Decoupling of microbial glucose uptake and mineralization in soil

ARTICLE IN PRESS

Soil Biology & Biochemistry 40 (2008) 616–624 www.elsevier.com/locate/soilbio

Decoupling of microbial glucose uptake and mineralization in soil Paul W. Hillb, John F. Farrara, David L. Jonesb, a

School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK School of the Environment and Natural Resources, University of Wales, Bangor, Gwynedd LL57 2UW, UK

b

Received 22 February 2007; received in revised form 22 August 2007; accepted 18 September 2007 Available online 9 October 2007

Abstract The rate of organic matter turnover in soil is a critical component of the terrestrial carbon cycle and is frequently estimated from measurements of respiration. For estimates to be reliable requires that isotopically labelled substrate uptake into the soil microbial biomass and its subsequent mineralization occurs almost simultaneously (i.e. no time delay). Here we investigated this paradigm using glucose added to an agricultural soil. Immediately after collection from the field, various concentrations of 14C-labeled glucose (1 mM to 10 mM) were added to soil and the depletion from the soil solution measured at 1–60 min after substrate addition. 14CO2 production from the mineralization of glucose was simultaneously measured. The microbial uptake of glucose from soil solution was concentrationdependent and kinetic analysis suggests the operation of at least two distinct glucose transport systems of differing affinity. At glucose concentrations reflecting those naturally present in the soil solution (54710 mM), the half-time (t1/2) of exogenous glucose was extremely rapid at ca. 30 s. At higher glucose concentrations (100 mM to 10 mM), the t1/2 values for the high-affinity carrier were altered little, but increasing proportions of glucose were taken up by the low affinity transport system. Glucose mineralization by the soil microbial community showed a significant delay after its uptake into the microbial biomass suggesting a decoupling of glucose uptake and subsequent respiration, possibly by dilution of glucose in labile metabolite pools. By fitting a double first order kinetic equation to the mineralization results we estimated the t1/2 for the first rapid phase of respiration at natural soil solution glucose concentrations to be 6–8 min, but at least 87% of the added glucose was retained in the microbial biomass prior to mineralization. Our results suggest that in this soil the soil solution glucose pool turns over 100–1000 times each day, an order of magnitude faster than when determined from measurements of mineralization. These results imply that traditional isotopic based measurements of substrate turnover measured using CO2 may vastly underestimate their rate of cycling in soil. r 2007 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Carbon cycling; Carbon dioxide; Dissolved organic carbon; Heterotrophic respiration; Mineralization; Rhizosphere; Sugars

1. Introduction Knowledge of the factors that regulate carbon dynamics in soil is crucial for the design of sustainable agricultural systems and for predicting the impacts of climate change on ecosystem functioning. The evolution of CO2 from the soil surface has frequently been used to simultaneously measure all the mineralization processes occurring in the soil profile (Hanson et al., 2000; Kuzyakov, 2006). Based upon the known inputs of organic materials into soil (e.g. plant litter, animal wastes etc.) it is clear that total soil Corresponding author. Tel.: +44 1248 382579; fax: +44 1248 354997.

E-mail addresses: [email protected], [email protected] (D.L. Jones). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.09.008

respiration is an integrator of many thousand mineralization pathways operating simultaneously in the plant-soil system. Whilst measurements of total soil respiration have provided vital clues to understanding soil responses to environmental perturbation (Ho¨gberg et al., 2001), the exact processes operating in soil that give rise to this response remain poorly understood (Kuzyakov, 2002; van Hees et al., 2005). Based upon the chemical composition of plants, the dominant input of C into most soil ecosystems will be sugars, amino acids and organic acids or their polymers (Stevenson, 1985). Inputs of C into soil (e.g. rhizodeposition) are processed by fungi and bacteria (Leake et al., 2006). These organisms require that polymers such as proteins, hemicellulose, lignin and cellulose are firstly broken down extracellularly to smaller units prior to

