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Induced N-limitation of bacterial growth in soil: Effect of carbon loading and N status in soil
Soil Biology & Biochemistry 74 (2014) 11e20
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Induced N-limitation of bacterial growth in soil: Effect of carbon loading and N status in soil Pramod N. Kamble a, b, c, Erland Bååth a, * a
Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62 Lund, Sweden P. G. Department of Environmental Science, PVP College Pravaranagar, University of Pune, Pune, India c K.T.H.M. College Nashik, University of Pune, Pune, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2013 Received in revised form 22 January 2014 Accepted 20 February 2014 Available online 10 March 2014
Application of C-rich plant residues can change the soil system from C-limitation for microbial growth to limitation by other nutrients. However, the initial nutrient status of the soil may interact with the added amount of residues in determining limitation. We studied this interactive effect in soils from the Harvard Forest LTER, where annual addition of N since 1988 has resulted in soils with different N-status: No N (Unfertilized), 50 (Low N) and 150 (High N) kg N ha1. We hypothesized that adding C-rich substrate would change the soil from being C- to being N-limited for bacterial growth and that the extent of Nlimitation would be higher with increasing substrate additions, while becoming less evident in soil with increasing N-status. We compared the effect of adding two C-rich substrates, starch (0, 10, 20, 40 mg g1 soil) and straw (0, 20, 40, 80 mg g1), incubating the soils for up to 3 and 4 weeks for starch and straw, respectively. Nutrient limitations were studied by measuring bacterial growth 3 days after adding C as glucose and N as NH4NO3 in a full factorial design. Initially bacterial growth in all soils was C-limited. As hypothesized, adding C-rich substrates removed the C-limitation, with lower amounts of starch and straw needed in the unfertilized and Low N soils than in the High N soil. Combinations of different Nstatus of the soil and amendment levels of starch and straw could be found, where bacterial growth appeared close to co-limited both by available C and N. However, at even higher amendment levels, presumable resulting in N-limitation, bacterial growth still responded less by adding N then C-limited soils by adding C. Thus, in a C-limited soil there appeared to be N available immediate for growth, while in an N-limited soil, easily available C was not immediately available. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: N-deposition C-rich substrate Induced-N limitation Bacterial growth Leucine incorporation Limiting factors
1. Introduction Growth of soil microorganisms is mainly regulated by the amount of substrate available for growth and physiochemical environmental factors like temperature, moisture content and pH. However, the microbial activity will also be regulated by nutrient limitation, where for example carbon rich substrate, like straw or cellulose, will not be decomposed at maximum rate in nitrogen limiting situations (Henriksen and Breland, 1999; Allison and Vitousek, 2005). Bacterial growth in soil appears, however, to be most commonly limited by the availability of carbon (C) (Nordgren, 1992; Joergensen and Scheu, 1999; Aldén et al., 2001; Ekblad and Nordgren, 2002; Demoling et al., 2007; Göransson et al., 2011),
* Corresponding author. Tel.: þ46 46 222 42 64. E-mail addresses: [email protected] (P.N. Kamble), [email protected] (E. Bååth). http://dx.doi.org/10.1016/j.soilbio.2014.02.015 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
but nitrogen (N) and phosphorus (P) limitation has also been reported (Aldén et al., 2001; Cleveland et al., 2002; Rinnan et al., 2007; Reed et al., 2011). Boreal forests are described as a nutrient poor system, with plant growth generally being limited by lack of N (Tamm, 1991; Ekblad and Nordgren, 2002). However, soil bacterial growth appeared to be limited by C also in these habitats (Ekblad and Nordgren, 2002; Demoling et al., 2008; Kamble et al., 2013). The extent of C-limitation can, however, vary depending on the N-status of the soil. Thus, in unfertilized boreal forest soil, only a small increase in bacterial growth was found after adding glucose as easily available C-source (to test for C-limitation), while in Nfertilized soils a much larger growth response was found, indicating increased N-availability in such soils (Demoling et al., 2008; Kamble et al., 2013). A similar tendency was found by comparing soils with different anthropogenic N-deposition, where the growth response after adding glucose was larger in soils with higher Ndeposition (Demoling et al., 2008).
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The addition of N may thus alter the response of the bacterial community after adding C, although not altering the primary limiting substance. However, the microbial community can become N-limited by adding carbon rich substrates to the soil. This is wellknown after adding straw or pure cellulose (e.g. Knapp et al., 1983; Cochran et al., 1988; Wang and Bakken, 1997; Henriksen and Breland, 1999) and is usually measured by increased decomposition or respiration when the carbon-rich substrate is added together with extra N (Henriksen and Breland, 1999; Allison and Vitousek, 2005; Güsewell and Freeman, 2005; Rousk and Bååth, 2007). This usually implies long-term measurements. Furthermore, using respiration may not always result in easy interpretable results after alleviating N-limitations (Schimel and Weintraub, 2003). Aldén et al. (2001) described a method to determine nutrient limitation in soil based on estimations of bacterial growth increases after only a few days in response to adding nutrients alleviating limitation. They also showed that this method could be used to demonstrate how adding straw altered a soil system from being C-limited to one being N-limited for bacterial growth. However, this has not been tested on a larger scale using different C-rich substrates and soils differing in N-status. We have studied the interactive effect of C-rich substrate additions on bacterial growth limitations in soils with different N-status. We took advantage of one of the longest N-fertilization studies of boreal forests, the Chronic Nitrogen Amendment Study at the Harvard Forest Long Term Ecological Research (LTER) site in central Massachusetts, USA. Since 1988 soils have been subjected to three different fertilization regimes (unfertilized, low N and high N additions), resulting in soils with different N-status. Dissolved inorganic nitrogen (ammonium þ nitrate) was around 25 and 75 times higher compared to the unfertilized control in the low N and High N treatment, respectively (McDowell et al., 2004). Our hypotheses were: 1) C-rich substrate addition (straw and starch) will change the soils from being C-limited to N-limited, 2) This change will be dependent of level of substrate added, with increasing extent of Nlimitation with increasing level of substrate addition, and the Nstatus of the soil, with decreasing extent of N-limitation with increasing N-status of the soil, 3) There will thus be a combination of substrate addition and N-status of the soil that results in colimitation or close to co-limitation by both C and N for bacterial growth. Finally, we wanted to compare if bacterial growth changed to the same extent after alleviating C or N limitation by adding easily available C or N, respectively. 2. Material and methods 2.1. Soil and experimental setup for induced N limitation Soils from the Harvard Forest Long Term Ecological Research (LTER) site in central Massachusetts, USA, were used. The Chronic Nitrogen Amendment Study was established in 1988 to study longterm effects of increased N deposition on the boreal forest ecosystem (Aber and Magill, 2004). The forest is dominated by black (Quercus velutina) and red (Quercus rubra) oak mixed with black birch (Betula lenta), red maple (Acer rubrum) and American beech (Fagus grandiflora). Soils are of the Gloucester series (fine loamy, mixed, mesic, Typic Dystrochrepts, USDA). Since 1988 these soils have been treated annually with nitrogen amendments as a proxy of increased anthropogenic N-deposition. Three different rates of nitrogen were used: No N (Unfertilized), 50 (Low N) and 150 (High N) kg N ha1 annually (applied as six equal applications over the growing season) as NH4NO3eN in four replicates plots. Soil samples were collected from replicate 5 5 m sub-plots within each treatment plot (30 30 m) (Magill et al., 1997; Aber and Magill, 2004). Two 8 cm diameter and 10 cm long cores were
removed from each sub plot, separated into the organic and mineral horizon, and bulked by soil horizon. Samples were sieved (2 mm) and stored field moist at 4 C until analyzed. Two of the replicates from the organic soil we used in the present study. pH was 5.1, 5.0 and 4.4 in Unfertilized, Low N and High N soils, organic matter (as loss on ignition, 4 h at 600 C) 0.47, 0.45 and 0.38 g g1 soil, and the water content kept at 60% of dry weight. The C/N ratio did not differ between treatments (24.7 in Unfertilized, 26.3 in Low N and 23.7 in High N; Turlapati et al., 2013). To induce nitrogen limitation, dried wheat straw and soluble starch (Merck) were applied. Straw (C/N ¼ 75), at 20, 40 and 80 mg g1 of soil, and starch, at 10, 20, 40 mg g1 of soil, were added to all three soils in duplicate (Fig. 1). Straw was cut and milled before use and the <0.25 mm size fraction was used. A treatment with no substrate addition was also included, resulting in 7 different treatments in 3 soils with different N-status. The 42 jars (3 soils 7 treatments 2 replicates) containing 25 g of soil were then incubated at room temperature (22 C). Starch, which was assumed to be a more easily available C source then straw, was added in lower amounts and was sampled after 1, 2, and 3 weeks of incubation, while straw was sampled after 2, 3 and 4 weeks. 2.2. Measurement of limiting nutrients for bacterial growth At each sampling time, the nutrient limitation for bacterial growth was measured (Fig. 1) according to Aldén et al. (2001), as modified by Demoling et al. (2007). Only limitation of C and N were studied, since earlier results had indicated that these two were the primary and secondary limiting nutrient, respectively, in these soils, with the addition of extra P having no effect on bacterial growth in combination with the chosen levels of C and N (Kamble et al., 2013). First, 1 g of soil was weighed into 50 mL vials with lids. Glucose (5 mg g1 equivalent to 2 mg g1 glucose-C) and nitrogen (0.142 mg g1 NH4NO3 equivalent to 0.05 mg g1 NH4NO3eN) were then added in a full factorial design (No addition control (No), C, N and CN combined). Thus, for each of the two substrates, straw and starch, 12 combinations of substrate level (4 different including an unamended control) and soils (3 different) with two replicates were tested for limiting factors in a 2 2 factorial design, resulting in 96 measurements per substrate at each sampling time. After a 72 h incubation period with the added glucose-C and N at room temperature (22 C), bacterial growth was measured using the leucine incorporation method (Kirchman et al., 1985; Bååth, 1994; Bååth et al., 2001), which is using bacterial protein synthesis as a proxy of bacterial growth. Alleviating a nutrient limitation will increase the bacterial growth rate eventually resulting in increased bacterial numbers. Thus, the effect of adding a limiting
Soils with different extent of C-limitation (Unfert., Low N, High N)
Add starch or straw at different conc. to induce N-limitation Incubate 1-3 weeks for starch, 2-4 weeks for straw
Full factorial test for limiting factors by adding C (glucose) and N (NH4NO3) Measure bacterial growth after 3 days as Leu incorporation Fig. 1. Experimental set-up.
