Research Article

1 of 9 Analytical Science Advances doi.org/10.1002/ansa.202300054

Received: 21 October 2023

Revised: 14 December 2023

Accepted: 16 December 2023

In situ simultaneous measuring method for the determination

of key processes of soil organic carbon cycling: Soil microbial
respiration using laser spectrometry

Hongzhao Yuan1 Zhen He1 Liping Zhang1 Jiurong Wang1 Zhenke Zhu2
Tida Ge2

1
Key Laboratory of Agro-ecological Processes
in Subtropical Region, Institute of Subtropical Abstract
Agriculture, Chinese Academy of Sciences,
Rationale: Soil microbial heterotrophic C-CO2 respiration is important for C cycling.
Changsha, China
2
State Key Laboratory for Managing Biotic and
Soil CO2 differentiation and quantification are vital for understanding soil C cycling and
Chemical Threats to the Quality and Safety of CO2 emission mitigation. Presently, soil microbial respiration (SR) quantification mod-
Agro-products, Key Laboratory of
Biotechnology in Plant Protection of Ministry
els are based on native soil organic matter (SOM) and require consistent monitoring of
of Agriculture and Zhejiang Province, Institute δ13 C and CO2 .
of Plant Virology, Ningbo University, Ningbo,
China
Methods: We present a new apparatus for achieving in situ soil static chamber
incubation and simultaneous CO2 and δ13 C monitoring by cavity ring-down spec-
Correspondence
troscopy (CRDS) coupled with a soil culture and gas introduction module (SCGIM) with
Hongzhao Yuan, Key Laboratory of
Agro-ecological Processes in Subtropical multi-channel. After a meticulous five-point inter-calibration, the repeatability of CO2
Region, Institute of Subtropical Agriculture,
and δ13 C values by using CRDS-SCGIM were determined, and compared with those
Chinese Academy of Sciences, Mapoling,
Changsha, Hunan, China. obtained using gas chromatography (GC) and isotope ratio mass spectrometry (IRMS),
Email: yuanhongzhao@isa.ac.cn
respectively. We examined the method regarding quantifying SR with various concen-
Funding information trations and enrichment of glucose and then applied it to investigate the responses of
Project of the Chinese Academy of Sciences SR to the addition of different exogenous organic materials (glucose and rice residues)
for Technical Talent; National Natural Science
Foundation of China, Grant/Award Numbers: into paddy soils during a 21-day incubation.
42177330, 41771334, U21A2007; Project of Results: The CRDS-SCGIM CO2 and δ13 C measurements were conducted with high
Natural Science Foundation of Hunan
Province, Grant/Award Number: 2020JJ4655; precision (< 1.0 µmol/mol and 1‰, respectively). The optimal sampling interval and the
Project of the Chinese Academy of Sciences amount added were not exceeded 4 h and 200 mg C/100 g dry soil in a 1 L incubation
for Instrument Function Development
bottle, respectively; the 13 C-enrichment of 3%–7% was appropriate. The total SR rates
observed were 0.6–4.2 µL/h/g and the exogenous organic materials induced -49%–28%
of priming effects in native SOM mineralisation.
Conclusions: Our results show that CRDS-SCGIM is a method suitable for the quan-
tification of soil microbial CO2 respiration, requiring less extensive lab resources than
GC/IRMS.

KEYWORDS
cavity ring-down spectroscopy, in situ, multi-channel, priming effect, soil microbial respiration

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© 2023 The Authors. Analytical Science Advances published by Wiley-VCH GmbH