ARTICLE IN PRESS P.W. Hill et al. / Soil Biology & Biochemistry 40 (2008) 616–624

being taken up into the cell (e.g. mono- and di-saccharides, peptides, amino acids etc.; Barraclough, 1997; Jennings, 1995). These low molecular weight (MW) breakdown products are generally water soluble and consequently most C cycling through soil passes at some point through the soil solution dissolved organic C (DOC) pool. After uptake from solution by the soil microbial community the substrate can be partitioned into both catabolic and anabolic pathways. The relative partitioning is dependent upon both the type and rate of substrate supply (Jones et al., 2004). From studies in a limited number of ecosystems it appears that the concentration of individual low MW substrates in soil are very low, typically ranging from 0.1 to 50 mM (van Hees et al., 2005; Ryan et al., 2001). Exceptions to this may occur in the rhizosphere in response to root exudation where concentrations up to 10 mM have been predicted to occur, particularly for organic acids when plants are experiencing P deficiency (Veneklaas et al., 2003). Higher concentrations may also be present in patches of soil containing decomposing plant and animal residues (Hodge, 2004). Rapid utilization of low MW substrates has also been reported with both plant roots and soil microbial populations competing for this labile C resource in soil (Bardgett et al., 2003; Kuzyakov and Jones, 2006). The measurements of substrate turnover in soil have typically relied on the addition of isotopically labeled substrates (e.g. 13C, 14C) to soil in the laboratory and measurement of the subsequent rates of 14CO2 evolution. Using this approach, half-time (t1/2) values for amino acids, sugars and organic acids have been found to typically range from 20 min to 4 h (Jones, 1999; van Hees et al., 2005; Boddy et al., 2007). While this approach gives an estimate of C residence time in soil, it may not accurately reflect longevity in the solution phase due to a delay between microbial uptake and mineralization. For some aspects of soil C cycling (e.g. in the rhizosphere) it is the longevity of the compound in the solution phase which is critical in determining its functional significance and not its residence time in the microbial community (Jones et al., 2003). A further complication may be in the use of non-uniformly isotopically labeled substrates which may lead to an underestimate of transformation rates in soil (e.g. if only one point in the compound is labeled; Lucas and Jones, 2006). Our aim was to investigate the suitability of using CO2 production as a proxy for C substrate removal in soil. This study was undertaken in a temperate agricultural soil using glucose as a model substrate. This substrate was chosen as it represents a major component of both root exudation and SOM decomposition. 2. Materials and methods

617

Table 1 Background soil characteristics Parameter Total soil C (g kg1) Total soil N (g kg1) Soil solution DOC (mmol C l1) Soil solution glucose (mM) Soil water (g g1 soil DW) Soil respiration (mmol CO2 kg1 DW min1) pH Electrical conductivity (mS cm1)

3072 3.070.2 5.070.4 54710 0.370.01 2.170.07 6.970.02 4474

Values represent means7SEM (n ¼ 3).

and the mean annual soil temperature at 10 cm depth is 11 1C. The soil is classified as a Eutric Cambisol, and was first sampled in late-April, 2006 (at 09:00 am). At this time, it supported a newly planted crop of flax (Linum usitatissimum L.; shoots 5–8 cm in height). In addition, the same field was repeat sampled in mid-April, 2007 (at 08:30 am) when it supported a crop of wheat (Triticum aestivum L.; shoot height 10–15 cm). On both occasions approximately 1 kg of soil was collected from each of three points located 2 m apart from the Ahp horizon (0–10 cm), placed in gas-permeable plastic bags and transferred immediately to the laboratory. These samples represented the three replicates for all experiments. The soil had a fine crumb structure and it passed freely through a 2 mm sieve. Sieving ensured homogenization and removed interference from earthworms, stones and roots. Soil bulk density and moisture content were assessed by oven drying soil at 80 1C. Dry, root-free soil was analysed for C and N content using a CHN 2000 analyser (Leco Corp., St Joseph, MI, USA). Soil pH and electrical conductivity were measured in a 1:1 (v/v) soil-distilled water extract. Soil respiration was measured with a PP-Systems SR1 soil respirometer (PP-Systems, Hitchin, UK) at 20 1C. Unless stated, all presented results are those from the 2006 sampling. 2.2. Extraction of soil solution Soil solution was extracted by adapting the centrifugaldrainage procedure described by Giesler and Lundstro¨m (1993). Briefly, 1.7 g of field-moist soil was placed in a 1.5 ml microcentrifuge tube in which a hole had been pierced at the bottom. This tube was then placed into another intact microcentrifuge tube and the pair centrifuged (4000g, 1 min, 20 1C). During centrifugation the soil solution passed into the lower tube. Soil solution samples were stored frozen at 20 1C prior to analysis. Small samples of soil were used so that the soil solution could be recovered extremely rapidly.

