Interaction of C cycle with other cycles
Six major elements-H, C, N, O, S and P- constitute the major building blocks for all biological macromolecules (Schlesinger WH, 1997). As a result of constitutional needs of organisms for these elements to build their tissues, the elemental cycles of C, N and other elements are interlinked at the molecular level (Sterner RW & Elser JJ 2002). For example the anthropogenic additions of N in atmosphere are deposited back on ground in the forms that are readily available to plants thereby stimulating productivity and enhancing the uptake of CO2 from the atmosphere. In contrast, the increase in plant biomass production under elevated CO2 strongly depends on the availability of mineral N i.e. more the N is available; higher would be the production of plant biomass (Figure 1.5). Moreover, the stimulation of plant growth by elevated CO2 should lead to sequestration of soil available nutrients and hence to progressive N limitation. The progressive N limitation hypothesis suggests that the increased plant biomass and plant fixation of CO2 under elevated CO2 subsides in the long term.
Similarly in soils, one of the reasons of difficulty to understand and theorize the dynamics of biogeochemical cycles is the inter-dependence of these cycles through soil organisms. The presence of P and N in important quantities in DNA, RNA, ATP and proteins underlines its key role in soil micro-organisms (Fagerbakke et al., 1996). Any increase in microbial biomass in soil due to additional availability of C for example under living roots, will strongly be dependent on the availability of P. Soil organic matter, a major component of soil C (Batjes 1996), is another point of interlink for various biogeochemical cycles due to its chemistry. Soil organic matter (including fresh plant-derived C and soil humified recalcitrant C) is a major source of energy as well as various nutrients for soil microbes for cellular activity and growth. The understanding of dynamics of C cycle in soil without the knowledge about other biogeochemical cycles, like N and P, is hence difficult. Figure 1.5. Theoretical demonstration of CO2-N interactions. Biomass increase with a 50% increase in CO2 concentration is greater at higher than lower N supply rates. Moreover elevated CO2 can diminish N availability (Progressive N limitation), further stopping biomass increase at elevated CO2 (taken from Reich et al., 2006)
The importance of availability of mineral N for storing C in soils has recently been observed at ecosystem level. It seems that, when mineral N availability is weak, the soil does not store excess C coming from increased plant production under elevated CO2. Moreover, turnover of soil C stocks seems to be increased in this condition (Hungate et al., 2009). In contrast, when mineral N is relatively high in availability, the total stock of C appears to go up due to reduced turnover of soil C stocks (van Groenigen et al., 2006; Hungate et al., 2009).
The fact that the amount of organic C stored in the soil does not necessarily increase, and can even decrease, after an input of fresh organic matter had been demonstrated in lab incubations (Fontaine et al, 2004a and b). These incubation experiments show that certain microbial species (Fontaine et al., 2003; Blagodatskaya et al., 2007) are able to degrade recalcitrant soil organic matter by using energy-rich substrates present in the fresh organic matter, a co-metabolism named priming effect in soil science (Blagodatskaya and Kuzyakov 2008). Although the mechanisms at play are not understood, the availability of nutrients for soil microbes controls the intensity of the priming effect. When the availability of nutrient is high, the priming effect is relatively low and the supply of fresh organic matter increase the reserve of C in soil. When the availability of nutrients is low, the priming effect may be so high that the reserve of C in soil decrease with the supply of fresh organic matter. Therefore, priming effect provides another good example of how C cycle in soil is interlinked with cycles of other nutrients.
State of the art
The acceleration in mineralization of recalcitrant SOM after the supply of fresh (labile) C i.e. priming effect is the cornerstone of this thesis. Thus it would be pertinent to understand in detail what this phenomenon is, its historical background, its precise definition in the context of this study, its importance for SOM mineralization and the methods to measure.
Introduction to priming effect
The extra release of soil-derived C as CO2 or soil nitrogen as NH4+ or as NO3- from soils amended with substrates in comparison to non-amended soils was termed as ‘priming effect’ (PE). Löhnis (1926) first suggested the PE by studying the N input-output balance of fields amended with green manure of legumes. He showed that the N balance could only be explained by considering a significant acceleration of native soil organic matter mineralization induced by the green manure. No progress was made on the subject until 1946 when isotopic techniques source partitioning of CO2 evolution from soil and Broadbent and Norman (1946) showed that the CO2 evolution from the soil can increase from 4 to 11 fold after addition of 13C labelled plant residues. The term “priming effect” was however introduced by Bingemann et al. (1953). Since its introduction by Bingemann et al. in 1953, the term priming effect has also been used in other context and for describing other processes (Jenkinson et al., 1985; Dalenberg and Jager 1989). The focus of this thesis is SOM mineralization we refer to the original definition of priming effect given by by Bingeman et al. (1953), “the extra decomposition of native soil organic matter in a soil receiving an organic matter amendment” in this manuscript. Since priming effect in this work was studied in the presence of living roots (i.e. rhizosphere), it will be termed as rhizosphere priming effect (RPE).