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The effect of adding glucose-C and N on bacterial growth (the determination of C and N limitation) was evaluated in two ways. First, for each combination of Soil Substrate Sampling time the bacterial growth after adding C, N and CN was compared to the no addition control (No). Second, a simplified way, only comparing the effect of adding C to that of adding N, was used. One common way of standardizing the data, following Elser et al. (2009a), is by calculating the log ratio of growth after adding C, N or CN divided with the no addition control (No) in the first comparison, or by growth after C addition divided with growth after adding N (the Log C/N ratio; the second comparison). However, this has the disadvantage that soils with initially low bacterial growth (e.g. the High N soils without starch or straw addition) will tend to have higher ratios then soils with high initial growth rates, like after adding 80 mg straw g1 soil, even if the addition of C or N results in the same absolute increase in bacterial growth. Instead we used the delta values, that is, bacterial growth after adding C, N or CN minus growth in the no addition control (first comparison) or bacterial growth after adding C minus growth after adding N (denoted D Ce N limitation index; second comparison). The rational behind this is that the addition of a fixed amount of for example glucose will give the same absolute increase in bacterial growth irrespective of the initial growth unless there is N limitation, and vice versa for N. Using the D CeN limitation index as way of comparing C and N addition will result in values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0, due to no growth increase after either C or N application, is suggesting a co-limitation or close to a co-limitation by both C and N. A value near 0 could also be found if both C and N application is increasing growth to the same extent. However, this was not the case in the present study. The D CeN limitation index was statistically evaluated using a 2-factor ANOVA (factors Soil and Substrate addition level) for each substrate and sampling time. Using the Log C/N ratio, sensu Elser et al. (2009a), gave similar main results as using D CeN limitation index, and are presented as Supplementary information.
Leucine incorporation measured in the short-term test of limiting factors with no N- or C-addition reflected the bacterial
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nutrient can both increase the growth rate and the biomass. The leucine incorporation method as performed here does not differentiate between these two mechanisms, but both will result in increased leucine incorporation. Briefly, 1 g of soil was mixed with 20 mL distilled water and vortexed on a multivortex shaker for 3 min. The soil suspension was centrifuged for 10 min at 3000 rpm and 1.5 mL of the supernatant (the bacterial suspension) was sampled. These suspensions were transferred to 2 mL micro centrifugation tubes. Then 275 nM Leu (2 ml L-4, 5-3H-Leucine, 37 MBq mL1, 1.48e2.22 TBq mmol1, Perkin Elmer, USA, diluted with non-radioactive leucine) was added to the bacterial suspension. After 2 h of incubation at 22 C, growth was terminated by using 75 mL 100% trichloroacetic acid. Subsequently samples were subjected to washing and incorporated leucine was estimated in a scintillator following the procedures described in Bååth et al. (2001). Leu incorporation per h into bacteria extracted from 1 g of wet soil was used as a proxy of bacterial growth.
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Starch addition (mg g ) Fig. 2. The effect of starch amendments on bacterial growth, estimated as leucine incorporation into extracted bacteria, in soils with different N status: Unfertilized (black circle), Low N (red triangles), and High N (blue squares) soils. A) 1 week after amendment, B) 2 weeks after amendment, C) 3 weeks after amendment. Bars denote SE (n ¼ 2). Note the brake in the y-axis in A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
growth in the original soils, and the subsequent effect of adding straw or starch. Without any straw and starch addition, bacterial growth was significantly lower in the High N soil compared to the Unfertilized and Low N soils during the whole experiment (Figs. 2
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and 3; 0 mg g1 addition). There was, however, no difference in bacterial growth between the Unfertilized and Low N soils. Both addition of straw and starch increased bacterial growth during the whole incubation period (Figs. 2 and 3), with the bacterial growth increasing approximately proportionally with the rate
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of substrate addition, especially in the case of straw addition. Calculating the mean bacterial growth over all soils and incubation times resulted in similar increase in bacterial growth, irrespective of starch or straw was added. Adding 20 mg g1 soil resulted in a mean bacterial growth increase compared to the unamended soils of 30 and 40 pmol Leu h1 g1 for starch and straw, respectively, while adding 40 mg g1 soil increased this to 70 and 60 pmol Leu h1 g1, respectively. Highest bacterial growth was found after adding straw at 80 mg g1, with a mean increased bacterial growth over the entire incubation period compared to unamended soil of 150 pmol Leu h1 g1. An effect of starch addition on bacterial growth was found already at the first sampling occasion, after 1 week (Fig. 2). The bacterial growth then decreased slightly over time. Adding starch to the low N soil increased bacterial growth slightly more then in the Unfertilized soil, with a mean increase in bacterial growth due to starch addition over all times being around 1.5 times higher in the Low N compared to the Unfertilized treatment. Bacterial growth in the High N soil also increased due to the starch addition. Over the whole 3-week period, adding starch at the two highest concentrations (20 and 40 mg g1) resulted in 2 times larger growth increase in the High N soil compared to the Unfertilized soil. Thus, high levels of starch addition appeared to increase bacterial growth most in the High N soil and least in the Unfertilized soil. Straw addition usually resulted in highest values at the first measurement occasion, after 2 weeks incubation, followed by a gradual decrease in bacterial growth (Fig. 3). For example, with 80 mg1 straw g1 soil, bacterial growth in the Unfertilized and Low N soils was around 270 pmol Leu h1 g1 after 2 weeks, around 230 after 3 weeks, decreasing to 160 pmol Leu h1 g1 after 4 weeks. There was no interaction between straw addition and N status of the soils, and the increase in bacterial growth with level of straw addition was similar in all soils. Thus, irrespective of straw addition, it was the same difference in bacterial growth between High N and the other two soils, the Unfertilized and Low N soils, which in turn did not differ.
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Straw addition (mg g ) Fig. 3. The effect of straw amendments on bacterial growth, estimated as leucine incorporation into extracted bacteria, in soils with different N status: Unfertilized (black circle), Low N (red triangles), and High N (blue squares) soils. A) 2 week after amendment, B) 3 weeks after amendment, C) 4 weeks after amendment. Bars denote SE (n ¼ 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
There were no major differences in the effect of adding C and N to study nutrient limitation between the incubation times (1e3 weeks for starch, Fig. 4, and 2e4 weeks for straw, Fig. 5). In the unamended soil (0 mg g1 starch or straw), bacterial growth was Climited in all soils, with the extent of growth response with Caddition increasing in the order Unfertilized < Low N ¼ High N. After adding low amounts of starch (10 mg g1), the limitation test indicated that the bacteria were still C limited, although the growth response to only C was usually less than in the nonamended control soils (Fig. 4). With the amendment of 20 mg g1 starch, a positive growth response to C-addition was found only in the High N soil, while for the other two soils only adding C in combination with N gave any growth response. Using the highest level of starch, there was no bacterial growth response to adding only C in any of the soils, that is, no evidence for C-limitation. Bacterial growth did not respond much to N addition except for the amendment of 40 mg g1 soil in the High N soil after 2 and 3 weeks. Similar results as for adding starch were found after adding straw (Fig. 5). Increasing the amount of added straw decreased the bacterial growth response after adding only C, especially in the Unfertilized and Low N soils. For example, adding 20 mg g1 straw resulted in a bacterial growth increase after adding C only in the High N soil, while at the higher amendment levels of straw, no evidence of C-limitation was found in any of the soils. At the highest levels of straw addition (80 mg g1), adding glucose-C actually resulted in lower bacterial growth then in the control with no C or
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Fig. 4. Bacterial growth 72 h after adding C (Glucose) and N (NH4NO3) to starch amended soils with different N status: Unfertilized (open bars), Low N (hatched bars), and High N (filled bars) soils. Soils were amended with 0, 10, 20 and 40 mg starch g1 soil and incubated for 1, 2 and 3 weeks at 22 C. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil). The effect of adding C, N or CN was expressed as D bacterial growth calculated as the difference to bacterial growth in the control (No addition). A value close to zero thus indicates no extra growth on the added nutrients. Bars denote SE (n ¼ 2).