Anal Sci Adv. 2024;5:2300054. wileyonlinelibrary.com/journal/ansa 1 of 9

https://doi.org/10.1002/ansa.202300054

2 of 9 Analytical Science Advances
With the increasing concentrations of greenhouse gases—primarily
that of CO2 —in the atmosphere, global warming has become a central
issue in climate research. Specifically, CO2 has caused a sharp increase
in global extreme weather conditions.1 Soil CO2 efflux mainly from soil
microbial heterotrophic C-CO2 respiration is an important component
of the C cycle and the second largest flux in terrestrial ecosystems.2,3
Biological and physical processes involved in soil CO2 production and
transport are not yet fully understood and have thus received con-
siderable research attention, especially with regard to the C cycle of
terrestrial ecosystems.4,5 Plant residues are primary C sources for soil.
According to Lu et al.6 and Johnson et al.,7 plant residues are primary
sources for soil microbial metabolic and catabolic activities that con-
tribute to soil respiration and the formation of soil organic C (SOC).
Recently, the use of stable C isotope tracers has improved the under-
standing of belowground allocation and dynamics of plant residues
and has led to the quantification of soil CO2 produced by microbial
activities.8–10
Current models used for calculating soil microbial CO2 respira-
tion require large quantities of C isotope ratio (δ13 C) and CO2 gas
concentration data collected using soil incubation bottles at a cer-
tain temperature during the culture period. Gas chromatography (GC)
and isotope ratio mass spectrometry (IRMS) are the traditional meth-
ods for measuring CO2 concentration and δ13 C, respectively.11,12
However, they are time-consuming, expensive, and require signifi-
cant resources and technical expertise. These disadvantages cause
low sampling frequency and lead to insufficient information. Recent
efforts have utilised quantum cascade laser absorption and cavity
ring-down spectroscopy (CRDS) that use near-infrared laser absorp-
tion spectrometers to measure δ13 C and CO2 concentration.13,14 Such
instruments have several advantages over traditional GC/IRMS instru-
ments. Specifically, laser spectrometers do not have issues related
to intermolecular scrambling caused by ionisation during IRMS mea-
surements that require a technically complicated correction. For this
reason, they have been widely used in the field of environmental
research.15,16 Laser instruments require a continuous throughput of
an autogas sampler module to realise in situ continuous δ13 C and CO 2 ogy, to generate linear calibration curves and convert the raw [CO2 ]
concentration monitoring or estimate soil microbial respiration (SR).
Gas sampler modules are mostly designed for field experimental condi-
tions without soil sample culture sections,16,17 or have an open double
circuit design, which can easily cause cross-contamination between
labelled and unlabelled samples, thereby affecting the accuracy of the
results. Berryman et al.18 developed a flask (syringe) adaptor that
allows the collection and storage of small aliquots (20‒30 mL air) for
injection into CRDS instruments and broadens the range of CRDS
applications regarding measuring soil-respired CO2. However, it still
requires manual sampling and procedures.18 Hence, methods such as
these are not suitable for simultaneous monitoring of SR.
Given the vital role that farmland ecosystems play in the global car-
bon cycle, it is of great significance to study carbon cycles and the
regional balance of carbon.19,20 The input of easily available organic
substances in soil strongly changes the turnover of native soil organic
matter (SOM), that is, causes priming effects (PEs). Thus, a better
understanding of the response of soil microbial utilisation to the input
Research Article
doi.org/10.1002/ansa.202300054
of different forms of rice residues (shoots and roots) and their PEs on
native SOM mineralisation is critical for the understanding of global C
cycling and ecological functions of farmland ecosystems. In this study,
we investigated the application of an apparatus that integrates a laser
spectrometer with an automatic soil culture and gas introduction mod-
ule (SCGIM), hereinafter CRDS-SCGIM, for conducting δ13 C and CO2
measurements as an alternative to traditional GC/IRMS. Furthermore,
we conducted a series of experiments based on various concentra-
tions and enrichment of 13 C-labelled substrates (glucose and plant
residues) to validate the applicability of the newly established method
for quantifying SR in agricultural soils.
1 EXPERIMENTAL SECTION
1.1 Reagents and materials
Solutions were prepared with ultrapure water (Milli-Q water of > 18.2
MOhm; Milli-Q, Millipore Corp.). Premium grade reagents were pur-
chased from Sinopharm Chemical Reagent Co., Ltd unless otherwise
stated.
1.2 Inter-calibration and standard gas
preparation
Before measurement, inter-calibrations of δ values and CO2 concen-
tration were conducted for each standard gas to achieve reliable
accuracy under the manual mode of the CRDS instrument.
1.3 CO2 standard gas for concentration analysis
Standard gas concentrations (Sd1–Sd5) of 300, 500, 1500 3000 and
4000 µmol/mol were obtained from the National Institute of Metrol-
values obtained from the CRDS-SCGIM into true values. The obtained
linear calibration curve (Figure S1) was y = 1.0059x + 6.6381, with a
root mean squared error (RMSE) of 20.1.
1.4 CO2 standard gases for stable isotope
analysis
American Chemical Society (ACS) grade NaHCO3 (i.e. natural
abundance of 13 C as well as 99.0% 13 C-labelled NaHCO3 ) was
purchased from Cambridge Isotope Laboratories, Inc. and prepared
as a 50 µmol/mL solution. Subsequently, the solution with natural
abundance of 13 C was used as the diluent of the 13 C-labelled solution
to prepare a series of isotopically enriched NaHCO3 solutions and
cover a broad range of 13 C amounts ranging from −6‰ to 3522‰
(approximately 5%). Liberation of CO2 was accomplished by placing
1 mL of pure phosphoric acid and 1 mL of NaHCO3 solution in an