What is the rationale behind this priming effect induced by soil microorganisms? According to energy limitation theory (Fontaine et al., 2005; 2007), despite the presence of large soil organic carbon stocks in most soils, the soil microbes are unable to use it for assimilation and growth because the direct and indirect (cell maintenance) cost on synthesizing extracellular enzymes that mineralize SOM exceeds the return the microbes could get in terms of energy and nutrients. However, some microbial species mineralize recalcitrant SOM in co-metabolism using the fresh organic matter as a source of energy (Fontaine et al., 2003; Blagodatskaya et al., 2007). This degradation of recalcitrant SOM would permit microorganisms to access to the large reserve of nutrients that were held up SOM.
Measuring priming effect
The isotopic labelling, which permit separating soil C and fresh C mineralization, is the sole current reliable technique to quantify the priming effect.. Other approaches have been proposed to estimate the priming effect when the labelling approach is not possible but the results are highly disputable (Kuzyakov 2010).
In lab incubation studies, C labelled simple components of root exudates like glucose, fructose, alanine etc. (Hamer and Marschner 2005) or the extraction of labelled plant material like cellulose (Fontaine et al., 2007) or labelled plant litter (Conde et al., 2005) is added to unlabelled soil. The CO2 efflux from such soils as well as controls are measured and separated into soil-derived (unlabelled) and added-substrate derived (labelled) by isotopic mass-balance equations. The difference of soil-derived CO2 between treated (substrate-added) and control soil is the amount of priming effect. This is very excellent method, easy to control the conditions but it excludes the living roots thus neglecting the rhizospheric processes like rhizodeposition, root absorption of N and root-induced breaking of aggregates.
The measurement of priming in living soil-plant systems is measured by continuous or pulse labelling of plants by exposing them to an atmosphere with constant ratios of 14C- or 13C- CO2 to total CO2 over a certain period (Kuzyakov et al., 2001; Dijkstra et al., 2007). The root-derived (labelled) and soil-derived (unlabelled) CO2-C is separated from total soil (soil plus roots) CO2 efflux using isotopic mass balance equations. The difference of soil-derived CO2 between treated (planted) and control soil is the amount of priming effect.
Another method to separate soil-derived and root-derived CO2 efflux is by using the natural 13C abundance of soils and plants. The 13C natural abundance method is based on the differential discrimination of the heavier 13C isotope during CO2 assimilation by plants with different types of photosynthesis i.e. C3 or C4 plants. The soils developed under C3 or C4 vegetation contain SOM with 13C of -27 or 13 ‰ respectively (Cheng 1996). The natural abundance method is based on cultivation of C4 on a C3 or vice versa and estimation of the contribution of root-derived CO2 according the 13C value in the CO2 evolved.
The current method for measuring soil respiration and hence the priming effect induced by plants can only be used for single-stem plants like tree seedlings or annual plants with strong single stems like sunflower. Indeed, the soil-part must be separated from above ground plant part in order to avoid the assimilation of released soil CO2 by the plant. To this end, a paraffin wax is applied around stem separating soil and plant compartments (Figure 1.6). The CO2 efflux from soil (soil plus root) is then trapped in an alkali or directly measured by Gas Chromatography Mass Spectrometry (GCMS). This method can not be used for multi-tillered plants like herbaceous species signifying that the priming effect induced by plant grasses is not known.
Figure 1.6. The method used to trap soil CO2 efflux in alkali excluding the trapping of CO2 from aboveground plant parts. (Cheng 1996).
Why priming effect is important
Plant roots are an important of organic matter-labile C in the form of exudation, sloughed off roots cells, mucilage collectively called as ‘rhizodeposition’ and dead root litter-in soil.
Living roots, being a source of labile C, have been found to induce rhizosphere priming effects. The importance of this effect can be gauged from the fact that the increase in SOM mineralization in planted soils can reach to three-fold of SOM mineralization in bare soils under similar temperature and moisture conditions (Zhu and Cheng 2011). RPE have also been shown to induce net C loss from a soil (Fontaine et al., 2004; Dijkstra et al., 2007). Moreover, various biotic and abiotic factors that have been shown to modulate the rate of RPE suggest the importance of studying this phenomenon to devise future strategies for favouring net positive sequestration under living plants and not the net positive SOM mineralization.