N addition (the No treatment). Except for the highest rate of straw amendment little or no effect of adding N alone was found. Adding CN in combination always resulted in large growth increases, usually around 150e250 pmol Leu h1 g1 in the Unfertilized and Low N soil. However, in the High N soils, lower values were found after CN addition, especially in the treatments with no addition or low levels of starch addition (Fig. 4). However, adding starch or straw resulted in increased bacterial growth responses after adding CN with increasing amendment rates, with similar increased bacterial growth in all three soils with different N-status after adding the highest rate of starch, 40 mg1 g1. To summarize the results we calculated a D CeN limitation index (Figs. 6 and 7), which is the difference of bacterial growth after adding C minus the response after adding N. A positive value suggests C-limitation, while a negative value suggests N-limitation. A
value around zero indicates that neither C nor N alone limits growth, but C and N in combination, that is co-tolerance or at least close to co-tolerance of two nutrients (but see Discussion for other interpretations). No major differences between incubation times were seen, and thus results general for all times are reported. For both starch (Fig. 6) and straw (Fig. 7) addition there were significant statistical interaction between the soils with different N-status and the amendment levels except at the first measuring occasion. The D CeN limitation index increased with increasing N-status of the soil (unfertilized < Low N < High N soils) in the non-amended soils, and in the two lowest level of starch (10 and 20 mg g1) and the lowest level of straw amendment (10 mg g1). Up to these substrate amendment rates the D CeN limitation index decreased compared to the unamended soils, indicating lower extent of C-limitation with increasing substrate addition. With 20 mg g1 starch or straw
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Fig. 5. Bacterial growth 72 h after adding C (Glucose) and N (NH4NO3) to straw amended soils with different N status: Unfertilized (open bars), Low N (hatched bars), and High N (filled bars) soils. Soils were amended with 0, 20, 40 and 80 mg straw g1 soil and incubated for 2, 3 and 4 weeks at 22 C. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil). The effect of adding C, N or CN was expressed as D bacterial growth calculated as the difference to bacterial growth in the control (No addition). A value close to zero thus indicates no extra growth on the added nutrients. Bars denote SE (n ¼ 2).
the D CeN limitation index in the unfertilized soil was close to zero during all measurement occasions indicating close to co-limitation of both C and N for bacterial growth, while in the High N soils a Climitation of the bacterial growth was still evident. For the highest level of starch (40 mg g1), and the two highest levels of straw addition (40 and 80 mg g1), a different pattern was found. There was no effect of N-status of the soils, and in almost all cases a D CeN limitation index close to or below zero was found, suggesting colimitation or even N-limitation for bacterial growth. However, the negative values for the D CeN limitation index, suggesting N-limitation, were never as large as the positive values suggesting Climitation. For example, highest values for the D CeN limitation index was around 100e150 pmol Leu h1 g1 in the High N soil with low substrate additions (Fig. 6). Most negative values for the D CeN limitation index were usually only around 50 pmol
Leu h1 g1 or less, except for the highest level of straw amendment after 2 weeks (Fig. 7). 4. Discussion 4.1. Bacterial growth in soil and direct effect of starch and straw addition Without substrate addition bacterial growth was lowest in the High N soil. This has been found earlier in these soils (Kamble et al., 2013) and decreased bacterial growth appears to be a common response to increased N availability in N-poor boreal forest soils (Demoling et al., 2008). The decreased bacterial growth also appears to be part of a common decreased total activity of soil microbes in response to N-fertilization of boreal forests (Arnebrant
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et al., 1996; Thirukkumaran and Parkinson, 2000; Sjöberg et al., 2003; Bowden et al., 2004; Knorr et al., 2005; Janssens et al., 2010). So far no common explanation for this phenomenon exists (Liu and Greaver, 2010), but the fact that ligninolytic enzymes are inhibited by adding N is one possibility (Pregitzer et al., 2008; Zak et al., 2008). Since these enzymes appear to be key enzymes, possibly rate limiting ones, in degrading recalcitrant organic matter, the effect of N-addition may thus be a decrease in substrate availability. Alleviating substrate limitation increased microbial activity, in that both adding straw (Fig. 3) and starch (Fig. 2) increased bacterial growth. Previous work has also shown that adding similar type of substrates (e.g. straw or cellulose) results in increased bacterial growth (Rousk and Bååth, 2007; Meidute et al., 2008). It was expected that starch would be more favored by bacteria, since earlier results indicate that starch is preferable metabolized by bacteria (Rinnan and Bååth, 2009), while straw is more favored by fungi (Henriksen and Breland, 1999; Bossuyt et al., 2001; Six et al., 2006; Rousk and Bååth, 2007). However, a rapid response of bacteria after adding wheat residues has also been reported (Marschner et al., 2011). In the present study we did not find any major difference in the bacterial growth response to starch or straw, if the same level of amendment was compared.
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Starch addition (mg g ) Fig. 6. The D CeN limitation index for bacterial growth 72 h after adding C (Glucose) to that after adding N (NH4NO3) to soils with different nitrogen status: Unfertilized (black circles), Low N (red triangles), and High N (blue squares). The soils were incubated for 1 (A), 2 (B) and 3 (C) weeks at 22 C after amending with different amounts of starch. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil), where the D CeN limitation index was calculated as the difference in bacterial growth after adding C and adding N. Values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0 suggesting a co-limitation or close to a co-limitation by both C and N. Bars denote SE (n ¼ 2). ANOVA statistics with A ¼ amendment level of starch and S ¼ soils with different N status. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Our first hypothesis was that additions of large amounts of Crich substrate would turn the soil from being C-limited for bacterial growth to N-limited. The effect on C-limitation, from a growth response after adding glucose-C to no growth response with increasing starch or straw additions, was most easily seen in the High N soil. In the unamended High N soil, adding glucose-C always resulted in increased bacterial growth indicating C-limitation; this was not the case after adding the highest levels of starch (Fig. 4) or straw (Fig. 5). Similar results, with a shift from C-limitation to no measurable C-limitation, were also observed by Aldén et al. (2001) after adding straw to soil. Theoretically the soils should turn into being N-limited after adding enough starch or straw, that is, result in a growth increase after adding easily available N. The extent of growth increases after adding N was minor, however. Even in the highest levels of straw and starch amendments little positive effects on bacterial growth was found after adding N, although it was higher than for adding C, resulting in negative D CeN limitation indices (Figs. 6 and 7). There are several possible explanations for this lack of clear effects of adding N even if the soil was expected to be N-limited for bacterial growth. First, the soils may have been co-limited by both C and N even at the highest amendment rates of straw and starch, and thus both nutrients had to be added in order to alleviate the existing limitation (independent nutrient co-limitation sensu Saito et al., 2008). Such situations have been described for N and P limitation in aquatic habitats (Elser et al., 2009b). Ekblad and Nordgren (2002) also observed that after the addition of sucrose to a soil, the microbial activity measured using respiration rate appeared to be limited by both C and N. In our study co-limitation was, however, most likely only the case with intermediate levels of substrate amendments, for example the case after adding 20 or 40 mg straw g1 to the Unfertilized and Low N soils (Fig. 5). Colimitation by both C and N is therefore unlikely to be the case for the highest starch and straw amendments, especially in soils, which from the beginning were low in available N. Another explanation for the lack of clear effects on bacterial growth in high amended soils after adding N is that the response will be in fungal instead of in bacterial growth. This could be the case for straw, which will favor fungal growth (Rousk and Bååth,
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Straw addition (mg g ) Fig. 7. The D CeN limitation index for bacterial growth 72 h after adding C (Glucose) to that after adding N (NH4NO3) to soils with different nitrogen status: Unfertilized (black circles), Low N (red triangles), and High N (blue squares). The soils were incubated for 1 (A), 2 (B) and 3 (C) weeks at 22 C after amending with different amounts of straw. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil), where the D CeN limitation index was calculated as the difference in bacterial growth after adding C and adding N. Values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0 suggesting a co-limitation or close to a co-limitation by both C and N. Bars denote SE (n ¼ 2). ANOVA statistics with A ¼ amendment level of straw and S ¼ soils with different N status.
