period, after the new glucose addition, glucose mineralisation was again lower in the control sand, but comparably low in the charred wood samples (48 and 52 %, respectively; Figure 1b). No significant difference in glucose mineralisation (p < 0.05) between these experiments occurred. In the charred rye and charred maize samples, the second glucose addition led to a glucose mineralisation of 65 % and 77 %, respectively. After 60 days of incubation, mineralisation of added glucose decreased in the order of the treatments “charred maize” (82 %) > “charred rye” (76 %) > “charred oak wood” (67 %) > control (60 %).

Mineralisation of charred materials

Table 2 shows that for all controls without glucose addition, greater mineralisation of BC was observed during the shorter first incubation period, where nearly twice as much BC was mineralised than between days 26 and 60. The charred maize and rye residues were more susceptible to mineralisation than the charred wood. At the end of incubation, 0.78 % of maize-derived BC, 0.72 % of rye-derived BC, and 0.26 % of wood-derived BC had been mineralised (Table 2).

Table 2: Cumulative BC mineralisation and priming effect during the two incubation periods (standard deviation in parenthesis, n = 3).

Incubation BC mineralised Additional BC Priming BC mineralised Total BC miner- period in the control mineralised in effect in the control alised in glu- glucose-treated cose-treated samples samples

[d] CO2-C [mg] CO2-C [mg] [%] [%] [%]

Maize 0-26 16.0 5.8 +36 0.53 (0.03) 0.73 (0.11)

26-60 7.5 7.5 +100 0.25 (0.00) 0.50 (0.02)

Rye 0-26 13.9 7.1 +51 0.46 (0.05) 0.70 (0.06)

26-60 7.7 8.6 +112 0.26 (0.02) 0.54 (0.07)

Wood 0-26 5.2 3.8 +73 0.17 (0.04) 0.29 (0.04)

26-60 2.8 5.3 +189 0.09 (0.00) 0.27 (0.04) Chapter 5 Interactive priming of black carbon and glucose mineralisation 119

A significant acceleration of BC mineralisation was observed when glucose had been added (Table 2). Consequently, glucose primed the decomposition of BC. In all treatments, the second glucose addition caused a stronger positive priming effect than the first and more than doubled the BC mineralisation (Table 2). Also, the absolute amount of additional BC mineralised increased after the second glucose addition, whereas the absolute amount of BC mineralisation in the control declined during the 2nd incubation period (Table 2).

To better understand the underlying mechanisms, we considered the time course of additional (BC-derived) CO2-C release in conjunction with the respiration rates. In the samples with charred maize and rye, the sharpest increase in additional CO2-C evolution occurred during the 6 days after the first glucose addition. Mineralisation rates also peaked during this time interval, reaching a maximum of ca. 0.9 mg CO2-C h-1 at day 4. The respiration rates showed a second peak immediately after the 2nd -1 glucose addition. With 0.45 mg CO2-C h , this peak was lower than the first but, it exhibited a more pronounced tailing (Figures 2a and 2b). Afterwards, respiration rates declined, but until the end of incubation they remained significantly (p < 0.001) higher than in the controls. In the samples with charred wood, the largest amounts of additionally evolved CO2-C also occurred soon after the two glucose additions (Fig- ure 2c). However, additional CO2-C production did not gradually decline as observed for the other samples but stopped between day 12 and 26 and between day 38 and 60 and even became negative. The different rates of additional CO2-C production in these samples compared with those containing charred maize and rye coincided with a different development of CO2 respiration rates. The more gradual increase in additional C respiration at the beginning of the experiment correlated with a larger tailing of the first peak of CO2 respiration rates. In contrast, less prolonged production of additional C after the 2nd glucose addition correlated with a smaller tailing of the 2nd peak of respiration rates (Figure 2c). Overall, there was a significant (r = 0.94, p < 0.001) linear relationship between the amount of mineralised glucose and the priming of BC mineralisation (Figure 3). The regression line has been fitted through zero, since no priming effect can be observed when glucose is lacking in the system. Nega- tive priming happened during later stages of the incubation of charred wood with glucose (Figure 2c), when mineralisation of BC was smaller than in the control Chapter 5 Interactive priming of black carbon and glucose mineralisation 120

Control with Glucose additional C 1.0 16 a) Maize 0.8 12 ] ] g 1 - m 0.6 [ d g h C e - t m 2 [ 8 a l O C u - C 2 l 0.4 m a O u C on i acc 4 t 0.2 i dd a

0.0 0 0 10 20 30 40 50 60 Time [d] 1.0 16 b) Rye

0.8 ] 12 g m ] [ 1 - d C e h - t

0.6 2 a g l O u m C [ 8 l m C a u - n 2 0.4 o O i acc t i C

4 dd

0.2 a

0.0 0 0 10 20 30 40 50 60 Time [d]

1.0 16 c) Wood

0.8 ] 12 g m ] [ 1 - d C e - t

0.6 2 a g h l O u m C [ 8 l m C a u -

2 0.4 on O i acc t i C

4 dd

0.2 a

0.0 0 0 10 20 30 40 50 60 Time [d]

Figure 2: Change in respiration rates of the controls and glucose-amended treatments as well as cumulative release of additional (BC-derived) CO2-C for: a) charred maize residues; b) charred rye residues; c) charred wood. Chapter 5 Interactive priming of black carbon and glucose mineralisation 121

5 y = 0.15x ] y 0.15x g 2 m R² R= 0 =.808***.* [ 4 C - 2 3 O Maize C l a 2 Rye on i t i 1 d Wood d a 0

-1 0 10 20 30 40

glucose CO2-C [mg]

Figure 3: Correlation between glucose mineralisation and additional mineralisation of BC material, for all analysed time intervals during the incubation experiment.

Discussion

After 60 days 0.78, 0.72 and 0.26 % of the charred maize, rye and wood were mineralised in the controls. Apparently, some microorganisms were able to live with BC as sole C source. Different pH values in the charred maize and rye treatments did not significantly affect the BC. However, charred maize and rye residues were more susceptible to degradation than charred wood, presumably due to the more gentle charring conditions. Baldock and Smernik (2002) observed in incubation experiments with thermally treated Pinus resinosa sapwood (temperatures between 200 and 350 °C) that mineralisation of the altered wood was lower than 2 % after 120 d. Wood that was charred at temperatures below 200 °C was more degradable as it contained less aryl C. Also in this study, the most resistant BC material (charred wood) had the highest aryl C content and the largest C/N ratio (Table 1).

The charred materials in our study were as degradable as soil organic matter in different horizons of a Haplic Podzol, of which between 0.2 % (Bs horizon) and 0.5 % (Oa horizon) were mineralised after 26 days of incubation (unpublished results). Mean residence times for charred straw residues and charred wood of 39 and 76 Chapter 5 Interactive priming of black carbon and glucose mineralisation 122 years, respectively, were calculated by a fit of loss of BC in the controls using a two- component first-order decay equation. These are minimum turnover times since they are based on an incubation experiment of only 60 days where probably more easily degradable moieties of BC were degraded preferentially. However, from day 35 to 40 until the end of incubation no further decline in mineralisation rates was observed. The calculated mean residence times are slightly higher than suggested by Shneour (1966) for graphitised carbon (2 % decomposed in 96 days) but in accordance with Bird et al. (1999), who suggested that BC can be significantly degraded on decadal to centennial timescales in subtropical soils. Assuming that no black carbon was trans- located by wind erosion in significant amount, these authors suggested a half-life of \HDUVIRUFDUERQLVHGSDUWLFOHVLQWKHIUDFWLRQ PDQGDKDOI-life of < 50 \HDUVI RUO DUJH FDUERQLVHG SDUWLFOHVL Q WKHIU DFWLRQ ! P EDVHG RQ PHDVXUe- ments of oxidation-resistant elemental carbon in samples from a plot protected against fire over 50 years and one with continued burning. We therefore conclude that our model experiment reflected realistic BC degradation dynamics.

In total, the two glucose additions accelerated BC mineralisation by 58, 72 and 115 % relative to the control. These observed positive priming effects must be due to enhanced turnover of the BC materials, since the additionally released CO2-C largely exceeded the amount of microbial C and DOC introduced with the inoculum. As this was only 0.04 mg (determined with the CFE-method; unpublished results), maximum CO2-C evolution of inoculum-derived C could only contribute between 0.3 % and 0.5 % to the observed additional CO2-C evolution. After the second glucose addition, the proportion of BC primed was even higher than after the first addition, although the absolute amount of BC mineralised in the controls during the second incubation period was lower than during the first. This finding indicates that the pool of degradable pyrogenic compounds was not depleted during the first phase of incubation and we suggest that a more adapted microbial population had been established in the glucose amended samples to account for this additional BC mineralisation.

The observed positive priming effects of glucose on the charred materials were strong compared with other materials. Hamer and Marschner (2002) detected a posi- -1 WLYHSULPLQJHIIHFWRIZKHQJJOXFRVH-C mg C where added to peat. -1 :KHQJOXFRVHZDVDGGHGLQDORZHUFRQFHQWUDWLRQ JJOXFRVH-C mg C) no sig- Chapter 5 Interactive priming of black carbon and glucose mineralisation 123 nificant effect was observed. Also, the mineralisation of lignin could not be significantly influenced by these different glucose additions. Some authors even observed a retardation of organic residue fractions and soil organic matter mineralisation after glucose addition (Szolnoki et al., 1963; Degens and Sparling, 1996). According to Fontaine et al. (2003), the addition of soluble, easily available substrates should not induce strong priming effects, because such substrates would only enhance the development of fast growing r-strategist microorganisms, which are not able to degrade complex organic matter. However, the close correlation between glucose and additional BC mineralisation (Figure 3) suggests that BC degradation can be explained to a large extent by co-metabolism. BC materials are porous in nature, i.e., degradation might begin from outer as well as from inner surfaces. Whether degradation from the latter is possible will depend on the size of the inner pores. If these were very small, organic molecules diffused into the BC particle would have been physically protected from decay. Such a stabilisation mechanism in inner voids of BC has, e.g., been proposed for a range of hydrophobic organic pollutants (Pignatello and Xing, 1996; Luthy et al., 1997; Jonker and Koelmans, 2002). If also true for this study, we should therefore expect a stabilisation of glucose inside BC. However, stabilisation of glucose or its metabolites at the charred materials did not occur.