2.1. Field sites and sampling regime 2.3. Substrate depletion from the soil Soil was obtained from temperate oceanic agricultural land located in Abergwyngregyn, Gwynedd, North Wales (531140 N, 41010 W; Table 1). The annual rainfall is 1250 mm

For the substrate depletion experiments, 1.7 g of fieldmoist soil (bulk density 1 g cm3) was placed in a 1.5 ml

ARTICLE IN PRESS P.W. Hill et al. / Soil Biology & Biochemistry 40 (2008) 616–624

618

microcentrifuge tube as described above and 0.34 ml of a uniformly 14C-labeled glucose solution (0.5 kBq ml1; 1 mM to 10 mM; Amersham Biosciences UK Ltd., Chalfont St. Giles, UK) added to the surface. Infiltration of the glucose solution into the soil took o3 s. On the first occasion (2006), the soil solution was recovered from the labeled soil as described above at varying times after 14C-glucose addition (1, 5, 10, 20, 40 and 60 min). On the second occasion (2007) it was recovered only after 1 min. The amount of 14C-glucose remaining in the soil solution at each sampling event was determined by liquid scintillation counting using a Wallac 1404 scintillation counter (Wallac EG&G, Milton Keynes, UK) and Optiphase HiSafe 3 scintillation fluid (Wallac EG&G). All the glucose depletion studies were carried out in triplicate at 2071 1C within 6 h of collection of the soil from the field. To account for any non-biological changes in soil solution 14C glucose concentration (e.g. mixing and dilution in the intrinsic soil solution), the same experiments were also carried out on field-moist soil that had been autoclaved (121 1C, 20 min) and allowed to cool (10 min) prior to performing the 14Cglucose depletion studies.

collected 10 d apart from different areas of the field site. Both batches gave extremely similar results and therefore only results from the second set of experiments are presented here. All experiments were conducted in triplicate. Data were subjected to ANOVA with LSD post hoc test (SPSS version 12.0; SPSS Inc., Chicago, IL) with Po0.05 used as the upper limit for statistical significance. The 14C-glucose solution added to the soil will become diluted to some extent by mixing with unlabeled water already present in the soil. This dilution can easily be calculated by assuming complete mixing of the added 14Csolution (0.34 ml) with that present in the soil (0.3570.02 ml in 1.7 g of soil in 2006 samples). However, this mixing will not be instantaneous and therefore an alternative dynamic approach is needed to calculate the rate of glucose mixing in soil. Consequently, the rate of mixing of the added 14C-glucose with the native soil solution was evaluated by comparing the theoretical 14Cactivity of recovered soil solution with that of soil solution recovered from autoclaved soils (Fig. 1). To describe this, a first order single exponential decay was fitted to the experimental data, where R ¼ y0 þ a expðbtÞ,

2.4. Substrate mineralization

(1) 14

To determine the rate of CO2 evolution after the addition of the 14C-labeled glucose to the soil, 1.7 g of soil was placed in a 10 cm3 glass vessel and 0.34 ml of 14C labeled glucose added to the soil as described above. The vessel was then sealed and moist air (2071 1C) passed over the soil at a rate of 600 ml min1. The outflow from the vessel was bubbled through Oxosol scintillation fluid (National Diagnostics Ltd, Hessle, UK) to trap the 14 CO2 evolved. The trap was changed after 1, 5, 10, 20, 40 and 60 min and the 14C determined by liquid scintillation counting as described previously. 14CO2 capture efficiency (8074%; mean7SEM; n ¼ 6) was determined by addition of 3.9 kBq of NaH14CO3 solution (50 ml) to the glass vessel and subsequently adding 2 ml of 1 M HCl by injection through a suba seal. Measured 14C activities were corrected for NaOH capture efficiency by multiplying all the values by a factor of 1.25. The same experiments were also carried out on field-moist soil that had been autoclaved and allowed to cool as described above. 2.5. Chemical analysis Soil solution samples were analysed for DOC using a Shimadzu TOC-V-TN analyser (Shimadzu Corp., Kyoto, Japan). Glucose was determined fluorometrically with an Amplexs Red glucose assay kit (Invitrogen Corp., Carlsbad, CA). 2.6. Statistical and data analysis In 2006, the substrate depletion experiments described above were repeated with two independent batches of soil

where R is the amount of C recoverable after mixing, y0 is the asymptote to which the actual recovered 14C fell after mixing with the intrinsic soil water, t is the time after substrate addition and a and b are coefficients of the exponential decay. The resulting equation was used to calculate the maximum soil solution 14C activity at any given time after substrate addition. The mixing rate was in turn used in the determination of the uptake rates described below. The carrier systems operating during glucose uptake were investigated with an Eadie–Hofstee plot. Straight Percent deviation from perfect mixing of added 14C-glucose with soil solution