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evacuated special 1 L reaction bottle and flushing it with zero-grade
air. Subsequently, the generated standard gases were calibrated using
a continuous-flow IRMS instrument (MAT 253; Thermo Fisher Scien-
tific) coupled with a COMBI-PAL autosampler using 12-mL headspace
vials and an automated trace gas pre-concentrator cryo-focusing unit
(Thermo Fisher Scientific). The calibration values could be traced to the
international standard Peedee belemnite using delta units (δ‰). The
CRDS instrument was inter-calibrated for determining and normalis-
ing the δ values to the international isotope ratio scales using the linear
standard curve (Figure S2) of y = 1.0319x + 19.292 with an RMSE
of 78.4. This also enabled CRDS-SCGIM to produce accurate results
when the samples were within the range of the standard δ values.
1.5 Internal laboratory reference gas
Compressed air (396 ± 1 µmol/mol; −5.5 ± 0.3 ‰) was obtained from
Saizhong Gas Co., Ltd. and used as the internal lab reference gas (IR).
13 C-C H O
1.6 at% of 6 12 6 solution
99.0 at% of 13 C-C6 H12 O6 purchased from Sigma-Aldrich was prepared
into 5 g C/L and was subsequently diluted into 3.0 at% and 5.0 at% of
13 C by natural abundance of C
6 H12 O6 (5 g C/L).
1.7 Equipment details and configuration
The utilised G2201-i isotope analyser (Picarro Inc.) used near-infrared
CRDS technology to enable high-precision C isotope ratio (δ13 C)
determination and analysis of CO2 gas at a concentration range of
100–4000 µmol/mol, with δ13 C values below 5000 ‰ (∼6.5 at%). The
maximum drift (GT. 24 h; 1 h mean) was less than 0.5 ‰ (δ13 C).
1.8 Automatic SCGIM
This device (patent No. 201911117976.6; licence to manufacture:
Changsha Tianxin Qinyang Laboratory Equipment Co., Ltd.) provides
automatic control of the start-up and switching of various valves,
is multi-channel (up to 48 channels) for samples, and can carry out
on-line independent collection and analysis alongside automatic com-
pletion of the soil sample culture bottle vacuum and gas replacement.
The structure of SCGIM (Figure 1) was composed of three modules:
soil incubation module, gas sampling and import module, and gas dis-
placement module. The soil incubation module maintains a constant
temperature environment for the soil culture and can hold at least 48
1L soil culture bottles. The bottle caps are designed to include multiple
gas sampling/displacement) channels and gas pressure balance chan-
nels. Each of these channels corresponds to a separate valve controlled
by a Programmable Logic Controller. The gas sampling/displacement
channels are connected with the other two modules, respectively. The
air pressure balance channel together with the built-in buffer bag iden-
Research Article
doi.org/10.1002/ansa.202300054
tified by a unique ID number corresponds to another independent
valve, connected with the vacuum pump or the atmosphere output.
Once the SCGIM and CRDS were readied, the gas sampling and
import module of SCGIM were connected to the CRDS. Prior to begin-
ning the SR monitoring program, the user can set the measurement
parameters, including the bottle IDs, the sampling or interval time, and
the gas replacement times. During the first cycle run, the gas in an
identified bottle was drawn into the spectrometer for analysis. Simul-
taneously, the buffer bag inside of the bottle filled with outside air
pushed in due to the air pressure difference, to balance the pressure of
the tested bottle. Following the completion of the sampling sequence,
the gas displacement module comes into action. The remaining gas in
the bottle was removed by the vacuum pump functioning for 30 s, fol-
lowed by the entry of zero-grade air gas lasting 30 s. These series of
steps were repeated several times for each bottle. Once the sequence
reached completion, the system switched to the interval period. Dur-
ing the interval period, the CO2 efflux was held in the culture bottles
until the sampling program began. Therefore, the on-line independent
analysis of multiple samples can be carried out with the entire process
being automatically controlled, thereby also reducing the cumbersome
manual operation process.
1.9 Measurements of δ13 C and CO2
concentrations using CRDS-SCGIM
The purpose of the SCGIM unit is to enable the controlled trans-
port of CO2 samples from incubation bottles into the connected
CRDS instrument. To maximise the precision of the concentration and
δ13 C measurements, several additional experiments were conducted.
Firstly, the IR was used as a model for the continuous flow through the
CRDS for 24 h, and adjusted Allan variance analysis was used to test
the reproducibility of the CRDS results. Specifically, an Allan deviation
graph was used to validate the instrument drift and obtain the result-
ing optimum averaging time of the CRDS-SCGIM measurements across
varying sampling times (1–10 min). After the CRDS-SCGIM system was
readied online, all SCGIM channels were tested for repeatability using
IR samples (including sampling and gas replacement). The repeatabil-
ity tests for the concentration and isotopic measurements (n = 6) of
CRDS-SCGIM were conducted using three selected standard samples
(std1, std3 and std5). First, during the sampling period, the samples in
the bottles entered the SCGIM unit and subsequently the CRDS instru-
ment at a speed of 25 mL/min for 3 min. At the end of the measurement
period, the mean and standard deviations of the sample measurement
period were reported by the coordinator software on the CRDS instru-
ment. Upon termination of the measurement operation, the SCGIM
unit automatically switched to the bypass and standby modes, and
the culture bottles were treated for gas replacement for three cycles
(1 min per cycle) using tested samples (IR or std1, std3 and std5). For
comparison with GC/IRMS, an additional sampling procedure was con-
ducted simultaneously, that is, 30 mL of gas was sampled into 12-mL
pre-evacuated septa-capped glass vials (Labco Limited) using a syringe.
The CO2 concentrations and δ13 C values of the vials were measured