The glucose mineralisation during the whole incubation period was lowest in the control sand. All the charred materials enhanced glucose mineralisation, i.e., there was also a positive priming effect of added BC on glucose mineralisation and not only vice versa. Nguyen and Guckert (2001) observed that short term utilisation of glucose was larger in soils planted with maize than in unplanted soils. They concluded that there was more carbon available for the microorganisms due to rhizodeposition. However, all BC materials studied here were highly aromatic (Table 1) and so like lignins, hardly provided enough additional substrate for promotion of the glucose-degrading microbial community. We propose that the charred materials offered more surfaces for the growth of the microorganisms. Chenu et al. (2001) observed that the addition of glucose increased the amount of bacteria and fungi on the surface of clayey soil aggregates, whereas in a sandy soil microbial numbers were increased both inside and on the aggregate surfaces. Possibly, the same happened here with increased microbial growth at outer and possibly inner pores of the charred residues. Chapter 5 Interactive priming of black carbon and glucose mineralisation 124

Conclusions

Mineralisation of BC is stimulated by glucose additions. The second glucose addition exhibits a stronger priming effect on BC degradation than the first, because of a better adaptation of the microorganisms to BC decay. The close correlation between additional BC and glucose mineralisation suggests that co-metabolism presumably is one important mechanism in BC decay, but further research is needed to elucidate this idea. Moreover, it is important to gain more insight into the microbial community and enzymes active during BC degradation. In addition, we worked with an artificial system, so further experiments are necessary to elucidate the mechanisms of BC degradation in soils under varying environmental conditions. However, since glucose mineralisation was not retarded but enhanced by the presence of the charred materials, we have to reject the hypothesis that the presence of BC will retard the decomposition of labile substrates: there may be potentially also an interactive priming of porous relatively stable (BC) and labile (glucose) carbon sources in the environment.

Acknowledgements

This project is financially supported by the German Foundation of Research (DFG).

It is part of the priority program 1090 “Soils as source and sink of CO2 – mechanisms and regulation of organic matter stabilisation in soils”. We thank Dr. H. Knicker (München, Germany) for solid-state 13C NMR analysis, Dr. M. W. I. Schmidt (Zürich, Switzerland) for providing the charred wood material and the two anonymous reviewers.

Chapter 5 Interactive priming of black carbon and glucose mineralisation 125

References

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Chenu, C., Hassink, J. and Bloem, J., 2001. Short-term changes in the spatial distribution of microorganisms in soil aggregates as affected by glucose addition. Biology and Fertility of Soils, 34: 349-356.

Cooney, D.O., 1998. Adsorption design for wastewater treatment. Lewis Publishers, Boca Raton.

Degens, B. and Sparling, G., 1996. Changes in aggregation do not correspond with changes in labile organic C fractions in soil amended with 14C-glucose. Soil Biology & Biochemistry, 28: 453-462.

Fontaine, S., Mariotti, A. and Abbadie, L., 2003. The priming effect of organic matter: a question of microbial competition? Soil Biology & Biochemistry, 35: 837-843.

Glaser, B. and Amelung, W., 2003. Pyrogenic carbon in native grassland soils along a climosequence in North America. Global Biogeochemical Cycles, 17: art. no. 1064.

Goldberg, E.D., 1985. Black carbon in the environment - properties and distribution. John Wiley and Sons, New York. Chapter 5 Interactive priming of black carbon and glucose mineralisation 126

Hofrichter, M., Ziegenhausen, D. and Sorge, S., 1999. Degradation of lignite (low- rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system. Applied Microbiology and Biotechnology, 52: 78-84.

Jonker, M.T.O. and Koelmans, A.A., 2002. Sorption of polycyclic aromatic hydrocarbons and polychlorinated biphenyls to soot and soot-like materials in the aqueous environment: mechanistic considerations. Environmental Science & Technology, 36 : 3725-3734.

Kuzyakov, Y., Friedel, J.K. and Stahr, K., 2000. Review of mechanisms and quantification of priming effects. Soil Biology & Biochemistry, 32: 1485-1498.

Luthy, R.G., Aiken, G.R., Brusseau, M.L., Cunningham, S.D., Gschwend, P.G., Pignatello, J.J., Reinhard, M., Traina, S.J., Weber Jr., W.J. and Westall, J.C., 1997. Sequestration of hydrophobic organic contaminants by geosorbents. Environmental Science & Technology, 31: 3341-3347.

Merbach, W., Schmidt, L. and Wittenmayer, L.H., 1999. Die Dauerdüngungsver- suche in Halle (Saale). Teubner, Stuttgart.

Nguyen, C. and Guckert, A., 2001. Short-term utilisation of 14C-[U]glucose by soil microorganisms in relation to carbon availability. Soil Biology & Biochemistry, 33: 53-60.

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Paul, E.A. and Clark, F.E., 1996. Soil microbiology and biochemistry. Academic Press, San Diego. Pignatello, J.J. and Xing, B., 1996. Mechanisms of slow sorption of organic chemicals to natural particles. Environmental Science & Technology, 30: 1-11. Chapter 5 Interactive priming of black carbon and glucose mineralisation 127

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Chapter 6

Isotopic 13C fractionation during the mineralisation of organic substrates

Co-authors: Waltraud Dalhus, Bernd Marschner, Ulrike Schulte and Gerd Gleixner

Rapid Communications in Mass Spectrometry (2004), in review.

Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 130

Abstract

In incubation experiments with organic substrates of different quality and /13C signature we examined the isotopic 13C fractionation during mineralisation. Dissolved organic carbon (DOC) extracted from forest floor material of a Dystric Cambisol

(C3) and DOC extracted from maize straw (C4) were incubated in sand for 51 days at 20 °C. The respective solid materials were incubated 29 days. During incubation the

CO2 evolved was trapped in KOH and total CO2 evolution was monitored hourly while C isotopic contents were determined at different time intervals. To assess molecular changes during degradation, samples of the solid substrates were analysed before and after incubation by pyrolysis-GC/MS-IRMS. During the early stages of 13 decomposition CO2 of forest floor origin was usually C enriched, whereas CO2 of maize origin was 13C depleted as compared to the initial substrates. With ongoing 13 GHFRPSRVLWLRQWKH/ C values of the CO2 approached those of the initial substrates, suggesting that factors controlling isotopic fractionation changed with time. After incubation forest floor DOC was slightly more 13C depleted than forest floor material compared to the initial substrates, since more forest floor DOC (28.8 %) was mineralised than forest floor material (4.7 %). Due to its low degradability, pyrolysis products of forest floor before and after incubation differed not significantly in amount as well as 13C signature. For maize the 13C mass balance was not correct. Compared to the measured /13C value of the residual maize after incubation the 13C depletion of evolved CO2 was too high. The cause for this could not be identified. However, the observed isotopic 13C fractionation was that high that it will be significant even if we 13 have overestimated it. Probably, the high C depletion in CO2 of maize was due to discrimination against 13C during microbial metabolism rather than preferential substrate utilisation. The relative amount of lignin- as well as polysaccharide-derived pyrolysis products decreased accompanied by an increase of 13C in the pyrolysis products after incubation. Our results show that isotopic 13C fractionation during organic matter mineralisation has to be taken into account when calculating their turnover in soils.

Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 131

Introduction

Using the natural differences in the 13C/12C isotopic ratio of plant materials and soil organic matter is a common tool to determine their fates and turnover times (Licht- fouse, 1997; John et al., 2003). Due to their different photosynthetic pathways C3 13 13 plants are more depleted in C than C4 plants having average / C values of -27 ‰ 13 and -ÅUHVSHFWLYHO\ %RXWWRQ +RZHYHUWKH/ C values also differ between different plant components. Lipids and lignin are depleted in 13C compared to the bulk plant material whereas sugars, amino acids and hemicelluloses are enriched (Boutton, 1996). During microbial breakdown of organic substrates isotopic fractionation can occur in two directions. If 13C enriched substrates are utilised preferen- 13 tially the evolved CO2 is enriched in C and the residual organic matter is depleted in 13C (Ågren et al., 1996; Schweizer et al., 1999). On the other hand, metabolic reac- 13 13 tions discriminate against C so that respired CO2 may also be depleted in C causing the microbial biomass and metabolites to become enriched in 13C (Blair et al., 13 1985). During microbial succession a further enrichment of C in the evolved CO2 is possible when these 13C enriched microbial residues are metabolised in the food chain (Macko and Estep, 1984; Accoe et al., 2002). Therefore, isotopic fractionation during the mineralisation of different organic substrates is commonly observed (Mary et al., 1992; Schweizer et al., 1999; Santrucková et al., 2000a; Fernandez and Cadisch, 2003; Kristiansen et al., 2004). However, there are also studies in which no isotopic fractionation occurred or is considered to be negligible (Cheng, 1996; Ek- blad and Högberg, 2000; Nyberg et al., 2000). Hence, it is still uncertain when and where an isotopic fractionation will occur and which factors control the magnitude of this fractionation.

The objective of this study was to determine isotopic fractionation during the mineralisation of two distinctly different organic substrates (maize straw and forest floor material from a mixed temperate forest) and their respective water soluble components, since the latter fraction is generally considered to contain the most bioavail- able fraction of organic matter (Zsolnay, 1996; Marschner and Kalbitz, 2003). To gain more insight into fractionation processes, on-line pyrolysis - gas chromatography/mass spectrometry - isotope ratio mass spectrometry (Py-GC/MS-IRMS) was Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 132 used to determine the 13C values of pyrolysis products before and after incubation of the solid organic materials.

Materials and methods

Organic substrates of C3 and C4 plant origin

Zea mays L. plants (C4) were collected in June 2001 before maturity from a field in Münster (Germany). Stems and leaves were cut in 1 cm pieces, dried at 30 °C and finely ground (< 2 mm). Samples of the Oa layer of a Dystric Cambisol under a mixed stand of Fagus sylvatica L. and Quercus robur L. (both C3) were collected at

Steigerwald (Northern Bavaria, Germany) in October 2000 and air-dried. The Corg contents were 41.8 % and 35.6 %, respectively. The C/N ratio of maize was higher than that of forest floor material (23.0 and 18.7, respectively; C/N-analyser Vario max). For maize a pH of 6.8 and for forest floor material a pH of 3.9 was determined potentiometrically in 0.01 M CaCl2 at a substrate:solution ratio of 1:5 (w/v).

From both substrates, dissolved organic carbon (DOC) extracts were obtained with deionised water at a ratio of 1:10 (w/v). The suspensions were kept at approximately 5 °C and stirred periodically. After 24 h they were first filtered under vacuum through a Whatman glas fibre filter (1 m) and then through a thoroughly washed Sartorius cellulose nitrate filter (0.45 m). In the filtrates DOC was determined with a TOC analyser (Shimadzu 5050). For the maize the concentration was 6228 mg DOC l-1 and 472 mg DOC l-1 for the forest floor. This corresponds to 17.5 and 1.1 % of the total Corg content of the materials. The DOC extracts were freeze-dried for further analysis. The abbreviations maize, forest floor, maize DOC and forest floor DOC are used for sample identification.

Incubation

For incubation, forest floor (10 g dw) was mixed with 1 ml nutrient solution (12.3 g -1 KH2PO4 + 9.4 g K2HPO4 + 11.7 g NH4NO3 l ). Maize (3.6 g dw) was thoroughly Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 133 mixed with 46.4 g sand (acid washed and ignited, Baker) and 1 ml nutrient solution. Sand (50 g) with addition of 1 ml nutrient solution served as control. All samples were adjusted to approximately 60 % water holding capacity (WHC) with deionised water. In case of the forest floor extract, 381 ml extract containing 180 mg DOC were added to 50 g sand and then freeze-dried, since previous experiments showed that adding freeze-dried DOC to sand was not practical. Maize extract corresponding to 100 mg DOC was added to 30 g sand. Furthermore a higher concentrated nutrient solution was added to adjust to a C:N ratio of 1:5. Water content had to be kept to a minimum for incubation (between 20 and 30 % of WHC), because rewetted DOC tended to be sticky.

