- Email: [email protected]
The effects of glucose loading rates on bacterial and fungal growth in soil
Soil Biology & Biochemistry 70 (2014) 88e95
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
The effects of glucose loading rates on bacterial and fungal growth in soil Stephanie Reischke*, Johannes Rousk, Erland Bååth Section of Microbial Biology, Department of Biology, Lund University, Ecology Building, SE-223 62 Lund, Skåne, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2013 Received in revised form 9 December 2013 Accepted 15 December 2013 Available online 24 December 2013
Microbial activity in soil is usually limited by the availability of carbon (C). Adding an easily available C source, like glucose, has therefore been a common approach to study alleviation of resource limitations. Most such studies have relied on respiration to study microbial dynamics, with few following the explicit growth response. We determined the response in bacterial and fungal growth, as well as respiration, to additions of glucose (0.5e32 mg C g1 soil) during up to 6 days, using leucine incorporation for bacterial growth and acetate-in-ergosterol incorporation for fungal growth. A concentration of 2 mg glucose-C g1 soil, where the fungal contribution appeared to be small, was also studied with a high time resolution. Adding glucose resulted in an initial lag phase of stable respiration and bacterial growth. Bacterial growth was similar to the unamended control, while respiration was 8 fold higher during this period. The 14-h lag phase was followed by an exponential increase for both respiration and bacterial growth, with a similar intrinsic growth rate (m) of around 0.25 h1. After the exponential phase, bacterial growth decreased exponentially. The respiration initially decreased even more rapidly than bacterial growth. At concentrations exceeding 4 mg glucose-C g1 the relative stimulation of fungal growth surpassed that of bacteria, with the highest amendment rates, 32 mg C g1, resulting in mainly fungal growth. Lower loading rates than 4 mg glucose-C g1 appeared to stimulate mainly bacterial growth. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Substrate loading rates Glucose Bacterial growth Fungal growth Respiration 3 H-leucine incorporation 14 C-acetate incorporation Mineralisation Decomposition
1. Introduction Microbial activity in soil is limited by the availability and quality of resources, where especially carbon (C) limitation has been found to be common in soil (Joergensen and Scheu, 1999; Ilstedt and Singh, 2005; Demoling et al., 2007). One common approach to study microorganisms in soil has therefore been to add an easily available Csource, like glucose. Saturating the microbial C-demand with glucose in soil has resulted in a number of useful observations. For instance, the level of the instantaneous increase in respiration rate is proportional to the microbial biomass in soil (the substrate-induced respiration, or SIR rate; Anderson and Domsch, 1978; Höper, 2006), and the exponential increase in respiration rate occurring within a day or so after addition can be used to estimate the intrinsic rate of the increase in microbial biomass induced by the glucose addition (Stenström et al., 1998; Blagodatskaya et al., 2009; Wutzler et al., 2012). The physiological state of the microbial biomass can be estimated using kinetic modelling based on respiration data after adding glucose (Blagodatskaya et al., 2007). High glucose
* Corresponding author. Tel.: þ46 46 222 37 63. E-mail address: [email protected] (S. Reischke). 0038-0717/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2013.12.011
amendment rates are also used to estimate the microbial availability of nutrients in soil (Nordgren, 1992; Ilstedt et al., 2007). The microbial response, measured as respiration, to substantial additions of glucose usually results in three phases. The first is the lag phase, were no observable extra growth on the substrate can be measured. This is followed by an exponential increase in activity due to growth on the added substrate until peak activity, when the C-resource is exhausted. The third phase is the decline in activity after peak rates. Glucose addition induces an almost instant increase in microbial respiration (Anderson and Domsch, 1978; Ehlers et al., 2010). The level of respiration response increases with the loading rate of glucose until the microbial C-demand is saturated. If a surplus of glucose above that needed for saturation is added, this saturation level of respiration will stay constant for around 4e15 h at temperatures w20 C (Anderson and Domsch, 1973, 1978; Blagodatskaya et al., 2007). This initial stable C-saturated microbial respiration, the SIR level, has been interpreted as a lag phase, where the growth rate of the microbial community remains unchanged from the rate before the substrate addition (Anderson and Domsch, 1985; Nordgren et al., 1988). However, it can also be interpreted as an apparent lag phase, where a small fraction of the microorganisms starts to increase their growth rate immediately on
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
the added glucose. This increase will not be detectable during the apparent lag phase, since it is masqued by the respiration of the larger fraction of microorganisms whose growth rate does not increase from glucose, but it can be estimated using a kinetic model for growth (Stenström et al., 1998; Blagodatskaya et al., 2007; Wutzler et al., 2012). If sufficient glucose is added not to be exhausted during the lag phase, and there is no other limiting nutrient, the stable SIR level is followed by an exponential increase in respiration. This exponential phase will continue until respiration rate peaks at the point when glucose is exhausted. The additional microbial respiration reflects growth of the microbes on the added C source, and the intrinsic growth rate, m, for this period of exponential growth can be calculated (Nordgren et al., 1988; Stenström et al., 1998; Blagodatsky et al., 2000; Blagodatskaya et al., 2007). In the third phase, after the respiration peak, respiration will drastically decrease again, to gradually converge with the respiration rate before the addition (Anderson and Domsch, 1973; Teklay et al., 2006). This phase of declining respiration has not received explicit study to date. There are reports that have monitored microbial growth by measuring changes in biomass, however, with conflicting results. Marstorp and Witter (1999) followed the increase in the DNA concentration as a proxy for growth and found a good correlation between the initiation and extent of the exponential response of respiration and the increase in DNA. Anderson and Martens (2013), on the other hand, found discrepancies between respiration and changes in DNA content, with lower growth rates estimated from the DNA concentration than those inferred from respiration data. One reason for these incongruities is that changes in biomass, especially during the initial lag phase of constant respiration and the early exponential phase, are difficult to estimate with sufficient precision. Only one study to date has measured bacterial growth directly after adding glucose using the leucine incorporation technique (Iovieno and Bååth, 2008). Although they found that during the lag phase in respiration bacterial growth was stable, their sampling scheme did not have sufficient time resolution to compare growth during the exponential phase and the period after peak respiration. So far no direct estimates of fungal growth after adding glucose to soil have been made. However, fungal and bacterial biomass estimations after prolonged incubations with different loading rates of easily available C have indicated that higher substrate concentrations will favour fungal over bacterial biomass synthesis (Griffiths et al., 1999). We have compared respiration, bacterial growth and fungal growth after the addition of a wide range of glucose concentrations over a 6 day period to cover all three growth phases. First, we followed the development of respiration and growth after adding one concentration of glucose (2 mg C g1 soil) at a high time resolution. We specifically tried to answer the following questions: Does the lag phase in respiration coincide with a concomitantly stable rate of bacterial or fungal growth? Is the exponential growth phase in respiration explained by bacterial or fungal growth? Secondly, we compared a range of different loading rates of glucose. The chosen range of glucose additions, covering 0.5e32 mg glucose-C g1 soil, matched the range used by Griffiths et al. (1999) to compare bacterial and fungal biomass responses. In this second part we addressed the questions: Is there a shift between bacterial and fungal growth with different glucose loading rates in soil? Is the respiration response due to different organism groups depending on the loading rates? 2. Material and methods 2.1. Soil preparation and incubation conditions Freshly collected soil was sieved using a 2.8 mm mesh and stored at 4 C until used. The soil is classified as sandy loamy brown
89
earth soil (Cambisol, FAO; Inceptisol, USDA) from managed grassland from south-eastern Sweden. Soil parameters were: pH(H2O) ¼ 6.7 0.3 (mean se, n ¼ 3), soil organic matter content ¼ 13.5 0.8%, and water content ¼ 26.2 3.5%. In each experiment 100 g soil were weighed into 180 ml containers and amended with different glucose concentrations (see below). To avoid inducing nutrient limitation by other resources than C, NH4NO3 and KH2PO4 were added at a final concentration of 0.1 mg N and P per 2 mg C added to the soil as glucose. After the glucose addition the soil samples were homogenized by shaking 1 min and mixing with a spatula for 30 s, and then incubated at 20 C in a temperature controlled room. 2.2. Experimental setup Two experiments were conducted. Microbial activity was measured as respiration, bacterial growth and fungal growth during each experiment. In experiment 1, 2.0 mg glucose-C g1 soil was added and the microbial activity was measured every 2 h during the lag and the exponential phase, and with longer time intervals later, over totally 169 h. Fungal activity was only followed for 72 h. To achieve a time-resolution of every second hour, glucose was added to three separate sets of soil at three different time points: in the morning, in the afternoon and in the evening. An unamended control was also included, but with a less intensive sampling schedule. Two replicates were started at each of the three time points, and all presented values are the mean of the duplicates. Soil pH was measured after 2 and 24 h and was found to be minimally affected by the addition (<0.1 pH unit). In experiment 2 seven glucose concentrations (0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 mg glucose-C g1 soil) were added to the soil in addition to an unamended control. This experiment was performed at two separate occasions. The lower range of concentrations (0e 4 mg glucose-C g1 soil) was first examined for respiration and microbial growth during 146 h. Subsequently, the higher range of concentrations (8e32 mg glucose-C g1 soil) was studied, where respiration and microbial growth were followed for 183 h. To ensure reproducible experimental conditions the unamended control and 2.0 mg glucose-C g1 soil treatments were included also in this second batch. Since this experiment did not focus on the lag phase, only a few samples were taken before the exponential growth increase. Soil pH was measured after 2 and 24 h. Adding the 16 mg glucose-C g1 soil treatment (with nutrients) decreased pH to 6.5 and adding 32 mg glucose-C g1 soil to pH 6.3, while pH decreased with <0.2 units for the other treatments. 2.3. Microbial measurements of respiration and growth 2.3.1. Respiration Respiration was measured by transferring 1.0 g soil into a 20 ml glass vial and purging the headspace with pressurized air. The vial was closed with a crimp lid and incubated for 2 h at 20 C. Afterwards the headspace CO2 concentration was analysed using a gas chromatograph with a thermal conductivity detector. 2.3.2. Bacterial growth To estimate bacterial growth, 1.0 g soil was transferred into a 50 ml centrifuge tube and the leucine (Leu) incorporation method (Kirchman et al., 1985) in bacteria extracted from soil using the homogenization/centrifugation techniques (Bååth, 1994) with modifications (Bååth et al., 2001) was applied. We used a mixture of radio-labelled Leu, [3H]Leu (37 MBq ml1, 5.74 TBq mmol1, Perkin Elmer, USA) and unlabelled Leu, resulting in 275 nM Leu in the bacterial suspension, and a 2 h incubation at 20 C. After washing out unincorporated Leu (Bååth et al., 2001), the amount of
90
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
incorporated radioactivity was determined by using a liquid scintillator. The incorporation of 3H-leucine, expressed as pmol Leu incorporated in extracted bacteria g1 soil h1, was used as a proxy for bacterial growth.