14

100

50

0

-50

-100 0

10

20 30 40 50 Time after substrate addition (min)

60

Fig. 1. Deviation from perfect mixing when a 14C-glucose solution (10 mM) is added to a heat-sterilized (autoclaved) soil containing non-14C labeled water. A 0.34 ml of a 14C-glucose solution was added to 1.7 g soil containing 0.35 ml of soil water. The dashed line at 0% represents the point at which perfect mixing has occurred throughout the soil. Values above the line indicate that proportionally more of the 14C-glucose is recovered by our centrifugal-drainage procedure (i.e. the two solutions have not mixed perfectly yet). Values represent means7SEM (n ¼ 3).

ARTICLE IN PRESS P.W. Hill et al. / Soil Biology & Biochemistry 40 (2008) 616–624

lines were fitted to portions of the data to estimate the Michaelis–Menten kinetic parameters Km and Vmax according to V ¼ V max  K m  V =S,

(2)

where V is the rate of glucose uptake, S is soil solution glucose concentration, Vmax is the maximum rate of glucose uptake, and Km is the Michaelis constant describing the glucose concentration at which half maximal uptake occurs. To determine the half-life (t1/2) of glucose in the soil solution, a single first order exponential decay was fitted to data for the lowest glucose concentrations (1 and 10 mM) according to g ¼ y01 þ a1 expðb1 tÞ,

(3)

14

where g is the C remaining in the soil solution, y01 is an asymptote, b1 is the exponential coefficient describing depletion by the soil microbial community, a1 describes the size of pools and t is time. The t1/2 of the soil solution glucose pool (a1) can then be defined as t1=2 ¼ lnð2Þ=b1 .

(4)

14

Depletion of C from the soil solution after the addition of higher concentrations of glucose was better described by a double first-order exponential decay equation: g ¼ ½a1 expðb1 tÞ þ ½a2 expðb2 tÞ.

(5)

Half-times for the soil solution glucose pools a1 and a2 were calculated according to Eq. (4). We assumed that the two exponentials parts of the equation represented uptake of glucose by two independent carrier systems (Jennings, 1995). Previous studies have shown that low MW substrate mineralization in soils over time is biphasic (Chotte et al., 1998; Saggar et al., 1999; Jones et al., 2005; van Hees et al., 2005; Boddy et al., 2007). The first rapid phase of 14CO2 production is attributable to the immediate use of the substrate in catabolic processes (i.e. respiration), the remaining substrate being taken up and immobilized in the microbial biomass (i.e. formation of new biomass or storage polymers). The slower second phase of 14CO2 production is then attributable to the subsequent turnover of the soil microbial community or storage polymers leading to the production of 14CO2. Consequently, the halflife of the glucose in soil can be calculated from the 14CO2 evolution results using the double first-order exponential decay equation (Paul and Clark, 1996): g ¼ ½a3 expðb3 tÞ þ ½a4 expðb4 tÞ,

(6)

where b3 is the exponential coefficient describing the primary mineralization phase, b4 is the exponential coefficient describing the secondary mineralization of the microbial biomass, and a3 and a4 describe the size of pools. The t1/2 of the soil solution glucose pool (a3) can therefore be defined as t1=2 ¼ lnð2Þ=b3 .

(7)