All samples except the forest floor were inoculated with a mixed inoculum obtained from air-dried forest floor material and air-dried Ap horizon of an arable field. Both soil materials were mixed (1:1 w/w ), rewetted to 60 % WHC and stored at 15 °C two weeks prior to extraction. The inoculum was obtained by shaking the soil mixture with 4 mM CaCl2-solution at a ratio of 1:3 (w/v) for 30 minutes. Subsequently the suspension was filtered through a 5 m Sartorius filter. The maize samples and the control received 1 and the DOC samples 2 ml inoculum.

Each sample was placed in a 250 ml incubation vessel in triplicate and incubated at 20 °C in a Respicond-apparatus (Nordgren Innovations). The solid materials were incubated for 29 days and the DOC samples for 51 days. During the incubation CO2- evolution was recorded hourly by the changes in electrical conductivity in 10 ml of a 0.6 M KOH solution placed inside the incubation vessels (Nordgren, 1988). After the incubation the samples were dried at 40 °C and finely ground to determine Corg content and /13C value. The three replicates of the forest floor and maize straw were mixed for subsequent Py-GC/MS-IRMS.

13 For the C analysis, the CO2 absorbed in the KOH solution periodically was precipitated with BaCl2, immediately vacuum filtrated through a Sartorius 0.45 m cellulose nitrate filter and thoroughly washed with deionised water. During filtration a contamination with atmospheric CO2 was prevented with a soda lime trap in the incom- ing air-stream. The BaCO3 samples were dried at 60 °C and homogenised in a mor- tar. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 134

Carbon isotope (13C) analysis

The carbon isotopic composition of all samples before and after incubation was determined on-line using a C/N analyser (Carlo Erba 1110) coupled to a Finnigan MAT Delta C mass spectrometer. The /13C values were expressed relative to the international PDB (PeeDee Belemnite) standard

13 / C [‰] = [(Rsample-RPDB) / RPDB] x 1000 where R is the isotopic ratio of 13C/12C. As international laboratory reference the USGS-24 standard was used. The precision of this analysis is about ± 0.4 ‰.

13 The / C values of the BaCO3 samples were determined with a more precise off-line method (precision about ± 0.1 ‰). The theoretical background for the off-line preparation refers to Wachter & Hayes (1985) and comprises the reaction of the BaCO3 3 minerals with enriched phosphoric acid (! §JFP ) to induce CO2 release, which can be subsequently analysed in a mass spectrometer. The preparation was carried out according to Swart et al. (1991), who developed the sealed vessel method includ- 13 ing vacuum distillation. The / C values of the CO2 were measured with a Finnigan MAT Delta S and expressed relative to the international PDB standard. As an international laboratory reference the NBS-19 standard was used.

Pyrolysis-GC/MS-IRMS analysis

Forest floor and maize substrates before and after incubation were analysed by pyro- lytical degradation coupled on-line to mass spectrometry and isotope-ratio mass spectrometry. Dried and ground samples were pyrolysed in a ferromagnetic tube with a Curie temperature of 500 °C (0316 Fischer pyrolyser). The pyrolysis products were transferred on-line to a Hewlett-Packard 5890 gas chromatograph and separated on a BPX 5 column. The column outlet was coupled by a fixed splitter to an ion-trap mass spectrometer (ThermoQuest GCQ) operated at 70 eV. The remaining GC eluates after the fixed splitter were transferred to a combustion furnace converting the pyro- 13 lysis products to CO2, N2 and H227KH/ C value was determined using an isotope ratio mass spectrometer (Finnigan MAT Delta plus XL). The software ISODAT 7.0 13 )LQQLJDQ0$7 ZDVDSSOLHGIRUFDOFXODWLQJWKH/ C value of individual compounds. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 135

Only properly resolved peaks with an amplitude higher than 100 mV were used for data interpretation. Pyrolysis products were identified by comparison with reference spectra using GCQ identification software. Relative amounts of the pyrolysis prod- 13 XFWVZ HUH FDOFXODWHG XVLQJ WKHP ] WUDFHR IW KH, 506 FKURPDWRJUDPI RU / C analysis. For comparison, the amounts of the pyrolysed samples were normalised to the Corg content of the respective substrate before incubation to consider the C degradation during incubation. Then the peak areas were related to those calculated values. A more detailed description of the analytical method is given elsewhere (Gleixner et al., 1999; Kracht, 2001; Gleixner et al., 2002).

Results

Mineralisation of organic substrates

The dissolved organic substrates were incubated nearly twice as long as the solid ones, since their lag phases of respiratory inactivity were much longer (Figure 1) and 13 a certain minimum amount of evolved CO2 was needed for C analysis. Until day 16 of incubation 15 % of maize DOC and forest floor DOC were mineralised, thereafter more maize DOC than forest floor DOC was mineralised so that at the end of incubation a total of 68 % maize DOC and 29 % forest floor DOC were mineralised. The solid organic substrates were incubated for 29 days. Here also, much more maize was mineralised than litter. At the end of incubation a total of 56.5 % maize-C and only 4.7 % forest floor-C were mineralised (Figure 1). For both substrates, respiration rates peaked during the first 2 days of incubation. It must be noted, that during the first days of maize incubation, respiration rates were so high that the KOH solutions were saturated with CO2 before they could be exchanged for fresh solutions. As a consequence, CO2 release rates had to be interpolated between days 3 and 4 and between days 5 and 7 and for these periods no samples for 13C-analysis were available. After incubation, the C-loss in the maize straw samples was determined and showed that the interpolation of CO2 release rates was quantitatively correct. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 136

70 ]

C Maize DOC

d 60 e dd

a 50 f

o Maize %

[ 40 C - 2

O 30 C Forest floor DOC ve i 20 t a l u 10 m Forest floor u C 0 0 8 16 24 32 40 48 56 Time [d]

Figure 1: Cumulative CO2-C evolution during incubation of maize, maize DOC, forest floor and forest floor DOC (mean values, n = 3).

Isotopic signature of evolved CO2 during incubation

All examined samples showed changes in the isotopic signature of the evolved CO2 13 during incubation. The results were expressed as CO2-/ C relative to the proportion of C mineralised, in order to account for the different decomposition stages of substrates (Figure 2). Due to the different lag phases between the replicates of the DOC samples this seems more adequate than the temporal course of respiration rates. In the experiments with solid and dissolved organic forest floor substrates the evolved 13 CO2 was generally enriched in C relative to the original substrates (Figure 2a and 2b). This was most pronounced during the first 4 to 10 days of incubation when also the variability in the forest floor DOC replicates was very high. At the end of the 13 incubation, the cumulative / C value of the total CO2 was -25.7 ‰ for the forest 13 floor and -ÅIRUIRUHVWIORRU'2&DQGWKXVVWLOOZHOODERYHWKH/ C values of the original substrates (-27.3 ‰ and -26.3 ‰, respectively; Table 1). Consequently, the analysis of the remaining substrates showed a slight 13C depletion in the same 13 range as that calculated from the CO2 losses (Table 1). Corresponding to the higher DOC mineralisation (Figure 1) the 13C depletion was higher in the samples with forest floor DOC than with the forest floor material. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 137

-16 -8 a) Forest floor c) Maize -10 -19

-12 -22 -14

] -25 -16 ‰ [

C -28 -18 13

δ 0 1 2 3 4 0 15 30 45 60 75 - 2 O C -16 b) Forest floor DOC -8 d) Maize DOC

-10 -19

-12 -22 -14

-25 -16

-28 -18 0 10 20 30 0 15 30 45 60 75

Cumulative CO2-C [% of added C]

13 Figure 2: CO2-/ C signatures for the three replicates during mineralisation of a) forest floor, b) forest floor DOC, c) maize and d) maize DOC with reference to the isotopic 13C signature of the respective substrate before incubation (line).

Isotopic 13C fractionation also occurred during the mineralisation of the solid and dissolved maize substrates (Figures 2c and 2d). But in contrast to the forest floor, 13 13 CO2 was highly &GHSOHWHGGXULQJWKHILUVWGD\VRILQFXEDWLRQFRPSDUHGWRWKH/ C values of the original substrates (up to -5.2 ‰). With progressive mineralisation of 13 WKHV XEVWUDWHV WKH / C values of the CO2 increased to values around those of the original substrates and even became higher during the last days of incubation (Fig- 13 ures 2c and 2d). By the end of the LQFXEDWLRQV WKH FXPXODWLYH / C values of the evolved CO2 were calculated to be -13.7 ‰ for maize straw and -12.1 ‰ for maize 13 13 DOC, thus indicating a C depletion in CO2 and consequently a C enrichment in 13 WKHUHPDLQLQJVXEVWUDWH+RZHYHUWKH/ C value of the maize after incubation was lower than at the beginning and differed significantly from that calculated from the 13 cumulative CO2 data (Table 1). This discrepancy between observed and calculated /13C values can not be attributed to the data gap at the beginning of the incubation 13 when the KOH was saturated, since the C-EDODQFHZDVFRUUHFW7KHFDOFXODWHG/ C value in the remaining maize DOC was in the same range as that calculated for maize Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 138 but could not be verified by analysis because the C content was too low after incubation.

Table 1:0HDVXUHG/13C values of the substrates before (n = 2) and after incubation (n = 3, SD in SDUHQWKHVLV DVZHOODVFDOFXODWHG/13C values of the substrates after incubation using the cumulated 13 / C signature of the evolved CO2 during incubation (ND: not determined).

δ13C (‰) before incubation after incubation

measured measured calculated Forest floor -27.3 -27.5 (0.1) -27.4 (0.0) Forest floor DOC -26.3 -27.0 (0.1) -27.7 (0.5) Maize -11.9 -12.6 (0.1) -9.4 (0.5) Maize DOC -11.1 ND -9.1 (0.7)

Pyrolysis products and their isotopic signature

Only a few of the major peaks in the MS-IRMS chromatograms were identified. For the maize straw, these were 10 pyrolysis products before and after incubation (Table 2). Most of them originated from polysaccharides or lignin. Before incubation, most identified pyrolysis products were 13C depleted compared to the bulk plant material (-11.9 ‰). Products with lignin as precursor were generally more depleted than products of polysaccharides and proteins (Table 2). The highest 13C depletion was observed for 4-vinylphenol (-18.4 ‰). Since it is known from literature (Boutton, 1996) that lignin is usually 13C depleted compared to bulk plant material it is likely that lignin was the precursor for this product rather than protein. After incubation, the /13C signature of most pyrolysis products did not differ significantly from that before incubation. However, a significant 13C enrichment (between +1.5 and +3.1 ‰) was observed for 2-hydroxy-3-methyl-2-cyclopentenen-1-one, 3-methylphenol and 4- vinylphenol (Table 2). A comparison of the IRMS chromatograms before and after incubation indicated the development of a new pyrolysis product after incubation (not shown). This product had a /13C value of -12.3 ‰, but could not be identified. It is most likely that it is a chitin containing substance which would be attributed to microbial biomass.