2007). Earlier results also suggested that straw with extra N actually favored fungi even more then only adding straw, while bacteria were not affected by the extra N (Rousk and Bååth, 2007). However, since starch addition has been shown to favor bacterial growth (Rinnan and Bååth, 2009), this is an unlikely explanation in the case of starch addition. Although there usually will be easily available N present as ammonium or nitrate in a C-limited soil, the opposite is not necessarily the case. Thus in C-limited soils inorganic N will usually accumulate; an effect used to measure net nitrogen mineralization by incubating soil in the laboratory. However, in an N-limited soil, there might be no similar accumulation of easily available C. Instead, in an N-limiting situation easily available C might be respired (Schimel and Weintraub, 2003) or taken up as energy reserve, for example as triglycerides in fungi (Ekblad and Nordgren, 2002; Bååth, 2003) or as poly-beta-hydroxybutyric acid (PHB) in bacteria (Wang and Bakken, 1998), and thus not immediately available for growth. Furthermore, one has to bear in mind that limitation by a macromolecule, like starch or cellulose in straw, and an easily available substrate, like glucose, is not strictly the same. The former needs to be degraded into smaller monomers by extracellular enzymes in order to be available to the microorganisms. This may take some time, especially if the proper enzymes have to be induced. Alleviating N-limitation would immediately cause a C-limitation in this scenario, and there will be a time lag in production of available C from the macromolecules or soil organic matter, before extensive growth will be present. There may thus be a different time frame for a response after alleviating N-limitations compared to C-limitations in soil, where in the former case more than the 72 h used in the present study is needed to detect a response in bacterial growth. This may be a different situation compared to aquatic habitats, where addition of P and N often result in a large increase in bacterial growth within a few days (Elser et al., 1995; Rivkin and Anderson, 1997; Granéli et al., 2004), suggesting that in aquatic habitat there will directly be easily available C present in N or P limited situations. The accumulation of dissolved organic C has also been found under P-limitation in aquatic systems (Vadstein et al., 2003). There were significant interactions between substrate amendment levels and different N-status in the soil in determining C- and N-limitation of the bacterial community, substantiating our second hypothesis. Thus, there was a combination of soil and substrate addition that resulted in co-limitation in accordance with our third hypothesis. However, it is unlikely that strict co-limitation is very common in soil. Instead, a situation close to co-limitation is more likely. Adding the limited nutrient will in such a case increase bacterial growth, but then rapidly induce limitation of the secondary limiting nutrient, resulting in only a barely detected growth increase. However, adding both limiting nutrient will result in extensive growth as in the present study. Such a situation was also found in a subarctic heath soil (Rinnan et al., 2007). The extent of bacterial growth after adding glucose-C suggested increasing amounts of available N for bacterial growth with increasing N-fertilization rate (Figs. 4 and 5 with 0 mg g1 additions; see also Kamble et al., 2013). The amount of starch and straw needed to change the system into not being C-limited also indicated the same order of N-availability. This was most easily seen when comparing the D CeN limitation index (Figs. 6 and 7), with the High N soil tending to stay C-limited at higher substrate amendment levels than the Unfertilized soil, with presumably the least available N. Thus, comparing limiting nutrients for bacterial growth could be used in two ways to suggest the availability of N as the secondary limiting nutrient; by the extent of the direct growth increase after adding the primary limiting nutrient in an easily available form (glucose-C) or by the amount of the primary limiting nutrient added
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in a more recalcitrant form (starch-C and straw-C) needed to alter the soil situation from C-limitation to co-limitation by C and N. Although the use of the D CeN limitation index both was less time consuming (half of the treatments needed compared to a full factorial design), and appeared easy to interpret, it should be used with some caution. Similar problems will be the case if the Log C/N growth ratio is used. First, there will be the same index in the case of no growth response after C-addition and N-addition alone or if both treatments increase bacterial growth to the same extent. To differentiate between these two results, a control with neither Cnor N-addition has to be included (our No treatment). Even more important is the combined CN treatment included in the full factorial design. This is a positive control, which should increase bacterial growth to show that not another nutrient is limiting growth or that too low amounts of C and N have been added to increase growth, as discussed by Demoling et al. (2007). In most cases the combined addition of CN resulted in a clear growth increase, suggesting that this was not a problem in our study. Adding CN to the High N soils without starch or straw resulted, however, in a lower increased bacterial growth then adding CN to the other two soils, with even lower growth increase then adding C alone. This was also found earlier for this soil (Kamble et al., 2013), who suggested that high levels of N in the High N soil may be the reason for low bacterial growth. High levels of soil N have earlier been shown to reduce bacterial growth (Aldén et al., 2001). Adding starch or straw resulted in soils with increased bacterial growth after adding CN, resulting in similar bacterial growth increases at highest substrate loadings as in the other two, less N-rich, soils. This is in accordance with increasing C-rich substrate loadings resulting in immobilization of N, consequently with the presumed negative effect of high amounts of N on bacterial growth disappearing. However, another possibility is fungal growth being stimulated after CN addition in the High N soil, with fungi then outcompeting bacteria. Thus, fungal growth has to be studied to differentiate between these suggestions. 4.3. Conclusion We found that adding C-rich substrate, like starch and straw, resulted in increased bacterial growth, but at the same time switches the soil from being C-limited to being close to co-limited by both C and N or even N-limited. The extent of C- or N-limitation was an interaction between the amount of substrate added and the initial N-status of the soil. A more extensive C-limitation at lower substrate additions and higher N-status of the soil was found, while especially high substrate additions and low N status soil was more inclined to become co- or only N-limited. However, alleviating Climitation by adding easily available C (glucose) resulted in more extensive bacterial growth than alleviating N-limitation (induced by adding straw or starch) by adding inorganic N. Thus, in a Climited soil there appeared to be N available immediate for growth, while in an N-limited soil, easily available C was not immediately available. The present study has, however, concentrated on bacterial growth. To be able to properly evaluate nutrient limitation in soil, the response of fungal growth has to be included. Furthermore, we induced N-limitation by adding fresh C-rich substrate in excess and in the form of macromolecules. The response of the soil microorganisms to N-limitation, and to alleviating limitation, may be different in a soil naturally N-limited. Acknowledgments This study was supported by an Erasmus Mundi grant to P.N.K. and by the Swedish Research Council to E.B. (Project No. 20094503). We thank Dr. S.D. Frey for providing samples from the
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Chronic Nitrogen Amendment Study at the Harvard Forest Long Term Ecological (LTER) Site in Petersham, MA, USA. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2014.02.015. References Aber, J.D., Magill, A.H., 2004. Chronic nitrogen additions at the Harvard Forest (USA): the first 15 years of a nitrogen saturation experiment. Forest Ecology & Management 196, 1e5. Aldén, L., Demoling, F., Bååth, E., 2001. Rapid method of determining factors limiting bacterial growth in soil. Applied and Environmental Microbiology 67, 1830e 1838. Allison, S.D., Vitousek, P.M., 2005. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biology & Biochemistry 37, 937e944. Arnebrant, K., Bååth, E., Söderström, B., Nohrstedt, H.Ö., 1996. Soil microbial activity in eleven Swedish coniferous forests in relation to site fertility and nitrogen fertilization. Scandinavian Journal of Forest Research 11, 1e6. 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., 2003. The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi. Microbial Ecology 45, 373e383. Bååth, E., Petterson, 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. Bossuyt, H., Denef, K., Six, J., Frey, S.D., Merckx, R., Paustian, K., 2001. Influence of microbial populations and residue quality on aggregate stability. Applied Soil Ecology 16, 195e208. Bowden, R.D., Davidson, E., Savage, K., Arabia, C., Steudler, P., 2004. Chronic nitrogen additions reduce total soil respiration and microbial respiration in temperate forest soils at the Harvard Forest. Forest Ecology & Management 196, 43e56. 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Güsewell, S., Freeman, C., 2005. Nutrient limitation and enzyme activities during litter decomposition of nine wetland species in relation to litter N:P ratios. Functional Ecology 19, 582e593. Henriksen, T.M., Breland, T.A., 1999. Nitrogen availability effects on carbon mineralization, fungal and bacterial growth, and enzyme activities during decomposition of wheat straw in soil. Soil Biology & Biochemistry 31, 1121e1134. Janssens, I.A., Dieleman, W., Luyssaert, S., Subke, J.-A., Reichstein, M., Ceulemans, R., Ciais, P., Dolman, A.J., Grace, J., Matteucci, G., Papale, D., Piao, S.L., Schultze, E.-D., Tang, J., Law, B.E., 2010. Reduction of forest soil respiration in response to nitrogen deposition. Nature Geoscience 3, 315e322. 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 34, 341e354. Kamble, P.N., Rousk, J., Frey, S.D., Bååth, E., 2013. Bacterial growth and growthlimiting nutrients following chronic nitrogen additions to a hardwood forest. Soil Biology & Biochemistry 59, 32e37. Kirchman, D., K’nees, 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.