619

Exponential equations were fitted to the experimental results using a least squares iteration routine in Sigmaplot 14.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Isotopic pool dilution dynamics As glucose is not sorbed in this soil (Kuzyakov and Jones, 2006), we predicted that the added 14C-glucose would rapidly equilibrate throughout the entire soil. Initially, the added 14C-glucose solution will occupy the large soil macropores and this fraction of water will be preferentially recovered by our centrifugal-extraction procedure in comparison to the non-labeled water held in the remaining smaller pores. By monitoring the concentration of 14C-glucose in soil solution extracted from the autoclaved soils over time (where biotic removal is prevented) we were able to calculate the dynamics of isotopic pool dilution (Fig. 1). We found that a first-order exponential decay equation (Eq. (1)) fitted well to the change in 14C-glucose concentration in the sterile soil (r2 ¼ 0.98; Fig. 1). From this we calculated that the halftime required for the two solution pools to mix was 0.4970.01 min. Perfect mixing of the two solutions was achieved after approximately 15 min. Using Eq. (1) and the glucose results presented in Table 1, we calculated that the concentrations of glucose that would have been experienced by the soil microbial community at the start of the experiment (i.e. after isotopic pool dilution). For the added glucose concentrations of 1, 10, 100, 1000 and 10 000 mM we calculated that the resulting concentrations in soil at equilibrium were 22, 28, 82 and 621 mM, and 6 mM. However, based upon the glucose depletion rates from soil solution (see Section 3.2), it is clear that frequently these equilibrium concentrations were not actually achieved due to concomitant microbial removal. Consequently, for the determination of uptake kinetics, we have assumed that glucose was removed from the soil solution during the first minute by microbes experiencing the concentrations of the added solutions. 3.2. Glucose depletion from soil We chose glucose as a model substrate for this study as it probably represents the dominant C compound entering soil (in a monomeric or polymeric state) and therefore should be central to soil C cycling (Paul and Clark, 1996). Glucose removal rates from the soil solution were extremely rapid. The microbial community removed 9371 and 7877% (2006 and 2007, respectively; mean7 SEM; n ¼ 3) of the added glucose from the soil solution within 1 min when added at 1 mM (Fig. 2). This fell to 5571 and 1774% at the highest glucose concentration (10 mM). In absolute terms (i.e. nmol g1), however, the actual amount of glucose removed from solution increased with increasing glucose concentration (Table 2). At the

ARTICLE IN PRESS P.W. Hill et al. / Soil Biology & Biochemistry 40 (2008) 616–624

1200 1000 800 600 400 200 0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

V/S Fig. 2. Eadie–Hofstee plot of the rate of glucose uptake by soil microorganisms (V) against the ratio of the rate of glucose uptake to the concentration of added glucose solution (S) for soil sampled in 2007. Values represent means7SEM (n ¼ 3).

Table 2 Total glucose uptake and mineralization in the first minute after substrate addition, and the percent of glucose taken up which was respired in the first minute after substrate addition Rate of glucose uptake (mmol glucose C kg1 DW soil min1)

Rate of glucose mineralization (mmol glucose C kg1 DW soil min1)

Percentage of C taken up that is subsequently respired

2006 1 10 100 1000 10 000

0.970.005 7.970.07 6570.7 56673 53687112

0.0470.002 0.370.04 1.770.3 2371.6 12472.5

3.970.2 3.770.5 2.770.5 4.070.3 2.370.1

2007 1 10 100 1000 10 000

1.270.1 8.970.4 5077 275757 26377581

0.0370.003 0.370.02 2.870.3 1972.2 161726

2.670.5 3.770.4 6.071.3 7.771.5 6.370.5

Glucose concentration in solution (mM)

100–300 mM, whereas those for the low-affinity carrier were up to an order of magnitude greater. Corresponding estimates of Vmax were 20–40 nmol glucose g1 DW soil min1 for the high-affinity carrier and up to 14 mmol glucose g1 DW soil min1 for the low-affinity carrier. The temporal dynamics of glucose depletion from the soil solution were dependent upon the concentration of glucose supplied (Fig. 3). The depletion of 14C-glucose from the soil solution after addition of a 1 or 10 mM glucose solution was best described by a single exponential decay equation (r240.996 in all cases; Eq. (3), Fig. 3). The depletion of 14 C-glucose from the soil solution after the addition of a 1 or 10 mM glucose solution was better-fitted by a double exponential kinetic decay model (r240.996 in all cases; Eq. (5), Fig. 3). Both single and double exponential decay models fitted well to 100 mM data, but the double exponential decay model provided the best fit (r240.9997). The exponential coefficients and half-times (t1/2) describing the loss of glucose from the soil solution are presented in Table 3. The half-time for the glucose pool a1 was very short (o1 min) being significantly shorter at 1 mM than at either 10 or 100 mM (Pp0.007). They were not significantly different from those for 1 and 10 mM, but in this case a large proportion of the added substrate was allocated to the glucose pool a2, which possessed t1/2 values 40 or 300 times (1 and 10 mM, respectively) greater than those of pool a1. The half-time values for a2 were ca. 10 times longer for 10 mM than for 1 mM. 3.3. Substrate mineralization in soil