C I s o h t a o

13 p GLQJPHDQ/ p t

Table 2: List of pyrolysis products found in maize before and after incubation with correspon C values and peak areas (m/z 44 IRMS) i e c r 13

(significant at p * < 0.05, ** < 0.01, *** < 0.001). 6 C

f r ac 13 a a /13 / C value [‰] Area Area ti C value [‰] on

Product Origin a ti

before incubation o after incubation before incubation after incubation n d u r

i n

n = 5 n = 5 n = 4 n = 5 g t h e

Toluene Protein -11.5 -11.6 33.8 17.5*** m i n e r

2-Furancarbox-aldehyde Polysaccharide -13.4 -13.1 13.7 4.0*** a li s a

2-Methylfuran Polysaccharide -15.5 -14.2 15.9 7.4*** ti on o

Dimethylfuran Polysaccharide -12.5 -13.2 3.3 2.1* f o r g

Phenol Lignin -16.7 -16.1 22.5 16.3* a n i c s

2-Hydroxy-3-methyl-2- cyclopentenen-1-one Polysaccharide -12.0 -8.9* 16.0 6.1 u b s t r n = 2 a t e s 3-Methylphenol Polysaccharide -16.2 -13.6* 6.8 4.5 Lignin Protein Protein 4-Vinylphenol -18.4 -16.9* 34.7 13.1** Lignin 2-Methoxy-4-vinylphenol Lignin -17.5 -16.4 31.4 19.0* 2.6 Dimethoxyphenol Lignin -17.5 -15.3 8.8 7.6

a 139 Areas normalised to original Corg content (3 %) and the amount of the pyrolysed sample.

Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 140

The calculated relative amounts of pyrolysis products before and after incubation give only a rough estimate of the relative degradation of these substances (Table 2). During incubation, the relative amount of all products decreased between 13 % (2,6- dimethoxyphenol) and 71 % (2-furancarbox-aldehyde). The relative amount of typical lignin-derived pyrolysis products (2-methoxy-4-vinylphenol, 2,6- dimethoxyphenol) decreased by about 34 % accompanied by an increase of the 13 ZHLJKWHGDYHUDJH/ C value about 1 ‰. For typical polysaccharide-derived pyrolysis products (2-furancarbox-aldehyde, 2-methylfuran, 2-hydroxy-3-methyl-2- cyclopentenen-1-one) a relative decrease of 62 % accompanied by a slight but not 13 VLJQLILFDQWZ HLJKWHG DYHUDJHL QFUHDVHR IW KH / C value by about 0.7 ‰ was observed.

In the forest floor 21 pyrolysis products were identified before and after incubation (Table 3). Pyrolysis products with lignin as precursor were also usually 13C depleted compared to the bulk litter material. During incubation the 13C signature of the pyrolysis products did not change significantly (p > 0.05). Changes of the relative amount of pyrolysis products were also smaller than those in the maize samples. This is due to the low degradability of litter compared to maize (see section “Mineralisation of organic substrates”). New pyrolysis products were not detected.

C I

13 s h Table 3: List of pyrolysis products found in forest floor before and after incubation with corresponding mean / C values and peak areas (m/z 44 IRMS). o a t o p p t e i c r 6 13 13 a a 13

/ /

C value [‰] C value [‰] Area Area C Product Origin f

r before incubation after incubation before incubation after incubation ac ti on n = 4 n = 2 n = 4 n = 2 a ti o

1-Methylpyrrole Protein -27.4 -28.1 2.9 3.2 n d

1H-pyrrole Protein -24.7 -26.3 7.4 6.9 u r i n

Toluene Protein -31.8 -32.1 7.7 7.8 g t h

2-Furancarbox-aldehyde Polysaccharide -25.1 -25.2 11.5 11.0 e m i

2,4 Pentadienal Polysaccharide -28.2 -29.0 8.6 7.2 n e r

2-Methyl-2-cyclopenten-1-one Polysaccharide -27.7 -29.0 1.5 1.4 a li s n = 3 a ti 5-Methyl-2-furaldehyde Polysaccharide -24.8 -25.7 3.5 3.4 on o f

Phenol Protein, Lignin -30.3 -30.9 16.0 17.2 o r g

2-Hydroxy-3-methyl-2-cyclopenten-1-one Polysaccharide -22.9 -21.8 4.9 4.0 a n i 2-Methylphenol Polysaccharide -26.4 -28.3 5.4 6.0 c s u

Protein, Lignin b s t r a

3-Methylphenol Polysaccharide -30.7 -31.7 10.8 12.0 t e s

Protein, Lignin 2-Methoxyphenol Lignin -30.9 -31.6 14.8 15.8 4-Ethylphenol Alkylphenol -30.5 -32.1 3.0 3.2 2-Methoxy-4-methylphenol Lignin -29.2 -30.2 11.5 11.4 4-Vinylphenol Protein, Lignin -31.5 -31.9 8.4 9.0 n-Ethyl-n-methylphenol -24.3 -23.2 3.1 2.6 2-Methoxy-4-ethylphenol Lignin -27.6 -28.6 4.5 4.3 2-Methoxy-4-vinylphenol Lignin -31.0 -31.7 10.8 12.2 2.6-Dimethoxyphenol Lignin -31.7 -32.6 7.8 8.0 4-Allyl-2-Methoxyphenol Lignin -30.9 -32.1 7.9 8.3

1-(4-Hydroxy-3-methoxy-phenyl)-ethanone Lignin -32.3 -34.1 2.4 2.1 141 a Areas normalised to original Corg content (35.6 %) and the amount of the pyrolysed sample. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 142

Discussion

Isotopic fractionation during mineralisation of substrates

13 The C fractionation during mineralisation of the fresh C4 plant materials was dif- 13 ferent from that of the C3 forest floor materials. CO2 of maize origin was C depleted during the phase of highest respiration at the beginning of incubation compared to the initial isotopic composition of the substrate. With ongoing decomposi- 13 WLRQWKH/ C values of the CO2 approached those of the initial substrate. Similar isotopic fractionation patterns were observed for different fresh C4 and C3 plant materials (Zea maize, Lolium perenne, Pinus pinaster, Cocos nucifera, Brachiaria humidi- cola, Desmodium ovalifolium) (Schweizer et al., 1999; Fernandez et al., 2003; Kris- 13 tiansen et al., 2004). A C depletion of CO2 during early stages of mineralisation was also detected for root mucilage, roots and glucose extracted from maize (Mary et al., 1992).

13 This C depletion of CO2 during the phase of highest respiration at the beginning of incubation can not be explained by a preferential utilisation of easily degradable substrates, since those are usually 13C enriched compared to the bulk material (Boutton, 1996). Therefore it seems more likely that during this phase of intensive activity, the microbial metabolism selectively discriminates against 13C, as shown by Fernandez and Cadisch (2003). In their study, the CO2 that evolved during the initial mineralisation of glucose and albumin by white rot fungi was 13C-depleted by up to -5 and -7

‰, respectively and CO2 from lignin mineralisation even was depleted by up to -12 ‰ during the early stages of decomposition (Fernandez and Cadisch, 2003). Since lignin is degraded co-metabolically (Leonowicz et al., 2001) and many easily available C sources in maize are present, it seems likely that lignin degradation may also 13 contribute to the strong C depletion in CO2 observed in our study. This is supported by the relative decrease of lignin-derived pyrolysis products and the accompanying enrichment in 13C in the residues. The decrease of the relative amounts of typical polysaccharide-derived pyrolysis products was higher, but here isotopic fractionation was less pronounced. But it must be kept in mind that only few pyrolysis products were considered for this calculation. Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 143

During later stages of maize degradation other microorganisms utilising more 13C enriched substrates were probably active (Mary et al., 1992; Santrucková et al., 2000b). Fernandez and Cadisch (2003) showed that the degree of isotopic fractionation by the two white rot fungi they tested was different. Furthermore, the occurrence of a new 13C enriched pyrolysis product after incubation suggests that the amount of 13C enriched microbial metabolites and residues increases.

13 It is reported that the duration of the period with C depleted CO2 at the beginning of incubation was longer for quickly degradable plant materials with a high labile C content and shorter for slowly degradable plant materials (Fernandez et al., 2003).

13 CO2 of forest floor origin was C enriched as compared to the initial isotopic com- 13 SRVLWLRQRIWKHVXEVWUDWH+HUHWRRZLWKRQJRLQJGHFRPSRVLWLRQWKH/ C values of the CO2 approached those of the initial substrates. A similar trend was observed after three days incubation of different leaf litter as well as excrements of soil invertebrates fed on the respective leaf litter in most cases (Santrucková et al., 2000a). San- 13 trucková (2000b) observed a C enrichment in CO2 during 40 days of incubation of 13 DWURSLFDOJUDVVODQGVRLOFRPSDUHGWRWKH/ C of soil organic carbon (SOC), while in a temperate grassland soil this fractionation occurred only after 10 days incubation. All these materials have been subjected to microbial degradation before incubation and thus the supply of substrates for microorganisms is more limited than in the maize assay. Besides, microbial metabolites and residues enriched in 13C accumulated and may have been the main CO2 source (Schmidt and Gleixner, 1998; Accoe et al., 2002). However, during the mineralisation of SOC from the surface layer of a 13 IRUHVWVRLOXQGHU'RXJODVILUWKH/ C values of CO2 did not differ significantly from that of SOC (Lin et al., 1999). The same was observed for an arable soil, while an- 13 other arable soil showed a C enrichment of CO2 between day 7 and 35 of incuba- 13 tion (Liang et al., 1999). Besides, a C depletion of CO2 evolved during 117 days of incubation of faeces of maize fed sheep was reported (Kristiansen et al., 2004). Thus a different degree of more easily degradable components only partly explains the observed differences in isotopic fractionation patterns between fresh and altered organic materials. A further factor which must be considered is the different distribution of C atoms in C3 and C4 materials. It has been observed that discrimination dur- Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 144 ing decomposition of C3- and C4-derived sucrose by fungi was different (Henn and Chapela, 2000).

Isotopic fractionation during the mineralisation of DOC followed the same pattern as for the respective solid materials. However, the CO2 evolved from forest floor DOC was more 13C enriched than those of forest floor material during the whole incubation. This is probably due to the higher content of easily degradable 13C enriched substrates in the DOC leading to enhanced microbial metabolism. Before incubation, forest floor DOC was about 1 ‰ more 13C enriched than the solid substrate.

