Soil Biology & Biochemistry 83 (2015) 88e92

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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
Anke M. Herrmann*, Tobias Bo
€ lscher
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: The calorespirometric ratio, i.e. the ratio of heat production-to-CO2 production has been used to evaluate
Received 13 October 2014 metabolism and microbial carbon use efficiency in soil systems. But limited sample throughput and high
Received in revised form variability when evaluating microbial energetics and CO2 respiration separately hampered its applica-
20 January 2015
bility in soil science. In this study we tested if heat flows and CO2 respiration can be determined
Accepted 21 January 2015
Available online 7 February 2015
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
Keywords:
Calorespirometric ratio
comparison with previous studies, the reproducibility of the ratios was improved. Its non-destructive
Carbon respiration nature allows combination with the characterization of the chemical and biological composition in
Heat production soil systems. Used together these methods have the potential to improve our understanding of microbial
Microbial energetics communities, their processes and activities below-ground.
Calorimetry © 2015 Elsevier Ltd. All rights reserved.
Microbial C use efficiency

Basal and substrate-induced respiration approaches are microbial energetics and CO2 respiration, changes in calorespiro-
commonly used to (i) assess maintenance energy requirements metric ratios may indicate differences in microbial C use effi-
(Anderson and Domsch, 1985), (ii) examine the amount of micro- ciencies and metabolic pathways (Hansen et al., 2004; Herrmann
bial biomass (Anderson and Domsch, 1978), (iii) determine micro- et al., 2014) or a change in organic material undergoing decom-
bial carbon (C) use efficiency (Steinweg et al., 2008; Frey et al., position (Hansen et al., 2004). However, respiration and microbial
2013) and (iv) to evaluate the functional status of soils (Degens energetics are often obtained by separate samples and methods
and Harris, 1997; Campbell et al., 2003). Yet, a microbial ener- (Sparling, 1983; Herrmann et al., 2014) which often results in highly
getics approach is an emerging method in exploring microbial varying calorespirometric ratios. Barros et al. (2010, 2011) deter-
metabolism and microbial C use efficiencies in soil systems. mined the calorespirometric ratio solely based on calorimetry by
Isothermal calorimetry is used for the determination of calores- simultaneously monitoring the metabolic heat production in two
pirometric ratios (ratio of heat production-to-CO2 production) and separate soil samples. In this type of experiment one sample is
thermodynamic efficiency indices (Sparling, 1983; Barros et al., incubated in the presence of a vial with sodium hydroxide (NaOH)
2010, 2011; Harris et al., 2012). Isothermal calorimetry measures and the other sample contains no NaOH. The reaction of CO2 with
the net outcome of heat flows derived from all catabolic and NaOH is an exothermic one which releases
108.5 kJ mol
1 CO2
anabolic reactions in soils; it quantifies all microbial metabolic or
9.04 mJ mg
1 CO2eC (Criddle et al., 1991), and the amount of
processes, not only those accounted for by CO2 respiration mea- evolved CO2 can be calculated by the differences of heat released
surements. Recent research (Herrmann et al., 2014) therefore has between soils incubated with and without NaOH. However, the
demonstrated that such an approach is a more comprehensive sample throughput is limited and the method relies on the
methodology, providing complementary information for investi- assumption that the reaction between CO2 and NaOH does not
gating soil microbial metabolism than the respiratory approach introduce any experimental biases to heat dissipated from micro-
alone. Although very little is known about the link between soil bial 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
* Corresponding author. Tel.: þ46 18 67 1561; fax: þ46 18 67 3476.
necessitating substantial numbers of treatment replicates. An
E-mail address: Anke.Herrmann@slu.se (A.M. Herrmann).

http://dx.doi.org/10.1016/j.soilbio.2015.01.020
0038-0717/© 2015 Elsevier Ltd. All rights reserved.
 €lscher / Soil Biology & Biochemistry 83 (2015) 88e92
A.M. Herrmann, T. Bo 89