2
Respiration (µg CO -C g-1 h-1)
50
2.3.3. Fungal growth and ergosterol concentration To estimate fungal growth, 1.0 g soil was transferred into a 10 ml glass tube and the acetate-in-ergosterol incorporation, which was originally developed for aquatic environments (Newell and Fallon, 1991) and later modified for soil by Bååth (2001), was measured. We used a mixture of 20 ml 1-[14C]acetic acid (sodium salt, 2.04 GBq mmol1, 7.4 MBq ml1, Perkin Elmer, USA) and unlabelled acetate resulting in a final acetate concentration of 220 mM, and an incubation time of 2 h at 20 C. To separate and quantify the ergosterol, we used an HPLC with a UV detector (282 nm) and a fraction collector (Rousk and Bååth, 2007). The incorporated radioactivity of the ergosterol fraction was measured by using liquid scintillation. The amount of incorporated 14C-acetate, expressed as pmol g1 soil h1, was used as a proxy for fungal growth, while the ergosterol concentration was used as a proxy of fungal biomass.
40
cap dec
30
20
10
Bacterial growth (pmol Leu g-1 h-1)
50
100
150
-1
where Y is respiration or bacterial growth, A and B the part of the biomass not growing and growing on the added glucose, respectively, m is the intrinsic growth rate and t is the time after adding glucose (Stenström et al., 1998; Blagodatsky et al., 2000). The lag phase was then calculated as
(2)
200
B
600 500 400 300 200 100 0
0
50
100
150
200
C
-1
Fungal growth (pmol Ac g h )
(1)
using a parametric bootstrap procedure with 10000 bootstrap samples to calculate SE (Efron, 1979). We also used a simple two part model, assuming a real lag phase, with a constant activity during the lag phase and calculating the intrinsic growth rate (m) from the linear slope of the log-transformed data during the exponential phase (“the linear approach”). The lag phase was then determined by the time-point when the modelled curves for the lag and exponential phase intersected. This calculation was used in experiment 2 (different loading rates), since insufficient data were available to be able to fit Eq. (1) with sufficient precision. After the peak rate, the decrease in bacterial growth was modelled using a negative exponential equation, since bacterial death is known to be logarithmic. Respiration decreased very rapidly immediately after peak respiration (Fig. 1), followed by a slower decrease. Lacking any mechanistic hypothesis for this interval, data points were here connected by a line, and not modelled. As an index for the microbial growth efficiency, we used the ratio between respiration and growth (dividing the respiration (mg CO2 g1 h1) by the bacterial growth (pmol Leu g1 h1 into extracted bacteria)), where high values indicate low efficiency, and vice versa. We only estimated the microbial growth efficiency in a situation dominated by bacterial growth (experiment 1). Since we have not established conversion factors from leucine incorporation
A
fast dec
0
Different time intervals of the respiration and bacterial growth curves after the addition of 2 mg glucose-C g1 soil (Fig. 1) were modelled with different equations. The initial stable phase for bacterial growth and respiration after adding glucose can be modelled either as a real or as an apparent lag phase. The latter was modelled for the combined lag and exponential phase until peak activity at 22 h as
lag phase ¼ lnðA=BÞ=m
comb 2 mgequ C
0
3. Calculations
Y ¼ A þ Bemt
0Control resp
30
20
10
0
0
50
100
150
200
Time (h) Fig. 1. Respiration (A), bacterial growth as Leu incorporation (B) and fungal growth as Ac-in-ergosterol incorporation (C) after the addition of 2 mg glucose-C g1 soil (:) and an unamended control (C) during incubation at 20 C. The lag and exponential phase was modelled using Eq. (1). Respiration: y ¼ 11.6 þ 0.16e0.24x (R2 ¼ 0.99); bacterial growth: y ¼ 44.5 þ 2.2e0.25x (R2 ¼ 0.98). After peak bacterial growth the decline was modelled by y ¼ 549e0.0043x (R2 ¼ 0.79).
to biomass production in soil, we standardized the data to a mean value of one over time for the unamended control treatment. 4. Results 4.1. High temporal resolution of the 2 mg glucose-C g1 addition Respiration increased to values 8-fold those in the unamended control immediately after adding 2 mg glucose-C g1 soil; a level that remained constant during the first 14 h (Fig. 1A). Up to 22 h respiration then increased exponentially, with a calculated growth rate, m, of 0.14 h1 using the linear approach. Estimating the growth rate with Eq. (1) resulted in a m of 0.24 0.014 h1 and a lag phase of 17 1.9 h. Peak respiration occurred after 22 h at values 4-fold
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
4.2. Effects of glucose loading rates on fungal and bacterial growth The first measurement, 8 h after glucose additions, showed up to 8-fold higher respiration rates compared to the control (Fig. 4A, B). The two highest loading rates, 16 and 32 mg C g1 soil, resulted in lower respiration during the lag phase, being 50 and 30%, respectively, of that in treatments with <8 mg C g1 soil. The highest loading rates also had longer lag phases, with 16 and 32 mg C g1 soil resulting in 16 and 25 h lag phases, respectively, compared to around 11e13 h below 4 mg C g1 soil. Addition of 2 mg glucoseC g1 or more resulted in increased respiration after the lag phase. This was not found for the lowest concentrations (0.5 and 1 mg glucose-C g1). Growth rate, m, estimated with the linear approach, did not vary with glucose concentration, seen as almost parallel increases in respiration during the exponential phase (Fig. 4A, B). Peak respiration increased with glucose concentration, ranging from around a 50-fold higher rate than in the control for the 2 and 4 mg C g1 treatments, and up to a 240-fold higher rate for the highest addition. After peak respiration, the rates of all treatments, except the 32 mg C g1 addition, decreased rapidly towards the control value (Fig. 4A, B), but still up to 14-fold higher values were found at the end of the study (>140 h). Bacterial growth showed similar values as the control during the first 8e12 h for glucose concentrations up to 16 mg C g1 soil (Fig. 4C, D). Adding 16 mg C g1 decreased growth to around 60% of that in the other glucose treatments, while the highest glucose addition (32 mg C g1) showed very low bacterial growth over the entire incubation time (Fig. 4D). The lag phase was stable up to 4 mg C g1 soil (8e12 h), but increased to 19 h for 8 mg C g1 soil and around 35 h for 16 mg C g1 soil. Except for the 0.5 mg C g1
A
2
-1
Cum. respiration (mg CO -C g )
1
-1
Cum. bacterial growth (nmol Leu g )
Control 0 2000 2 mg C
0.8
0.6
0.4
0.2
0 0 60
50
100
150
200
50
100
150
200
100
150
200
B
50 40 30 20 10 0 0 2.5
C
-1
Cum. fungal growth (nmol Ac g )
higher than during the preceding lag phase. Respiration then decreased rapidly within the next 6 h followed by a slower decline over the next 140 h (Fig. 1A). After 168 h incubation the glucose treated soil still respired at a 2-fold higher rate than in the unamended control. Bacterial growth in the glucose-amended soil did not change compared to the control during the first 12e14 h (Fig. 1B). During the next 10 h (up to 22 h) bacterial growth showed an exponential increase, with a calculated m of 0.17 h1 using the linear approach. Using Eq. (1) resulted in a m of 0.25 0.024 h1 and a lag phase of 13 3.3 h, with no significant differences to corresponding values for respiration. At peak values after 22 h, bacterial growth was 9fold higher than the control. Bacterial growth then decreased exponentially with a slope of 0.0043 0.00061 h1. Bacterial growth still was 6-fold higher compared to the control at the end of the experiment. Fungal growth was marginally affected by the 2 mg glucoseC g1 soil treatment, but values tended to exceed those of the control more than 20 h after addition (Fig. 1C). Cumulative data indicated that fungal growth (Fig. 2C) only increased to 1.3 times those of the control after 72 h compared to bacterial growth, which increased 6-fold (Fig. 2B) and respiration 10-fold that in the control (Fig. 2A). Thus, the elevated respiration after adding glucose appeared mostly to be related to bacterial and not fungal growth. With minimal fungal contribution, bacterial growth could be assumed to reflect total microbial growth and thus we used the ratio of respiration-to-bacterial growth as a proxy for changes in the bacterial C-use efficiency (Fig. 3). The increased respiration and stable leucine incorporation during the lag phase resulted in very high values, suggesting low bacterial C-use efficiency. The ratio then declined during the exponential phase (14e24 h), but was still higher than in the control. After peak respiration, during the exponential decline of bacterial growth, the relative respiration to bacterial growth ratio was lower than in the unamended control.
91
2
1.5
1
0.5
0 0
50
Time (h) Fig. 2. Cumulative respiration (A), bacterial growth as Leu incorporation (B) and fungal growth as Ac-in-ergosterol incorporation (C) after the addition of 2 mg glucoseC g1 soil (:) and an unamended control (C) during the incubation at 20 C for 169 h (A and B) and 78 h (C).
treatment all other glucose addition treatments showed an exponential increase after the lag phase (Fig. 4C, D). Growth rate, m, estimated with the linear approach, varied between 0.15 and 0.19 h1 for the treatments with 1e4 mg glucose-C g1, while growth rates decreased to 0.11 h1 and 0.025 h1 for 8 and 16 mg C g1 soil, respectively. At 32 mg C g1 m was not possible to estimate. Peak bacterial growth was 5e20-fold higher than in the control. Bacterial growth declined in the latter part of the experiment, but was still 3e7-fold higher compared to the control at the end of the experiment. Fungal growth was almost constant after glucose additions below 8 mg C g1 over the whole incubation period, with only minor increases compared to the control (Fig. 4E, F). However, using 32 mg glucose-C g1 we obtained the highest level of fungal growth (10-fold higher than in the control) with m estimated to 0.065 h1
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
Relative respiration:bacterial growth
92
14 12 10 8 6 4 2 0
0
50
100
150
200
Time (h) Fig. 3. Relative ratio between respiration and bacterial growth as Leu incorporation following the addition of 2 mg glucose-C g1 soil (:) and an unamended control (C) over 169 h at 20 C.