The different direction of fractionation for maize and forest floor DOC may also be due to their different degradability, as explained above for the solid materials. This is consistent with results from Kalbitz et al. (2003b) who observed that the isotopic composition of DOC with low degradability originating from forest floors and fens changed only little during 90 days of incubation and was stronger for DOC of high degradability. However, for maize straw DOC a mineralisation of 89 % (Kalbitz et 13 DOD DFFRPSDQLHGE\DGHFUHDVHLQWKH/ C value of DOC about -4 ‰ (Kalbitz et al., 2003b) was reported, while in our study an increase of +2 ‰ was calculated.

Determination of the isotopic composition of CO2

One problem that remains unresolved in this study is the discrepancy between measured and calculated δ13C values in the maize residues. Assuming that the rather straightforward and reliable 13C-analysis of the substrates before and after incubation is correct, the maize residue was 13C depleted by -0.7 ‰ after incubation. Thus, the 13 total CO2 evolved during incubation should have been enriched in C. Instead, the 13 calculated δ C value of the cumulative CO2 was also lower than that of the initial substrate.

Therefore, the question arises if isotopic fractionation occurred in the incubation vessels or during sample preparation. The isotopic composition of the CO2 was determined by trapping it in KOH, precipitating it with BaCl2 and then measuring the 13 12 C/ C ratio of the precipitated BaCO3. A kinetic isotopic fractionation due to incomplete absorption of CO2 in the KOH solution, as suggested by other authors (Fritz et al., 1985; Cheng, 1996), can be excluded. Own control experiments showed Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 145 that CO2 was trapped quantitatively in the KOH eight hours after the release of known amounts of CO2 from CaCO3. According to Böttcher et al. (1991) this absorption time should be sufficient to prevent isotopic fractionation in systems where the ratio of surface area of absorption solution to volume of the gas phase is 0.05 as it is the case in our system. Isotopic fractionation due to the higher diffusion coefficients 12 13 of CO2 relative to CO2 from the sample also is unlikely, since the maize was incubated in a thin layer (< 2 cm) of very porous medium grained sand.

Contamination by atmospheric CO2 during incubation or during sample preparation is also quite unlikely, since its relatively high δ13C value of -7.8 ‰ to -12 ‰ would 13 cause a C enrichment in the CO2 and not a depletion. Even if a rather unrealistically 13 low δ C value of -28 ‰ is assumed for anthropogenic indoor CO2 (Boutton, 1991), a contamination with 445 mg CO2 would be necessary to explain the observed discrepancies. Such high contamination of the samples with atmospheric CO2 is impos- sible, because the sorption capacity of the 10 mL of 0.6 M KOH is only around 132 mg CO2 and BaCO3 was generally precipitated when the KOH solution was saturated close to 75 %.

We therefore are unable to explain the observed discrepancies in the 13C values of the maize samples. Still, the isotopic fractionation during the initial decomposition phase of maize straw has been observed by others (Fernandez et al., 2003; Kristian- sen et al., 2004) and it is so pronounced that we have no doubt about its direction, possibly only about its magnitude.

Conclusions

Although the incorrect 13C mass balance for the maize sample showed that analytical problems in isotope ratio measurement of CO2 by absorption in KOH solution exist, our results indicate that a pronounced isotopic 13C fractionation occurred during the 13 mineralisation of organic matter. The high C depletion of CO2 during the early decomposition stages of maize and maize DOC is most probable due to a selective discrimination against 13C during microbial metabolism. In contrast, forest floor and its 13 DOC extract showed a C enrichment in the evolved CO2, most likely from the mi- Chapter 6 Isotopic 13C fractionation during the mineralisation of organic substrates 146 crobial utilisation of 13C enriched metabolites and cell fragments due to the absence of easily degradable fresh substrates. During incubation the degree of isotopic fractionation changed, thus suggesting that the factors controlling the fractionation changed, too. The results show that isotopic fractionation during short-term incuba- 13 tion experiments can strongly affect the δ C values of the evolved CO2 which thus is an unreliable tool for the determination of the turnover of specific substrate in soils. 13 Separate measurements of the C signature of respired CO2 from each tested substrate and soil have to be carried out. Furthermore, it still has to be clarified, if the commonly practiced CO2 absorption in KOH or NaOH solution is appropriate for determining the isotopic ratio of CO2 during microbial respiration.

Acknowledgements

This project is financially supported by the German Foundation of Research (DFG).

It is part of the priority program 1090 “Soils as source and sink of CO2 – mechanisms and regulation of organic matter stabilisation in soils”. We thank for the tech- nical assistance of Steffen Rühlow (Jena) in pyrolysis-GC/MS-IRMS analysis and

Beate Gehnen (Bochum) in off-line preparation of the BaCO3 samples.

References

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Chapter 7

Epilogue

Chapter 7 Epilogue 152

Occurrence of priming effects

The mineralisation of all examined organic materials (lignin, peat, SOM, black carbon) was influenced by the addition of at least one of the tested substrates. The data clearly indicated that not only SOM of arable soils is affected, but also SOM of surface and subsoil horizons of forest soils. The acceleration of the mineralisation of organic carbon is a common process immediately after the addition of water soluble organic substrates, that are easily available for microorganisms. This may suggest that stabilisation of organic materials in soils is at least partially controlled by the lack of easily available organic substrates.

Positive priming effects ranging from +7 to +157 % were measured 26 days after substrate addition. Highest positive priming effects were mainly observed in samples with organic matter of low biodegradability: charred materials, SOM of forest soil horizons and peat. In general, correlations between priming effects and soil physical and chemical properties and SOM properties were not detected, except between priming effects induced by alanine and the C/N ratio (Chapter 3). The wider the C/N ratio, the higher was the priming effect after alanine addition. This suggests that N presumably is a further factor limiting mineralisation of SOM.

Some data suggest that labile as well as stable pools of SOM are affected by priming. On the one hand, the mineralisation of charred materials, which are believed to contribute to the refractory SOM pool, was accelerated by glucose addition (Chapter 5). Besides, priming effects were repeatedly inducible (Chapter 4, Chapter 5). Even during a four month incubation alanine additions to the Oa horizon of the Cambisol induced further positive priming effects after each addition, independent of the time of pre-incubation (Chapter 4). Priming effects were highest during the first 4 to 6 days after the respective substrate addition. Without new input of easily available C, the priming effects in most cases subsided during incubation and became negative towards the end of incubation. Sometimes repeated additions induced stronger priming effects than the initial additions. However, in some cases it was also observed that repeated oxalic acid additions stopped inducing priming effects (Chapter 4).

The combined addition of fructose and alanine enhanced SOC mineralisation stronger than the single additions (Chapter 4). It has not only been shown that the Chapter 7 Epilogue 153 composition of soluble substrates is important for occurrence and magnitude of priming effects but also the amount of the soluble substrate added. However, general statements on the effects of different substrate amounts on organic matter mineralisation cannot be given. Sometimes strong priming effects may occur at low substrate concentrations while in other cases higher substrate concentrations are needed to induce strong priming effects (Chapter 2, Chapter 4).

In most cases with positive priming effects, at the end of incubation more carbon from the added substrate remained in the sample than was lost due to additional organic matter mineralisation. Thus, the addition of soluble organic substrates generally increased the organic carbon content. But in some cases the additional organic matter mineralisation was not compensated by the non-mineralised substrate remaining in the sample. This net loss of carbon was mainly observed after the addition of alanine and was highest in the Oa horizon of the Cambisol. Sometimes oxalic acid also induced a net loss of carbon.

Negative priming effects only occurred after the addition of oxalic acid or catechol and ranged from -6 to -43 % after 26 days of incubation. The mineralisation of lignin as well as that of SOM of arable and forest soils was retarded by catechol additions. Oxalic acid induced negative priming effects on lignin and SOM of some forest soils. However, both substrates also induced positive priming effects in some samples. After repeated substrate additions negative priming effects subsided or even became positive, while at the same time more substrate was mineralised (Chapter 4).

It must be concluded, that the stimulation or retardation of SOC mineralisation by additions of soluble organic substrates is an important process for the carbon cycle in soils. The combined and repeated substrate additions which reflect more realistic soil environmental conditions than separate and single additions clearly point out the relevance of priming effects in soils.

It can be expected that priming effects under field conditions are highly variable and show seasonal changes, since concentrations of easily available carbon in soil solutions vary and contain a mixture of different carbon types (Kalbitz et al., 2000). Es- pecially in the neighbourhood of root tips, where easily available substrates are released into the soil solution, high priming effects should be expected. This is supported by experiments with planted soils (Fu and Cheng, 2002; Cheng et al., 2003). Chapter 7 Epilogue 154

The mineralisation of SOC in the planted soils was between 39 and 300 % higher than in the unplanted control. Furthermore, a wavelike distribution of bacterial populations along roots has been observed. This has been attributed to growth and death cycles of microorganisms in response to the moving nutrient source, the root tip (van Bruggen et al., 2000). It is probable that priming effects change concomitantly.

Mechanisms of priming effects

Turnover of microbial biomass

In the first experiments on lignin and peat mineralisation microbial biomass was not measured. The maximal inoculum-derived microbial biomass was estimated. For the lignin system it could be excluded that the observed positive priming effects were only due to enhanced turnover of the native microbial biomass. In the peat system this was not that clear, since also peat-borne biomass was present which could not be estimated (Chapter 2). In the experiments with soil samples the amount of microbial biomass was measured before incubation. This allows a more precise estimation of the contribution of its turnover to additional CO2 evolution. As presented in Chapter 3, in most cases apparent priming can be excluded. Direct evidence for real priming effects was given in the experiments with repeated measurement of native and new microbial biomass during incubation (Chapter 4). It was observed that the turnover of native Cmic maximally explained 35 % of the additional CO2 evolution. In experiments on black carbon mineralisation maximal 0.5 % of the additional CO2 evolution was due to turnover of inoculum-derived microbial biomass.

It can be concluded, that the turnover of native soil microbial biomass after substrate additions contributes to the observed additional CO2 evolution, but is by far not sufficient to explain the total effect. Thus, the addition of soluble organic substrates indeed accelerated the turnover of organic matter.

Chapter 7 Epilogue 155

Co-metabolism

A possible mechanism of positive priming effects discussed is co-metabolism. Or- ganic matter is considered to be degraded cometabollically if another organic substrate is required by microorganisms for their degradation (Paul and Clark, 1996). This organic substrate is a carbon and energy source for microorganisms and thus enhances their growth and enzyme production. When priming effects are only due to increased metabolic activity from an increased microbial biomass, it should be expected that the additional mineralisation of organic matter decreases after exhaustion of the substrate.

A significant linear relationship between substrate and organic matter mineralisation was only observed in the experiments with black carbon. The higher was the glucose mineralisation, the higher was the additional mineralisation of the respective charred material (Chapter 5). This suggests that co-metabolism may be an important mechanism in BC mineralisation. However, BC mineralisation also occurred in the controls. It is generally believed that lignin is degraded co-metabolically (Kirk and Far- rell, 1987; Leonowicz et al., 2001). But here, a relationship between substrate and additional organic matter mineralisation was not detected. This was mainly due to the fact that one point did not fit to the regression line. Between days 6 and 12 of incubation in many cases the organic matter mineralisation was retarded although substrate was mineralised.