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F I G U R E 1 Schematic diagram of the automatic soil culture and gas introduction device. The green, blue and orange arrowheads represent the
gas flow direction of the culture bottles during the different modules’ working process, respectively.
using a gas chromatograph (7890A; Agilent Technologies) and IRMS
instrument (MAT253), respectively, as described earlier.
1.10 Soil sample collection and preparation of
13 C-plant residues
Stagnic anthrosol developed from highly weathered granite was col-
lected from a rice field (28◦ 33′ 04″ N, 113◦ 19′ 52″ E) located at the
Changsha Research Station for Agricultural and Environmental Moni-
toring. Moist soil samples were collected from the plow layer (0–20 cm)
and sieved (< 4 mm) to remove coarse plant residues. The soil con-
tained 18.1 g/kg of organic C, 1.80 g/kg of total N, 0.43 g/kg of total
P and had a pH of 5.56 (1:2.5 soil to water ratio). The rice cultivation
and continuous 13 CO labelling were performed according to meth-
2
ods reported by Zhu et al.10 in an automatically controlled gas-tight
growth chamber system (110 × 250 × 180 cm) for 30 days. Labelled
rice plants and soils were destructively sampled at the end of labelling.
Rice shoots were removed from the shoot bases, and roots were sepa-
rated from the soil by washing with deionised water. Shoots and roots
were dried at 60◦ C for 48 h and cut into < 2 mm pieces. Prior to analy-
sis for C content and δ13 C, dry shoot and root samples were ground into
fine powder and measured using MAT253 equipped with an elemental
analyser (Flash 2000; Thermo Fisher Scientific).
1.11 Optimal parameter of the method for SR
assessment
To validate the method’s applicability, several experiments were con-
ducted to assess sampling interval time, 13 C-labelled concentrations,
Research Article
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and 13 C-labelled enrichment in the SR process. For optimization of the
above parameters, we used C6 H12 O6 as a model owing to it having the
simplest structure among all the tested substrates and being easily uti-
lized by soil microorganisms with the most rapid PEs. Therefore, the
experiments were divided into three parts. Part I, soils with similar C
addition amount (200 mg C kg−1 of dry soil) and 13 C-labelled enrich-
ment (∼3.0 at%) were analyzed for SR measurement under different
sampling times of 2, 4 and 8 h; Part II, soils with similar 13 C-labelled
enrichment (∼3.0 at%) but different C addition amounts (200, 400
and 800 mg of C/kg of dry soil); and Part III, soils with 13 C-C6 H12 O6
addition of 3%, 5% and 7% 13 C-labelled enrichment (Glu-C) under
similar C addition amount (200 mg C kg−1 of dry soil). Simultane-
ously, soil without the addition of substrates (CK) was prepared as the
control.
All treatments with three replications, each up to 36 bottles, were
in a completely randomized design, and prepared with 150 g of fresh
soil (100 g oven-dry weight) weighed into 1 L bottles. For the Part III
treatments, Glu-C was added to the soil and mixed thoroughly. Subse-
quently, bottles were sealed using stoppers with a balanced airbag and
gas-path connector. The prepared containers were subsequently incu-
bated at 25 ◦ C in the dark for 24 h while conducting SR analysis. The
SCGIM parameters were set as described above with an interval time
of 4 h except for the otherwise specified sampling time of 3 min and gas
replacement of 3 min.
1.12 Incubation experiment for soil microbial
respiration
The newly established method was used to assess soil microbial respi-
ration of different treated soils and measure the most important soil