using the linear approach. Fungal growth declined rapidly after peak activity (Fig. 4F). We also found a clear fungal growth response in the 16 mg glucose-C g1 treatment, increasing to peak levels after 42 h and then decreasing until converging with the values of the control treatment towards the end of the study. Cumulative respiration increased with higher loading rates of glucose (Fig. 5). There was a shift with increasing substrate loading rates from a situation dominated by bacterial growth to a situation dominated by fungal growth. At 8 mg glucose-C g1 soil both bacterial and fungal growth appeared to contribute to the respiration. At and below 4 mg C g1 soil bacteria was the main responding organism group, while at 16 and especially at 32 mg C g1 fungal growth dominated the respiration response. The ergosterol content at the end of the experiment was used as an alternative way of expressing accumulated fungal growth (Fig. 5). Both the ergosterol content and the cumulative fungal growth, estimated from Ac-in-ergosterol incorporation, showed corroborative results with almost no growth below 8 mg added glucose-C g1, and then increasing with higher glucose loading rates. 5. Discussion 5.1. Comparing dynamics of microbial growth to respiration Adding 2 mg glucose-C g1 soil resulted in a lag phase, an exponential phase and a declination phase; the phases mostly coincidental for bacterial growth and respiration (Fig. 1). In contrast, fungi appeared to only react to a minor degree to the addition, and with a different time dynamic. It is widely held that bacteria are the principal group to rapidly react to an increased concentration of labile C-substrate (Moore et al., 2005; Paterson et al., 2008). Data on incorporation of low amounts of 13C-glucose into PLFAs indicative of bacteria and fungi in grassland and agricultural soils at near neutral pHs have also often found such results (Ziegler et al., 2005; Dungait et al., 2013), although in other habitats fungi may be more important (Rinnan and Bååth, 2009). Thus, we conclude that at this level of added glucose to this soil, bacteria initially dominated the decomposition and mineralisation of the added substrate. The degree of the response of bacterial growth and respiration to the glucose differed after addition, however. During the lag phase, we found no change in bacterial growth rates compared to
the control, while respiration was constantly high (at the SIR level). This implied an uncoupling between respiration and bacterial growth (see below on discussion on C-use efficiency). The lack of bacterial growth increase on the added glucose during the lag phase is consistent with the results of Iovieno and Bååth (2008), and studies on biomass changes (e.g. Marstorp and Witter, 1999) as well as plate counts (Anderson and Domsch, 1985) after adding glucose. The lag phase was followed by an exponential increase in respiration and bacterial growth (Figs. 1 and 2). Marstorp and Witter (1999) compared respiration with kinetics of dsDNA formation and also found that the exponential increase in respiration was reflected in an exponential increase in microbial biomass. Our estimated growth rates (m) of 0.14e0.17 h1 (using the linear approach) or 0.24e0.25 h1 (using Eq. (1)) were within the values found by Blagodatskaya et al. (2007; ranging between 0.10 and 0.35 h1) and those compiled by Anderson and Martens (2013; m ranging between 0.14 and 0.41 h1). The use of Eq. (1) has earlier been shown to result in higher estimates of m than using the linear approach (Blagodatskaya and Kuzyakov, 2013), which is consistent with our results. More important is that m calculated from respiration and bacterial growth data during the exponential phase matched, irrespective of method of calculation. Peak respiration and bacterial growth also coincided in time. Similar m and time of peak activities for bacterial growth and respiration data suggest that bacteria dominated the use of glucose during the exponential phase. The final decline in respiration has received less study than the exponential increase. This decrease appeared to be composed of two parts; an initial very rapid decrease followed by a slower decrease. This two-part pattern in respiration after peak values was also found after rewetting soils dried for a long period (Meisner et al., 2013). Bacterial growth during the declination phase was tentatively modelled by a negative exponential equation, similar to the decline in bacterial growth after peak values following the wetup of a dry soil (Meisner et al., 2013). Respiration initially decreased much more rapidly than bacterial growth. Similar observations have been made in pure culture studies, where respiration per cell has been found to rapidly decrease by up to two orders of magnitudes during the transition from exponential to early stationary phase (Riedel et al., 2013). After the rapid respiration decrease, the rate of the decline of respiration began to align with that for bacterial growth, although it was still almost twice as fast. During this period, glucose in the soil will be exhausted (Anderson and Domsch, 1985; Thiet et al., 2006; Sawada et al., 2008). Growth higher than in the control will then be due to glucose taken up into storage products (Bååth, 2003), recycling of the biomass already formed while growing on glucose, or growth on soil organic matter released through priming (Blagodatskaya and Kuzyakov, 2008). Assuming that bacteria are the main group respiring and growing on glucose during the initial time period of experiment 1, the high respiration-to-bacterial growth during the lag phase (Fig. 3) suggests an immediate uncoupling of growth and respiration, either due to waste metabolism of excess energy (Neijssel and Tempest, 1976) or due to glucose being mainly used for catabolism during this period. However, part of the explanation could also be that glucose is taken up and used for storage, that is, without growth, but still with a respiration cost (Bremer and Kuikman, 1994; Sawada et al., 2008). During the exponential phase the respiration-to-bacterial growth ratio decreased, suggesting increasing C-use efficiency during this period. During the declining phase the respiration-to-bacterial growth ratio decreased to values even lower than in the unamended control. However, we do not think this suggests that the microbial community has become even more C-use efficient than in the control soil. Rather, the microbial
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
200
A
Control 0.5 1.0 2.0 4.0
150
B
Control 2.0 8.0 16.0 32.0
150
2
Respiration (µg CO -C g-1 h-1)
200
93
100
100
50
50
0
1000
50
100
150
200
0
0
1000
C
800
800
600
600
400
400
200
200
50
100
150
200
50
100
150
200
100
150
200
D
-1
-1
(pmol Leu g h )
Bacterial growth in extracted bacteria
0
0
0
Fungal growth (pmol Ac g-1 h-1)
35
50
100
150
200
0
250
E
30
0
F
( )
200 25 150
20 15
100
10 50 5 0
0
50
100
150
Time (h)
200
0
0
50
Time (h)
Fig. 4. Respiration, bacterial growth as Leu incorporation and fungal growth as Ac-in-ergosterol incorporation measured at two separate occasions with up to 4 mg added glucoseC g1 soil (A, C and E) and with higher concentrations (B, D and F). In A, C and E, 0, 0.5, 1, 2 and 4 mg glucose-C g1 soil were added and microbial activity was measured during 146 h. In B, D and F, 0, 2, 8, 16 and 32 mg glucose-C g1 soil were added and microbial activity was measured during 195 h. The data-point within the parenthesis was considered an outlier.
growth in the control soil will be composed of both bacterial and fungal contributions, and using only bacterial growth would underestimate C-use efficiency. This underscores the importance of including both fungal and bacterial growth when estimating microbial C-use efficiencies, although we might have periods where mostly bacteria or fungi are growing, as determined by different loading rates (as here shown) or differences in soil pHs (BárcenasMoreno et al., 2011). Summarizing, respiration-to-bacterial growth can vary substantially during a substrate-induced growth event, as found for pure culture studies (Riedel et al., 2013). 5.2. Effect of loading rates With increasing glucose loading rates the microbial community growth progressively shifted from one dominated by bacteria to
one dominated by fungi (Figs. 4 and 5). These findings corroborate those of Griffiths et al. (1999), who showed that higher substrate loading rates of easily available substrate selected for fungi. A possible explanation could be a high osmolarity in the soil solution at high substrate loading rates. Fungi are generally considered to be able to handle high osmotic stress better than bacteria (Griffin, 1972), and fungal growth at high loading rates could be due to fast-growing osmo-tolerant fungi (e.g. yeast) (van der Wal et al., 2006). Griffiths et al. (1999) also suggested that organisms dominating at high substrate loading rates are better able to cope with the relatively lower availability of other nutrients, which might occur under these conditions. The hyphal growth form and the more flexible stoichiometric homeostasis of fungi (Strickland and Rousk, 2010) may be important for this aspect. A negative effect of altered osmotic potential at higher loading rates was evident in
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
Relative cumulative values after 146 h
94
concentrations. This would be at concentrations that earlier have been suggested to trigger microbial activity in soil (De Nobili et al., 2001).
resp Respiration
50
Bacterial growth bg Fungal growth fg
erg Ergosterol
40
Acknowledgements
30
This work was supported by grant from the Swedish Research Council to E.B. (grant no 621-2012-3450) and to J.R. (grant no 6212011-5719). This work was part of Lund University Centre for Studies of Carbon Cycle and Climate Interactions (LUCCI).
20 References
10
0
0
1
10 -1
Glucose concentration (mg C g ) Fig. 5. Relative values of cumulative respiration, bacterial growth as Leu incorporation, and fungal growth as Ac-in-ergosterol incorporation at a glucose concentration range from 0 to 32 mg C g1 soil. Ergosterol concentrations as a proxy of fungal biomass at the end of the incubation are also shown. The data were standardized to 1 for the unamended control. Note the log scale on the x-axis.