12 A further indication of co-metabolism is the ratio of the additional amount of CO2- C evolved to the amount of mineralised substrate. This ratio is calculated for all analysed time intervals during the incubation. In the case of co-metabolism, the ratio should be more or less constant during time. This was only the case for the charred materials. Here, during the first days after the respective glucose addition the ratio was about 0.15 (Table 1). However, in the lignin system the low alanine addition led 12 14 to increasing ratios of additional CO2-C to CO2-C which were about 20 after 12 days of incubation (Table 1). Chapter 7 Epilogue 156

Table 1: Ratio of additional organic carbon to substrate mineralisation for some samples showing positive priming effects. All soil samples with positive priming effects after alanine addition are presented as well as the lignin system after low and high alanine addition. The charred materials received glucose additions at day 0 and 26 of incubation.

12 14 Incubation period Additional CO2-C / CO2-C

[d] Podzol Oa Podzol EA Podzol Bs

0 - 4 0.12 0.33 0.19 4 - 6 0.08 0.15 0.16 6 - 12 0.10 0.18 0.51 12 - 19 -0.08 -0.05 0.64 19 - 26 -0.62 -0.28 -0.30

Podzol Bw Cambisol Oa Cambisol A

0 - 4 0.35 0.47 0.14 4 - 6 0.18 2.33 1.16 6 - 12 -0.04 2.54 1.29 12 - 19 0.46 2.06 2.77 19 - 26 -0.29 3.46 2.95

Maize NPK Lignin high Lignin low

0 - 4 0.30 -0.80 3.54 4 - 6 0.12 0.61 1.14 6 - 12 -0.38 0.27 0.73 12 - 19 -0.62 3.96 20.56 19 - 26 -1.04 2.89 19.92

Charred Maize Charred Rye Charred Wood

0 - 4 0.18 0.19 0.11 4 - 6 0.12 0.17 0.15 6 - 12 0.07 0.12 0.15 12 - 19 0.12 0.33 -0.15 19 - 26 0.40 0.34 -0.43 26 - 32 0.13 0.16 0.16 32 - 34 0.05 0.22 0.10 34 - 38 0.12 0.19 0.06 38 - 40 0.17 0.16 -0.01 40 - 46 0.44 0.11 0.07 46 - 53 0.15 0.04 -0.12 53 - 60 -0.04 0.25 -0.08 Chapter 7 Epilogue 157

Also in the Oa and A horizon of the Cambisol with alanine addition this ratio was much higher during later incubation periods (Table 1). Although 44 % and 26 % of the total priming effects, respectively, occurred during the first four days, the ratio of 12 14 additional CO2-C / CO2-C then increased steadily to values as high as 3.5, thus indicating that despite a very low alanine mineralisation rate, priming effects persisted. Possibly extracellular enzymes were stabilised in these horizons and thus maintained their activity over a longer time as suggested by Bending et al. (2002) or there is enough available SOC for the activated microorganisms to keep growing. The latter assumption is supported by Fontaine et al. (2004), who observed that positive priming after cellulose addition to a savannah soil was maintained until the end of a 70 days incubation although most cellulose was exhausted by day 13. They concluded that the cellulose supply increased the populations of SOM feeding microbes, which were able to survive on SOM after the cellulose was exhausted.

In the maize and maize NPK soil, an opposite trend was observed. While initially 12 14 about 0.3 mg of additional CO2-C was released for every mg of CO2-C from fructose, alanine or oxalic acid, this ratio decreased and became even negative in the course of incubation (shown exemplary in Table 1 for maize NPK with alanine). Negative values are observed when the mineralisation of organic matter was retarded during the respective time interval compared to the control. In other cases, e.g. the Bw horizon of the Podzol with alanine (Table 1) strong changes of this ratio between the different incubation periods occurred. So apparently, a simple relationship between substrate mineralisation dynamics and priming effects does not exist.

Furthermore, the increase in Cmic after combined fructose and alanine addition to the Bs horizon of the Podzol by a factor of 1.4 seemed to be too small to explain the observed seven-fold increase of SOC mineralisation during the first 4 days of incubation by co-metabolism.

Thus, in most cases a general increase in total microbial biomass and their metabolic activity is not sufficient to explain positive priming effects. It is likely, that different mechanisms control priming effects induced by the different substrates, while the influence of the respective mechanisms may depend on the soil environmental conditions. Chapter 7 Epilogue 158

Further possible mechanisms

Especially the results with repeated substrate additions (Chapter 4) suggest that the composition of the microbial community and structural changes induced by the respective substrate are important for the observed priming effects. This assumption is supported by findings in other studies. For example, Bell et al. (2003) observed that additions of 14C-labelled wheat straw induced higher positive priming effects in arable soils with the higher fungi to bacteria ratio. Using denaturing gradient gel electrophoresis (DGGE) profiles, Falchini et al. (2003) showed that oxalic acid and glutamic acid additions to soil favoured the development of a few new bacterial species, whereas glucose addition enhanced growth of formerly existing bacteria. Since glutamic acid as well as glucose induced positive priming effects, Falchini et al. (2003) expected that different microbial communities were responsible for the additional mineralisation of SOM. Waldrop and Firestone (2004b) demonstrated that the presence of different plant communities did not alter the microbial community responsible for decomposition of relatively labile organic substrates but did alter the profiles of microbial communities responsible for decomposition of the more recalcitrant substrates, pine litter and indigenous SOM. Baudoin et al. (2003) observed that artificial root exudates caused changes in the substrate utilisation patterns and the genetic structure of the microbial communities. Structural changes in the composition of the microbial community and their functional diversity were proven even several millimetres away from roots (Kandeler et al., 2002).

It is also conceivable that the substrate addition abolishes the energy limitation of the microorganisms. This enables them to produce energetically expensive enzymes involved in the degradation of the more complex structures of SOM.

Suitability of natural 13C abundance for studying priming effects

It has been shown that the use of 14C isotope techniques is a suitable tool for determining priming effects of simple organic substrates. However, in soil solutions these substrates are always present together with many other complex organic substrates. To examine the influence of “real” soil solutions on the mineralisation of SOC it is Chapter 7 Epilogue 159

13 QHFHVVDU\ WRXVHWKHQDWXUDOGLIIHUHQFHVLQWKH/ C signature of organic matter, as explained in Chapter 1. During the incubation of organic matter extracted from maize 13 VWUDZDQGIURPIRUHVWIORRUDVZHOODVRIWKHUHVSHFWLYHVROLGPDWHULDOVWKH/ C values 13 of respired CO2GLIIHUHGIURPWKH/ C value of the respective initial material (Chap- ter 6). This isotopic fractionation was most pronounced within the phase of highest mineralisation at the beginning of incubation. Therefore, in experiments with substrate treated soils it is not possible to calculate the proportion of respired substrate directly from the amount and the isotopic composition of CO2 evolved. It is necessary to include isotopic fractionation factors for both substrate and SOC mineralisation dependent on the incubation time. These factors have to be determined in independent experiments. However, it is unknown to what extent the isotopic fractionation for the respective organic material is the same when incubated in combination with another organic material. Since it has been shown that the decomposition of different organic materials is an interdependent process, it seems possible that also isotopic fractionation will be influenced. Besides, it has to be clarified, why there was a discrepancy in the 13C mass balance for the incubated maize samples, as dis- 13 cussed in Chapter 6. Here, it seems XVHIXOWRGHWHUPLQHWKH/ C signature of the CO2 directly in the gas phase and compare these values with those obtained by the precipitation method. After elimination of methodical problems, it should be possible to elucidate the influence of organic carbon present in the soil solution on the mineralisation of SOC.

Conclusions

Dissolved organic substrates play an important role in the carbon cycle of soils. All tested substrates (glucose, fructose, glycine, alanine, oxalic acid, acetic acid and catechol) are able to accelerate the mineralisation of organic matter. Oxalic acid and catechol in some cases also retard the mineralisation. Priming effects are ubiquitously occurring in soils: in arable soils as well as in surface and subsurface horizons of forest soils. Factors controlling the occurrence of priming effects still remain uncertain. Relationships between the examined physical and chemical soil properties, SOM properties and priming effects were not detected. Only the magnitude of the Chapter 7 Epilogue 160 priming effect induced by alanine depended on the C/N ratio of SOM. Different types of organic matter are affected by priming. The mineralisation of lignin, peat and charred materials can be strongly accelerated. Since priming effects were not depressed after extended pre-incubation of a soil sample, it seems unlikely that only the labile SOC-pool is susceptible. Priming effects can persist over one month. In most cases they were highest within the first 4 to 6 days after substrate addition and then subsided. However, repeated substrate additions in general induced further priming effects. Besides, a combined substrate addition induced a higher priming effect than the single substrate additions. This points out the relevance of easily available organic substrates for the turnover of SOM. In the case of positive priming, the turnover of microbial biomass explained maximal 35 % of the additional CO2 evolution. Therefore, the observed positive priming effects indeed were attributed to an enhanced mineralisation of SOM. Co-metabolism is one possible mechanism responsible for positive priming effects. The enhanced mineralisation of black carbon may be attributed largely to co-metabolism, while in the soil samples this mechanism seems to be of minor importance. It is expected that substrates induce changes of the microbial community structure and of their functional diversity. The addition of DOM extracted from soils and / or plant residues to soils with another natural 13C abundance will allow the examination of priming effects under more natural conditions.

Further research

Further research is needed to understand mechanisms of priming effects in soils and thus to better understand the carbon cycle in soils.

As discussed above, it seems useful to examine enzyme activities during incubation studies on priming effects to track changes in the functional diversity of the microbial community. Especially during the first four to six days repeated measurements on enzyme activities should be carried out, since this was generally the period in which strongest priming effects occurred. A microplate fluorimetric assay developed by Marx et al. (2001) enables to analyse a large number of soil samples and enzymes in a short time. The activities of different hydrolytic eQ]\PHV IRUH [DPSOH - Chapter 7 Epilogue 161

JOXFRVLGDVH -FHOORELDVH -JDODFWRVLGDVH -xylosidase and phosphatase, involved in C, N or P transformations can be assessed with this method.

Supplementary it seems promising to track changes in the microbial community structure using DGGE profiles (Sørensen et al., 2002). The use of different poly- merase chain reaction (PCR) primers should enable to amplify rRNA sequences typical of different groups of microoorganisms such as proteobacteria and actinomycetes and thus to distinguish between more r- and more K-strategist microorganisms. The first group of microorganisms is considered to respond rapidly to the addition of easily available substrates, while it is expected that only the latter slowly growing one is able to degrade SOM (Fontaine et al., 2003).

