- Email: [email protected]
Simultaneous screening of microbial energetics and CO2 respiration in soil samples from different ecosystems
Soil Biology & Biochemistry 83 (2015) 88e92
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Simultaneous screening of microbial energetics and CO2 respiration in soil samples from different ecosystems € lscher Anke M. Herrmann*, Tobias Bo Department of Chemistry & Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O.Box 7015, SE-750 07 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 October 2014 Received in revised form 20 January 2015 Accepted 21 January 2015 Available online
The calorespirometric ratio, i.e. the ratio of heat production-to-CO2 production has been used to evaluate metabolism and microbial carbon use efficiency in soil systems. But limited sample throughput and high variability when evaluating microbial energetics and CO2 respiration separately hampered its applicability in soil science. In this study we tested if heat flows and CO2 respiration can be determined simultaneously in the same soil sample without any measurable experimental biases. Heat outputs were not significantly different when CO2 respiration was determined concurrently by means of a colorimetric method. Our method provides a simple, cheap and rapid screening of the calorespirometric ratio, and in comparison with previous studies, the reproducibility of the ratios was improved. Its non-destructive nature allows combination with the characterization of the chemical and biological composition in soil systems. Used together these methods have the potential to improve our understanding of microbial communities, their processes and activities below-ground. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Calorespirometric ratio Carbon respiration Heat production Microbial energetics Calorimetry Microbial C use efficiency
Basal and substrate-induced respiration approaches are commonly used to (i) assess maintenance energy requirements (Anderson and Domsch, 1985), (ii) examine the amount of microbial biomass (Anderson and Domsch, 1978), (iii) determine microbial carbon (C) use efficiency (Steinweg et al., 2008; Frey et al., 2013) and (iv) to evaluate the functional status of soils (Degens and Harris, 1997; Campbell et al., 2003). Yet, a microbial energetics approach is an emerging method in exploring microbial metabolism and microbial C use efficiencies in soil systems. Isothermal calorimetry is used for the determination of calorespirometric ratios (ratio of heat production-to-CO2 production) and thermodynamic efficiency indices (Sparling, 1983; Barros et al., 2010, 2011; Harris et al., 2012). Isothermal calorimetry measures the net outcome of heat flows derived from all catabolic and anabolic reactions in soils; it quantifies all microbial metabolic processes, not only those accounted for by CO2 respiration measurements. Recent research (Herrmann et al., 2014) therefore has demonstrated that such an approach is a more comprehensive methodology, providing complementary information for investigating soil microbial metabolism than the respiratory approach alone. Although very little is known about the link between soil
* Corresponding author. Tel.: þ46 18 67 1561; fax: þ46 18 67 3476. E-mail address: [email protected] (A.M. Herrmann). http://dx.doi.org/10.1016/j.soilbio.2015.01.020 0038-0717/© 2015 Elsevier Ltd. All rights reserved.
microbial energetics and CO2 respiration, changes in calorespirometric ratios may indicate differences in microbial C use efficiencies and metabolic pathways (Hansen et al., 2004; Herrmann et al., 2014) or a change in organic material undergoing decomposition (Hansen et al., 2004). However, respiration and microbial energetics are often obtained by separate samples and methods (Sparling, 1983; Herrmann et al., 2014) which often results in highly varying calorespirometric ratios. Barros et al. (2010, 2011) determined the calorespirometric ratio solely based on calorimetry by simultaneously monitoring the metabolic heat production in two separate soil samples. In this type of experiment one sample is incubated in the presence of a vial with sodium hydroxide (NaOH) and the other sample contains no NaOH. The reaction of CO2 with NaOH is an exothermic one which releases 108.5 kJ mol1 CO2 or 9.04 mJ mg1 CO2eC (Criddle et al., 1991), and the amount of evolved CO2 can be calculated by the differences of heat released between soils incubated with and without NaOH. However, the sample throughput is limited and the method relies on the assumption that the reaction between CO2 and NaOH does not introduce any experimental biases to heat dissipated from microbial decomposition. This assumption has not yet been validated. Moreover, the limited sample throughput is a major drawback of the proposed method as high sample throughput is preferable in soil science because soils are structurally heterogeneous media necessitating substantial numbers of treatment replicates. An
€lscher / Soil Biology & Biochemistry 83 (2015) 88e92 A.M. Herrmann, T. Bo
