Discussion

My experiments demonstrated that the common soil pre-treatments of sieving and drying can alter the outcomes of lab incubations to investigate the effects of substrate addition to soils. The substantial changes in basal respiration in response to common homogenisation techniques deserve particular attention, because basal soil respiration is a common functional measure, which is used to assess differences in ecosystem function and soil microbial activity e.g. across different sites and after land-use changes (Creamer et al., 2014; Gülser & Erdoǧan, 2008; Moyano, Kutsch, & Schulze, 2007).

In experiment I, the most striking differences in basal soil respiration between Fintact and Dsieved occurred within the first six months of incubation (Figure

4.1). The sharp peak in basal soil respiration from Dsieved cores following rewetting

and disturbance (sieving and drying) indicates that a large proportion of the soil organic carbon was available for immediate microbial use (Fraser et al., 2016). By

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contrast, the steady increase in basal soil respiration from the Fintact cores during

the first three months, and the subsequent decline, likely reflected the gradual use and depletion of available C pools. These differences in basal soil respiration among treatments during the first six months of my experiment are particularly relevant as many lab studies of soil processes are short-term and are rarely longer than this six-month period. Although basal respiration from Dsieved+roots was

consistently slightly higher than Dsieved microcosms (Figure 4.1), soil treatment

had a much greater effect on basal respiration than decomposing roots.

In experiment II, the higher basal soil respiration in the Dsieved microcosms

compared to Fintact microcosms (Figure 4.4) indicates substantial changes in soil C

dynamics, which persisted throughout the 30-day incubation period. Hence, sieving and drying soils is also likely to influence the response of soils to experimental treatments. The pattern of soil respiration after litter addition was similar for all soil treatments, but the magnitude of the response was higher in disturbed soils compared to Fintact cores. Sieving and litter addition had a

comparably strong influence on soil respiration, which has potential consequences for the interpretation of experimental results. Importantly, sieving amplified the effect of litter addition, which has implications for determining the relevance of experimental results, especially when comparing treatment effect sizes among different soils, ecosystems, and studies (see also Chapter 3). Furthermore, as microcosms without substrate addition treatments are used as experimental controls in incubation experiments, these differences in basal respiration after soil pre-treatments may further confound results as you must assume that a change in the control will also equally affect the response to treatments, especially in

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comparative studies between different soils that are likely to vary in their response to disturbance (see Chapter 3).

The influence of soil pre-treatment on the response of the soils to subsequent experimental treatments may also be soil-specific, because soils vary in their resistance and resilience to disturbance according to the conditions experienced

in situ. For example, drying is less likely to affect the response of soils from arid climates, because they naturally experience large fluctuations in soil water content and are likely to have drought tolerant microbial communities (Zornoza et al., 2006, 2007). By contrast, temperate forest soils such as those used in my experiments may be more affected by sieving and drying because the microbial community is fungal-dominated (Bardgett et al., 2005; Fierer et al., 2009; Grayston et al., 2004; Joergensen & Wichern, 2008) making it less adapted to drought and more susceptible to damage from sieving, such as the destruction of fungal hyphae.

The increase in ion exchange rates in disturbed microcosms compared to Fintact microcosms may also help explain the difference in soil respiration between

soil pre-treatments, with the release of available nutrients after sieving and drying likely driving these changes (Kristensen et al., 2000; Petersen & Klug, 1994; Thomson et al., 2010). Nitrogen in particular is bound to organic compounds (Bingham & Cotrufo, 2016) that are likely to have been directly affected by the physical disturbance of sieving. In addition, the effects of drying and rewetting are likely to have resulted in the release of nitrogen from the cytoplasmic contents from lysed microbial biomass (Birch, 1958; Fierer & Schimel, 2002, 2003). The increased availability of N in disturbed microcosms (Figure 4.2) would result in higher soil respiration both before and after litter additions by alleviating nutrient

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limitation, increasing microbial activity and rates of C cycling (Allison & Vitousek, 2005).

The increased availability of total nitrogen in disturbed soils was largely due to greater availability of NO3- (Figure 4.2). This is likely a reflection of the

increasing de-stabilization of soil organic compounds by sieving and dryings, releasing soil organic nitrogen that was physically protected within soil aggregates, or chemically bound to minerals (Lopez-Sangil & Rovira, 2013), making it available for microbial mineralization. On the other hand, there was an increase in NH4+ in the microcosms with dried soils but no difference between

Fintact and Fsieved. This may be due to the rapid mineralisation of NH4+ into NO3- by

nitrifying bacteria (Davidson, Hart, & Firestone, 1992; Mobarry, Wagner, Urbain, Rittmann, & Stahl, 1996) after soil sampling, storage and the application of soil pre-treatments. The higher availability of ammonium in dried compared to sieved soils could be explained by the sensitivity and lack of resilience of nitrifying bacteria to drought. Nitrifying bacteria such as Nitrosomonas and Nitrobacter are gram-negative (Mobarry et al., 1996), which tent to be more susceptible to drought stress than gram-positive bacteria (Schimel, Balser, & Wallenstein, 2007). This could potentially lead to a (marginal) accumulation of NH4+ nitrogen in the soil

matrix, which is left un-nitrified as a result of the higher susceptibility of nitrifying bacteria to drought conditions.

The availability of Ca2+_{, Fe and Al also increased after sieving and drying}

compared to Fintact microcosms (Figure 4.2). These ions are important for soil

structure and the formation of soil aggregates and the chemical and physical protection of nutrients (Bronick & Lal, 2005). The increase in the exchange rates of these ions therefore indicates that the soil pre-treatments have affected soil

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aggregate complexes, which could increase the availability of soil nutrients to the microbial community.