Microbial utilization and mineralization of [¹⁴C]glucose added in six orders of concentration to soil

Abstract

The substrate availability for microbial biomass (MB) in soil is crucial for microbial biomass activity. Due to the fast microbial decomposition and the permanent production of easily available substrates in the rooted top soil mainly by plants during photosynthesis, easily available substrates make a very important contribution to many soil processes including soil organic matter turnover, microbial growth and maintenance, aggregate stabilization, CO₂ efflux, etc. Naturally occurring concentrations of easily available substances are low, ranging from 0.1 μM in soils free of roots and plant residues to 80 mM in root cells. We investigated the effect of adding ¹⁴C-labelled glucose at concentrations spanning the 6 orders of magnitude naturally occurring concentrations on glucose uptake and mineralization by microbial biomass. A positive correlation between the amount of added glucose and its portion mineralized to CO₂ was observed: After 22 days, from 26% to 44% of the added 0.0009 to 257 μg glucose C g⁻¹ soil was mineralized. The dependence of glucose mineralization on its amount can be described with two functions. Up to 2.6 μg glucose C g⁻¹ soil (corresponds to 0.78% of initial microbial biomass C), glucose mineralization increased with the slope of 1.8% more mineralized glucose C per 1 μg C added, accompanied by an increasing incorporation of glucose C into MB. An increased spatial contact between micro-organisms and glucose molecules with increasing concentration may be responsible for this fast increase in mineralization rates (at glucose additions <2.6 μg C g⁻¹). At glucose additions higher than 2.6 μg C g⁻¹ soil, however, the increase of the glucose mineralization per 1 μg added glucose was much smaller as at additions below 2.6 μg C g⁻¹ soil and was accompanied by decreasing portions of glucose ¹⁴C incorporated into microbial biomass. This supports the hypothesis of decreasing efficiency of glucose utilization by MB in response to increased substrate availability in the range 2.6–257 μg C g⁻¹ (=0.78–78% of microbial biomass C). At low glucose amounts, it was mainly stored in a chloroform-labile microbial pool, but not readily mineralized to CO₂. The addition of 257 μg glucose C g⁻¹ soil (0.78 μg C glucose μg⁻¹ C micro-organisms) caused a lag phase in mineralization of 19 h, indicating that glucose mineralization was not limited by the substrate availability but by the amount of MB which is typical for 2nd order kinetics.

Introduction

Microbial biomass (MB) in soil is usually limited by available carbon (C) (Schimel and Weintraub, 2003), even though total soil organic carbon (SOC) content is high. This C limitation is caused by low availability of much of the SOC (e.g. Morita, 1988). On the other hand, easily available substrates like sugars, amino acids or organic acids with fast turnover (Jones, 1998) are continuously added to soil by root exudation and plant residue decomposition (Mary et al., 1993, Kuzyakov, 2002), by addition of plant residues (De Nobili et al., 2001) or by organic fertilizers (Leifeld et al., 2002, Bol et al., 2003). Several authors studied the turnover of these substances in soil in respect to their assimilation by micro-organisms, their incorporation into microbial biomass and their mineralization to CO₂ (Wu et al., 1993, Nguyen and Guckert, 2001). Chemically defined low molecular substances like sugar monomers or amino acids as model substances for readily available substrates are easier to apply than complex natural materials (Van Veen et al., 1985, Nguyen and Henry, 2002, Hamer and Marschner, 2005). They are commonly used to estimate the parameters that influence the substrate behaviour in soils (Falchini et al., 2003). More complex materials such as root mucilage (Mary et al., 1992, Mary et al., 1993) and plant residues (Cochran et al., 1988, Chotte et al., 1998) have nevertheless been used in some of these studies.