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Knapp, E.B., Elliott, L.F., Campbell, G.S., 1983. Microbial respiration and growth during the decomposition of wheat straw. Soil Biology & Biochemistry 15, 319e 323. Knorr, M., Frey, S.D., Curtis, P.S., 2005. Nitrogen additions and litter decomposition: a meta analysis. Ecology 86, 3252e3257. Liu, L.L., Greaver, T.L., 2010. A global perspective on belowground carbon dynamics under nitrogen enrichment. Ecology Letters 13, 819e828. Magill, A.H., Aber, J.D., Hendricks, J.J., Bowden, R.D., Melillo, J.M., Steudler, P.A., 1997. Biogeochemical response of forest ecosystems to simulated chronic nitrogen deposition. Ecological Applications 7, 402e415. McDowell, W.H., Magill, A.H., Aitkenhead-Peterson, J.A., Aber, J.D., Merriam, J.L., Kaushal, S.S., 2004. Effects of chronic nitrogen amendment on dissolved organic matter and inorganic nitrogen in soil solution. Forest Ecology and Management 196, 29e41. Marschner, P., Umar, S., Baumann, K., 2011. The microbial community composition changes rapidly in the early stages of decomposition of wheat residue. Soil Biology & Biochemistry 43, 445e451. Meidute, S., Demoling, F., Bååth, E., 2008. Antagonistic and synergistic effects of fungal and bacterial growth in soil after adding different carbon and nitrogen sources. Soil Biology & Biochemistry 40, 2334e2343. Nordgren, A., 1992. A method for determining microbially available N and P in an organic soil. Biology and Fertility of Soils 13, 1179e1192. Pregitzer, K.S., Burton, A.J., Zak, D.R., Talhelm, A.F., 2008. Simulated chronic nitrogen deposition increases carbon storage in Northern Temperate forests. Global Change Biology 14, 142e153. Reed, S.C., Vitousek, P.M., Cleveland, C.C., 2011. Are patterns in nutrient limitation belowground consistent with those aboveground: results from a 4 million year chronosequence. Biogeochemistry 106, 323e336. Rivkin, R.B., Anderson, M.R., 1997. Inorganic nutrient limitation of oceanic bacterioplankton. Limnology and Oceanography 42, 730e740. Rinnan, R., Bååth, E., 2009. Differential utilization of carbon substrates by bacteria and fungi in tundra soil. Applied and Environmental Microbiology 75, 3611e 3620. Rinnan, R., Michelsen, A., Bååth, E., Jonasson, S., 2007. Mineralization and carbon turnover in subarctic heath soil as affected by warming and additional litter. Soil Biology & Biochemistry 39, 3014e3023.
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Induced N-limitation of bacterial growth in soil: Effect of carbon loading and N status in soil Pramod N. Kamble a, b, c, Erland Bååth a, * a
Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62 Lund, Sweden P. G. Department of Environmental Science, PVP College Pravaranagar, University of Pune, Pune, India c K.T.H.M. College Nashik, University of Pune, Pune, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2013 Received in revised form 22 January 2014 Accepted 20 February 2014 Available online 10 March 2014
Application of C-rich plant residues can change the soil system from C-limitation for microbial growth to limitation by other nutrients. However, the initial nutrient status of the soil may interact with the added amount of residues in determining limitation. We studied this interactive effect in soils from the Harvard Forest LTER, where annual addition of N since 1988 has resulted in soils with different N-status: No N (Unfertilized), 50 (Low N) and 150 (High N) kg N ha1. We hypothesized that adding C-rich substrate would change the soil from being C- to being N-limited for bacterial growth and that the extent of Nlimitation would be higher with increasing substrate additions, while becoming less evident in soil with increasing N-status. We compared the effect of adding two C-rich substrates, starch (0, 10, 20, 40 mg g1 soil) and straw (0, 20, 40, 80 mg g1), incubating the soils for up to 3 and 4 weeks for starch and straw, respectively. Nutrient limitations were studied by measuring bacterial growth 3 days after adding C as glucose and N as NH4NO3 in a full factorial design. Initially bacterial growth in all soils was C-limited. As hypothesized, adding C-rich substrates removed the C-limitation, with lower amounts of starch and straw needed in the unfertilized and Low N soils than in the High N soil. Combinations of different Nstatus of the soil and amendment levels of starch and straw could be found, where bacterial growth appeared close to co-limited both by available C and N. However, at even higher amendment levels, presumable resulting in N-limitation, bacterial growth still responded less by adding N then C-limited soils by adding C. Thus, in a C-limited soil there appeared to be N available immediate for growth, while in an N-limited soil, easily available C was not immediately available. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: N-deposition C-rich substrate Induced-N limitation Bacterial growth Leucine incorporation Limiting factors
1. Introduction Growth of soil microorganisms is mainly regulated by the amount of substrate available for growth and physiochemical environmental factors like temperature, moisture content and pH. However, the microbial activity will also be regulated by nutrient limitation, where for example carbon rich substrate, like straw or cellulose, will not be decomposed at maximum rate in nitrogen limiting situations (Henriksen and Breland, 1999; Allison and Vitousek, 2005). Bacterial growth in soil appears, however, to be most commonly limited by the availability of carbon (C) (Nordgren, 1992; Joergensen and Scheu, 1999; Aldén et al., 2001; Ekblad and Nordgren, 2002; Demoling et al., 2007; Göransson et al., 2011),
* Corresponding author. Tel.: þ46 46 222 42 64. E-mail addresses: [email protected] (P.N. Kamble), [email protected] (E. Bååth). http://dx.doi.org/10.1016/j.soilbio.2014.02.015 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
but nitrogen (N) and phosphorus (P) limitation has also been reported (Aldén et al., 2001; Cleveland et al., 2002; Rinnan et al., 2007; Reed et al., 2011). Boreal forests are described as a nutrient poor system, with plant growth generally being limited by lack of N (Tamm, 1991; Ekblad and Nordgren, 2002). However, soil bacterial growth appeared to be limited by C also in these habitats (Ekblad and Nordgren, 2002; Demoling et al., 2008; Kamble et al., 2013). The extent of C-limitation can, however, vary depending on the N-status of the soil. Thus, in unfertilized boreal forest soil, only a small increase in bacterial growth was found after adding glucose as easily available C-source (to test for C-limitation), while in Nfertilized soils a much larger growth response was found, indicating increased N-availability in such soils (Demoling et al., 2008; Kamble et al., 2013). A similar tendency was found by comparing soils with different anthropogenic N-deposition, where the growth response after adding glucose was larger in soils with higher Ndeposition (Demoling et al., 2008).