alternative to the use of NaOH to trap evolved CO2 is the use of a colorimetric method for the determination of CO2 respiration (Rowell, 1995) which was further developed by Campbell et al. (2003) into a rapid microtiter plate method (MicroResp™). The reaction here is between CO2 and bicarbonate and it is an endothermic reaction of þ17.8 kJ mol1 or þ1.48 mJ mg1 CO2eC respired (Chang and Cruickshank, 2005). This is only a sixth of the absolute value between the reaction of CO2 and NaOH. The minimal heat consumption between CO2 and bicarbonate suggests that the colorimetric method may be combined with the microbial energetics approach for rapid screening of calorespirometric ratios in the same soil sample. Here, the heat outputs in soil samples are solely derived from microbial metabolism and CO2 respired is determined on the basis of the colorimetric approach (Rowell, 1995; Campbell et al., 2003). The aim of this study was to evaluate if simultaneous measurements of microbial energetics and CO2 respiration can be determined in soils without any measurable experimental biases. The rationale is that there are no significant changes in heat outputs when bicarbonate CO2-traps are incorporated into microbial energetic measurements, and that the proposed method would then allow rapid screening of the calorespirometric ratio in soils. Soils were sampled from six Swedish long-term field experiments under differing land-use management systems (Table 1). At each site 20 sub-samples were taken, thoroughly mixed and combined to one sample. Samples were sieved to 2 mm, homogenized, plant material removed and stored at 20 C until September 2013. Soils were then adjusted to 45% of their water holding capacity (WHC) and pre-incubated for 10 days at 25 C to allow any disturbance due to soil preparation (i.e. sieving, freeze-thawing etc.) to subside. For each experimental site, sixteen aliquots of soil (5 g dry-weight) were placed into 20 mL glass reaction vessels and samples were split into two sets. One set (eight aliquots of soil) was amended with 60 ml per g of soil of Milli-Q water whereas the other set received 60 ml per g of soil of D-glucose (30 mg C substrate per mL soil water) (Campbell et al., 2003). All the solution additions brought the soil moisture content up to 60% WHC. In half of each set, CO2 respiration was determined in addition to heat production by a colorimetric method that relies on the change in the pH due the reaction between evolved CO2 and bicarbonate (Rowell, 1995; Campbell et al., 2003). CO2-traps were prepared in breakable microtiter assembly strips (ThermoScientific, Vantaa, Finland). Each
89
Fig. 1. Schematic representation of the experimental design. Either Milli-Q water or glucose was added as substrate to soil samples at time ¼ 0. CO2-traps are pink at the start of the incubation period and the color changes to yellow depending on the amount of carbon respired during the incubation. A calibration curve of absorbance versus headspace equilibrium CO2 concentration is used to estimate how much carbon is respired from the system. Heat (Q Sample) is dissipated during the experiment and this is determined by isothermal calorimetry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
strip comprises eight microtiter wells (300 ml total volume per well) which are detachable into single CO2-trap wells. They can be reassembled in a 96-well plate carriage to be read when required. All samples (with and without CO2-traps) were then sealed and introduced into a TAM Air calorimeter (TA Instruments Sollentuna, Sweden) and heat production and CO2 evolution were measured over 5 h at 25 C. A schematic representation of one reaction vessel with a CO2-trap is given in Fig. 1. Heat output was recorded between 1.5 h and 5 h as the calorimeter requires at least 45 min until the heat signal can be considered to be correct. For CO2 measurements, the CO2-traps were read immediately before and after 5 h incubation at 25 C with a plate reader (SPECTRAmax® Plus384, Molecular Devices, Wokingham, U.K.) at 572 nm. A calibration curve of absorbance () versus headspace equilibrium CO2 concentration (y) in reaction vials was fitted to a power decay model (R2 ¼ 0.99) as follows: y ¼ 3.19x3.0. Preliminary work showed that CO2
Table 1 Soil characteristics; arable and grassland soils: A-horizon 0e10 cm; forest system: E-horizon 0e10 cm. Location
Soil type (FAO)
Province Uppland Eutric Cambisol Arable soila
Grasslanda
Eutric Cambisol a
Forest system
Sandy Podzol
€sterbotten Province Va Eutric Cambisol Arable soilb Grasslandb
Eutric Cambisol
Forest systemb
Haplic Podzol
a b c d
Coordinates
Soil managementc
Field experiment
Soil C (%)
Soil N (%)
Soil pH (H2O)
Detailed information
Fors: 60 200 N, 17 290 E
Crop rotation: Barley, oats, spring oilseed, winter wheat, oats, winter wheat Semi-natural pasture
Since 1963
1.8
0.20
7.3
Carlgren and Mattsson (2001)
3.5
0.25
6.1
Sindhøj et al. (2006).
Pinus sylvestris L. forest
Grazing since at least 1720 Since 1986
1.3
0.06
4.2
Persson (1980).
Barley annually
Since 1965
2.7
0.18
5.9
€ Bergkvist and Oborn (2011).
Five year green fallow followed by one year barleyd Picea abies forest; unfertilized control soil
Since 1965
3.7
0.25
5.3
€ Bergkvist and Oborn (2011).