the lower bacterial growth during the lag phase with increasing glucose addition rates, as also observed when optimising glucose concentrations to measure SIR-responses (Rousk et al., 2009a). This putative osmotic effect was also evidenced in the longer lag phase in respiration especially after adding 16 and 32 mg glucose-C g1. This was also observed when adding large amounts of glucose when using respiration kinetics to measure microbial available phosphorus, prompting the use of perlite as a substrate carrier (Ilstedt et al., 2007). Low pH has been shown to favour fungal compared to bacterial growth (Rousk et al., 2009b, 2011; Bárcenas-Moreno et al., 2011). However, we do not think that decreased pH due to glucose treatments (with nutrients) could explain the shift to fungal growth at high glucose loading rates. The decrease was at most only around half a pH unit, and the pH range (6.3e6.7) is within an interval where pH-related effects on bacterial growth have been found to be limited (Rousk et al., 2009b, 2011). Although initially bacterial growth was inhibited at high loading rates, at 8 and 16 mg glucose-C g1 soil bacterial growth eventually increased after a prolonged lag phase (Fig. 4D). This may be due to fungi consuming glucose, thus altering the osmotic potential creating conditions conducive for bacterial growth. Fungal growth preceding bacterial growth, apparently altering the environmental conditions, has previously been observed during the colonization of burned forest soils in relation to pH (Bárcenas-Moreno et al., 2011). Another explanation is that growth of bacteria at high glucose concentrations was due to a different community, being more osmo-tolerant than the one growing at lower substrate loading rates. That the concentration of added substrate affects the resulting microbial community has been shown repeatedly (e.g. Griffiths et al., 1999; Pennanen et al., 2004). No exponential phase was observed in the respiration measurements at the lowest concentrations added, although there was a bacterial growth response also in these treatments. This suggests that bacterial growth on glucose is not only possible when sufficient glucose is added to trigger an exponential response in respiration. Since low concentrations are more likely to prevail in the natural soil environment, this highlights the need for studying respiration and growth kinetics in the low range of substrate
Anderson, J.P., Domsch, K.H., 1973. Quantification of bacterial and fungal contributions to soil respiration. Archiv für Mikrobiologie 93, 113e127. Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215e 221. Anderson, T.H., Domsch, K.H., 1985. Maintenance carbon requirements of activelymetabolizing microbial-populations under in situ conditions. Soil Biology & Biochemistry 17, 197e203. Anderson, T.H., Martens, R., 2013. DNA determinations during growth of soil microbial biomasses. Soil Biology & Biochemistry 57, 487e495. Bååth, E., 1994. Measurement of protein-synthesis by soil bacterial assemblages with the leucine incorporation technique. Biology and Fertility of Soils 17, 147e 153. Bååth, E., 2001. Estimation of fungal growth rates in soil using 14C-acetate incorporation into ergosterol. Soil Biology & Biochemistry 33, 2011e2018. Bååth, E., 2003. The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi. Microbial Ecology 45, 373e383. Bååth, E., Pettersson, M., Söderberg, K.H., 2001. Adaptation of a rapid and economical microcentrifugation method to measure thymidine and leucine incorporation by soil bacteria. Soil Biology & Biochemistry 33, 1571e1574. Bárcenas-Moreno, G., Rousk, J., Bååth, E., 2011. Fungal and bacterial recolonisation of acid and alkaline forest soils following artificial heat treatments. Soil Biology & Biochemistry 43, 1023e1033. Blagodatskaya, E.V., Blagodatsky, S.A., Anderson, T.H., Kuzyakov, Y., 2007. Priming effects in Chernozem induced by glucose and N in relation to microbial growth strategies. Applied Soil Ecology 37, 95e105. Blagodatskaya, E.V., Blagodatsky, S.A., Anderson, T.H., Kuzyakov, Y., 2009. Contrasting effects of glucose, living roots and maize straw on microbial growth kinetics and substrate availability in soil. European Journal of Soil Sciences 60, 186e197. Blagodatskaya, E., Kuzyakov, Y., 2008. Mechanisms of real and apparent priming effects and their dependence on soil microbial biomass and community structure: critical review. Biology and Fertility of Soils 45, 115e131. Blagodatskaya, E., Kuzyakov, Y., 2013. Active organisms in soil: critical review of estimation criteria. Soil Biology & Biochemistry 67, 192e211. Blagodatsky, S.A., Heinemeyer, O., Richter, J., 2000. Estimating the active and total soil microbial biomass by kinetic respiration analysis. Biology and Fertility of Soils 32, 73e81. Bremer, E., Kuikman, P., 1994. Microbial utilization of 14C[U]glucose in soil is affected by the amount and timing of glucose additions. Soil Biology & Biochemistry 26, 511e517. De Nobili, M., Contin, M., Mondini, C., Brookes, P.C., 2001. Soil microbial biomass is triggered into activity by trace amounts of substrate. Soil Biology & Biochemistry 33, 1163e1170. Demoling, F., Figueroa, D., Bååth, E., 2007. Comparison of factors limiting bacterial growth in different soils. Soil Biology & Biochemistry 39, 2485e2495. Dungait, J.A.J., Kemmitt, S.J., Michallon, L., Guo, S., Wen, Q., Brookes, P.C., Evershed, R.P., 2013. The variable response of soil microorganisms to trace concentrations of low molecular weight organic substrates of increasing complexity. Soil Biology & Biochemistry 64, 57e64. Efron, B., 1979. Bootstrap methods: another look at the jackknife. Annals of Statistics 7, 1e26. Ehlers, K., Bakken, L.R., Frostegård, Å., Frossard, E., Bünemann, E.K., 2010. Phosphorus limitation in a Ferralsol: impact on microbial activity and cell internal P pools. Soil Biology & Biochemistry 42, 558e566. Griffin, D.M., 1972. Ecology of Soil Fungi. Chapman and Hall, London. Griffiths, B.S., Ritz, K., Ebblewhite, N., Dobson, G., 1999. Soil microbial community structure: effects of substrate loading rates. Soil Biology & Biochemistry 31, 145e153. Höper, H., 2006. Substrate-induced respiration. In: Bloem, J., Hopkins, D.W., Benedetti, A. (Eds.), Microbiological Methods for Assessing Soil Quality. CABI Publishing, Oxfordshire, pp. 84e92. Ilstedt, U., Singh, S., 2005. Nitrogen and phosphorus limitations of microbial respiration in a tropical phosphorus-fixing acrisol (ultisol) compared with organic compost. Soil Biology & Biochemistry 37, 1407e1410. Ilstedt, U., Singh, S., Nordgren, A., 2007. Using perlite as a substrate carrier for measuring microbial available phosphorus by respiration kinetics in soils. Biology and Fertility of Soils 43, 503e510.
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95 Iovieno, P., Bååth, E., 2008. Effect of drying and rewetting on bacterial growth rates in soil. Fems Microbiology Ecology 65, 400e407. Joergensen, R.G., Scheu, S., 1999. Response of soil microorganisms to the addition of carbon, nitrogen and phosphorus in a forest Rendzina. Soil Biology & Biochemistry 31, 859e866. Kirchman, D., Knees, E., Hodson, R., 1985. Leucine incorporation and its potential as a measure of protein-synthesis by bacteria in natural aquatic systems. Applied and Environmental Microbiology 49, 599e607. Marstorp, H., Witter, E., 1999. Extractable dsDNA and product formation as measures of microbial growth in soil upon substrate addition. Soil Biology & Biochemistry 31, 1443e1453. Meisner, A., Bååth, E., Rousk, J., 2013. Microbial growth responses upon rewetting soil dried for four days or one year. Soil Biology & Biochemistry 66, 188e192. Moore, J.C., McCann, K., de Ruiter, P.C., 2005. Modeling trophic pathways, nutrient cycling, and dynamic stability in soils. Pedobiologia 49, 499e510. Neijssel, O.M., Tempest, D.W., 1976. Role of energy-spilling reactions in growth of Klebsiella-Aerogenes Nctc-418 in aerobic chemostat culture. Archives of Microbiology 110, 305e311. Newell, S.Y., Fallon, R.D., 1991. Toward a method for measuring instantaneous fungal growth-rates in field samples. Ecology 72, 1547e1559. Nordgren, A., 1992. A method for determining microbially available-N and available-P in an organic soil. Biology and Fertility of Soils 13, 195e199. Nordgren, A., Bååth, E., Söderström, B., 1988. Evaluation of soil respiration characteristics to assess heavy-metal effects on soil-microorganisms using glutamicacid as a substrate. Soil Biology & Biochemistry 20, 949e954. Paterson, E., Osler, G., Dawson, L.A., Gebbing, T., Sim, A., Ord, B., 2008. Labile and recalcitrant plant fractions are utilised by distinct microbial communities in soil: independent of the presence of roots and mycorrhizal fungi. Soil Biology & Biochemistry 40, 1103e1113. Pennanen, T., Caul, S., Daniell, T.J., Griffiths, B.S., Ritz, R., Whatley, R.W., 2004. Community-level responses of metabolically-active soil organisms to the quantity and quality of substrate inputs. Soil Biology & Biochemistry 36, 841e 848. Riedel, T.E., Berelson, W.M., Nealson, K.H., Finkel, S.E., 2013. Oxygen consumption rates of bacteria under nutrient-limited conditions. Applied and Environmental Microbiology 79, 4921e4931.