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The addition of N may thus alter the response of the bacterial community after adding C, although not altering the primary limiting substance. However, the microbial community can become N-limited by adding carbon rich substrates to the soil. This is wellknown after adding straw or pure cellulose (e.g. Knapp et al., 1983; Cochran et al., 1988; Wang and Bakken, 1997; Henriksen and Breland, 1999) and is usually measured by increased decomposition or respiration when the carbon-rich substrate is added together with extra N (Henriksen and Breland, 1999; Allison and Vitousek, 2005; Güsewell and Freeman, 2005; Rousk and Bååth, 2007). This usually implies long-term measurements. Furthermore, using respiration may not always result in easy interpretable results after alleviating N-limitations (Schimel and Weintraub, 2003). Aldén et al. (2001) described a method to determine nutrient limitation in soil based on estimations of bacterial growth increases after only a few days in response to adding nutrients alleviating limitation. They also showed that this method could be used to demonstrate how adding straw altered a soil system from being C-limited to one being N-limited for bacterial growth. However, this has not been tested on a larger scale using different C-rich substrates and soils differing in N-status. We have studied the interactive effect of C-rich substrate additions on bacterial growth limitations in soils with different N-status. We took advantage of one of the longest N-fertilization studies of boreal forests, the Chronic Nitrogen Amendment Study at the Harvard Forest Long Term Ecological Research (LTER) site in central Massachusetts, USA. Since 1988 soils have been subjected to three different fertilization regimes (unfertilized, low N and high N additions), resulting in soils with different N-status. Dissolved inorganic nitrogen (ammonium þ nitrate) was around 25 and 75 times higher compared to the unfertilized control in the low N and High N treatment, respectively (McDowell et al., 2004). Our hypotheses were: 1) C-rich substrate addition (straw and starch) will change the soils from being C-limited to N-limited, 2) This change will be dependent of level of substrate added, with increasing extent of Nlimitation with increasing level of substrate addition, and the Nstatus of the soil, with decreasing extent of N-limitation with increasing N-status of the soil, 3) There will thus be a combination of substrate addition and N-status of the soil that results in colimitation or close to co-limitation by both C and N for bacterial growth. Finally, we wanted to compare if bacterial growth changed to the same extent after alleviating C or N limitation by adding easily available C or N, respectively. 2. Material and methods 2.1. Soil and experimental setup for induced N limitation Soils from the Harvard Forest Long Term Ecological Research (LTER) site in central Massachusetts, USA, were used. The Chronic Nitrogen Amendment Study was established in 1988 to study longterm effects of increased N deposition on the boreal forest ecosystem (Aber and Magill, 2004). The forest is dominated by black (Quercus velutina) and red (Quercus rubra) oak mixed with black birch (Betula lenta), red maple (Acer rubrum) and American beech (Fagus grandiflora). Soils are of the Gloucester series (fine loamy, mixed, mesic, Typic Dystrochrepts, USDA). Since 1988 these soils have been treated annually with nitrogen amendments as a proxy of increased anthropogenic N-deposition. Three different rates of nitrogen were used: No N (Unfertilized), 50 (Low N) and 150 (High N) kg N ha1 annually (applied as six equal applications over the growing season) as NH4NO3eN in four replicates plots. Soil samples were collected from replicate 5 5 m sub-plots within each treatment plot (30 30 m) (Magill et al., 1997; Aber and Magill, 2004). Two 8 cm diameter and 10 cm long cores were
removed from each sub plot, separated into the organic and mineral horizon, and bulked by soil horizon. Samples were sieved (2 mm) and stored field moist at 4 C until analyzed. Two of the replicates from the organic soil we used in the present study. pH was 5.1, 5.0 and 4.4 in Unfertilized, Low N and High N soils, organic matter (as loss on ignition, 4 h at 600 C) 0.47, 0.45 and 0.38 g g1 soil, and the water content kept at 60% of dry weight. The C/N ratio did not differ between treatments (24.7 in Unfertilized, 26.3 in Low N and 23.7 in High N; Turlapati et al., 2013). To induce nitrogen limitation, dried wheat straw and soluble starch (Merck) were applied. Straw (C/N ¼ 75), at 20, 40 and 80 mg g1 of soil, and starch, at 10, 20, 40 mg g1 of soil, were added to all three soils in duplicate (Fig. 1). Straw was cut and milled before use and the <0.25 mm size fraction was used. A treatment with no substrate addition was also included, resulting in 7 different treatments in 3 soils with different N-status. The 42 jars (3 soils 7 treatments 2 replicates) containing 25 g of soil were then incubated at room temperature (22 C). Starch, which was assumed to be a more easily available C source then straw, was added in lower amounts and was sampled after 1, 2, and 3 weeks of incubation, while straw was sampled after 2, 3 and 4 weeks. 2.2. Measurement of limiting nutrients for bacterial growth At each sampling time, the nutrient limitation for bacterial growth was measured (Fig. 1) according to Aldén et al. (2001), as modified by Demoling et al. (2007). Only limitation of C and N were studied, since earlier results had indicated that these two were the primary and secondary limiting nutrient, respectively, in these soils, with the addition of extra P having no effect on bacterial growth in combination with the chosen levels of C and N (Kamble et al., 2013). First, 1 g of soil was weighed into 50 mL vials with lids. Glucose (5 mg g1 equivalent to 2 mg g1 glucose-C) and nitrogen (0.142 mg g1 NH4NO3 equivalent to 0.05 mg g1 NH4NO3eN) were then added in a full factorial design (No addition control (No), C, N and CN combined). Thus, for each of the two substrates, straw and starch, 12 combinations of substrate level (4 different including an unamended control) and soils (3 different) with two replicates were tested for limiting factors in a 2 2 factorial design, resulting in 96 measurements per substrate at each sampling time. After a 72 h incubation period with the added glucose-C and N at room temperature (22 C), bacterial growth was measured using the leucine incorporation method (Kirchman et al., 1985; Bååth, 1994; Bååth et al., 2001), which is using bacterial protein synthesis as a proxy of bacterial growth. Alleviating a nutrient limitation will increase the bacterial growth rate eventually resulting in increased bacterial numbers. Thus, the effect of adding a limiting
Soils with different extent of C-limitation (Unfert., Low N, High N)
Add starch or straw at different conc. to induce N-limitation Incubate 1-3 weeks for starch, 2-4 weeks for straw
Full factorial test for limiting factors by adding C (glucose) and N (NH4NO3) Measure bacterial growth after 3 days as Leu incorporation Fig. 1. Experimental set-up.
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The effect of adding glucose-C and N on bacterial growth (the determination of C and N limitation) was evaluated in two ways. First, for each combination of Soil Substrate Sampling time the bacterial growth after adding C, N and CN was compared to the no addition control (No). Second, a simplified way, only comparing the effect of adding C to that of adding N, was used. One common way of standardizing the data, following Elser et al. (2009a), is by calculating the log ratio of growth after adding C, N or CN divided with the no addition control (No) in the first comparison, or by growth after C addition divided with growth after adding N (the Log C/N ratio; the second comparison). However, this has the disadvantage that soils with initially low bacterial growth (e.g. the High N soils without starch or straw addition) will tend to have higher ratios then soils with high initial growth rates, like after adding 80 mg straw g1 soil, even if the addition of C or N results in the same absolute increase in bacterial growth. Instead we used the delta values, that is, bacterial growth after adding C, N or CN minus growth in the no addition control (first comparison) or bacterial growth after adding C minus growth after adding N (denoted D Ce N limitation index; second comparison). The rational behind this is that the addition of a fixed amount of for example glucose will give the same absolute increase in bacterial growth irrespective of the initial growth unless there is N limitation, and vice versa for N. Using the D CeN limitation index as way of comparing C and N addition will result in values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0, due to no growth increase after either C or N application, is suggesting a co-limitation or close to a co-limitation by both C and N. A value near 0 could also be found if both C and N application is increasing growth to the same extent. However, this was not the case in the present study. The D CeN limitation index was statistically evaluated using a 2-factor ANOVA (factors Soil and Substrate addition level) for each substrate and sampling time. Using the Log C/N ratio, sensu Elser et al. (2009a), gave similar main results as using D CeN limitation index, and are presented as Supplementary information.
Leucine incorporation measured in the short-term test of limiting factors with no N- or C-addition reflected the bacterial
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nutrient can both increase the growth rate and the biomass. The leucine incorporation method as performed here does not differentiate between these two mechanisms, but both will result in increased leucine incorporation. Briefly, 1 g of soil was mixed with 20 mL distilled water and vortexed on a multivortex shaker for 3 min. The soil suspension was centrifuged for 10 min at 3000 rpm and 1.5 mL of the supernatant (the bacterial suspension) was sampled. These suspensions were transferred to 2 mL micro centrifugation tubes. Then 275 nM Leu (2 ml L-4, 5-3H-Leucine, 37 MBq mL1, 1.48e2.22 TBq mmol1, Perkin Elmer, USA, diluted with non-radioactive leucine) was added to the bacterial suspension. After 2 h of incubation at 22 C, growth was terminated by using 75 mL 100% trichloroacetic acid. Subsequently samples were subjected to washing and incorporated leucine was estimated in a scintillator following the procedures described in Bååth et al. (2001). Leu incorporation per h into bacteria extracted from 1 g of wet soil was used as a proxy of bacterial growth.
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Starch addition (mg g ) Fig. 2. The effect of starch amendments on bacterial growth, estimated as leucine incorporation into extracted bacteria, in soils with different N status: Unfertilized (black circle), Low N (red triangles), and High N (blue squares) soils. A) 1 week after amendment, B) 2 weeks after amendment, C) 3 weeks after amendment. Bars denote SE (n ¼ 2). Note the brake in the y-axis in A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
growth in the original soils, and the subsequent effect of adding straw or starch. Without any straw and starch addition, bacterial growth was significantly lower in the High N soil compared to the Unfertilized and Low N soils during the whole experiment (Figs. 2
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and 3; 0 mg g1 addition). There was, however, no difference in bacterial growth between the Unfertilized and Low N soils. Both addition of straw and starch increased bacterial growth during the whole incubation period (Figs. 2 and 3), with the bacterial growth increasing approximately proportionally with the rate
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of substrate addition, especially in the case of straw addition. Calculating the mean bacterial growth over all soils and incubation times resulted in similar increase in bacterial growth, irrespective of starch or straw was added. Adding 20 mg g1 soil resulted in a mean bacterial growth increase compared to the unamended soils of 30 and 40 pmol Leu h1 g1 for starch and straw, respectively, while adding 40 mg g1 soil increased this to 70 and 60 pmol Leu h1 g1, respectively. Highest bacterial growth was found after adding straw at 80 mg g1, with a mean increased bacterial growth over the entire incubation period compared to unamended soil of 150 pmol Leu h1 g1. An effect of starch addition on bacterial growth was found already at the first sampling occasion, after 1 week (Fig. 2). The bacterial growth then decreased slightly over time. Adding starch to the low N soil increased bacterial growth slightly more then in the Unfertilized soil, with a mean increase in bacterial growth due to starch addition over all times being around 1.5 times higher in the Low N compared to the Unfertilized treatment. Bacterial growth in the High N soil also increased due to the starch addition. Over the whole 3-week period, adding starch at the two highest concentrations (20 and 40 mg g1) resulted in 2 times larger growth increase in the High N soil compared to the Unfertilized soil. Thus, high levels of starch addition appeared to increase bacterial growth most in the High N soil and least in the Unfertilized soil. Straw addition usually resulted in highest values at the first measurement occasion, after 2 weeks incubation, followed by a gradual decrease in bacterial growth (Fig. 3). For example, with 80 mg1 straw g1 soil, bacterial growth in the Unfertilized and Low N soils was around 270 pmol Leu h1 g1 after 2 weeks, around 230 after 3 weeks, decreasing to 160 pmol Leu h1 g1 after 4 weeks. There was no interaction between straw addition and N status of the soils, and the increase in bacterial growth with level of straw addition was similar in all soils. Thus, irrespective of straw addition, it was the same difference in bacterial growth between High N and the other two soils, the Unfertilized and Low N soils, which in turn did not differ.