Furthermore, it is still uncertain which pools of SOM are affected by priming. It is generally believed that organic matter associated with sand-sized particles is typically younger, shows larger C/N ratios and is more labile than SOM associated with smaller soil particles (Stemmer et al., 1999; Kahle et al., 2002). Plant derived organic matter is usually rapidly converted and recovered in the sand fraction, whereas SOM in the clay fraction comprises mainly products of microbial metabolism (Christensen, 2001). In general, cell numbers and microbial biomass are higher in the smaller fractions, while the enzyme activity largely depends on the enzyme investigated (Kan- deler et al., 1999). In free air carbon dioxide enrichment studies (FACE) with 13C depleted CO2, it has been shown that about 60 % of the new soil C was incorporated in the sand fraction (van Kessel et al., 2000; Hagedorn et al., 2003a). Availability of SOM was lowest in the silt fraction (Nelson et al., 1994). Rumpel et al. (2004) observed a close positive relationship between the C enrichment in fractions < 6.3 m and the age of bulk SOM determined by radiocarbon dating. All these results indicate that composition and properties of SOM in different particle size fractions (sand, silt, clay) differ. Therefore, it should be further investigated, whether SOM in all particle size fractions is affected by priming and whether SOM in the sand fraction is more affected since more young and labile organic matter is present. Furthermore, the combination of 14C and 13C isotope techniques should enable to elucidate whether old or new organic matter in the fractions is primarily subject to priming. For this purpose soil samples are needed, consisting of younger SOM with a different /13C Chapter 7 Epilogue 162 signature compared to older SOM as it is the case in FACE experiments where plants 13 are grown under a highly C depleted CO2 atmosphere.

In this context it might also be promising to examine the influence of N additions on the occurrence of priming effects. According to Waldrop and Firestone (2004a) N additions stimulated the utilisation of older soil carbon (> 14 years). They attributed this to a greater peroxidase activity. In contrast, results from Hagedorn et al. (2003b) indicate that N fertilisation reduced the mineralisation of old and humified SOC (> 4 years). Besides, the role of N for the occurrence and magnitude of priming effects is still uncertain, as presented above.

Further experiments using catechol as substrate should be carried out. On the one hand it would be promising to examine the effects of repeated catechol additions on the mineralisation of charred materials. It is possible that this monomeric phenol stimulates the production of phenol oxidases (e.g. laccase) and peroxidases. These enzymes are considered to be important for coal degradation or liquefaction (Fakoussa and Hofrichter, 1999). Therefore, it is expected that catechol accelerates the mineralisation of charred materials. On the other hand, catechol is a substrate mainly retarding the mineralisation of SOM, although microorganisms seem to adapt to repeated catechol additions which sometimes leads to positive priming (Chapter 4). Experiments with combined additions of catechol and those organic substrates causing positive priming effects, would be more realistic. Maybe negative priming effects induced by catechol will be compensated by positive priming effects induced by the other substrate.

In this thesis all incubation experiments were carried out at 20 °C. Also in most other studies about priming effects temperatures between 20 and 27 °C were used. Fur- thermore, most soils originate from temperate regions of the northern hemisphere. However, it is known that changes in the temperature induce changes in the microbial community structure accompanied by a shift in organic matter mineralisation (Andrews et al., 2000). Dalias et al. (2001) suggest that increasing temperatures not only accelerate mineralisation rates of plant residues incorporated in soils but also change decomposition processes so that material produced at higher temperatures is more recalcitrant than that produced at lower temperatures. Waldrop and Firestone (2004a) observed that greater quantities of older carbon were respired at higher tem- Chapter 7 Epilogue 163 peratures (20 and 35 °C) compared to 5 °C. According to Vasconcellos (1994), acceleration of SOM mineralisation after glucose addition (1 mg C g-1 soil) was in most cases more than twice as high at 35 °C than at 15 °C. At 35 °C priming effects in temperate soils were higher than in tropical soils. Also for Antarctic soil incubated at 12 °C a significant positive priming effect after the addition of Deschampsia antarc- tica was observed (Malosso et al., 2004). Thus, to understand the importance of priming effects on a global scale, it is necessary to investigate these effects in soils of different climatic regions under typical temperature conditions.

References

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Summary

Soils contain the largest active terrestrial carbon pool and play an important role in the global carbon cycle. The mineralisation of soil organic carbon (SOC) leads to the release of CO2 into the atmosphere. At the same time CO2 is incorporated into soil organic carbon by plants and microorganisms. On the background of increasing atmospheric CO2 levels there is much interest whether soils act as net carbon sink or source in the carbon cycle. Although conceptual models about stabilisation mechanisms of organic carbon in soils exist, quantitative knowledge about the processes is scarce.

Most soil organic carbon is not available for microorganisms. The addition of plant residues or simple organic substrates such as sugars or amino acids is known to influence the mineralisation of SOC. On the one hand, the mineralisation of SOC can be enhanced. This phenomenon is known as positive priming effect. On the other hand a retardation of SOC mineralisation, i.e. a negative priming effect, can occur. Since all microbial uptake mechanisms require an aqueous environment, it is expected that dissolved organic substrates are the most important C-source for microorganisms in soils. The aim of this thesis was to quantify the influence of different soluble organic substrates typically occurring in soil solutions on the mineralisation of different types of organic matter.

In laboratory incubation experiments the effects of glucose, fructose, alanine, glycine, oxalic acid, acetic acid or catechol on the mineralisation of lignin, peat, SOC in soil samples from different soil types or charred materials were examined. All soluble organic substrates used have been uniformly 14C-labelled to differentiate between the mineralisation of substrate and that of SOC. In most cases samples received sub- -1 -1 strate additions of 13.3 µg substrate-C mg Corg. Also lower (2.7 µg substrate-C mg -1 Corg) and higher (20 µg substrate-C mg Corg) substrate additions were tested. During 14 the incubation the total CO2 production was measured hourly. The amount of CO2 was determined at various time intervals. The CO2 was trapped in KOH solution. To gain more insight into mechanisms of priming effects, in some experiments changes in the amount of the native and new microbial biomass were determined using the chloroform-fumigation-extraction method. Furthermore, experiments with repeated Summary 168 and combined substrate additions were carried out. The suitability of the natural 13C abundance for examining the influence of natural, complex solutions of organic carbon on SOC mineralisation was tested.

The mineralisation of lignin and peat was affected by the addition of all seven 14C- labelled organic substrates. Most substrate additions accelerated the mineralisation of lignin and peat. A clear relationship between the amount of added substrate and the magnitude of the priming effect did not exist. Increases in organic matter mineralisation with the amount of added substrate occurred proportionally as well as non pro- -1 portionally. The low addition (2.7 µg substrate-C mg Corg) of glycine and oxalic acid stronger enhanced the peat mineralisation than the high addition (13.3 µg sub- -1 strate-C mg Corg). After 26 days of incubation the strongest positive priming effect (+157 %) was observed in the peat system with the low amount of oxalic acid. The high alanine addition caused the strongest increase in lignin mineralisation (+40 %). Only glucose often did not significantly influence lignin and peat mineralisation. Negative priming effects only were found in the lignin system with oxalic acid and catechol addition at both concentrations.

The effects of fructose, alanine, oxalic acid and catechol on the mineralisation of SOC from different horizons of two forest soils (Haplic Podzol and Dystric Cambi- sol) and one arable soil (Haplic Phaeozem) under maize and rye cultivation with and -1 without NPK fertilisation were investigated (13.3 µg substrate-C mg Corg) over 26 days. In almost all soils, priming effects were induced by one or several of the added substrates. The strongest positive priming effects occurred with fructose and alanine in the Bs horizon of the Haplic Podzol (+91 % and +85 %, respectively). In general, positive priming effects were more pronounced in forest soils that contain SOC of low biodegradability. However, a prediction on occurrence and magnitude of priming effects based on soil properties or SOM composition was not possible. Only between the priming effects induced by alanine and the C/N ratio a significant positive relationship was observed. Catechol and oxalic acid caused negative as well as positive priming effects.

The combined addition of fructose and alanine to the Bs horizon of the Haplic Pod- zol induced a higher positive priming effect (+127 %) than the single substrate additions. During the first 4 days, seven times more SOC was mineralised than in the Summary 169 control while the microbial biomass only increased by a factor of 1.4. This indicates that the observed priming effect can not be solely attributed to co-metabolism. Be- sides, the turnover of microbial biomass explained maximally 35 % of the additional

CO2 evolution. When added repeatedly, most substrates induced higher positive priming effects than single additions. After extended pre-incubation of a soil sample, priming effects were not depressed. Thus, it seems unlikely that only the labile SOC pool is susceptible to priming. With repeated substrate additions, substrate mineralisation increased. The increased catechol utilisation was accompanied by subsiding negative priming effects, which in one case even became positive.

The mineralisation of charred maize and rye residues (thermally altered at 350 °C) and charred oak wood (thermally altered at 800 °C) was strongly enhanced by glu- -1 cose additions (20 µg glucose-C mg Corg at day 0 and 26). In the controls without glucose addition, between 0.3 % (wood) and 0.8 % (maize) of the initial charred materials were mineralised after 60 days and 0.6 % to 1.2 % when glucose was added. The second glucose addition induced a stronger positive priming effect than the first one. A close correlation (r = 0.94, p < 0.001) between glucose and additional black carbon mineralisation suggests that black carbon mineralisation may be for the most part due to co-metabolism. Also glucose mineralisation was enhanced by the presence of charred material.

During the incubation of maize straw and forest floor material as well as dissolved 13 organic carbon extracted from these materials the δ C values of respired CO2 differed from the δ13C value of the respective inital material. This isotopic fractionation was that pronounced within the phase of highest mineralisation at the beginning of incubation that it cannot be solely due to the analytical problems in isotope ratio 13 measurement of CO2 by absorption in KOH. Thus isotopic C fractionation during organic matter mineralisation has to be taken into account when calculating priming effects in soils.

It has been shown that the interdependent decomposition of dissolved and solid organic materials is an important process in the carbon cycle of soils. The stabilisation of organic materials in soils is at least partially controlled by the lack of easily available organic substrates. Zusammenfassung 170

Zusammenfassung

Böden enthalten den größten aktiven terrestrischen Kohlenstoff-Pool und spielen eine wichtige Rolle im globalen Kohlenstoffkreislauf. Die Mineralisation von organischem Bodenkohlenstoff (SOC) führt zur Freisetzung von CO2 in die

Atmosphäre. Zur gleichen Zeit wird CO2 durch Pflanzen und Mikroorganismen in organischen Bodenkohlenstoff umgewandelt. Vor dem Hintergrund ansteigender atmosphärischer CO2-Gehalte ist es von großem Interesse, ob Böden als Kohlenstoff- Senke oder -Quelle im Kohlenstoffkreislauf fungieren. Obwohl konzeptionelle Modelle über Stabilisierungsmechanismen von organischem Kohlenstoff in Böden existieren, ist das quantitative Wissen über die Prozesse gering.