Various soil properties affecting substrate turnover such as texture (Gregorich et al., 1991, Ladd and Amato, 1995) or CEC (Amato and Ladd, 1992) were identified in former studies. Many investigations focused on the chemical composition of the added substrates (e.g. Dalenberg and Jager, 1989, Falchini et al., 2003, Hamer and Marschner, 2005), the manner of its application (Sorensen et al., 1996) or the frequency of substrate addition or the time period between several substrate additions (Bremer and Kuikman, 1994). Most previous experiments added much higher amounts of substrates (60–700 mM) than the concentrations usually present in natural soils. Therefore, the results on assimilation by microbial biomass, decomposition to CO₂, as well as sorption by clay and organic particles may be not representative for behaviour of these substances in natural soils (Bremer and Kuikman, 1994, Van Hees et al., 2005). In a root-free soil, naturally occurring concentrations of easily available substances are less than 50 μM (van Hees et al., 2005). According to Jones (1998), 0.5–10 μM of the easily available substances in a root-free soil consist of organic acids, and 0.32–4.72 μM are amino acids (Monreal and McGill, 1985). Fischer et al. (2007) found soil solution concentrations of 8.2 μmol l⁻¹ for total amino acids and of 2.4 μmol l⁻¹ for total carbohydrates, dominated by glucose with 0.7 μmol l⁻¹. Concentrations of low molecular substances can increase up to 100 μM in a rooted soil (van Hees et al., 2005). In the rhizosphere, concentrations of 500 μM may be attained (van Hees et al., 2005). Of this 0.4–400 nmol g⁻¹ dry soil, up to 80 nmol g⁻¹ dry soil is composed of organic acids. The highest values were found in root cells and ranged from 10 to 20 mM for organic acids (Jones, 1998) and from 10 to 90 mM for mono- and disaccharide (Jones and Darrah, 1996, Ryan et al., 2001).

Due to fast microbial decomposition, such substrates have short mean residence time in soils ranging from hours for carboxylic acids (van Hees et al., 2005) and amino acids (Jones et al., 2005) to 31–162 days for glucose (Saggar et al., 1999). This yields a substantial contribution of easily available substances to soil respiration, which reflects the continuous input and fast decomposition of these organic substances (van Hees et al, 2005). Therefore, knowledge about the pathway of easily available substances through microbial biomass and its mineralization to CO₂ over the whole range of concentrations in soil is crucial. Easy available substances are essential as C and energy source for microbial growth (Mary et al., 1992). For example, the activation of micro-organisms from no-growth or starvation state to active state (Morita, 1988) by easily available substrates can be the main reason for accelerated SOM mineralization (Dalenberg and Jager, 1989, Kuzyakov et al., 2000, Hamer and Marschner, 2005).

Several authors showed the effect of the amount of added substrate on its microbial mineralization to CO₂ (Bremer and van Kessel, 1990, Mary et al., 1993, Bremer and Kuikman, 1994) and found positive correlations (Wu et al., 1993, Bremer and Kuikman, 1994, Marstop and Witter, 1999). However, if high amounts of easily available substrates were added, less C was incorporated into microbial biomass (Bremer and Kuikman, 1994, Marstop and Witter, 1999, Nguyen and Henry, 2002). One possible explanation for this effect is the impact on the energy state of the MB (e.g. Gottschal, 1992, Nguyen and Guckert, 2001). At low concentrations, easily available substrates are assimilated by MB and stored in a chloroform-labile pool (Hill et al., 2008), but the energy and C provided are insufficient for microbial growth (Bremer and Kuikman, 1994).

So far, studies on glucose mineralization used either only low or very high ranges of glucose concentrations to compare with naturally occurring concentrations of easily available substrates. Bremer and Kuikman (1994), for example, used glucose concentrations between 8.3 mM and 533 mM, Nguyen and Guckert (2001) concentrations between 8 μM and 13 mM. Hill et al. (2008) used glucose concentrations from 1 μM to 10 mM to reflect natural C concentrations in soil solution of root-free soil and the rhizosphere, but did not use glucose concentrations above than 10 mM although they pointed out that such concentrations can occur in soil by decomposition of plant and animal residues. Therefore, we investigated the dynamics of glucose mineralization and of its utilization by microbial biomass in relation to additions over the whole range of natural occurring substrate concentrations in the same experiment to avoid experimentally caused influences on glucose mineralization. A higher incubation temperature, for example, is known to result in an increased mineralization of the added substrate (Leifeld et al., 2002, Dioumaeva et al., 2003).

In the literature, long time intervals between samplings are often combined with long incubation periods (e.g. Bremer and Kuikman, 1994, 35 days with just three sampling times) or the incubation period is very short (Nguyen and Guckert, 2001, Hill et al., 2008). In our study, a high resolution at the beginning of the incubation when most of the glucose is mineralized was combined with a long-term monitoring of mineralization by longer time intervals. Accordingly, glucose concentrations from 0.29 μM (0.0009 μg glucose C g⁻¹ soil) to 80 mM (257 μg glucose C g⁻¹ soil) were added to a silt-loamy soil and traced during 22 days in increasing time intervals from 0.2 to 3 days.

We hypothesized (1) that substrate amounts ranging over 6 orders of magnitude would produce a significant, but varying effects on the assimilation and mineralization by microbial biomass and (2) that the effect of increasing glucose concentrations on its mineralization is a function of various mechanisms at different levels of glucose concentrations.