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Straw addition (mg g ) Fig. 3. The effect of straw amendments on bacterial growth, estimated as leucine incorporation into extracted bacteria, in soils with different N status: Unfertilized (black circle), Low N (red triangles), and High N (blue squares) soils. A) 2 week after amendment, B) 3 weeks after amendment, C) 4 weeks after amendment. Bars denote SE (n ¼ 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
There were no major differences in the effect of adding C and N to study nutrient limitation between the incubation times (1e3 weeks for starch, Fig. 4, and 2e4 weeks for straw, Fig. 5). In the unamended soil (0 mg g1 starch or straw), bacterial growth was Climited in all soils, with the extent of growth response with Caddition increasing in the order Unfertilized < Low N ¼ High N. After adding low amounts of starch (10 mg g1), the limitation test indicated that the bacteria were still C limited, although the growth response to only C was usually less than in the nonamended control soils (Fig. 4). With the amendment of 20 mg g1 starch, a positive growth response to C-addition was found only in the High N soil, while for the other two soils only adding C in combination with N gave any growth response. Using the highest level of starch, there was no bacterial growth response to adding only C in any of the soils, that is, no evidence for C-limitation. Bacterial growth did not respond much to N addition except for the amendment of 40 mg g1 soil in the High N soil after 2 and 3 weeks. Similar results as for adding starch were found after adding straw (Fig. 5). Increasing the amount of added straw decreased the bacterial growth response after adding only C, especially in the Unfertilized and Low N soils. For example, adding 20 mg g1 straw resulted in a bacterial growth increase after adding C only in the High N soil, while at the higher amendment levels of straw, no evidence of C-limitation was found in any of the soils. At the highest levels of straw addition (80 mg g1), adding glucose-C actually resulted in lower bacterial growth then in the control with no C or
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Fig. 4. Bacterial growth 72 h after adding C (Glucose) and N (NH4NO3) to starch amended soils with different N status: Unfertilized (open bars), Low N (hatched bars), and High N (filled bars) soils. Soils were amended with 0, 10, 20 and 40 mg starch g1 soil and incubated for 1, 2 and 3 weeks at 22 C. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil). The effect of adding C, N or CN was expressed as D bacterial growth calculated as the difference to bacterial growth in the control (No addition). A value close to zero thus indicates no extra growth on the added nutrients. Bars denote SE (n ¼ 2).
N addition (the No treatment). Except for the highest rate of straw amendment little or no effect of adding N alone was found. Adding CN in combination always resulted in large growth increases, usually around 150e250 pmol Leu h1 g1 in the Unfertilized and Low N soil. However, in the High N soils, lower values were found after CN addition, especially in the treatments with no addition or low levels of starch addition (Fig. 4). However, adding starch or straw resulted in increased bacterial growth responses after adding CN with increasing amendment rates, with similar increased bacterial growth in all three soils with different N-status after adding the highest rate of starch, 40 mg1 g1. To summarize the results we calculated a D CeN limitation index (Figs. 6 and 7), which is the difference of bacterial growth after adding C minus the response after adding N. A positive value suggests C-limitation, while a negative value suggests N-limitation. A
value around zero indicates that neither C nor N alone limits growth, but C and N in combination, that is co-tolerance or at least close to co-tolerance of two nutrients (but see Discussion for other interpretations). No major differences between incubation times were seen, and thus results general for all times are reported. For both starch (Fig. 6) and straw (Fig. 7) addition there were significant statistical interaction between the soils with different N-status and the amendment levels except at the first measuring occasion. The D CeN limitation index increased with increasing N-status of the soil (unfertilized < Low N < High N soils) in the non-amended soils, and in the two lowest level of starch (10 and 20 mg g1) and the lowest level of straw amendment (10 mg g1). Up to these substrate amendment rates the D CeN limitation index decreased compared to the unamended soils, indicating lower extent of C-limitation with increasing substrate addition. With 20 mg g1 starch or straw
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Fig. 5. Bacterial growth 72 h after adding C (Glucose) and N (NH4NO3) to straw amended soils with different N status: Unfertilized (open bars), Low N (hatched bars), and High N (filled bars) soils. Soils were amended with 0, 20, 40 and 80 mg straw g1 soil and incubated for 2, 3 and 4 weeks at 22 C. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil). The effect of adding C, N or CN was expressed as D bacterial growth calculated as the difference to bacterial growth in the control (No addition). A value close to zero thus indicates no extra growth on the added nutrients. Bars denote SE (n ¼ 2).
the D CeN limitation index in the unfertilized soil was close to zero during all measurement occasions indicating close to co-limitation of both C and N for bacterial growth, while in the High N soils a Climitation of the bacterial growth was still evident. For the highest level of starch (40 mg g1), and the two highest levels of straw addition (40 and 80 mg g1), a different pattern was found. There was no effect of N-status of the soils, and in almost all cases a D CeN limitation index close to or below zero was found, suggesting colimitation or even N-limitation for bacterial growth. However, the negative values for the D CeN limitation index, suggesting N-limitation, were never as large as the positive values suggesting Climitation. For example, highest values for the D CeN limitation index was around 100e150 pmol Leu h1 g1 in the High N soil with low substrate additions (Fig. 6). Most negative values for the D CeN limitation index were usually only around 50 pmol
Leu h1 g1 or less, except for the highest level of straw amendment after 2 weeks (Fig. 7). 4. Discussion 4.1. Bacterial growth in soil and direct effect of starch and straw addition Without substrate addition bacterial growth was lowest in the High N soil. This has been found earlier in these soils (Kamble et al., 2013) and decreased bacterial growth appears to be a common response to increased N availability in N-poor boreal forest soils (Demoling et al., 2008). The decreased bacterial growth also appears to be part of a common decreased total activity of soil microbes in response to N-fertilization of boreal forests (Arnebrant
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et al., 1996; Thirukkumaran and Parkinson, 2000; Sjöberg et al., 2003; Bowden et al., 2004; Knorr et al., 2005; Janssens et al., 2010). So far no common explanation for this phenomenon exists (Liu and Greaver, 2010), but the fact that ligninolytic enzymes are inhibited by adding N is one possibility (Pregitzer et al., 2008; Zak et al., 2008). Since these enzymes appear to be key enzymes, possibly rate limiting ones, in degrading recalcitrant organic matter, the effect of N-addition may thus be a decrease in substrate availability. Alleviating substrate limitation increased microbial activity, in that both adding straw (Fig. 3) and starch (Fig. 2) increased bacterial growth. Previous work has also shown that adding similar type of substrates (e.g. straw or cellulose) results in increased bacterial growth (Rousk and Bååth, 2007; Meidute et al., 2008). It was expected that starch would be more favored by bacteria, since earlier results indicate that starch is preferable metabolized by bacteria (Rinnan and Bååth, 2009), while straw is more favored by fungi (Henriksen and Breland, 1999; Bossuyt et al., 2001; Six et al., 2006; Rousk and Bååth, 2007). However, a rapid response of bacteria after adding wheat residues has also been reported (Marschner et al., 2011). In the present study we did not find any major difference in the bacterial growth response to starch or straw, if the same level of amendment was compared.