Der größte Teil von organischem Kohlenstoff in Böden ist für Mikroorganismen nicht verfügbar. Es ist bekannt, dass die Zugabe von Pflanzenresten oder einfachen organischen Substraten wie Zuckern oder Aminosäuren die Mineralisation von SOC beeinflusst. Einerseits kann die SOC-Mineralisation gesteigert werden. Dieses Phä- nomen ist bekannt als positiver Priming Effekt. Andererseits kann eine Verzögerung der SOC-Mineralisation, also ein negativer Priming Effekt, auftreten. Da alle mikrobiellen Aufnahmemechanismen eine wässrige Umgebung benötigen wird erwartet, dass gelöste organische Substrate die wichtigste C-Quelle für Mikroorganismen in Böden darstellen. Ziel dieser Doktorarbeit war es, den Einfluss verschiedener lösli- cher organischer Substrate, die typischerweise in Bodenlösungen vorkommen, auf die Mineralisation verschiedener Typen organischer Substanz zu quantifizieren.

In Inkubationsexperimenten im Labor wurden die Effekte von Glucose, Fructose, Alanin, Glycin, Oxalsäure, Essigsäure oder Brenzcatechin auf die Mineralisation von Lignin, Torf, SOC in Bodenproben verschiedener Bodentypen oder verkohlten Mate- rialien untersucht. Alle verwendeten löslichen organischen Substrate waren einheit- lich mit 14C markiert, um zwischen der Mineralisation von Substrat und SOC unter- scheiden zu können. In den meisten Fällen erhielten die Proben Substratzugaben von -1 -1 13,3 µg Substrat-C mg Corg. Auch geringere (2,7 µg Substrat-C mg Corg) und hö- -1 here (20 µg Substrat-C mg Corg) Substratzugaben wurden getestet. Während der

Inkubation wurde die gesamte produzierte CO2-Menge stündlich gemessen. Die 14 Menge an CO2 wurde in verschiedenen Zeitintervallen bestimmt. Das CO2 wurde Zusammenfassung 171 in KOH-Lösung aufgefangen. Um einen größeren Einblick in die Mechanismen von Priming Effekten zu gewinnen, wurden in einigen Experimenten Änderungen der Menge an nativer und neuer mikrobieller Biomasse mit der Chloroform- Fumigations-Extraktions Methode bestimmt. Außerdem wurden Experimente mit wiederholten und kombinierten Substratzugaben durchgeführt. Es wurde die Eignung der natürlichen 13C Verteilung für die Untersuchung des Einflusses natürlicher, kom- plexer Lösungen von organischem Kohlenstoff auf die SOC-Mineralisation getestet.

Die Mineralisation von Lignin und Torf wurde durch alle sieben 14C-markierten organischen Substrate beeinflusst. Die meisten Substratzugaben beschleunigten die Mineralisation von Lignin und Torf. Eine klare Beziehung zwischen der Menge an zugegebenem Substrat und der Größe des Priming Effektes bestand nicht. Anstiege der Mineralisation organischer Substanz mit der Menge an zugegebenem Substrat waren sowohl proportional als auch nicht proportional. Die geringe Zugabe (2,7 µg -1 Substrat-C mg Corg) von Glycin und Oxalsäure verstärkte die Torfmineralisation -1 mehr als die hohe Zugabe (13,3 µg Substrat-C mg Corg). Nach 26 Tagen Inkubation wurde der stärkste positive Priming Effekt (+157 %) im Torf-System mit der geringen Menge an Oxalsäure beobachtet. Die hohe Alaninzugabe verursachte den stärksten Anstieg der Ligninmineralisation (+40 %). Nur Glucose beeinflusste die Lignin- und Torfmineralisation oft nicht signifikant. Negative Priming Effekte wurden nur im Lignin-System mit Oxalsäure- und Brenzcatechinzugabe in beiden Konzentrationen gefunden.

Die Effekte von Fructose, Alanin, Oxalsäure und Brenzcatechin auf die SOC- Mineralisation verschiedener Horizonte von zwei Waldböden (Haplic Podzol und Dystric Cambisol) und eines Ackerbodens (Haplic Phaeozem) unter Mais- und Rog- genanbau mit und ohne NPK Düngung wurden 26 Tage lang untersucht (13,3 µg -1 Substrat-C mg Corg). In fast allen Böden wurden Priming Effekte durch eines oder mehrere der zugegebenen Substrate ausgelöst. Die stärksten positiven Priming Effek- te traten mit Fructose und Alanin im Bs-Horizont des Haplic Podzol auf (+91 % bzw. +85 %). Im Allgemeinen waren positive Priming Effekte in Waldböden mit schlecht abbaubarer organischer Bodensubstanz stärker ausgeprägt. Eine Vorhersage über Auftreten und Größe von Priming Effekten basierend auf Bodeneigenschaften und Zusammensetzung der organischen Bodensubstanz war jedoch nicht möglich. Nur Zusammenfassung 172 zwischen den Priming Effekten die durch Alanin ausgelöst wurden und dem C/N- Verhältnis wurde eine signifikante positive Beziehung beobachtet. Brenzcatechin und Oxalsäure verursachten sowohl negative als auch positive Priming Effekte.

Die kombinierte Zugabe von Fructose und Alanin zum Bs-Horizont des Haplic Pod- zol löste einen stärkeren positiven Priming Effekt aus (+127 %) als die einzelnen Substratzugaben. Innerhalb der ersten vier Tage wurde siebenmal mehr SOC mineralisiert als in der Kontrolle, während die mikrobielle Biomasse nur um den Faktor 1,4 zunahm. Dies deutet darauf hin, dass der beobachtete Priming Effekt nicht nur auf Cometabolismus zurückzuführen ist. Außerdem erklärte der Umsatz der mikrobiellen

Biomasse maximal 35 % der zusätzlichen CO2-Entwicklung. Die meisten Substrate lösten nach wiederholter Zugabe stärkere positive Priming Effekte aus als nach ein- maliger Zugabe. Nach ausgedehnter Vorinkubation einer Bodenprobe wurden Pri- ming Effekte nicht unterdrückt. Deshalb scheint es unwahrscheinlich, dass nur der labile SOC-Pool anfällig für Priming ist. Mit wiederholten Substratzugaben nahm die Substratmineralisation zu. Die zunehmende Brenzcatechinnutzung wurde durch ab- nehmende negative Priming Effekte begleitet, welcher in einem Fall sogar positiv wurde.

Die Mineralisation der verkohlten Mais- und Roggenrückstände (thermisch gealtert bei 350 °C) und des verkohlten Eichenholzes (thermisch gealtert bei 800 °C) wurde -1 durch Glucosezugaben stark gesteigert (20 µg Glucose-C mg Corg an Tag 0 und 26). In den Kontrollen ohne Glucosezugabe waren nach 60 Tagen zwischen 0,3 % (Holz) und 0,8 % (Mais) der anfänglichen verkohlten Materialien mineralisiert und 0,6 bis 1,2 % wenn Glucose zugegeben wurde. Die zweite Glucosezugabe löste einen stärke- ren positiven Priming Effekt aus als die erste. Eine enge Korrelation (r = 0,94, p < 0,001) zwischen Glucosemineralisation und zusätzlicher Mineralisation der verkohlten Materialien lässt vermuten, dass die Mineralisation der verkohlten Materialien zum größten Teil auf Cometabolismus zurückzuführen ist. Auch die Glucoseminera- lisation wurde durch die Anwesenheit der verkohlten Materialien gesteigert.

Während der Inkubation von Maisstroh und Auflage eines Waldbodens sowie des aus diesen Materialien gelösten organischen Kohlenstoffs unterschieden sich die 13 13 δ C-Werte des veratmeten CO2 von dem δ C-Wert des jeweiligen Ausgangsmateri- als. Diese isotopische Fraktionierung war während der Phase stärkster Mineralisation Zusammenfassung 173 zu Beginn der Inkubation so stark ausgeprägt, dass sie nicht nur auf die analytischen

Probleme bei der Isotopenverhältnismessung des in KOH absorbiertem CO2 zurück- zuführen ist. Daher muss die isotopische 13C-Fraktionierung während der Mineralisa- tion organischer Substanz bei der Berechnung von Priming Effekten in Böden be- rücksichtigt werden.

Es konnte gezeigt werden, dass die voneinander abhängige Zersetzung von gelösten und festen organischen Materialien einen wichtigen Prozess im Kohlenstoffkreislauf von Böden darstellt. Die Stabilisierung von organischem Kohlenstoff in Böden wird zumindest teilweise durch das Fehlen von leicht verfügbaren Substraten kontrolliert. Acknowledgements 174

Acknowledgements

I would like to thank Professor Dr. Bernd Marschner as advisor of this thesis for support and constructive criticism. PD Dr. Tim Mansfeldt and PD Dr. Frank Wisotzky are gratefully acknowledged as reviewers.

Many thanks to Waltraud Dalhus, Willi Gosda, Anne-Katrin Heine, Heidi Kerkhoff, Heike Ohm and Gerlind Wilde for their help in laboratory work.

I am grateful to Dr. Thilo Rennert (Bochum) for critically reading and improving the text.

Thanks also to the members of the Isotopic Laboratory of the University of Bochum, especially to Ute Heinrichs and Dr. Manfred Roth for their friendly assistance in working with 14C.

I thank Dr. Wulf Amelung (Berlin) and Sonja Brodowski (Bayreuth) for their good cooperation and Dr. Ulrike Schulte (Bochum) and Dr. Gerd Gleixner (Jena) for their kindly support in 13C isotope techniques.

This work was financially supported by the German Research Foundation (DFG) and was part of the priority program 1090 "Soils as source and sink of CO2 - mechanisms and regulation of organic matter stabilisation in soils".

Menden, April 2004 Ute Hamer Curriculum vitae 175

Curriculum vitae

Zur Person geboren am 01.05.1976 in Wickede (Ruhr) ledig

Schulbildung

1982-1986 Kath. Grundschule St. Michael Menden-Schwitten

1986-1995 Walburgisgymnasium Menden, Abschluss: Abitur

Hochschulstudium

1995-2000 Studium der Geographie an der Ruhr-Universität Bochum mit den Nebenfächern Geologie und Wasserwirt- schaft/Umwelttechnik und den Vertiefungsrichtungen Bo- denkunde/Bodenökologie und Landschaftsökologie

Abschluss: Diplom-Geographin

Thema der Diplomarbeit: Einfluss organischer Dünger auf Cu- und Zn-Bindungsformen und DOC-Eigenschaften in Re- kultivierungsböden aus Löß seit 15.09.00 Wissenschaftliche Mitarbeiterin in der Arbeitsgemeinschaft Bodenkunde und Bodenökologie des Geographischen Insti- tuts der Ruhr-Universität Bochum im Rahmen des DFG- Schwerpunktprogramms 1090 „Böden als Quelle und Senke

für CO2"

Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig verfasst habe und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe. Diese Ar- beit oder Teile davon wurden bei keiner anderen Fakultät oder Hochschule einge- reicht.

Menden, den 22.04.04 Ute Hamer