Values represent means7SEM (n ¼ 3) for samples taken in 2006 and 2007.

highest added glucose concentration (10 mM) the glucose uptake rates were equivalent to 64.4 and 31.6 mg C kg1 soil DW min1 (2006 and 2007 samples, respectively). Based upon a soil microbial biomass of 750 mg C kg1 soil DW, this equates to uptake rates of 0.087 and 0.042 mg glucose C mg1 biomass-C min1. When performed in 2006, the range of glucose concentrations used was not designed to produce an Eadie–Hofstee plot. However, the curvature of the plot suggested that a two-carrier transport system was operating. This was verified in 2007 when the depletion of a larger number of glucose concentrations was monitored (Fig. 2; Jennings, 1995). Estimates of Km for the high affinity transport system were of the order of

Following addition of the 14C-labeled glucose to the soil, there was an initial rapid phase of 14CO2 evolution followed by a secondary slower phase of evolution (Fig. 4). The double exponential decay equation gave a good fit to the biphasic experimental data at all glucose remaining in solution (% of total added)

V (nmol glucose-C g-1DW soil min-1)

1400

14C-glucose

620

100 1 µM 10 µM 100 µM 1 mM 10 mM

80

60

40

20

0 0

10

20

30

40

50

60

Time after substrate addition (min) Fig. 3. Amount of 14C-label remaining in soil solution after the addition of a 14C-labeled glucose pulse (1 mM to 10 mM) to an agricultural soil. Values represent means7SEM (n ¼ 3). Lines represent fits of single (1, 10 and 100 mM) or double (1 and 10 mM) first order kinetic equations to the experimental data.

ARTICLE IN PRESS P.W. Hill et al. / Soil Biology & Biochemistry 40 (2008) 616–624 Table 3 Coefficients of single (1 and 10 mM) and double (100 mM and 1 and 10 mM) first order curve fits to depletion of Glucose concentration in solution (mM)

y0

a1

b1

1 10 100 1000 10 000

1.470.1 1.470.1

98.670.1 98.670.1 90.573.0 58.371.8 63.770.9

2.970.1 1.770.1 1.370.1 2.970.5 2.070.2

a2

9.573.0 41.771.8 36.370.9

621

14

C from soil solution over time

b2

a1 t1/2 (min)

a2 t1/2 (min)

0.1270.03 0.0770.004 0.00770.0007

0.2470.01 0.4070.01 0.5370.02 0.2570.04 0.3570.03

7.272.2 9.870.5 105710.4

y0 is the asymptote to which 14C activity fell in single exponential curves, a1 and a2 are estimated pool sizes for fast and slow uptake routes, and b1 and b2 are the rate constants for the fast and slow uptake routes. t1/2 values are the half-times for pools a1 and a2 determined from b1 and b2. Values represent means7SEM (n ¼ 3).

14CO 2

evolution (% of total 14C-glucose added)

25

Soil microbes started to respire the glucose very rapidly after addition to the soil, respiring 2.470.2 or 2.170.2% (2006 and 2007, respectively) of the added 14C-glucose within 1 min of addition in the 1 mM treatment. This dropped to 0.8070.02 (2006) or 1.170.2% (2007) in the 10 mM treatment, although the actual quantity of glucose respired was much greater at higher concentrations (Table 2).

1 mM 10 mM 100 mM 1 mM 10 mM 100 mM autoclaved

20

15

10

4. Discussion 5

4.1. Kinetics of glucose uptake by the soil microbial community

0 0

10

20

30 40 Time (min)

50

60

Fig. 4. Cumulative 14CO2 evolution from an agricultural soil (2007 sampling) after the addition of a pulse of 14C-labeled glucose (1 mM to 10 mM). Values represent means7SEM (n ¼ 3). Lines represent fits of a double first order kinetic equation to the experimental data.

concentrations (r240.993 in all cases; Eq. (6), Fig. 4). The exponential coefficients and half-times (t1/2) of the glucose mineralization are presented in Table 4. Half-time values were not determined for the slower mineralization process described by b4 due to uncertainty over the connectivity between respiratory substrate pools (Boddy et al., 2007). There was no concentration-related trend in the exponential coefficients for 14CO2 evolution. A maximum of 19% of the added 14C-glucose was respired from the fast pool (a3), with half-times of 18 min or less. The remaining proportion was respired from the slower pool (a4). In 2006, values for the size and half-time of pool a3 appeared to be unusually large in the 1 mM treatment. To exclude the possibility of an experimental artefact, the mineralization experiment was repeated in 2007 and a similar result was found. Although repeatable, we are unable to offer an explanation for the apparent departure of the 1 mM treatments from the results for the other concentrations of glucose.