Since 1986
1.5
0.06
4.4
Linder (1995)
Nåntuna: 59 480 N, 17 380 E €draås: Ja 60 490 N, 16 300 E € b€ Ro acksdalen: 63 480 N, 20 140 E € b€ Ro acksdalen: 63 480 N, 20 140 E Flakaliden: 64 070 N, 19 270 E
Samples taken in April 2011. Samples taken in August 2012. Crop in italics is the present crop when soil samples were taken. Samples taken in the fifth year of green fallow.
90
€lscher / Soil Biology & Biochemistry 83 (2015) 88e92 A.M. Herrmann, T. Bo
production rates were constant throughout the 5 h incubation interval (data not shown). In total 96 samples were examined but only 24 samples could be analyzed simultaneously as only three TAM air calorimeters with 8 channels each were available. Therefore, the experiment was staggered over 4 days, i.e. samples and treatments were randomized and incubated in four separate calorimetry runs. The mean values are reported for the four technical replicates of each treatment (i.e. Milli-Q water with or without CO2-traps, Dglucose with or without CO2-traps) from the six long-term field experiments. The Student's t-test for independent variables was used to calculate differences in heat output rates between samples without and with CO2-traps as well as for differences in calorespirometric ratios between samples amended with Milli-Q water or glucose, respectively (Statsoft, 2000). Heat output ranged between 17 and 152 mJ g1 soil in Milli-Q water amended soils and increased to 306e1129 mJ g1 soil in
Table 2 Cumulative amounts of CO2 respired in soils amended with MilliQ-water (CO2eCMilli-Q water) or glucose (CO2eCGlucose) during a 5 h incubation at 25 C. Theoretical additional heat dissipated from Milli-Q water (DQMilli-Q water) and glucose (DQGlucose) amended soils due to the reaction between CO2 and bicarbonate. Mean values and standard errors of four technical replicates.
DQGlucose CO2eCMilli-Q water DQMilli-Q water CO2eCGlucose (mg C g1 soil) (mJ g1 soil) (mg C g1 soil) (mJ g1 soil) Province Uppland Arable land 0.9 ± 0.2 Grassland 3.3 ± 0.2 Forest system 0.8 ± 0.1 Province V€ asterbotten Arable land 1.6 ± 0.1 Grassland 1.1 ± 0.05 Forest system 3.5 ± 0.3
1.4 ± 0.3 4.9 ± 0.3 1.2 ± 0.1
13.3 ± 2.0 25.4 ± 3.0 5.7 ± 0.7
20 ± 3 38 ± 5 9 ± 1
2.4 ± 0.2 1.6 ± 0.1 5.2 ± 0.5
10.1 ± 1.8 7.6 ± 1.2 16.4 ± 1.5
15 ± 3 11 ± 2 24 ± 2
Fig. 2. Heat production (Q; mJ g1 soil) from soils amended with (A) Milli-Q water or (B) glucose over a 1.5e5 h time interval at 25 C. Mean values of four technical replicates and error bars indicate standard errors. Heat outputs are significantly different between Q without CO2-trap and Q with CO2-trap P < 0.05 (Student's t-test) when soil treatment is suffixed by *.
€lscher / Soil Biology & Biochemistry 83 (2015) 88e92 A.M. Herrmann, T. Bo
91
Fig. 3. Calorespirometric ratios of Milli-Q water (RatioMilli-Q) and glucose (RatioGlucose) amended soils. Mean values of four technical replicates and the error bars indicate standard errors. Ratios are significantly different between Milli-Q water and glucose amendments at P < 0.05, 0.01 or 0.001(Student's t-test) when soil treatments are suffixed by *, ** or ***, respectively. Soil treatment suffixed with ‘ assumes unequal variances when applying Student's t-test.