95
Rinnan, R., Bååth, E., 2009. Differential utilization of carbon substrates by bacteria and fungi in tundra soil. Applied and Environmental Microbiology 75, 3611e3620. Rousk, J., Bååth, E., 2007. Fungal and bacterial growth in soil with plant materials of different C/N ratios. FEMS Microbiology Ecology 62, 258e267. Rousk, J., Aldén Demoling, L., Bååth, E., 2009a. Contrasting short-term antibiotic effects on respiration and bacterial growth compromises the validity of the selective respiratory inhibition technique to distinguish fungi and bacteria. Microbial Ecology 58, 75e85. Rousk, J., Brookes, P.C., Bååth, E., 2009b. Contrasting soil pH effects on fungal and bacterial growth suggests functional redundancy in carbon mineralization. Applied and Environmental Microbiology 75, 1589e1596. Rousk, J., Brookes, P.C., Bååth, E., 2011. Fungal and bacterial growth responses to N fertilization and pH in the 150-year “Park Grass” UK grassland experiment. FEMS Microbiology Ecology 76, 89e99. Sawada, K., Funakawa, S., Kosak, T., 2008. Soil microorganisms have a threshold concentration of glucose to increase the ratio of respiration to assimilation. Soil Science and Plant Nutrition 54, 216e223. Stenström, J., Stenberg, B., Johansson, M., 1998. Kinetics of substrate-induced respiration (SIR): theory. Ambio 27, 35e39. Strickland, M.S., Rousk, J., 2010. Considering fungal:bacterial dominance in soils e methods, controls, and ecosystem implications. Soil Biology & Biochemistry 42, 1385e1395. Teklay, T., Nordgren, A., Malmer, A., 2006. Soil respiration characteristics of tropical soils from agricultural and forestry land-uses at Wondo Genet (Ethiopia) in response to C, N and P amendments. Soil Biology & Biochemistry 38, 125e133. Thiet, R.K., Frey, S.D., Six, J., 2006. Do growth yield efficiencies differ between soil microbial communities differing in fungal: bacterial ratios? Reality check and methodological issues. Soil Biology & Biochemistry 38, 837e844. van der Wal, A., van Veen, J.A., Pijl, A.S., Summerbell, R.C., de Boer, W., 2006. Constraints on development of fungal biomass and decomposition processes during restoration of arable sandy soils. Soil Biology & Biochemistry 38, 2890e2902. Wutzler, T., Blagodatsky, S.A., Blagodatskaya, E., Kuzyakov, Y., 2012. Soil microbial biomass and its activity estimated by kinetic respiration analysis e statistical guidelines. Soil Biology & Biochemistry 45, 102e112. Ziegler, S.E., White, P.M., Wolf, D.C., Thoma, G.J., 2005. Tracking the fate and recycling of 13C-labeled glucose in soil. Soil Science 170, 767e778.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
The effects of glucose loading rates on bacterial and fungal growth in soil Stephanie Reischke*, Johannes Rousk, Erland Bååth Section of Microbial Biology, Department of Biology, Lund University, Ecology Building, SE-223 62 Lund, Skåne, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2013 Received in revised form 9 December 2013 Accepted 15 December 2013 Available online 24 December 2013
Microbial activity in soil is usually limited by the availability of carbon (C). Adding an easily available C source, like glucose, has therefore been a common approach to study alleviation of resource limitations. Most such studies have relied on respiration to study microbial dynamics, with few following the explicit growth response. We determined the response in bacterial and fungal growth, as well as respiration, to additions of glucose (0.5e32 mg C g1 soil) during up to 6 days, using leucine incorporation for bacterial growth and acetate-in-ergosterol incorporation for fungal growth. A concentration of 2 mg glucose-C g1 soil, where the fungal contribution appeared to be small, was also studied with a high time resolution. Adding glucose resulted in an initial lag phase of stable respiration and bacterial growth. Bacterial growth was similar to the unamended control, while respiration was 8 fold higher during this period. The 14-h lag phase was followed by an exponential increase for both respiration and bacterial growth, with a similar intrinsic growth rate (m) of around 0.25 h1. After the exponential phase, bacterial growth decreased exponentially. The respiration initially decreased even more rapidly than bacterial growth. At concentrations exceeding 4 mg glucose-C g1 the relative stimulation of fungal growth surpassed that of bacteria, with the highest amendment rates, 32 mg C g1, resulting in mainly fungal growth. Lower loading rates than 4 mg glucose-C g1 appeared to stimulate mainly bacterial growth. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Substrate loading rates Glucose Bacterial growth Fungal growth Respiration 3 H-leucine incorporation 14 C-acetate incorporation Mineralisation Decomposition
1. Introduction Microbial activity in soil is limited by the availability and quality of resources, where especially carbon (C) limitation has been found to be common in soil (Joergensen and Scheu, 1999; Ilstedt and Singh, 2005; Demoling et al., 2007). One common approach to study microorganisms in soil has therefore been to add an easily available Csource, like glucose. Saturating the microbial C-demand with glucose in soil has resulted in a number of useful observations. For instance, the level of the instantaneous increase in respiration rate is proportional to the microbial biomass in soil (the substrate-induced respiration, or SIR rate; Anderson and Domsch, 1978; Höper, 2006), and the exponential increase in respiration rate occurring within a day or so after addition can be used to estimate the intrinsic rate of the increase in microbial biomass induced by the glucose addition (Stenström et al., 1998; Blagodatskaya et al., 2009; Wutzler et al., 2012). The physiological state of the microbial biomass can be estimated using kinetic modelling based on respiration data after adding glucose (Blagodatskaya et al., 2007). High glucose
* Corresponding author. Tel.: þ46 46 222 37 63. E-mail address: [email protected] (S. Reischke). 0038-0717/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2013.12.011
amendment rates are also used to estimate the microbial availability of nutrients in soil (Nordgren, 1992; Ilstedt et al., 2007). The microbial response, measured as respiration, to substantial additions of glucose usually results in three phases. The first is the lag phase, were no observable extra growth on the substrate can be measured. This is followed by an exponential increase in activity due to growth on the added substrate until peak activity, when the C-resource is exhausted. The third phase is the decline in activity after peak rates. Glucose addition induces an almost instant increase in microbial respiration (Anderson and Domsch, 1978; Ehlers et al., 2010). The level of respiration response increases with the loading rate of glucose until the microbial C-demand is saturated. If a surplus of glucose above that needed for saturation is added, this saturation level of respiration will stay constant for around 4e15 h at temperatures w20 C (Anderson and Domsch, 1973, 1978; Blagodatskaya et al., 2007). This initial stable C-saturated microbial respiration, the SIR level, has been interpreted as a lag phase, where the growth rate of the microbial community remains unchanged from the rate before the substrate addition (Anderson and Domsch, 1985; Nordgren et al., 1988). However, it can also be interpreted as an apparent lag phase, where a small fraction of the microorganisms starts to increase their growth rate immediately on
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
the added glucose. This increase will not be detectable during the apparent lag phase, since it is masqued by the respiration of the larger fraction of microorganisms whose growth rate does not increase from glucose, but it can be estimated using a kinetic model for growth (Stenström et al., 1998; Blagodatskaya et al., 2007; Wutzler et al., 2012). If sufficient glucose is added not to be exhausted during the lag phase, and there is no other limiting nutrient, the stable SIR level is followed by an exponential increase in respiration. This exponential phase will continue until respiration rate peaks at the point when glucose is exhausted. The additional microbial respiration reflects growth of the microbes on the added C source, and the intrinsic growth rate, m, for this period of exponential growth can be calculated (Nordgren et al., 1988; Stenström et al., 1998; Blagodatsky et al., 2000; Blagodatskaya et al., 2007). In the third phase, after the respiration peak, respiration will drastically decrease again, to gradually converge with the respiration rate before the addition (Anderson and Domsch, 1973; Teklay et al., 2006). This phase of declining respiration has not received explicit study to date. There are reports that have monitored microbial growth by measuring changes in biomass, however, with conflicting results. Marstorp and Witter (1999) followed the increase in the DNA concentration as a proxy for growth and found a good correlation between the initiation and extent of the exponential response of respiration and the increase in DNA. Anderson and Martens (2013), on the other hand, found discrepancies between respiration and changes in DNA content, with lower growth rates estimated from the DNA concentration than those inferred from respiration data. One reason for these incongruities is that changes in biomass, especially during the initial lag phase of constant respiration and the early exponential phase, are difficult to estimate with sufficient precision. Only one study to date has measured bacterial growth directly after adding glucose using the leucine incorporation technique (Iovieno and Bååth, 2008). Although they found that during the lag phase in respiration bacterial growth was stable, their sampling scheme did not have sufficient time resolution to compare growth during the exponential phase and the period after peak respiration. So far no direct estimates of fungal growth after adding glucose to soil have been made. However, fungal and bacterial biomass estimations after prolonged incubations with different loading rates of easily available C have indicated that higher substrate concentrations will favour fungal over bacterial biomass synthesis (Griffiths et al., 1999). We have compared respiration, bacterial growth and fungal growth after the addition of a wide range of glucose concentrations over a 6 day period to cover all three growth phases. First, we followed the development of respiration and growth after adding one concentration of glucose (2 mg C g1 soil) at a high time resolution. We specifically tried to answer the following questions: Does the lag phase in respiration coincide with a concomitantly stable rate of bacterial or fungal growth? Is the exponential growth phase in respiration explained by bacterial or fungal growth? Secondly, we compared a range of different loading rates of glucose. The chosen range of glucose additions, covering 0.5e32 mg glucose-C g1 soil, matched the range used by Griffiths et al. (1999) to compare bacterial and fungal biomass responses. In this second part we addressed the questions: Is there a shift between bacterial and fungal growth with different glucose loading rates in soil? Is the respiration response due to different organism groups depending on the loading rates? 2. Material and methods 2.1. Soil preparation and incubation conditions Freshly collected soil was sieved using a 2.8 mm mesh and stored at 4 C until used. The soil is classified as sandy loamy brown
89
earth soil (Cambisol, FAO; Inceptisol, USDA) from managed grassland from south-eastern Sweden. Soil parameters were: pH(H2O) ¼ 6.7 0.3 (mean se, n ¼ 3), soil organic matter content ¼ 13.5 0.8%, and water content ¼ 26.2 3.5%. In each experiment 100 g soil were weighed into 180 ml containers and amended with different glucose concentrations (see below). To avoid inducing nutrient limitation by other resources than C, NH4NO3 and KH2PO4 were added at a final concentration of 0.1 mg N and P per 2 mg C added to the soil as glucose. After the glucose addition the soil samples were homogenized by shaking 1 min and mixing with a spatula for 30 s, and then incubated at 20 C in a temperature controlled room. 2.2. Experimental setup Two experiments were conducted. Microbial activity was measured as respiration, bacterial growth and fungal growth during each experiment. In experiment 1, 2.0 mg glucose-C g1 soil was added and the microbial activity was measured every 2 h during the lag and the exponential phase, and with longer time intervals later, over totally 169 h. Fungal activity was only followed for 72 h. To achieve a time-resolution of every second hour, glucose was added to three separate sets of soil at three different time points: in the morning, in the afternoon and in the evening. An unamended control was also included, but with a less intensive sampling schedule. Two replicates were started at each of the three time points, and all presented values are the mean of the duplicates. Soil pH was measured after 2 and 24 h and was found to be minimally affected by the addition (<0.1 pH unit). In experiment 2 seven glucose concentrations (0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 mg glucose-C g1 soil) were added to the soil in addition to an unamended control. This experiment was performed at two separate occasions. The lower range of concentrations (0e 4 mg glucose-C g1 soil) was first examined for respiration and microbial growth during 146 h. Subsequently, the higher range of concentrations (8e32 mg glucose-C g1 soil) was studied, where respiration and microbial growth were followed for 183 h. To ensure reproducible experimental conditions the unamended control and 2.0 mg glucose-C g1 soil treatments were included also in this second batch. Since this experiment did not focus on the lag phase, only a few samples were taken before the exponential growth increase. Soil pH was measured after 2 and 24 h. Adding the 16 mg glucose-C g1 soil treatment (with nutrients) decreased pH to 6.5 and adding 32 mg glucose-C g1 soil to pH 6.3, while pH decreased with <0.2 units for the other treatments. 2.3. Microbial measurements of respiration and growth 2.3.1. Respiration Respiration was measured by transferring 1.0 g soil into a 20 ml glass vial and purging the headspace with pressurized air. The vial was closed with a crimp lid and incubated for 2 h at 20 C. Afterwards the headspace CO2 concentration was analysed using a gas chromatograph with a thermal conductivity detector. 2.3.2. Bacterial growth To estimate bacterial growth, 1.0 g soil was transferred into a 50 ml centrifuge tube and the leucine (Leu) incorporation method (Kirchman et al., 1985) in bacteria extracted from soil using the homogenization/centrifugation techniques (Bååth, 1994) with modifications (Bååth et al., 2001) was applied. We used a mixture of radio-labelled Leu, [3H]Leu (37 MBq ml1, 5.74 TBq mmol1, Perkin Elmer, USA) and unlabelled Leu, resulting in 275 nM Leu in the bacterial suspension, and a 2 h incubation at 20 C. After washing out unincorporated Leu (Bååth et al., 2001), the amount of
90
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
incorporated radioactivity was determined by using a liquid scintillator. The incorporation of 3H-leucine, expressed as pmol Leu incorporated in extracted bacteria g1 soil h1, was used as a proxy for bacterial growth.