Among biotic factors, plant biomass (Dijkstra et al., 2006), photosynthesis (Kuzyakov and Cheng 2001), plant phenology (Fu and Cheng 2002) have been related with rate of RPE. Plant biomass and photosynthesis have been linked with RPE suggesting that the increased rate of rhizodeposition under increased plant biomass or photosynthesis increases the rate of RPE (Kuzyakov and Cheng 2001; Dijkstra et al., 2006). For annual plants, it has been shown that the RPE is higher during vegetative growth stages of plant in comparison to reproductive (or later) stages presumably due to high amount of exudation from young roots during early age of plant (Fu and Cheng 2002).
Abiotic factors like soil nutrient status, especially the availability of mineral N has also been found to influence the magnitude of RPE. Under high availability of mineral N, lesser amount of priming effect was observed and vice versa (Fontaine et al., 2004a). High availability of N reduces the N limitation and the competition for N between microbial biomass and plant roots. Under high soil fertility the microbes are more adapted to assimilate root exudates as they are less inclined to mineralize high-cost recalcitrant SOM for nutrients when the later are available almost free of cost. The atmospheric CO2 levels, soil and air temperature and moisture are expected to change in future climates (IPCC 2007) and all of them have been found to modulate the magnitude of RPE. For example, in low fertility soils the sustained growth responses of forest to elevated CO2 are maintained by enhanced rates of N cycling fuelled by inputs of root-derived exudates and enhanced RPE (Phillips et al., 2011). The increased temperature has been found to increase RPE suggesting the increased temperature sensitivity of SOM mineralization due to RPE (Zhu and Cheng 2011). Higher soil moisture contents (not anaerobic conditions) also facilitate the RPE presumably by increased root exudation (Dijkstra and Cheng 2007).
In summary, the future climatic changes that can influence RPE directly or indirectly by changing soil nutrient status, soil moisture, biomass production etc. underscore the importance of rhizosphere priming effect and the need to study it.
The importance of rhizosphere priming effect (RPE) vis-à-vis C and N cycles in grasslands is not clear owing perhaps to the absence of a method to directly measure RPE induced by grasses. After detailed literature review, six knowledge gaps were identified to work on for this thesis:
What is the effect of grazing on RPE? Grazing is central to the management of grasslands and has been shown to accelerate the decomposition of plant litter present in soil (Klumpp et al., 2009). However, the effect of grazing on mineralization of SOM is unclear. The increased availability of soluble C, higher net N mineralization and increased microbial biomass in soil after clipping the plant leaves were considered an evidence to suggest that the plant clipping could accelerate SOM mineralization i.e. increased RPE thereby releasing mineral N from recalcitrant SOM for plant uptake (Hamilton and Frank 2001). If this hypothesis is true, then clipping (a simulation of mowing or grazing minus animal excreta) could decrease soil C stocks on two fronts i.e. first, due to reduced overall C input into soil due to reduced aboveground production and rhizodeposits and second, by stimulating the mineralization of already existing soil C (SOM). However, the studies where soil CO2 efflux was measured after plant clipping do not support the hypothesis of increased RPE after plant clipping. In contrast, these studies have shown that the plant clipping can reduce soil CO2 efflux by 20-50 % (Craine et al., 1999; Bahn et al., 2006). Thus, the impact of grazing on recalcitrant soil organic matter must be studied though a direct approach.
Are plants with deep roots able to reactivate deep ancient C and N cycle trough a priming effect? The SOM mineralization has been found to accelerate in the presence of living roots of annual plants and tree seedlings (Cheng et al., 2003; Dijkstra et al., 2007) and in some cases to result in net soil C loss (Dijkstra et al., 2007). Most of the studies aiming to find out the effect of living roots on soil C stocks and soil-derived CO2 efflux are limited to upper 20 or 30 cm of soil. However, of the 1600 Gt of organic C held in the top meter of world soils, about half is in the 25-100 cm layer (Jobbàgy and Jackson 2000). Moreover, some recent studies focussing on C sequestration in soils have suggested that deep soils may not be sequestering C or even losing some of it (Carter 2005). The C loss from deep soils suggests that the labile C deposition may be accelerating the mineralization of deep soil C. A recent study in lab conditions (Fontaine et al., 2007) have actually shown that the labile C addition to soil sampled from deep soil (80 cm depth) could accelerate SOM mineralization and destabilize ~ 2500 years old C. The SOM dynamics in deep soils in the presence of living roots is an important question to study to predict its feedbacks as well as viability of deep soil for C sequestration in future climates in which temperatures have been predicted to rise.