glucose amended soils (Fig. 2a and b). CO2 respired ranged between 0.9 and 3.5 mg C g1 in Milli-Q water amended soil with an increase to 5.7 and 25.4 mg C g1 when glucose was added to soils (Table 2). Using the respiration values and assuming an endothermic reaction of þ17.8 kJ mol1 or þ1.48 mJ per mg CO2eC respired suggest a 2e7% decrease in heat outputs (cf. Table 2 and Fig. 2). Such values were, however, within the standard error of observed heat outputs. Changes in heat outputs due to the colorimetric respiration method were therefore not detectable (Fig. 2). Thus, simultaneous measurements of respiration did not introduce any measurable experimental bias when determining microbial energetics in a wide range of soils under different land-use management systems. The calorespirometric ratios ranged from 31 up to 100 mJ mg1 CO2eC in our study with differences between glucose and Milli-Q water amended soil systems (Fig. 3). We observed a significant increase in the calorespirometric ratio when the forest systems and the arable soil in the province Uppland were amended with glucose in comparison with Milli-Q water additions (Fig. 3). An increase in the ratio in these systems may indicate that more reduced substrates, i.e. substrates with a higher energy content, are decomposed and/or more incomplete decomposition processes are taking place resulting in intermediate products, i.e. intermediary catabolism with CO2 not being the decomposition product (Hansen et al., 2004; Herrmann et al., 2014). That there is a shift towards more reduced substrates undergoing decomposition is unlikely as decomposition is dominated by glucose breakdown in the carbonsubstrate amended treatments. Calorespirometric ratios derived from aerobic degradation of carbohydrates, e.g. glucose, are usually € et al., between 260 and 460 kJ mol1 (Hansen et al., 2004; Wadso 2004) which corresponds to 21.7 up to 38.3 mJ mg1 CO2eC. In soils, such ratios vary substantially ranging from 150 up to 1200 kJ mol1 equivalent to 12.5 up to 100 mJ mg1 CO2eC (Sparling, 1983; Barros et al., 2010; Harris et al., 2012). Since the forest soils are nutrient poor systems and the Uppland arable soil samples were taken prior annual mineral fertilizer addition, we hypothesize that the addition of glucose, an easily available substrate for most microorganisms, may induce a more inefficient microbial metabolism in these soil samples. Such inefficient metabolism may be due to incomplete decomposition processes with CO2 not being the decomposition end product but waste heat
is dissipated from the samples (intermediary catabolism). In other words, more heat is wasted per unit CO2 respired in nutrient poor soil samples, which is reflected by the higher calorespirometric ratios upon glucose addition. Although, the present experiment was not designed to test this hypothesis, we nevertheless consider that this observation warrants further investigation. The method here provides a simple, cheap and rapid screening of the calorespirometric ratio with improved reproducibility of the ratios (cf. 8e26% coefficient of variances (CV) versus up to 50% CV; see Herrmann et al., 2014, ratios calculated from Fig. 1b and d). Its non-destructive nature allows for combination with characterization of the chemical composition of native soil organic matter and/ or microbial community composition methods. In combination with labeled substrates, we believe that exploring the calorespirometric ratio in soils may improve our understanding of physiological life strategies, i.e. evaluation of biochemical pathways (Dijkstra et al., 2011; Apostel et al., 2013) and microbial C use efficiencies (Harris et al., 2012; Frey et al., 2013).
Acknowledgments We thank the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas 2012-530) for € is thanked for valuable comments on financial support. Lars Wadso the manuscript. We also sincerely acknowledge two anonymous reviewers who provided insightful comments which assisted us in developing and improving this paper. Shelagh Green is thanked for language review of the final manuscript.
References Anderson, J.P.E., Domsch, K.H., 1978. Physiological method for quantitative measurement of microbial biomass in soils. Soil Biology and Biochemistry 10, 215e221. Anderson, T.H., Domsch, K.H., 1985. Determination of eco-physiological maintenance requirements of soil microorganisms in a dormant state. Biology and Fertility of Soils 1, 81e89. Apostel, C., Dippold, M., Glaser, B., Kuzyakov, Y., 2013. Biochemical pathways of amino acids in soil: assessment by position-specific labeling and 13C-PLFA analysis. Soil Biology and Biochemistry 67, 31e40.
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€lscher / Soil Biology & Biochemistry 83 (2015) 88e92 A.M. Herrmann, T. Bo
~o n, J.A., Proupín, J., Villanueva, M., Hansen, L.D., Barros, N., Salgado, J., Rodríguez-An 2010. Calorimetric approach to metabolic carbon conversion efficiency in soils. Journal of Thermal Analysis and Calorimetry 99, 771e777. Barros, N., Fejoo, S., Hansen, S.D., 2011. Calorimetric determination of metabolic heat, CO2 rates and the calorespirometric ratio of soil basal metabolism. Geoderma 160, 542e547. € Bergkvist, G., Oborn, I., 2011. Long-term field experiments in Sweden e what are they designed to study and what could they be used for?. In: Stockdale, E.A., Watson, C. (Eds.), Making Crop Rotation Fit for the Future. Aspects of Applied Biology, vol. 113, pp. 75e85. Campbell, C.D., Chapman, S.J., Cameron, C.M., Davidson, M.S., Potts, J.M., 2003. A rapid microtiter plate method to measure carbon dioxide evolved from carbon substrate amendments so as to determine the physiological profiles of soil microbial communities by using whole soil. Applied and Environmental Microbiology 69, 3593e3599. Carlgren, K., Mattsson, L., 2001. Swedish soil fertility experiments. Acta Agriculturae Scandinavica, Section B - Soil and Plant Science 51, 49e76. Chang, R., Cruickshank, B., 2005. Chemistry, Eighth ed. Mc Graw Hill Higher Education, New York, USA. Criddle, R.S., Fontana, A.J., Rank, D.R., Paige, D., Hansen, L.D., Breidenbach, R.W., 1991. Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry. Analytical Biochemistry 194, 413e417. Degens, B.P., Harris, J.A., 1997. Development of a physiological approach to measuring the catabolic diversity of soil microbial communities. Soil Biology and Biochemistry 29, 1309e1320. Dijkstra, P., Thomas, S.C., Heinrich, P.L., Koch, G.W., Schwartz, E., Hungate, B.A., 2011. Effect of temperature on metabolic activity of intact microbial communities: evidence for altered metabolic pathway activity but not for increased mainté nance respiration and reduced carbon use efficiency. Soil Biology and Biochemistry 43, 2023e2031.