2
Respiration (µg CO -C g-1 h-1)
50
2.3.3. Fungal growth and ergosterol concentration To estimate fungal growth, 1.0 g soil was transferred into a 10 ml glass tube and the acetate-in-ergosterol incorporation, which was originally developed for aquatic environments (Newell and Fallon, 1991) and later modified for soil by Bååth (2001), was measured. We used a mixture of 20 ml 1-[14C]acetic acid (sodium salt, 2.04 GBq mmol1, 7.4 MBq ml1, Perkin Elmer, USA) and unlabelled acetate resulting in a final acetate concentration of 220 mM, and an incubation time of 2 h at 20 C. To separate and quantify the ergosterol, we used an HPLC with a UV detector (282 nm) and a fraction collector (Rousk and Bååth, 2007). The incorporated radioactivity of the ergosterol fraction was measured by using liquid scintillation. The amount of incorporated 14C-acetate, expressed as pmol g1 soil h1, was used as a proxy for fungal growth, while the ergosterol concentration was used as a proxy of fungal biomass.
40
cap dec
30
20
10
Bacterial growth (pmol Leu g-1 h-1)
50
100
150
-1
where Y is respiration or bacterial growth, A and B the part of the biomass not growing and growing on the added glucose, respectively, m is the intrinsic growth rate and t is the time after adding glucose (Stenström et al., 1998; Blagodatsky et al., 2000). The lag phase was then calculated as
(2)
200
B
600 500 400 300 200 100 0
0
50
100
150
200
C
-1
Fungal growth (pmol Ac g h )
(1)
using a parametric bootstrap procedure with 10000 bootstrap samples to calculate SE (Efron, 1979). We also used a simple two part model, assuming a real lag phase, with a constant activity during the lag phase and calculating the intrinsic growth rate (m) from the linear slope of the log-transformed data during the exponential phase (“the linear approach”). The lag phase was then determined by the time-point when the modelled curves for the lag and exponential phase intersected. This calculation was used in experiment 2 (different loading rates), since insufficient data were available to be able to fit Eq. (1) with sufficient precision. After the peak rate, the decrease in bacterial growth was modelled using a negative exponential equation, since bacterial death is known to be logarithmic. Respiration decreased very rapidly immediately after peak respiration (Fig. 1), followed by a slower decrease. Lacking any mechanistic hypothesis for this interval, data points were here connected by a line, and not modelled. As an index for the microbial growth efficiency, we used the ratio between respiration and growth (dividing the respiration (mg CO2 g1 h1) by the bacterial growth (pmol Leu g1 h1 into extracted bacteria)), where high values indicate low efficiency, and vice versa. We only estimated the microbial growth efficiency in a situation dominated by bacterial growth (experiment 1). Since we have not established conversion factors from leucine incorporation
A
fast dec
0
Different time intervals of the respiration and bacterial growth curves after the addition of 2 mg glucose-C g1 soil (Fig. 1) were modelled with different equations. The initial stable phase for bacterial growth and respiration after adding glucose can be modelled either as a real or as an apparent lag phase. The latter was modelled for the combined lag and exponential phase until peak activity at 22 h as
lag phase ¼ lnðA=BÞ=m
comb 2 mgequ C
0
3. Calculations
Y ¼ A þ Bemt
0Control resp
30
20
10
0
0
50
100
150
200
Time (h) Fig. 1. Respiration (A), bacterial growth as Leu incorporation (B) and fungal growth as Ac-in-ergosterol incorporation (C) after the addition of 2 mg glucose-C g1 soil (:) and an unamended control (C) during incubation at 20 C. The lag and exponential phase was modelled using Eq. (1). Respiration: y ¼ 11.6 þ 0.16e0.24x (R2 ¼ 0.99); bacterial growth: y ¼ 44.5 þ 2.2e0.25x (R2 ¼ 0.98). After peak bacterial growth the decline was modelled by y ¼ 549e0.0043x (R2 ¼ 0.79).
to biomass production in soil, we standardized the data to a mean value of one over time for the unamended control treatment. 4. Results 4.1. High temporal resolution of the 2 mg glucose-C g1 addition Respiration increased to values 8-fold those in the unamended control immediately after adding 2 mg glucose-C g1 soil; a level that remained constant during the first 14 h (Fig. 1A). Up to 22 h respiration then increased exponentially, with a calculated growth rate, m, of 0.14 h1 using the linear approach. Estimating the growth rate with Eq. (1) resulted in a m of 0.24 0.014 h1 and a lag phase of 17 1.9 h. Peak respiration occurred after 22 h at values 4-fold
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
4.2. Effects of glucose loading rates on fungal and bacterial growth The first measurement, 8 h after glucose additions, showed up to 8-fold higher respiration rates compared to the control (Fig. 4A, B). The two highest loading rates, 16 and 32 mg C g1 soil, resulted in lower respiration during the lag phase, being 50 and 30%, respectively, of that in treatments with <8 mg C g1 soil. The highest loading rates also had longer lag phases, with 16 and 32 mg C g1 soil resulting in 16 and 25 h lag phases, respectively, compared to around 11e13 h below 4 mg C g1 soil. Addition of 2 mg glucoseC g1 or more resulted in increased respiration after the lag phase. This was not found for the lowest concentrations (0.5 and 1 mg glucose-C g1). Growth rate, m, estimated with the linear approach, did not vary with glucose concentration, seen as almost parallel increases in respiration during the exponential phase (Fig. 4A, B). Peak respiration increased with glucose concentration, ranging from around a 50-fold higher rate than in the control for the 2 and 4 mg C g1 treatments, and up to a 240-fold higher rate for the highest addition. After peak respiration, the rates of all treatments, except the 32 mg C g1 addition, decreased rapidly towards the control value (Fig. 4A, B), but still up to 14-fold higher values were found at the end of the study (>140 h). Bacterial growth showed similar values as the control during the first 8e12 h for glucose concentrations up to 16 mg C g1 soil (Fig. 4C, D). Adding 16 mg C g1 decreased growth to around 60% of that in the other glucose treatments, while the highest glucose addition (32 mg C g1) showed very low bacterial growth over the entire incubation time (Fig. 4D). The lag phase was stable up to 4 mg C g1 soil (8e12 h), but increased to 19 h for 8 mg C g1 soil and around 35 h for 16 mg C g1 soil. Except for the 0.5 mg C g1
A
2
-1
Cum. respiration (mg CO -C g )
1
-1
Cum. bacterial growth (nmol Leu g )
Control 0 2000 2 mg C
0.8
0.6
0.4
0.2
0 0 60
50
100
150
200
50
100
150
200
100
150
200
B
50 40 30 20 10 0 0 2.5
C
-1
Cum. fungal growth (nmol Ac g )
higher than during the preceding lag phase. Respiration then decreased rapidly within the next 6 h followed by a slower decline over the next 140 h (Fig. 1A). After 168 h incubation the glucose treated soil still respired at a 2-fold higher rate than in the unamended control. Bacterial growth in the glucose-amended soil did not change compared to the control during the first 12e14 h (Fig. 1B). During the next 10 h (up to 22 h) bacterial growth showed an exponential increase, with a calculated m of 0.17 h1 using the linear approach. Using Eq. (1) resulted in a m of 0.25 0.024 h1 and a lag phase of 13 3.3 h, with no significant differences to corresponding values for respiration. At peak values after 22 h, bacterial growth was 9fold higher than the control. Bacterial growth then decreased exponentially with a slope of 0.0043 0.00061 h1. Bacterial growth still was 6-fold higher compared to the control at the end of the experiment. Fungal growth was marginally affected by the 2 mg glucoseC g1 soil treatment, but values tended to exceed those of the control more than 20 h after addition (Fig. 1C). Cumulative data indicated that fungal growth (Fig. 2C) only increased to 1.3 times those of the control after 72 h compared to bacterial growth, which increased 6-fold (Fig. 2B) and respiration 10-fold that in the control (Fig. 2A). Thus, the elevated respiration after adding glucose appeared mostly to be related to bacterial and not fungal growth. With minimal fungal contribution, bacterial growth could be assumed to reflect total microbial growth and thus we used the ratio of respiration-to-bacterial growth as a proxy for changes in the bacterial C-use efficiency (Fig. 3). The increased respiration and stable leucine incorporation during the lag phase resulted in very high values, suggesting low bacterial C-use efficiency. The ratio then declined during the exponential phase (14e24 h), but was still higher than in the control. After peak respiration, during the exponential decline of bacterial growth, the relative respiration to bacterial growth ratio was lower than in the unamended control.
91
2
1.5
1
0.5
0 0
50
Time (h) Fig. 2. Cumulative respiration (A), bacterial growth as Leu incorporation (B) and fungal growth as Ac-in-ergosterol incorporation (C) after the addition of 2 mg glucoseC g1 soil (:) and an unamended control (C) during the incubation at 20 C for 169 h (A and B) and 78 h (C).
treatment all other glucose addition treatments showed an exponential increase after the lag phase (Fig. 4C, D). Growth rate, m, estimated with the linear approach, varied between 0.15 and 0.19 h1 for the treatments with 1e4 mg glucose-C g1, while growth rates decreased to 0.11 h1 and 0.025 h1 for 8 and 16 mg C g1 soil, respectively. At 32 mg C g1 m was not possible to estimate. Peak bacterial growth was 5e20-fold higher than in the control. Bacterial growth declined in the latter part of the experiment, but was still 3e7-fold higher compared to the control at the end of the experiment. Fungal growth was almost constant after glucose additions below 8 mg C g1 over the whole incubation period, with only minor increases compared to the control (Fig. 4E, F). However, using 32 mg glucose-C g1 we obtained the highest level of fungal growth (10-fold higher than in the control) with m estimated to 0.065 h1
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
Relative respiration:bacterial growth
92
14 12 10 8 6 4 2 0
0
50
100
150
200
Time (h) Fig. 3. Relative ratio between respiration and bacterial growth as Leu incorporation following the addition of 2 mg glucose-C g1 soil (:) and an unamended control (C) over 169 h at 20 C.