Table of contents :
CHAPTER 1 GENERAL INTRODUCTION
I. CONTEXT OF STUDY
I.1. GLOBAL CARBON CYCLE
I.2. SOIL ORGANIC CARBON
I.3. INTERACTION OF C CYCLE WITH OTHER CYCLES
II. STATE OF THE ART
II.1. INTRODUCTION TO PRIMING EFFECT
II.2. MEASURING PRIMING EFFECT
II.3. WHY PRIMING EFFECT IS IMPORTANT
II.4. KNOWLEDGE GAPS
III. OBJECTIVES OF THESIS
III.1. HYPOTHESES OF THESIS
III.2. APPROACHES OF THE STUDY
IV. THESIS LAYOUT
CHAPTER 2 PLANT CLIPPING DECELERATES THE MINERALIZATION OF RECALCITRANT SOIL ORGANIC MATTER UNDER MULTIPLE GRASSLAND SPECIES
III. MATERIALS & METHODS:
III.1. SOIL SAMPLING AND PLANT SOWING
III.2. LABELLING SYSTEM & MESOCOSM
III.3. RESPIRATION MEASUREMENT
III.4. SOIL AND PLANT ANALYSES
III.5. PLFA MEASUREMENTS
III.6. STATISTICAL ANALYSES
IV.1. PLANT BIOMASS AND ISOTOPIC COMPOSITION
IV.2. RESPIRATION FLUXES
IV.3. SOLUBLE C & MINERAL NITROGEN IN SOIL
IV.4. MICROBIAL BIOMASS & COMMUNITY COMPOSITION
CHAPTER 3 LIVING ROOTS INDUCE THE MINERALIZATION OF 15, 000 YEARS OLD ORGANIC C FROM DEEP SOIL OF A TEMPERATE GRASSLAND
III. MATERIALS & METHODS
III.1. SOIL SAMPLING AND PLANT SOWING
III.2. RESPIRATION MEASURES
III.3. SOIL CARBON FRACTIONS
III.4. SOIL NITROGEN
III.5. PLFA MEASUREMENTS
III.6. STATISTICAL ANALYSES
IV.1. SOM MINERALIZATION & RHIZOSPHERE PRIMING EFFECT
IV.2. MINERAL NITROGEN IN SOIL
IV.3. ROOT LITTER AND SOIL ORGANIC C
IV.4. MICROBIAL BIOMASS & COMMUNITY COMPOSITION
CHAPTER 4 TEMPERATURE RESPONSE OF ORGANIC C MINERALIZATION: A NEW EXPERIMENTALLY TESTED THEORY BASED ON ENZYME INACTIVATION AND MICROBIAL ENERGY LIMITATION RECONCILES THE RESULTS OF LAB AND ECOSYSTEM EXPERIMENTS
IV. EXPERIMENTAL TEST
V. MATERIALS AND METHODS
V.1. SOIL SAMPLING AND EXPERIMENTAL DESIGN
V.2. LABELLING SYSTEM & MESOCOSM
V.3. RESPIRATION MEASUREMENT
V.4. STATISTICAL ANALYSIS
VI.1. EFFECT OF PLANT ON SOIL RESPIRATION
VI.2. EFFECT OF TEMPERATURE ON FRESH C RESPIRATION
VI.3. TESTING THE THEORY: EFFECT OF TEMPERATURE ON RECALCITRANT C MINERALIZATION IN PLANTED AND BARE SOILS
CHAPTER 5 ARTICLES IN PREPARATION
PART 1: THE EFFECT OF SIX GRAMINEAE AND ONE LEGUMINOUS SPECIES ON SOIL ORGANIC MATTER DYNAMICS IN TEMPERATE GRASSLAND
PART 2: DOES HIGHER AVAILABILITY OF N DECREASE SOIL ORGANIC C MINERALIZATION AND STORE MORE ORGANIC C?
PART 3: DECOUPLING THE EFFECT OF ROOT EXUDATES, MYCORRHIZAE AND ROOTS ON SOIL C PROCESSES IN THREE TEMPERATE GRASSLAND SPECIES
CHAPTER 6 ROLE OF PLANT RHIZOSPHERE ACROSS MULTIPLE SPECIES, GRASSLAND MANAGEMENT AND TEMPERATURE ON MICROBIAL COMMUNITIES AND LONG-TERM SOIL ORGANIC MATTER DYNAMICS
I. GENERAL DISCUSSION
ANNEX OF THE THESIS
ACELLULAR RESPIRATION: RECONSTITUTION OF AN OXIDATIVE
METABOLISM BY ENZYMES RELEASED FROM DEAD CELLS