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 endo-
thermic reaction of þ17.8 kJ mol
1 or þ1.48 mJ mg
1 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 ener-
getics 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 eval-
uate 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 Fig. 1. Schematic representation of the experimental design. Either Milli-Q water or
changes in heat outputs when bicarbonate CO2-traps are incorpo- glucose was added as substrate to soil samples at time ¼ 0. CO2-traps are pink at the
rated into microbial energetic measurements, and that the pro- start of the incubation period and the color changes to yellow depending on the
posed method would then allow rapid screening of 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
calorespirometric ratio in soils. is respired from the system. Heat (Q Sample) is dissipated during the experiment and
Soils were sampled from six Swedish long-term field experi- this is determined by isothermal calorimetry. (For interpretation of the references to
ments under differing land-use management systems (Table 1). At colour in this figure legend, the reader is referred to the web version of this article.)
each site 20 sub-samples were taken, thoroughly mixed and com-
bined to one sample. Samples were sieved to 2 mm, homogenized,
plant material removed and stored at
20  C until September 2013. strip comprises eight microtiter wells (300 ml total volume per well)
Soils were then adjusted to 45% of their water holding capacity which are detachable into single CO2-trap wells. They can be
(WHC) and pre-incubated for 10 days at 25  C to allow any reassembled in a 96-well plate carriage to be read when required.
disturbance due to soil preparation (i.e. sieving, freeze-thawing All samples (with and without CO2-traps) were then sealed and
etc.) to subside. For each experimental site, sixteen aliquots of introduced into a TAM Air calorimeter (TA Instruments Sollentuna,
soil (5 g dry-weight) were placed into 20 mL glass reaction vessels Sweden) and heat production and CO2 evolution were measured
and samples were split into two sets. One set (eight aliquots of soil) over 5 h at 25  C. A schematic representation of one reaction vessel
was amended with 60 ml per g of soil of Milli-Q water whereas the with a CO2-trap is given in Fig. 1. Heat output was recorded between
other set received 60 ml per g of soil of D-glucose (30 mg C substrate 1.5 h and 5 h as the calorimeter requires at least 45 min until the
per mL soil water) (Campbell et al., 2003). All the solution additions heat signal can be considered to be correct. For CO2 measurements,
brought the soil moisture content up to 60% WHC. In half of each the CO2-traps were read immediately before and after 5 h incuba-
set, CO2 respiration was determined in addition to heat production tion at 25  C with a plate reader (SPECTRAmax® Plus384, Molecular
by a colorimetric method that relies on the change in the pH due Devices, Wokingham, U.K.) at 572 nm. A calibration curve of
the reaction between evolved CO2 and bicarbonate (Rowell, 1995; absorbance () versus headspace equilibrium CO2 concentration
Campbell et al., 2003). CO2-traps were prepared in breakable mi- (y) in reaction vials was fitted to a power decay model (R2 ¼ 0.99) as
crotiter assembly strips (ThermoScientific, Vantaa, Finland). Each follows: y ¼ 3.19x
3.0. 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) Coordinates Soil managementc Field experiment Soil C Soil N Soil pH Detailed information
(%) (%) (H2O)

Province Uppland
Arable soila Eutric Cambisol Fors: Crop rotation: Barley, oats, Since 1963 1.8 0.20 7.3 Carlgren and Mattsson (2001)
60 200 N, 17 290 E spring oilseed, winter
wheat, oats, winter wheat
Grasslanda Eutric Cambisol Nåntuna: Semi-natural pasture Grazing since at 3.5 0.25 6.1 Sindhøj et al. (2006).
59 480 N, 17 380 E least 1720
Forest system a
Sandy Podzol €draås:
Ja Pinus sylvestris L. forest Since 1986 1.3 0.06 4.2 Persson (1980).
60 490 N, 16 300 E
Province Va€sterbotten
Arable soilb Eutric Cambisol Ro€ b€
acksdalen: Barley annually Since 1965 2.7 0.18 5.9 €
Bergkvist and Oborn (2011).
63 480 N, 20 140 E
Grasslandb Eutric Cambisol Ro€ b€
acksdalen: Five year green fallow followed Since 1965 3.7 0.25 5.3 €
Bergkvist and Oborn (2011).
63 480 N, 20 140 E by one year barleyd
Forest systemb Haplic Podzol Flakaliden: Picea abies forest; unfertilized Since 1986 1.5 0.06 4.4 Linder (1995)
64 070 N, 19 270 E control soil
a
Samples taken in April 2011.
b
Samples taken in August 2012.
c
Crop in italics is the present crop when soil samples were taken.
d
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 in- Table 2