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CO2 efflux through an incubation experiment in exactly the same fash-
ion as the soil respiration experiments conducted by Zhu et
ensure that the CO2 efflux concentration in the culture bottles was
homogenous, with constituents in the appropriate range of instrument
measurement (not exceeding 4000 µmol/mol), the sampling interval
time was shortened to 4 h; correspondingly, the sampling frequency
was increased during the incubation period. During the interval period,
the CO2 efflux was held in the culture bottles until the sampling pro-
gram started. The experiment consisted of four treatments with three
replications each in a completely randomised design: (1) paddy soil
without additions (CK); (2) paddy soil with 13 C-C6 H12 O6 addition (Glu-
C); (3) paddy soil with 13 C-labelled shoots (Shoot-C); and (4) paddy soil
with 13 C-labelled roots (Root-C). For CK, 150 g of fresh soil (100 g
oven-dry weight) was directly weighed into 1 L containers. For the
Glu-C, Shoot-C, and Root-C treatments, 5.0 at% of
6 H12 O6 and
13 C-labelled shoots or roots were applied to 100 g (oven-dried weight)
of unlabelled soil for a final concentration of 200 mg C kg−1 of dry soil
and mixed thoroughly. Subsequently, samples were placed into 1 L cul-
ture bottles with 100 mL deionised water to create a 1−2-cm thick
water layer. The prepared culture bottles were sealed using stoppers
with a balanced airbag and gas-path connector.
The containers were subsequently incubated at 25 ◦ C in the dark
while conducting the soil microbial respiration analysis. The SCGIM
parameters were set as described above with an interval time of 4 h,
sampling time of 3 min, and gas replacement of 3 min using IR. For
comparison with GC/IRMS, an additional sampling procedure was con-
ducted simultaneously, that is, 30 mL of gas from the culture bottles
was sampled into 12-mL pre-evacuated septa-capped glass vials (Labco
Limited) using a syringe; this procedure was performed 10 times (on
days 1, 2, 3, 5, 7, 9, 11, 13, 15 and 20) during the 21-days incubation.
The CO2 concentrations and δ13 C values of the vials were measured
using a gas chromatograph (7890A; Agilent Technologies) and IRMS
instrument (MAT253), respectively, as described earlier.
1.13 Data analyses and statistics
The total soil microbial respiration rate (SRRt , µL CO2 /h/g air-dried soil)
was calculated as follows:
SRRt = (C1 −C0 ) ×Vg∕4∕100,
where C1 and C0 are the final and initial CO2 concentrations
µmol/mol (µL/L) of the 4-h incubation, respectively, and Vg (L) is the
headspace volume of the bottle with the soil sample.
The two-pool C isotope mixing model used in the current study, as
described by Zhu et al.,10 is as follows:
( )
fS = 𝛿 13 C addtion − 𝛿 13 C material ∕(𝛿 13 C control
where fS is the fraction of CO2 -C evolved from soil in the presence
of organic material. δ13 Caddtion and δ13 Ccontrol are the δ13 C values of
the total CO2 -C evolved from the incorporated organic material and
Research Article
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control (soil only) treatments, respectively. δ13 Cmaterial is the δ13 C value
al.10 To of the labelled glucose, shoot, and root.
The PE of native SOC induced by organic material input was
calculated as follows:
( )
PE = fS Ct,soc − Cc,soc × 100∕Cc,soc , (3)
where Ct,soc is the total quantity (mg C (kg soil)−1 ) of CO2 -C evolved
from the soil in the presence of organic material, and Cc,soc is the
quantity (mg C kg−1 soil) of CO2 -C evolved from the control soil.
All statistical analyses were performed using the SPSS statistical
package (v. 20.0) for Windows. Pearson’s product-moment correlation
test was used to determine the correlation between the two meth-
ods. Linear regression was used to describe the relationship between
13 C-C
the CRDS-SCGIM and IRMS/GC measurements of CO2 concentration
and δ13 C. T-tests were used to determine whether the means of the
concentration measurements for each standard gas (n = 6) were signif-
icantly different and greater than the inherent measurement error of
the measurement techniques (p < 0.05). An F-test was conducted for
the comparison of standard deviations to determine if CRDS-SCGIM
was more precise than IRMS/GC.21
2 RESULTS AND DISCUSSION
2.1 Repeatability of CRDS-SCGIM for
concentration and isotopic measurements
Theoretically, the time needed by the CRDS instrument to achieve
accuracy according to the manufacturer’s specifications is much longer
than that of traditional methods (IRMS); however, longer sampling
times cause lower precision because of instrumental drift.16 The Allan
variance analysis showed that the precisions of the CO2 concentration
and 13 C values of the IR gas were 10% of the Allan standard devia-
tion at 2 min and 10 s, respectively, while both significantly increased
after 1 h (Figure S3). Furthermore, repeatability of the CRDS-SCGIM
measurements of CO2 concentrations and 13 C values of the IR gas at
various sampling times (1–10 min) was quantified as 0.1−0.3 µmol/mol
and 0.6%–1.5%, respectively. These values were significantly affected
by the sampling time, and the optimum values were achieved at 3 min
(1) (Figure S4), which was, therefore, used for subsequent measurements.
Reproducibility tests were also conducted for selected standard
gases that were determined using the CRDS-SCGIM method. The pre-
cision of the CO2 concentration measurements was 0.8–2.0 µmol/mol
and increased with increasing CO2 concentration (Figure 2A). These
results agreed well with those of Pang et al.10 in terms of the higher
signal-to-noise ratio at a higher concentration of std3. Moreover, the
repeatability of the δ13 C measurements of the other three selected
− 𝛿 13 C material ), (2) standard gases was 1‰–5‰ (Figure 1B), which worsened with increas-
ing 13 C values, thereby significantly increasing the uncertainty of the
signal.22
One of the objectives of this study was to determine if the SCGIM
could be applied to CRDS for measuring the CO2 concentrations and