Frey, S.D., Lee, J., Melillo, J.M., Six, J., 2013. The temperature response of soil microbial efficiency and its feedback to climate. Nature Climate Change 3, 1e4. Hansen, L.D., Macfarlane, C., McKinnon, N., Smith, B.N., Criddle, R.S., 2004. Use of calorespirometric ratios, heat per CO2 and heat per O2, to quantify metabolic paths and energetics of growing cells. Thermochimica Acta 422, 55e61. Harris, J.A., Ritz, K., Coucheney, E., Grice, S.M., Lerch, T.Z., Pawlett, M., Herrmann, A.M., 2012. The thermodynamic efficiency of soil microbial communities subject to long-term stress is lower than those under conventional input regimes. Soil Biology and Biochemistry 47, 149e157. Herrmann, A.M., Coucheney, E., Nunan, N., 2014. Isothermal microcalorimetry provides new insight into terrestrial carbon cycling. Environmental Science and Technology 48, 4344e4352. Linder, S., 1995. Foliar analyses for detecting and correcting nutrient imbalances in Norway spruce. Ecological Bulletins 44, 178e190. Persson, T., 1980. Structure and function of northern coniferous forests e an ecosystem study. In: Ecological Bulletins 32. Stockholm. Sweden. Rowell, M.J., 1995. Colorimetric method for CO2 measurement in soil. Soil Biology and Biochemistry 7, 373e375. n, O., Ka €tterer, T., Gunnarsson, S., Pettersson, R., 2006. Projections Sindhøj, E., Andre of 30-year soil carbon balances for a semi-natural grassland under elevated CO2 based on measured root decomposability. Agriculture, Ecosystems and Environment 114, 360e368. Sparling, G.P., 1983. Estimation of microbial biomass and activity in soil using micro-calorimetry. Journal of Soil Science 34, 381e390. Statsoft, 2000. Statistica for Windows. Tulsa, OK, USA. Steinweg, J.M., Plante, A.F., Conant, R.T., Paul, E.A., Tanaka, D.L., 2008. Patterns of substrate utilization during long-term incubations at different temperatures. Soil Biology and Biochemistry 40, 2722e2728. €, L., Li, Y., Bjurman, J., 2004. Measurements on two mould fungi with a calWadso orespirometric method. Thermochimica Acta 422, 63e68.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Simultaneous screening of microbial energetics and CO2 respiration in soil samples from different ecosystems € lscher Anke M. Herrmann*, Tobias Bo Department of Chemistry & Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O.Box 7015, SE-750 07 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 October 2014 Received in revised form 20 January 2015 Accepted 21 January 2015 Available online
The calorespirometric ratio, i.e. the ratio of heat production-to-CO2 production has been used to evaluate metabolism and microbial carbon use efficiency in soil systems. But limited sample throughput and high variability when evaluating microbial energetics and CO2 respiration separately hampered its applicability in soil science. In this study we tested if heat flows and CO2 respiration can be determined simultaneously in the same soil sample without any measurable experimental biases. Heat outputs were not significantly different when CO2 respiration was determined concurrently by means of a colorimetric method. Our method provides a simple, cheap and rapid screening of the calorespirometric ratio, and in comparison with previous studies, the reproducibility of the ratios was improved. Its non-destructive nature allows combination with the characterization of the chemical and biological composition in soil systems. Used together these methods have the potential to improve our understanding of microbial communities, their processes and activities below-ground. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Calorespirometric ratio Carbon respiration Heat production Microbial energetics Calorimetry Microbial C use efficiency
Basal and substrate-induced respiration approaches are commonly used to (i) assess maintenance energy requirements (Anderson and Domsch, 1985), (ii) examine the amount of microbial biomass (Anderson and Domsch, 1978), (iii) determine microbial carbon (C) use efficiency (Steinweg et al., 2008; Frey et al., 2013) and (iv) to evaluate the functional status of soils (Degens and Harris, 1997; Campbell et al., 2003). Yet, a microbial energetics approach is an emerging method in exploring microbial metabolism and microbial C use efficiencies in soil systems. Isothermal calorimetry is used for the determination of calorespirometric ratios (ratio of heat production-to-CO2 production) and thermodynamic efficiency indices (Sparling, 1983; Barros et al., 2010, 2011; Harris et al., 2012). Isothermal calorimetry measures the net outcome of heat flows derived from all catabolic and anabolic reactions in soils; it quantifies all microbial metabolic processes, not only those accounted for by CO2 respiration measurements. Recent research (Herrmann et al., 2014) therefore has demonstrated that such an approach is a more comprehensive methodology, providing complementary information for investigating soil microbial metabolism than the respiratory approach alone. Although very little is known about the link between soil
* Corresponding author. Tel.: þ46 18 67 1561; fax: þ46 18 67 3476. E-mail address: [email protected] (A.M. Herrmann). http://dx.doi.org/10.1016/j.soilbio.2015.01.020 0038-0717/© 2015 Elsevier Ltd. All rights reserved.