terval (data not shown). In total 96 samples were examined but 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.
only 24 samples could be analyzed simultaneously as only three Theoretical additional heat dissipated from Milli-Q water (DQMilli-Q water) and
TAM air calorimeters with 8 channels each were available. There- glucose (DQGlucose) amended soils due to the reaction between CO2 and bicarbonate.
fore, the experiment was staggered over 4 days, i.e. samples and Mean values and standard errors of four technical replicates.
treatments were randomized and incubated in four separate calo- CO2eCMilli-Q water DQMilli-Q water CO2eCGlucose DQGlucose
rimetry runs. (mg C g
1 soil) (mJ g
1 soil) (mg C g
1 soil) (mJ g
1 soil)
The mean values are reported for the four technical replicates of
Province Uppland
each treatment (i.e. Milli-Q water with or without CO2-traps, D- Arable land 0.9 ± 0.2
1.4 ± 0.3 13.3 ± 2.0
20 ± 3
glucose with or without CO2-traps) from the six long-term field Grassland 3.3 ± 0.2
4.9 ± 0.3 25.4 ± 3.0
38 ± 5
experiments. The Student's t-test for independent variables was Forest system 0.8 ± 0.1
1.2 ± 0.1 5.7 ± 0.7
9 ± 1
Province V€asterbotten
used to calculate differences in heat output rates between samples
Arable land 1.6 ± 0.1
2.4 ± 0.2 10.1 ± 1.8
15 ± 3
without and with CO2-traps as well as for differences in calores- Grassland 1.1 ± 0.05
1.6 ± 0.1 7.6 ± 1.2
11 ± 2
pirometric ratios between samples amended with Milli-Q water or Forest system 3.5 ± 0.3
5.2 ± 0.5 16.4 ± 1.5
24 ± 2
glucose, respectively (Statsoft, 2000).
Heat output ranged between 17 and 152 mJ g
1 soil in Milli-Q
water amended soils and increased to 306e1129 mJ g
1 soil in

Fig. 2. Heat production (Q; mJ g
1 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 is dissipated from the samples (intermediary catabolism). In other
0.9 and 3.5 mg C g
1 in Milli-Q water amended soil with an increase words, more heat is wasted per unit CO2 respired in nutrient poor
to 5.7 and 25.4 mg C g
1 when glucose was added to soils (Table 2). soil samples, which is reflected by the higher calorespirometric
Using the respiration values and assuming an endothermic reaction ratios upon glucose addition. Although, the present experiment
of þ17.8 kJ mol
1 or þ1.48 mJ per mg CO2eC respired suggest a was not designed to test this hypothesis, we nevertheless consider
2e7% decrease in heat outputs (cf. Table 2 and Fig. 2). Such values that this observation warrants further investigation.
were, however, within the standard error of observed heat outputs. The method here provides a simple, cheap and rapid screening
Changes in heat outputs due to the colorimetric respiration method of the calorespirometric ratio with improved reproducibility of the
were therefore not detectable (Fig. 2). Thus, simultaneous mea- ratios (cf. 8e26% coefficient of variances (CV) versus up to 50% CV;
surements of respiration did not introduce any measurable exper- see Herrmann et al., 2014, ratios calculated from Fig. 1b and d). Its
imental bias when determining microbial energetics in a wide non-destructive nature allows for combination with characteriza-
range of soils under different land-use management systems. tion of the chemical composition of native soil organic matter and/
The calorespirometric ratios ranged from 31 up to 100 mJ mg
1 or microbial community composition methods. In combination
CO2eC in our study with differences between glucose and Milli-Q with labeled substrates, we believe that exploring the calores-
water amended soil systems (Fig. 3). We observed a significant pirometric ratio in soils may improve our understanding of physi-
increase in the calorespirometric ratio when the forest systems and ological life strategies, i.e. evaluation of biochemical pathways
the arable soil in the province Uppland were amended with glucose (Dijkstra et al., 2011; Apostel et al., 2013) and microbial C use effi-
in comparison with Milli-Q water additions (Fig. 3). An increase in ciencies (Harris et al., 2012; Frey et al., 2013).
the ratio in these systems may indicate that more reduced sub-
strates, i.e. substrates with a higher energy content, are decom-
posed and/or more incomplete decomposition processes are taking Acknowledgments
place resulting in intermediate products, i.e. intermediary catabo-
lism with CO2 not being the decomposition product (Hansen et al., We thank the Swedish Research Council for Environment,
2004; Herrmann et al., 2014). That there is a shift towards more Agricultural Sciences and Spatial Planning (Formas 2012-530) for
reduced substrates undergoing decomposition is unlikely as € is thanked for valuable comments on
financial support. Lars Wadso
decomposition is dominated by glucose breakdown in the carbon- the manuscript. We also sincerely acknowledge two anonymous
substrate amended treatments. Calorespirometric ratios derived reviewers who provided insightful comments which assisted us in
from aerobic degradation of carbohydrates, e.g. glucose, are usually developing and improving this paper. Shelagh Green is thanked for
between 260 and 460 kJ mol
1 (Hansen et al., 2004; Wadso € et al., language review of the final manuscript.
2004) which corresponds to 21.7 up to 38.3 mJ mg
1 CO2eC. In
soils, such ratios vary substantially ranging from 150 up to
1200 kJ mol
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