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F I G U R E 2 (A) Repeatability of CO2 concentration measurements
using the cavity ring-down spectroscopy–soil culture and gas
introduction module (CRDS–SCGIM) method (average of six
measurements; left axis) and standard deviation between the
measurements (right axis). (B) Repeatability of carbon stable isotope (δ
13 C) analysis for CO by the CRDS–SCGIM method (average of six
2 2.2
measurements; left axis) and standard deviation between the
measurements (right axis).
δ13 C values of multiple samples. No significant differences in 13 C val-
ues and CO2 concentrations between the CRDS method with and
without SCGIM were observed (Figure 3). Here, using an SCGIM pro-
vided a gas-tight channel and a gas pressure balance system, which
consisted of a balanced airbag and gas path control. When the sample
was pumped away from the bottles by the CRDS instrument, the same
volume of ambient air automatically entered into the balanced bag to
promptly fill the remainder of the incubation bottles. This step was cru-
cial for maintaining consistent SCGIM pressure during the sampling
period, realising automatic sampling, and introducing gas samples into
the CRDS instrument. The CRDS-SCGIM method achieved high accu-
racy, which was similar to that of the CRDS method without SCGIM.
Furthermore, the F-test of the standard deviations between differ-
ent methods was not significant (p > 0.05), indicating equal variance
between the three measurements. Lastly, the CRDS-SCGIM method
presented here was comparable with the GC/IRMS method. Therefore,
the use of a SCGIM for CRDS is feasible. In this way, CO2 concentration
and δ13 C can be measured at a high frequency without laborious proce-
dures in terms of collection and injection of gas samples into the CRDS
instrument using a syringe.
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F I G U R E 3 Comparisons of CO2 concentration (A) and carbon
stable isotope (B) measurements between the cavity ring-down
spectroscopy (CRDS) method with or without soil culture and gas
introduction module (SCGIM) and through gas chromatography (GC)
or isotope ratio mass spectrometry (IRMS) methods. Bars show
standard errors of the means (n = 6).
Validation of the method regarding
quantifying soil SR
We conducted a series of experiments adopted from the developed
CRDS-SCGIM method for the analysis of SR to obtain optimal param-
eter assessment. The CO2 concentrations and 13 C enrichment in
Glu-C addition treatments were all significantly higher than in the
Control soils and varied with varying sampling interval times, Glu-C
addition amounts, and 13 C enrichment (Figure 4A–C). The CO2 con-
centrations ranging from 1633 µmol/mol to 6605 µmol/mol, increased
with sampling interval time and Glu-C addition amount, except for
13 C enrichment. When the sampling interval time exceeded 4 h or
the Glu-C addition amount exceeded 200 mg, the CO2 concentra-
tion of SR in all tested soils exceeded 4000 µmol/mol. The CO2
enrichment of SR ranging from 432‰ to 2297‰ increased with
Glu-C addition amount and 13 C enrichment, but not with sampling
interval time. Considering the limitations of the instrument, it is rec-
ommended that optimal sampling interval time and Glu-C addition
amount do not exceed 4 h and 200 mg C/100 g soil in a 1 L incubation
bottle, respectively. The 13 C-enrichment of 3%–7% was considered
appropriate.
Furthermore, this newly established method was used to assess the
SR of the soil with different substrate additions and measure the most
important soil CO2 efflux through an incubation experiment following