microbial energetics and CO2 respiration, changes in calorespirometric ratios may indicate differences in microbial C use efficiencies and metabolic pathways (Hansen et al., 2004; Herrmann et al., 2014) or a change in organic material undergoing decomposition (Hansen et al., 2004). However, respiration and microbial energetics are often obtained by separate samples and methods (Sparling, 1983; Herrmann et al., 2014) which often results in highly varying calorespirometric ratios. Barros et al. (2010, 2011) determined the calorespirometric ratio solely based on calorimetry by simultaneously monitoring the metabolic heat production in two separate soil samples. In this type of experiment one sample is incubated in the presence of a vial with sodium hydroxide (NaOH) and the other sample contains no NaOH. The reaction of CO2 with NaOH is an exothermic one which releases 108.5 kJ mol1 CO2 or 9.04 mJ mg1 CO2eC (Criddle et al., 1991), and the amount of evolved CO2 can be calculated by the differences of heat released between soils incubated with and without NaOH. However, the sample throughput is limited and the method relies on the assumption that the reaction between CO2 and NaOH does not introduce any experimental biases to heat dissipated from microbial decomposition. This assumption has not yet been validated. Moreover, the limited sample throughput is a major drawback of the proposed method as high sample throughput is preferable in soil science because soils are structurally heterogeneous media necessitating substantial numbers of treatment replicates. An
€lscher / Soil Biology & Biochemistry 83 (2015) 88e92 A.M. Herrmann, T. Bo
alternative to the use of NaOH to trap evolved CO2 is the use of a colorimetric method for the determination of CO2 respiration (Rowell, 1995) which was further developed by Campbell et al. (2003) into a rapid microtiter plate method (MicroResp™). The reaction here is between CO2 and bicarbonate and it is an endothermic reaction of þ17.8 kJ mol1 or þ1.48 mJ mg1 CO2eC respired (Chang and Cruickshank, 2005). This is only a sixth of the absolute value between the reaction of CO2 and NaOH. The minimal heat consumption between CO2 and bicarbonate suggests that the colorimetric method may be combined with the microbial energetics approach for rapid screening of calorespirometric ratios in the same soil sample. Here, the heat outputs in soil samples are solely derived from microbial metabolism and CO2 respired is determined on the basis of the colorimetric approach (Rowell, 1995; Campbell et al., 2003). The aim of this study was to evaluate if simultaneous measurements of microbial energetics and CO2 respiration can be determined in soils without any measurable experimental biases. The rationale is that there are no significant changes in heat outputs when bicarbonate CO2-traps are incorporated into microbial energetic measurements, and that the proposed method would then allow rapid screening of the calorespirometric ratio in soils. Soils were sampled from six Swedish long-term field experiments under differing land-use management systems (Table 1). At each site 20 sub-samples were taken, thoroughly mixed and combined to one sample. Samples were sieved to 2 mm, homogenized, plant material removed and stored at 20 C until September 2013. Soils were then adjusted to 45% of their water holding capacity (WHC) and pre-incubated for 10 days at 25 C to allow any disturbance due to soil preparation (i.e. sieving, freeze-thawing etc.) to subside. For each experimental site, sixteen aliquots of soil (5 g dry-weight) were placed into 20 mL glass reaction vessels and samples were split into two sets. One set (eight aliquots of soil) was amended with 60 ml per g of soil of Milli-Q water whereas the other set received 60 ml per g of soil of D-glucose (30 mg C substrate per mL soil water) (Campbell et al., 2003). All the solution additions brought the soil moisture content up to 60% WHC. In half of each set, CO2 respiration was determined in addition to heat production by a colorimetric method that relies on the change in the pH due the reaction between evolved CO2 and bicarbonate (Rowell, 1995; Campbell et al., 2003). CO2-traps were prepared in breakable microtiter assembly strips (ThermoScientific, Vantaa, Finland). Each
89