As glucose is not sorbed to the soil used here (Kuzyakov and Jones, 2006), its removal from the soil solution can only occur due to either microbial uptake or abiotic mineralization (Majcher et al., 2000). As no depletion of 14 C from the soil solution or 14CO2 evolution was observed in the autoclaved treatments we assume that glucose depletion from the soil solution occurred almost entirely due to microbial uptake. It is clear from the curvature of the Eadie–Hofstee kinetic plot (Fig. 2) that microbial uptake of glucose from soil solution could be described by at least a two-component transport system. This is consistent with other reports of glucose uptake by two carriers of different affinity in a range of microorganisms, including those in soil (Scarborough, 1970; Jennings, 1995; Hora´k, 1997; Reifenberger et al., 1997; Nguyen and Guckert, 2001). It is likely that a high degree of functional redundancy exists for glucose use in the microbial community and that the kinetics observed here reflects the summation of many transport systems operating simultaneously (Jennings, 1995). Indeed, even within a single taxum significant variation in transporter expression may occur depending upon the prevailing environmental conditions (Boles and Hollenberg, 1997; Reifenberger et al., 1997). Our estimates of Km for both carriers were in the range of those determined for individual microbial species and for a microbial community in a clay-loam soil from northern France (0.01–50 mM; Scarborough, 1970;

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622

Table 4 Coefficients of double first order curve fits to depletion of

14

C from soil by microbial mineralization over time

a3

b3

a4

b4

a3 t1/2 (min)

2006 1 10 100 1000 10 000

10.471.4 10.471.3 8.071.1 19.072.9 4.470.6

0.08670.01 0.08970.02 0.10070.02 0.05170.01 0.19070.02

89.071.3 89.271.3 92.071.2 80.872.9 95.670.6

0.001570.0003 0.001670.0002 0.001370.0001 0.0005770.0004 0.001479  105

8.571.4 8.271.3 6.871.1 14.071.8 3.770.5

2007 1 10 100 1000 10 000

12.470.3 10.770.7 12.570.4 17.471.4 8.370.8

0.06670.005 0.11070.006 0.10070.005 0.04170.008 0.06470.003

87.170.3 88.870.7 87.270.5 82.371.4 91.470.8

0.001470.0001 0.001676.9  105 0.001370.0001 0.0002470.0002 0.001170.0001

10.670.8 6.470.4 6.770.3 18.173.3 10.970.5

Glucose concentration in solution (mM)

a3 and a4 are estimated pool sizes for fast and slow phases of mineralization, and b3 and b4 are the rate constants for the fast and slow phases of mineralization. t1/2 values are the half-times for pools a3 determined from b3. Values represent means7SEM (n ¼ 3) for both 2006 and 2007 samples.

Jennings, 1995; Boles and Hollenberg, 1997; Reifenberger et al., 1997; Nguyen and Guckert, 2001). 4.2. Dynamics of glucose uptake from soil solution The range of glucose concentrations employed here was chosen to reflect those likely to be found in soil. Low glucose concentrations might be expected to occur after dilution of soil water with rainfall (1–10 mM), whilst high concentrations may occur upon lysis of root cells in the rhizosphere (1–10 mM; Jones and Darrah, 1996). In all circumstances the relative rate of glucose depletion from the soil solution was extremely high and far greater than previously reported (van Hees et al., 2005). The doubleexponential depletion kinetics observed at the two highest glucose concentrations (1–10 mM) again demonstrate that soil microbes are able to utilize two uptake pathways. Due to the single exponential dynamics shown at low glucose concentrations (1–10 mM), our results suggest that under these conditions soil microbes take up glucose via a onecarrier system. However, it appears that a second transport system becomes important at a concentration somewhere between 10 and 100 mM. This coincides with the concentration of glucose found in soil solution (54 mM) and may indicate that soil microbial uptake of glucose is dominated by the high-affinity carrier. The lower affinity carrier may only become necessary at times of high glucose availability. The concentration of glucose at which the second transport system became important here (between 10 and 100 mM) is higher than that found for the clay loam mentioned above (Nguyen and Guckert, 2001). Nguyen and Guckert (2001) is the only other investigation measuring soil glucose uptake under comparable conditions. Assuming the water content of their experimental soil was the same as ours, they found biphasic uptake occurring at a glucose concentration below 10 mM. Half-times for microbial glucose uptake by the two carriers calculated from the