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F I G U R E 4 Effect of sampling interval times (A), Glu-C addition
amounts (B), and 13 C enrichment (C) on the CO2 concentration and
carbon stable isotope measurements of soil microbial respiration (SR)
under Control (no additions) and treatment (addition of
13 C-C H O ). Bars show the standard error of the mean (n = 3). The
6 12 6
standard error is not shown if the bar is smaller than the symbol. The
different capital or lower-case letters listed above bars represent
significant differences between treatments (P < 0.05, Duncan test,
one-way ANOVA).
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F I G U R E 5 Total soil microbial respiration rate (SRRt ) under CK
(no additions), Glu-C (addition of 13 C-C6 H12 O6 ), Shoot-C
(13 C–labelled shoots), and Root-C (13 C–labelled roots) soil treatments
throughout a 21-days incubation period with a sampling frequency of
six times per day. Bars show the standard error of the mean (n = 3).
The standard error is not shown if the bar is smaller than the symbol.
the same procedure as before.10 During the 21-day SR monitoring
experiment, the CO2 concentrations and 13 C enrichment of SR ranged
from 447 to 2313 µmol/mol and from −23.4‰ to 2207‰ (Figure
S4), respectively. the SRRt was observed ranging from 0.6 to 4.2 µL
CO2 /h/g soil, which initially increased sharply across all treatments and
subsequently decreased in an exponential manner over time (Figure 5).
Compared to the control, substrate addition increased the SRRs by
1.2–2.8 times and the addition of glucose caused higher quantities of
CO2 respiration throughout incubation with respect to corresponding
treatments that received additions of rice residues peaking almost
at the same time. In general, the input of labile organic matter (LOM)
stimulates microbial activity and higher enzyme production, resulting
in the decomposition of more organic substances and higher CO2
flux.23 The microbial respiratory response to substrate addition
strongly depended on the LOM quantity.24 Compared to glucose, the
quantity of LOM input accompanying the addition of crop residues was
much lower because labile metabolic organic components only occu-
pied about 20% of crop residues and consisted mostly of recalcitrant
organic C.25 With time, in environments where nutrients and labile
C are scarce, microbial activity and growth are limited, causing SRRt
values to decrease correspondingly in all treatments.26,27
Distinguishing CO2 produced from individual C pools is crucial when
investigating PE mechanisms. The use of 13 C-labelled material addi-
tions allowed the total respired CO2 to be partitioned into that derived
from the added material and that from SOM mineralisation.28,29 The
input of organic material caused inconsistent priming of SOC mineral-
isation during the 21-day period (Figure 6). The negative PE caused by
glucose addition initially decreased to the lowest point (−49% within
24 h); gradually increased to −0.5% until day 6; peaked at day 12
(27%); and then decreased over time, while remaining positive until the
end of the experiment. Similar phenomena were observed in the rice
residue treatments; however, the peak negative PEs (−7% to −28%)
were delayed by 2 days and lasted lower than those of Glu-C, which
can be explained by SOM being a highly recalcitrant substrate for
microorganisms to break down.26,27 In environments where nutrients
 Research Article
8 of 9 Analytical Science Advances doi.org/10.1002/ansa.202300054