Fig. 1. Schematic representation of the experimental design. Either Milli-Q water or glucose was added as substrate to soil samples at time ¼ 0. CO2-traps are pink at the start of the incubation period and the color changes to yellow depending on the amount of carbon respired during the incubation. A calibration curve of absorbance versus headspace equilibrium CO2 concentration is used to estimate how much carbon is respired from the system. Heat (Q Sample) is dissipated during the experiment and this is determined by isothermal calorimetry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
strip comprises eight microtiter wells (300 ml total volume per well) which are detachable into single CO2-trap wells. They can be reassembled in a 96-well plate carriage to be read when required. All samples (with and without CO2-traps) were then sealed and introduced into a TAM Air calorimeter (TA Instruments Sollentuna, Sweden) and heat production and CO2 evolution were measured over 5 h at 25 C. A schematic representation of one reaction vessel with a CO2-trap is given in Fig. 1. Heat output was recorded between 1.5 h and 5 h as the calorimeter requires at least 45 min until the heat signal can be considered to be correct. For CO2 measurements, the CO2-traps were read immediately before and after 5 h incubation at 25 C with a plate reader (SPECTRAmax® Plus384, Molecular Devices, Wokingham, U.K.) at 572 nm. A calibration curve of absorbance () versus headspace equilibrium CO2 concentration (y) in reaction vials was fitted to a power decay model (R2 ¼ 0.99) as follows: y ¼ 3.19x3.0. Preliminary work showed that CO2
Table 1 Soil characteristics; arable and grassland soils: A-horizon 0e10 cm; forest system: E-horizon 0e10 cm. Location
Soil type (FAO)
Province Uppland Eutric Cambisol Arable soila
Grasslanda
Eutric Cambisol a
Forest system
Sandy Podzol
€sterbotten Province Va Eutric Cambisol Arable soilb Grasslandb
Eutric Cambisol
Forest systemb
Haplic Podzol
a b c d
Coordinates
Soil managementc
Field experiment
Soil C (%)
Soil N (%)
Soil pH (H2O)
Detailed information
Fors: 60 200 N, 17 290 E
Crop rotation: Barley, oats, spring oilseed, winter wheat, oats, winter wheat Semi-natural pasture
Since 1963
1.8
0.20
7.3
Carlgren and Mattsson (2001)
3.5
0.25
6.1
Sindhøj et al. (2006).
Pinus sylvestris L. forest
Grazing since at least 1720 Since 1986
1.3
0.06
4.2
Persson (1980).
Barley annually
Since 1965
2.7
0.18
5.9
€ Bergkvist and Oborn (2011).
Five year green fallow followed by one year barleyd Picea abies forest; unfertilized control soil
Since 1965
3.7
0.25
5.3
€ Bergkvist and Oborn (2011).
Since 1986
1.5
0.06
4.4
Linder (1995)
Nåntuna: 59 480 N, 17 380 E €draås: Ja 60 490 N, 16 300 E € b€ Ro acksdalen: 63 480 N, 20 140 E € b€ Ro acksdalen: 63 480 N, 20 140 E Flakaliden: 64 070 N, 19 270 E
Samples taken in April 2011. Samples taken in August 2012. Crop in italics is the present crop when soil samples were taken. Samples taken in the fifth year of green fallow.
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production rates were constant throughout the 5 h incubation interval (data not shown). In total 96 samples were examined but only 24 samples could be analyzed simultaneously as only three TAM air calorimeters with 8 channels each were available. Therefore, the experiment was staggered over 4 days, i.e. samples and treatments were randomized and incubated in four separate calorimetry runs. The mean values are reported for the four technical replicates of each treatment (i.e. Milli-Q water with or without CO2-traps, Dglucose with or without CO2-traps) from the six long-term field experiments. The Student's t-test for independent variables was used to calculate differences in heat output rates between samples without and with CO2-traps as well as for differences in calorespirometric ratios between samples amended with Milli-Q water or glucose, respectively (Statsoft, 2000). Heat output ranged between 17 and 152 mJ g1 soil in Milli-Q water amended soils and increased to 306e1129 mJ g1 soil in
Table 2 Cumulative amounts of CO2 respired in soils amended with MilliQ-water (CO2eCMilli-Q water) or glucose (CO2eCGlucose) during a 5 h incubation at 25 C. Theoretical additional heat dissipated from Milli-Q water (DQMilli-Q water) and glucose (DQGlucose) amended soils due to the reaction between CO2 and bicarbonate. Mean values and standard errors of four technical replicates.