using the linear approach. Fungal growth declined rapidly after peak activity (Fig. 4F). We also found a clear fungal growth response in the 16 mg glucose-C g1 treatment, increasing to peak levels after 42 h and then decreasing until converging with the values of the control treatment towards the end of the study. Cumulative respiration increased with higher loading rates of glucose (Fig. 5). There was a shift with increasing substrate loading rates from a situation dominated by bacterial growth to a situation dominated by fungal growth. At 8 mg glucose-C g1 soil both bacterial and fungal growth appeared to contribute to the respiration. At and below 4 mg C g1 soil bacteria was the main responding organism group, while at 16 and especially at 32 mg C g1 fungal growth dominated the respiration response. The ergosterol content at the end of the experiment was used as an alternative way of expressing accumulated fungal growth (Fig. 5). Both the ergosterol content and the cumulative fungal growth, estimated from Ac-in-ergosterol incorporation, showed corroborative results with almost no growth below 8 mg added glucose-C g1, and then increasing with higher glucose loading rates. 5. Discussion 5.1. Comparing dynamics of microbial growth to respiration Adding 2 mg glucose-C g1 soil resulted in a lag phase, an exponential phase and a declination phase; the phases mostly coincidental for bacterial growth and respiration (Fig. 1). In contrast, fungi appeared to only react to a minor degree to the addition, and with a different time dynamic. It is widely held that bacteria are the principal group to rapidly react to an increased concentration of labile C-substrate (Moore et al., 2005; Paterson et al., 2008). Data on incorporation of low amounts of 13C-glucose into PLFAs indicative of bacteria and fungi in grassland and agricultural soils at near neutral pHs have also often found such results (Ziegler et al., 2005; Dungait et al., 2013), although in other habitats fungi may be more important (Rinnan and Bååth, 2009). Thus, we conclude that at this level of added glucose to this soil, bacteria initially dominated the decomposition and mineralisation of the added substrate. The degree of the response of bacterial growth and respiration to the glucose differed after addition, however. During the lag phase, we found no change in bacterial growth rates compared to
the control, while respiration was constantly high (at the SIR level). This implied an uncoupling between respiration and bacterial growth (see below on discussion on C-use efficiency). The lack of bacterial growth increase on the added glucose during the lag phase is consistent with the results of Iovieno and Bååth (2008), and studies on biomass changes (e.g. Marstorp and Witter, 1999) as well as plate counts (Anderson and Domsch, 1985) after adding glucose. The lag phase was followed by an exponential increase in respiration and bacterial growth (Figs. 1 and 2). Marstorp and Witter (1999) compared respiration with kinetics of dsDNA formation and also found that the exponential increase in respiration was reflected in an exponential increase in microbial biomass. Our estimated growth rates (m) of 0.14e0.17 h1 (using the linear approach) or 0.24e0.25 h1 (using Eq. (1)) were within the values found by Blagodatskaya et al. (2007; ranging between 0.10 and 0.35 h1) and those compiled by Anderson and Martens (2013; m ranging between 0.14 and 0.41 h1). The use of Eq. (1) has earlier been shown to result in higher estimates of m than using the linear approach (Blagodatskaya and Kuzyakov, 2013), which is consistent with our results. More important is that m calculated from respiration and bacterial growth data during the exponential phase matched, irrespective of method of calculation. Peak respiration and bacterial growth also coincided in time. Similar m and time of peak activities for bacterial growth and respiration data suggest that bacteria dominated the use of glucose during the exponential phase. The final decline in respiration has received less study than the exponential increase. This decrease appeared to be composed of two parts; an initial very rapid decrease followed by a slower decrease. This two-part pattern in respiration after peak values was also found after rewetting soils dried for a long period (Meisner et al., 2013). Bacterial growth during the declination phase was tentatively modelled by a negative exponential equation, similar to the decline in bacterial growth after peak values following the wetup of a dry soil (Meisner et al., 2013). Respiration initially decreased much more rapidly than bacterial growth. Similar observations have been made in pure culture studies, where respiration per cell has been found to rapidly decrease by up to two orders of magnitudes during the transition from exponential to early stationary phase (Riedel et al., 2013). After the rapid respiration decrease, the rate of the decline of respiration began to align with that for bacterial growth, although it was still almost twice as fast. During this period, glucose in the soil will be exhausted (Anderson and Domsch, 1985; Thiet et al., 2006; Sawada et al., 2008). Growth higher than in the control will then be due to glucose taken up into storage products (Bååth, 2003), recycling of the biomass already formed while growing on glucose, or growth on soil organic matter released through priming (Blagodatskaya and Kuzyakov, 2008). Assuming that bacteria are the main group respiring and growing on glucose during the initial time period of experiment 1, the high respiration-to-bacterial growth during the lag phase (Fig. 3) suggests an immediate uncoupling of growth and respiration, either due to waste metabolism of excess energy (Neijssel and Tempest, 1976) or due to glucose being mainly used for catabolism during this period. However, part of the explanation could also be that glucose is taken up and used for storage, that is, without growth, but still with a respiration cost (Bremer and Kuikman, 1994; Sawada et al., 2008). During the exponential phase the respiration-to-bacterial growth ratio decreased, suggesting increasing C-use efficiency during this period. During the declining phase the respiration-to-bacterial growth ratio decreased to values even lower than in the unamended control. However, we do not think this suggests that the microbial community has become even more C-use efficient than in the control soil. Rather, the microbial
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
200
A
Control 0.5 1.0 2.0 4.0
150
B
Control 2.0 8.0 16.0 32.0
150
2
Respiration (µg CO -C g-1 h-1)
200
93
100
100
50
50
0
1000
50
100
150
200
0
0
1000
C
800
800
600
600
400
400
200
200
50
100
150
200
50
100
150
200
100
150
200
D
-1
-1
(pmol Leu g h )
Bacterial growth in extracted bacteria
0
0
0
Fungal growth (pmol Ac g-1 h-1)
35
50
100
150
200
0
250
E
30
0
F
( )
200 25 150
20 15
100
10 50 5 0
0
50
100
150
Time (h)
200
0
0
50
Time (h)
Fig. 4. Respiration, bacterial growth as Leu incorporation and fungal growth as Ac-in-ergosterol incorporation measured at two separate occasions with up to 4 mg added glucoseC g1 soil (A, C and E) and with higher concentrations (B, D and F). In A, C and E, 0, 0.5, 1, 2 and 4 mg glucose-C g1 soil were added and microbial activity was measured during 146 h. In B, D and F, 0, 2, 8, 16 and 32 mg glucose-C g1 soil were added and microbial activity was measured during 195 h. The data-point within the parenthesis was considered an outlier.
growth in the control soil will be composed of both bacterial and fungal contributions, and using only bacterial growth would underestimate C-use efficiency. This underscores the importance of including both fungal and bacterial growth when estimating microbial C-use efficiencies, although we might have periods where mostly bacteria or fungi are growing, as determined by different loading rates (as here shown) or differences in soil pHs (BárcenasMoreno et al., 2011). Summarizing, respiration-to-bacterial growth can vary substantially during a substrate-induced growth event, as found for pure culture studies (Riedel et al., 2013). 5.2. Effect of loading rates With increasing glucose loading rates the microbial community growth progressively shifted from one dominated by bacteria to
one dominated by fungi (Figs. 4 and 5). These findings corroborate those of Griffiths et al. (1999), who showed that higher substrate loading rates of easily available substrate selected for fungi. A possible explanation could be a high osmolarity in the soil solution at high substrate loading rates. Fungi are generally considered to be able to handle high osmotic stress better than bacteria (Griffin, 1972), and fungal growth at high loading rates could be due to fast-growing osmo-tolerant fungi (e.g. yeast) (van der Wal et al., 2006). Griffiths et al. (1999) also suggested that organisms dominating at high substrate loading rates are better able to cope with the relatively lower availability of other nutrients, which might occur under these conditions. The hyphal growth form and the more flexible stoichiometric homeostasis of fungi (Strickland and Rousk, 2010) may be important for this aspect. A negative effect of altered osmotic potential at higher loading rates was evident in
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95
Relative cumulative values after 146 h
94
concentrations. This would be at concentrations that earlier have been suggested to trigger microbial activity in soil (De Nobili et al., 2001).
resp Respiration
50
Bacterial growth bg Fungal growth fg
erg Ergosterol
40
Acknowledgements
30
This work was supported by grant from the Swedish Research Council to E.B. (grant no 621-2012-3450) and to J.R. (grant no 6212011-5719). This work was part of Lund University Centre for Studies of Carbon Cycle and Climate Interactions (LUCCI).
20 References
10
0
0
1
10 -1
Glucose concentration (mg C g ) Fig. 5. Relative values of cumulative respiration, bacterial growth as Leu incorporation, and fungal growth as Ac-in-ergosterol incorporation at a glucose concentration range from 0 to 32 mg C g1 soil. Ergosterol concentrations as a proxy of fungal biomass at the end of the incubation are also shown. The data were standardized to 1 for the unamended control. Note the log scale on the x-axis.