data presented by Nguyen and Guckert (2001) were substantially longer than those found for our soil (2.7 and 69 min; Table 3). It is not possible to say whether these differences resulted from different experimental conditions or from a real difference between the soils. 4.3. Dynamics of glucose mineralization by the soil microbial community The biphasic exponential dynamics of 14CO2 evolution at all added glucose concentrations were similar to those reported previously (Boddy et al., 2007). Overall, the mineralization of the added glucose by the soil microbial community was extremely rapid. To our knowledge the half-times for the first phase of mineralization are the fastest yet reported, and are slightly less than half those reported for a nearby grassland soil (Boddy et al., 2007). It is striking that at some glucose concentrations microbes mineralized 42% of added 14C-glucose and 47% of that taken up by microbes (Fig. 2; Table 2) within 1 min of glucose addition to soil, despite probable isotope dilution in pre-existing microbial glucose pools. This suggests that soil microbes are severely C-limited. 4.4. Carbon turnover rates in soil The two treatments closest to the glucose concentrations found in our soil were 10 and 100 mM. At these concentrations the half-time in solution was ca. 30 s. From the rate of glucose uptake (Table 2) we estimate that in root-free soil most of the glucose pool turns over 0.08–0.7 times per minute. This suggests that the free glucose pool in soil may turn over 100–1000 times per day at summer temperatures. Microbial mineralization of glucose to 14CO2 was very fast, but necessarily slower than glucose uptake from the soil solution. A maximum of only 12.5% of added glucose

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was rapidly respired with half-times of 6–8 min when taken up at glucose concentrations close to those found naturally in the soil solution. The remainder was immobilized and cycled through the microbial biomass before mineralization. If we assume no connection between pools a3 and a4 (Kuzyakov and Demin, 1998), we can estimate half-times for cycling of glucose-C in the microbial biomass (pool a4) to be 7–9 h. Using the half-time for the first, fast component of mineralization (b3; Table 2) we can estimate glucose turnover in the soil solution at ca. 30 min (four half-times). From the flux of 14CO2, the soil solution glucose pool appears to turnover 0.003–0.03 times per minute. Thus estimates of soil solution substrate turnover times derived from measurements of mineralization may underestimate turnover times of labile portions of soil solution DOC by more than an order of magnitude. At steady state, the rates of glucose removal from the soil solution must be matched by new inputs of glucose from microbial/root turnover, microbial/root exudation and the hydrolysis of stable soil organic matter. Using the 10 and 100 mM treatments as the closest concentrations to natural soil solution glucose concentrations, we estimate that the rate of glucose input to soil was between 8 and 65 nmol glucose C g1 soil DW min1 (Table 2). Although, soil C fluxes were very likely still dominated by recent plant inputs retained in the microbial biomass and SOM, glucose exuded from plant roots would almost certainly have been removed from soil solution prior to the start of experiments. Thus, we estimate that at 20 1C between 0.5% and 4% of soil organic C (SOC) (including microbial biomass) is decomposed to glucose each day. Although we are not able to support our contention with measurements, we suggest that much of this flux is attributable to turnover of the soil microbial biomass and roots rather than stable SOM. Glucose concentrations in soil solution with living roots in situ may be higher than those measured here. Consequently, the flux of glucose through the soil DOC pool of soils under normal conditions (with plants growing in them) may also be underestimated. 5. Conclusions In this agricultural soil, the microbial depletion of glucose from solution was extremely rapid. Our results suggest that at low solution concentrations, glucose is taken up via a single high affinity transporter system. At higher glucose concentrations this high affinity transporter is joined by a low affinity transport system to maximize glucose capture from the soil. At natural soil solution concentrations, the half-time for glucose residence in this soil is ca. 30 s. From the rate of microbial glucose uptake we estimate that the pool of free glucose in the root-free soil used here turns over 100–1000 times per day. Determination of substrate turnover times from measurement of CO2 evolution rates may underestimate turnover times by an order of magnitude or more. We estimate that

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