DQGlucose CO2eCMilli-Q water DQMilli-Q water CO2eCGlucose (mg C g1 soil) (mJ g1 soil) (mg C g1 soil) (mJ g1 soil) Province Uppland Arable land 0.9 ± 0.2 Grassland 3.3 ± 0.2 Forest system 0.8 ± 0.1 Province V€ asterbotten Arable land 1.6 ± 0.1 Grassland 1.1 ± 0.05 Forest system 3.5 ± 0.3
1.4 ± 0.3 4.9 ± 0.3 1.2 ± 0.1
13.3 ± 2.0 25.4 ± 3.0 5.7 ± 0.7
20 ± 3 38 ± 5 9 ± 1
2.4 ± 0.2 1.6 ± 0.1 5.2 ± 0.5
10.1 ± 1.8 7.6 ± 1.2 16.4 ± 1.5
15 ± 3 11 ± 2 24 ± 2
Fig. 2. Heat production (Q; mJ g1 soil) from soils amended with (A) Milli-Q water or (B) glucose over a 1.5e5 h time interval at 25 C. Mean values of four technical replicates and error bars indicate standard errors. Heat outputs are significantly different between Q without CO2-trap and Q with CO2-trap P < 0.05 (Student's t-test) when soil treatment is suffixed by *.
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Fig. 3. Calorespirometric ratios of Milli-Q water (RatioMilli-Q) and glucose (RatioGlucose) amended soils. Mean values of four technical replicates and the error bars indicate standard errors. Ratios are significantly different between Milli-Q water and glucose amendments at P < 0.05, 0.01 or 0.001(Student's t-test) when soil treatments are suffixed by *, ** or ***, respectively. Soil treatment suffixed with ‘ assumes unequal variances when applying Student's t-test.
glucose amended soils (Fig. 2a and b). CO2 respired ranged between 0.9 and 3.5 mg C g1 in Milli-Q water amended soil with an increase to 5.7 and 25.4 mg C g1 when glucose was added to soils (Table 2). Using the respiration values and assuming an endothermic reaction of þ17.8 kJ mol1 or þ1.48 mJ per mg CO2eC respired suggest a 2e7% decrease in heat outputs (cf. Table 2 and Fig. 2). Such values were, however, within the standard error of observed heat outputs. Changes in heat outputs due to the colorimetric respiration method were therefore not detectable (Fig. 2). Thus, simultaneous measurements of respiration did not introduce any measurable experimental bias when determining microbial energetics in a wide range of soils under different land-use management systems. The calorespirometric ratios ranged from 31 up to 100 mJ mg1 CO2eC in our study with differences between glucose and Milli-Q water amended soil systems (Fig. 3). We observed a significant increase in the calorespirometric ratio when the forest systems and the arable soil in the province Uppland were amended with glucose in comparison with Milli-Q water additions (Fig. 3). An increase in the ratio in these systems may indicate that more reduced substrates, i.e. substrates with a higher energy content, are decomposed and/or more incomplete decomposition processes are taking place resulting in intermediate products, i.e. intermediary catabolism with CO2 not being the decomposition product (Hansen et al., 2004; Herrmann et al., 2014). That there is a shift towards more reduced substrates undergoing decomposition is unlikely as decomposition is dominated by glucose breakdown in the carbonsubstrate amended treatments. Calorespirometric ratios derived from aerobic degradation of carbohydrates, e.g. glucose, are usually € et al., between 260 and 460 kJ mol1 (Hansen et al., 2004; Wadso 2004) which corresponds to 21.7 up to 38.3 mJ mg1 CO2eC. In soils, such ratios vary substantially ranging from 150 up to 1200 kJ mol1 equivalent to 12.5 up to 100 mJ mg1 CO2eC (Sparling, 1983; Barros et al., 2010; Harris et al., 2012). Since the forest soils are nutrient poor systems and the Uppland arable soil samples were taken prior annual mineral fertilizer addition, we hypothesize that the addition of glucose, an easily available substrate for most microorganisms, may induce a more inefficient microbial metabolism in these soil samples. Such inefficient metabolism may be due to incomplete decomposition processes with CO2 not being the decomposition end product but waste heat
is dissipated from the samples (intermediary catabolism). In other words, more heat is wasted per unit CO2 respired in nutrient poor soil samples, which is reflected by the higher calorespirometric ratios upon glucose addition. Although, the present experiment was not designed to test this hypothesis, we nevertheless consider that this observation warrants further investigation. The method here provides a simple, cheap and rapid screening of the calorespirometric ratio with improved reproducibility of the ratios (cf. 8e26% coefficient of variances (CV) versus up to 50% CV; see Herrmann et al., 2014, ratios calculated from Fig. 1b and d). Its non-destructive nature allows for combination with characterization of the chemical composition of native soil organic matter and/ or microbial community composition methods. In combination with labeled substrates, we believe that exploring the calorespirometric ratio in soils may improve our understanding of physiological life strategies, i.e. evaluation of biochemical pathways (Dijkstra et al., 2011; Apostel et al., 2013) and microbial C use efficiencies (Harris et al., 2012; Frey et al., 2013).
Acknowledgments We thank the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas 2012-530) for € is thanked for valuable comments on financial support. Lars Wadso the manuscript. We also sincerely acknowledge two anonymous reviewers who provided insightful comments which assisted us in developing and improving this paper. Shelagh Green is thanked for language review of the final manuscript.
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