the lower bacterial growth during the lag phase with increasing glucose addition rates, as also observed when optimising glucose concentrations to measure SIR-responses (Rousk et al., 2009a). This putative osmotic effect was also evidenced in the longer lag phase in respiration especially after adding 16 and 32 mg glucose-C g1. This was also observed when adding large amounts of glucose when using respiration kinetics to measure microbial available phosphorus, prompting the use of perlite as a substrate carrier (Ilstedt et al., 2007). Low pH has been shown to favour fungal compared to bacterial growth (Rousk et al., 2009b, 2011; Bárcenas-Moreno et al., 2011). However, we do not think that decreased pH due to glucose treatments (with nutrients) could explain the shift to fungal growth at high glucose loading rates. The decrease was at most only around half a pH unit, and the pH range (6.3e6.7) is within an interval where pH-related effects on bacterial growth have been found to be limited (Rousk et al., 2009b, 2011). Although initially bacterial growth was inhibited at high loading rates, at 8 and 16 mg glucose-C g1 soil bacterial growth eventually increased after a prolonged lag phase (Fig. 4D). This may be due to fungi consuming glucose, thus altering the osmotic potential creating conditions conducive for bacterial growth. Fungal growth preceding bacterial growth, apparently altering the environmental conditions, has previously been observed during the colonization of burned forest soils in relation to pH (Bárcenas-Moreno et al., 2011). Another explanation is that growth of bacteria at high glucose concentrations was due to a different community, being more osmo-tolerant than the one growing at lower substrate loading rates. That the concentration of added substrate affects the resulting microbial community has been shown repeatedly (e.g. Griffiths et al., 1999; Pennanen et al., 2004). No exponential phase was observed in the respiration measurements at the lowest concentrations added, although there was a bacterial growth response also in these treatments. This suggests that bacterial growth on glucose is not only possible when sufficient glucose is added to trigger an exponential response in respiration. Since low concentrations are more likely to prevail in the natural soil environment, this highlights the need for studying respiration and growth kinetics in the low range of substrate
Anderson, J.P., Domsch, K.H., 1973. Quantification of bacterial and fungal contributions to soil respiration. Archiv für Mikrobiologie 93, 113e127. Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215e 221. Anderson, T.H., Domsch, K.H., 1985. Maintenance carbon requirements of activelymetabolizing microbial-populations under in situ conditions. Soil Biology & Biochemistry 17, 197e203. Anderson, T.H., Martens, R., 2013. DNA determinations during growth of soil microbial biomasses. Soil Biology & Biochemistry 57, 487e495. Bååth, E., 1994. Measurement of protein-synthesis by soil bacterial assemblages with the leucine incorporation technique. Biology and Fertility of Soils 17, 147e 153. Bååth, E., 2001. Estimation of fungal growth rates in soil using 14C-acetate incorporation into ergosterol. Soil Biology & Biochemistry 33, 2011e2018. Bååth, E., 2003. The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi. Microbial Ecology 45, 373e383. Bååth, E., Pettersson, M., Söderberg, K.H., 2001. Adaptation of a rapid and economical microcentrifugation method to measure thymidine and leucine incorporation by soil bacteria. Soil Biology & Biochemistry 33, 1571e1574. Bárcenas-Moreno, G., Rousk, J., Bååth, E., 2011. Fungal and bacterial recolonisation of acid and alkaline forest soils following artificial heat treatments. Soil Biology & Biochemistry 43, 1023e1033. Blagodatskaya, E.V., Blagodatsky, S.A., Anderson, T.H., Kuzyakov, Y., 2007. Priming effects in Chernozem induced by glucose and N in relation to microbial growth strategies. Applied Soil Ecology 37, 95e105. Blagodatskaya, E.V., Blagodatsky, S.A., Anderson, T.H., Kuzyakov, Y., 2009. Contrasting effects of glucose, living roots and maize straw on microbial growth kinetics and substrate availability in soil. European Journal of Soil Sciences 60, 186e197. Blagodatskaya, E., Kuzyakov, Y., 2008. Mechanisms of real and apparent priming effects and their dependence on soil microbial biomass and community structure: critical review. Biology and Fertility of Soils 45, 115e131. Blagodatskaya, E., Kuzyakov, Y., 2013. Active organisms in soil: critical review of estimation criteria. Soil Biology & Biochemistry 67, 192e211. Blagodatsky, S.A., Heinemeyer, O., Richter, J., 2000. Estimating the active and total soil microbial biomass by kinetic respiration analysis. Biology and Fertility of Soils 32, 73e81. Bremer, E., Kuikman, P., 1994. Microbial utilization of 14C[U]glucose in soil is affected by the amount and timing of glucose additions. Soil Biology & Biochemistry 26, 511e517. De Nobili, M., Contin, M., Mondini, C., Brookes, P.C., 2001. Soil microbial biomass is triggered into activity by trace amounts of substrate. Soil Biology & Biochemistry 33, 1163e1170. Demoling, F., Figueroa, D., Bååth, E., 2007. Comparison of factors limiting bacterial growth in different soils. Soil Biology & Biochemistry 39, 2485e2495. Dungait, J.A.J., Kemmitt, S.J., Michallon, L., Guo, S., Wen, Q., Brookes, P.C., Evershed, R.P., 2013. The variable response of soil microorganisms to trace concentrations of low molecular weight organic substrates of increasing complexity. Soil Biology & Biochemistry 64, 57e64. Efron, B., 1979. Bootstrap methods: another look at the jackknife. Annals of Statistics 7, 1e26. Ehlers, K., Bakken, L.R., Frostegård, Å., Frossard, E., Bünemann, E.K., 2010. Phosphorus limitation in a Ferralsol: impact on microbial activity and cell internal P pools. Soil Biology & Biochemistry 42, 558e566. Griffin, D.M., 1972. Ecology of Soil Fungi. Chapman and Hall, London. Griffiths, B.S., Ritz, K., Ebblewhite, N., Dobson, G., 1999. Soil microbial community structure: effects of substrate loading rates. Soil Biology & Biochemistry 31, 145e153. Höper, H., 2006. Substrate-induced respiration. In: Bloem, J., Hopkins, D.W., Benedetti, A. (Eds.), Microbiological Methods for Assessing Soil Quality. CABI Publishing, Oxfordshire, pp. 84e92. Ilstedt, U., Singh, S., 2005. Nitrogen and phosphorus limitations of microbial respiration in a tropical phosphorus-fixing acrisol (ultisol) compared with organic compost. Soil Biology & Biochemistry 37, 1407e1410. Ilstedt, U., Singh, S., Nordgren, A., 2007. Using perlite as a substrate carrier for measuring microbial available phosphorus by respiration kinetics in soils. Biology and Fertility of Soils 43, 503e510.
S. Reischke et al. / Soil Biology & Biochemistry 70 (2014) 88e95 Iovieno, P., Bååth, E., 2008. Effect of drying and rewetting on bacterial growth rates in soil. Fems Microbiology Ecology 65, 400e407. Joergensen, R.G., Scheu, S., 1999. Response of soil microorganisms to the addition of carbon, nitrogen and phosphorus in a forest Rendzina. Soil Biology & Biochemistry 31, 859e866. Kirchman, D., Knees, E., Hodson, R., 1985. Leucine incorporation and its potential as a measure of protein-synthesis by bacteria in natural aquatic systems. Applied and Environmental Microbiology 49, 599e607. Marstorp, H., Witter, E., 1999. Extractable dsDNA and product formation as measures of microbial growth in soil upon substrate addition. Soil Biology & Biochemistry 31, 1443e1453. Meisner, A., Bååth, E., Rousk, J., 2013. Microbial growth responses upon rewetting soil dried for four days or one year. Soil Biology & Biochemistry 66, 188e192. Moore, J.C., McCann, K., de Ruiter, P.C., 2005. Modeling trophic pathways, nutrient cycling, and dynamic stability in soils. Pedobiologia 49, 499e510. Neijssel, O.M., Tempest, D.W., 1976. Role of energy-spilling reactions in growth of Klebsiella-Aerogenes Nctc-418 in aerobic chemostat culture. Archives of Microbiology 110, 305e311. Newell, S.Y., Fallon, R.D., 1991. Toward a method for measuring instantaneous fungal growth-rates in field samples. Ecology 72, 1547e1559. Nordgren, A., 1992. A method for determining microbially available-N and available-P in an organic soil. Biology and Fertility of Soils 13, 195e199. Nordgren, A., Bååth, E., Söderström, B., 1988. Evaluation of soil respiration characteristics to assess heavy-metal effects on soil-microorganisms using glutamicacid as a substrate. Soil Biology & Biochemistry 20, 949e954. Paterson, E., Osler, G., Dawson, L.A., Gebbing, T., Sim, A., Ord, B., 2008. Labile and recalcitrant plant fractions are utilised by distinct microbial communities in soil: independent of the presence of roots and mycorrhizal fungi. Soil Biology & Biochemistry 40, 1103e1113. Pennanen, T., Caul, S., Daniell, T.J., Griffiths, B.S., Ritz, R., Whatley, R.W., 2004. Community-level responses of metabolically-active soil organisms to the quantity and quality of substrate inputs. Soil Biology & Biochemistry 36, 841e 848. Riedel, T.E., Berelson, W.M., Nealson, K.H., Finkel, S.E., 2013. Oxygen consumption rates of bacteria under nutrient-limited conditions. Applied and Environmental Microbiology 79, 4921e4931.
95
Rinnan, R., Bååth, E., 2009. Differential utilization of carbon substrates by bacteria and fungi in tundra soil. Applied and Environmental Microbiology 75, 3611e3620. Rousk, J., Bååth, E., 2007. Fungal and bacterial growth in soil with plant materials of different C/N ratios. FEMS Microbiology Ecology 62, 258e267. Rousk, J., Aldén Demoling, L., Bååth, E., 2009a. Contrasting short-term antibiotic effects on respiration and bacterial growth compromises the validity of the selective respiratory inhibition technique to distinguish fungi and bacteria. Microbial Ecology 58, 75e85. Rousk, J., Brookes, P.C., Bååth, E., 2009b. Contrasting soil pH effects on fungal and bacterial growth suggests functional redundancy in carbon mineralization. Applied and Environmental Microbiology 75, 1589e1596. Rousk, J., Brookes, P.C., Bååth, E., 2011. Fungal and bacterial growth responses to N fertilization and pH in the 150-year “Park Grass” UK grassland experiment. FEMS Microbiology Ecology 76, 89e99. Sawada, K., Funakawa, S., Kosak, T., 2008. Soil microorganisms have a threshold concentration of glucose to increase the ratio of respiration to assimilation. Soil Science and Plant Nutrition 54, 216e223. Stenström, J., Stenberg, B., Johansson, M., 1998. Kinetics of substrate-induced respiration (SIR): theory. Ambio 27, 35e39. Strickland, M.S., Rousk, J., 2010. Considering fungal:bacterial dominance in soils e methods, controls, and ecosystem implications. Soil Biology & Biochemistry 42, 1385e1395. Teklay, T., Nordgren, A., Malmer, A., 2006. Soil respiration characteristics of tropical soils from agricultural and forestry land-uses at Wondo Genet (Ethiopia) in response to C, N and P amendments. Soil Biology & Biochemistry 38, 125e133. Thiet, R.K., Frey, S.D., Six, J., 2006. Do growth yield efficiencies differ between soil microbial communities differing in fungal: bacterial ratios? Reality check and methodological issues. Soil Biology & Biochemistry 38, 837e844. van der Wal, A., van Veen, J.A., Pijl, A.S., Summerbell, R.C., de Boer, W., 2006. Constraints on development of fungal biomass and decomposition processes during restoration of arable sandy soils. Soil Biology & Biochemistry 38, 2890e2902. Wutzler, T., Blagodatsky, S.A., Blagodatskaya, E., Kuzyakov, Y., 2012. Soil microbial biomass and its activity estimated by kinetic respiration analysis e statistical guidelines. Soil Biology & Biochemistry 45, 102e112. Ziegler, S.E., White, P.M., Wolf, D.C., Thoma, G.J., 2005. Tracking the fate and recycling of 13C-labeled glucose in soil. Soil Science 170, 767e778.