F I G U R E 6 Priming effects (PE) of native soil organic C (SOC)

induced by organic material input under Glu-C (addition of
13 C-C H O ), Shoot-C (13 C–labelled shoots), and Root-C
6 12 6
(13 C–labelled roots) soil treatments throughout the course of a
21-days incubation period with a sampling frequency of six times per
day.

are abundant, inputs of labile C may first protect SOC mineralisation

because of the easy availability of energy for microbial growth and the
fact that PEs can be negatively correlated with the quantity of LOM
added to the soil.29,30 Additionally, there can be changes in the direc-
tion and magnitude of the PE on SOC mineralisation because of the
input of organic substrate that is accompanied by changes in soil nutri-
ents or the composition of the microbial community over time,23,31,32
such as switching from a negative to a positive PE rate on native
SOC,33 and an increase or decrease in the magnitude of the positive
PE rate.34
To validate the reliability of our method, we compared the results
with those obtained using traditional GC/IRMS. A highly significant
positive linear relationship (r2 > 0.996; p < 0.001; Figure 7) cov-
ering almost the full incubation period indicated that CRDS-SCGIM F I G U R E 7 Linear regressions of the CO2 concentration (A) and
δ13C values (B) determined by the cavity ring-down spectroscopy–soil
produced results that were consistent with those of the traditional
culture and gas introduction module (CRDS–SCGIM) method against
method. Furthermore, the CRDS-SCGIM method for soil microbial gas chromatography (GC) or isotope ratio mass spectrometry (IRMS).
respiration was more efficient than the traditional method.

3 CONCLUSIONS
The online determination of CO2 concentrations and δ13 C values in
the soil can be realised using automatic temperature-controlled soil
culture and gas introduction equipment combined with the G2201-
i analyser. The new method offers the advantages of high precision
and simple operation, and it can reduce the errors caused by manual
operations. The results of CO2 concentration and C isotope determi-
nation were consistent with those of the classical gas phase and mass
spectrometry methods. Therefore, the CRDS-SCGIM method is widely
applicable in the study of the microbially driven mechanism of soil C
turnover.
It is worth noting that the current SCGIM system is not suitable for
samples outside of the CRDS measuring range, thereby requiring addi-
tional automatic dilution and a data calibration processing module. In
addition, developing software is necessary for the collection and clas-
sification of the large quantities of data produced from the relatively
high sampling frequencies.
AUTHOR CONTRIBUTIONS
The manuscript was written with the contributions of all authors.
All authors have given approval to publish the final version of the
manuscript.
ACKNOWLEDGEMENTS
This study was financially supported by the National Natural Sci-
ence Foundation of China (grant nos. 42177330, 41771334 and
U21A2007), the Project of Natural Science Foundation of Hunan
Province (grant no. 2020JJ4655), and the Project of the Chinese
Academy of Sciences for Instrument Function Development, Techni-
cal talent. We would like to thank Editage (www.editage.cn) for English
language editing.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
Data are available upon request.

9 of 9 Analytical Science Advances
ORCID
Hongzhao Yuan https://orcid.org/0000-0001-8687-1672
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