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Starch addition (mg g ) Fig. 6. The D CeN limitation index for bacterial growth 72 h after adding C (Glucose) to that after adding N (NH4NO3) to soils with different nitrogen status: Unfertilized (black circles), Low N (red triangles), and High N (blue squares). The soils were incubated for 1 (A), 2 (B) and 3 (C) weeks at 22 C after amending with different amounts of starch. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil), where the D CeN limitation index was calculated as the difference in bacterial growth after adding C and adding N. Values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0 suggesting a co-limitation or close to a co-limitation by both C and N. Bars denote SE (n ¼ 2). ANOVA statistics with A ¼ amendment level of starch and S ¼ soils with different N status. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Our first hypothesis was that additions of large amounts of Crich substrate would turn the soil from being C-limited for bacterial growth to N-limited. The effect on C-limitation, from a growth response after adding glucose-C to no growth response with increasing starch or straw additions, was most easily seen in the High N soil. In the unamended High N soil, adding glucose-C always resulted in increased bacterial growth indicating C-limitation; this was not the case after adding the highest levels of starch (Fig. 4) or straw (Fig. 5). Similar results, with a shift from C-limitation to no measurable C-limitation, were also observed by Aldén et al. (2001) after adding straw to soil. Theoretically the soils should turn into being N-limited after adding enough starch or straw, that is, result in a growth increase after adding easily available N. The extent of growth increases after adding N was minor, however. Even in the highest levels of straw and starch amendments little positive effects on bacterial growth was found after adding N, although it was higher than for adding C, resulting in negative D CeN limitation indices (Figs. 6 and 7). There are several possible explanations for this lack of clear effects of adding N even if the soil was expected to be N-limited for bacterial growth. First, the soils may have been co-limited by both C and N even at the highest amendment rates of straw and starch, and thus both nutrients had to be added in order to alleviate the existing limitation (independent nutrient co-limitation sensu Saito et al., 2008). Such situations have been described for N and P limitation in aquatic habitats (Elser et al., 2009b). Ekblad and Nordgren (2002) also observed that after the addition of sucrose to a soil, the microbial activity measured using respiration rate appeared to be limited by both C and N. In our study co-limitation was, however, most likely only the case with intermediate levels of substrate amendments, for example the case after adding 20 or 40 mg straw g1 to the Unfertilized and Low N soils (Fig. 5). Colimitation by both C and N is therefore unlikely to be the case for the highest starch and straw amendments, especially in soils, which from the beginning were low in available N. Another explanation for the lack of clear effects on bacterial growth in high amended soils after adding N is that the response will be in fungal instead of in bacterial growth. This could be the case for straw, which will favor fungal growth (Rousk and Bååth,
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Straw addition (mg g ) Fig. 7. The D CeN limitation index for bacterial growth 72 h after adding C (Glucose) to that after adding N (NH4NO3) to soils with different nitrogen status: Unfertilized (black circles), Low N (red triangles), and High N (blue squares). The soils were incubated for 1 (A), 2 (B) and 3 (C) weeks at 22 C after amending with different amounts of straw. Bacterial growth was estimated as leucine incorporation into extracted bacteria (pmol Leu incorporated into extracted bacteria h1 g1 soil), where the D CeN limitation index was calculated as the difference in bacterial growth after adding C and adding N. Values >0 suggesting a C limited bacterial community, with the extent of C limitation increasing with a higher value of the expression. A value <0 suggests N limitation, while a value near 0 suggesting a co-limitation or close to a co-limitation by both C and N. Bars denote SE (n ¼ 2). ANOVA statistics with A ¼ amendment level of straw and S ¼ soils with different N status.
2007). Earlier results also suggested that straw with extra N actually favored fungi even more then only adding straw, while bacteria were not affected by the extra N (Rousk and Bååth, 2007). However, since starch addition has been shown to favor bacterial growth (Rinnan and Bååth, 2009), this is an unlikely explanation in the case of starch addition. Although there usually will be easily available N present as ammonium or nitrate in a C-limited soil, the opposite is not necessarily the case. Thus in C-limited soils inorganic N will usually accumulate; an effect used to measure net nitrogen mineralization by incubating soil in the laboratory. However, in an N-limited soil, there might be no similar accumulation of easily available C. Instead, in an N-limiting situation easily available C might be respired (Schimel and Weintraub, 2003) or taken up as energy reserve, for example as triglycerides in fungi (Ekblad and Nordgren, 2002; Bååth, 2003) or as poly-beta-hydroxybutyric acid (PHB) in bacteria (Wang and Bakken, 1998), and thus not immediately available for growth. Furthermore, one has to bear in mind that limitation by a macromolecule, like starch or cellulose in straw, and an easily available substrate, like glucose, is not strictly the same. The former needs to be degraded into smaller monomers by extracellular enzymes in order to be available to the microorganisms. This may take some time, especially if the proper enzymes have to be induced. Alleviating N-limitation would immediately cause a C-limitation in this scenario, and there will be a time lag in production of available C from the macromolecules or soil organic matter, before extensive growth will be present. There may thus be a different time frame for a response after alleviating N-limitations compared to C-limitations in soil, where in the former case more than the 72 h used in the present study is needed to detect a response in bacterial growth. This may be a different situation compared to aquatic habitats, where addition of P and N often result in a large increase in bacterial growth within a few days (Elser et al., 1995; Rivkin and Anderson, 1997; Granéli et al., 2004), suggesting that in aquatic habitat there will directly be easily available C present in N or P limited situations. The accumulation of dissolved organic C has also been found under P-limitation in aquatic systems (Vadstein et al., 2003). There were significant interactions between substrate amendment levels and different N-status in the soil in determining C- and N-limitation of the bacterial community, substantiating our second hypothesis. Thus, there was a combination of soil and substrate addition that resulted in co-limitation in accordance with our third hypothesis. However, it is unlikely that strict co-limitation is very common in soil. Instead, a situation close to co-limitation is more likely. Adding the limited nutrient will in such a case increase bacterial growth, but then rapidly induce limitation of the secondary limiting nutrient, resulting in only a barely detected growth increase. However, adding both limiting nutrient will result in extensive growth as in the present study. Such a situation was also found in a subarctic heath soil (Rinnan et al., 2007). The extent of bacterial growth after adding glucose-C suggested increasing amounts of available N for bacterial growth with increasing N-fertilization rate (Figs. 4 and 5 with 0 mg g1 additions; see also Kamble et al., 2013). The amount of starch and straw needed to change the system into not being C-limited also indicated the same order of N-availability. This was most easily seen when comparing the D CeN limitation index (Figs. 6 and 7), with the High N soil tending to stay C-limited at higher substrate amendment levels than the Unfertilized soil, with presumably the least available N. Thus, comparing limiting nutrients for bacterial growth could be used in two ways to suggest the availability of N as the secondary limiting nutrient; by the extent of the direct growth increase after adding the primary limiting nutrient in an easily available form (glucose-C) or by the amount of the primary limiting nutrient added
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in a more recalcitrant form (starch-C and straw-C) needed to alter the soil situation from C-limitation to co-limitation by C and N. Although the use of the D CeN limitation index both was less time consuming (half of the treatments needed compared to a full factorial design), and appeared easy to interpret, it should be used with some caution. Similar problems will be the case if the Log C/N growth ratio is used. First, there will be the same index in the case of no growth response after C-addition and N-addition alone or if both treatments increase bacterial growth to the same extent. To differentiate between these two results, a control with neither Cnor N-addition has to be included (our No treatment). Even more important is the combined CN treatment included in the full factorial design. This is a positive control, which should increase bacterial growth to show that not another nutrient is limiting growth or that too low amounts of C and N have been added to increase growth, as discussed by Demoling et al. (2007). In most cases the combined addition of CN resulted in a clear growth increase, suggesting that this was not a problem in our study. Adding CN to the High N soils without starch or straw resulted, however, in a lower increased bacterial growth then adding CN to the other two soils, with even lower growth increase then adding C alone. This was also found earlier for this soil (Kamble et al., 2013), who suggested that high levels of N in the High N soil may be the reason for low bacterial growth. High levels of soil N have earlier been shown to reduce bacterial growth (Aldén et al., 2001). Adding starch or straw resulted in soils with increased bacterial growth after adding CN, resulting in similar bacterial growth increases at highest substrate loadings as in the other two, less N-rich, soils. This is in accordance with increasing C-rich substrate loadings resulting in immobilization of N, consequently with the presumed negative effect of high amounts of N on bacterial growth disappearing. However, another possibility is fungal growth being stimulated after CN addition in the High N soil, with fungi then outcompeting bacteria. Thus, fungal growth has to be studied to differentiate between these suggestions. 4.3. Conclusion We found that adding C-rich substrate, like starch and straw, resulted in increased bacterial growth, but at the same time switches the soil from being C-limited to being close to co-limited by both C and N or even N-limited. The extent of C- or N-limitation was an interaction between the amount of substrate added and the initial N-status of the soil. A more extensive C-limitation at lower substrate additions and higher N-status of the soil was found, while especially high substrate additions and low N status soil was more inclined to become co- or only N-limited. However, alleviating Climitation by adding easily available C (glucose) resulted in more extensive bacterial growth than alleviating N-limitation (induced by adding straw or starch) by adding inorganic N. Thus, in a Climited soil there appeared to be N available immediate for growth, while in an N-limited soil, easily available C was not immediately available. The present study has, however, concentrated on bacterial growth. To be able to properly evaluate nutrient limitation in soil, the response of fungal growth has to be included. Furthermore, we induced N-limitation by adding fresh C-rich substrate in excess and in the form of macromolecules. The response of the soil microorganisms to N-limitation, and to alleviating limitation, may be different in a soil naturally N-limited. Acknowledgments This study was supported by an Erasmus Mundi grant to P.N.K. and by the Swedish Research Council to E.B. (Project No. 20094503). We thank Dr. S.D. Frey for providing samples from the
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