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\begin{document}

\title{Different root exudates C/N ratios accelerate CO2 emission from paddy
soil}



\author[1]{Guan Cai}%
\author[2]{Muhammad Shahbaz}%
\author[3]{Tida Ge}%
\author[4]{yajun Hu}%
\author[5]{Baozhen Li}%
\author[6]{Yuhuai Liu}%
\author[7]{Qiong Liu}%
\author[6]{ Olga  Shibistova}%
\author[1]{Leopold Sauheitl}%
\author[8]{Jinshui Wu}%
\author[9]{Georg Guggenberger}%
\author[3]{Zhenke Zhu}%
\affil[1]{Institute of Soil Science, Leibniz Universität Hannover, Hannover 30419, Germany}%
\affil[2]{Sveriges Lantbruksuniversitet Fakulteten for landskapsplanering tragards- och djurbruksvetenskap}%
\affil[3]{Institude of Subtropical Agriculture, The Chinese Academy of Sciences}%
\affil[4]{Key Laboratory of Agro-ecological Processes in Subtropical Region \& Changsha Research Station for Agricultural and Environmental Monitoring, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan, 410125, China}%
\affil[5]{Chinese Academy of Sciences}%
\affil[6]{Institute of Soil Science}%
\affil[7]{University of Bayreuth}%
\affil[8]{Chinese Acad Sci}%
\affil[9]{Leibniz University Hannover}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Root exudates can greatly modify microbial activity and soil organic
matter (SOM) mineralization. However, the mechanism of root exudation
and its stoichiometric ratio of C/N controlling upon paddy soil C
mineralization are poorly understand. In this study, we used a mixture
of glucose, oxalic acid, and alanine as root exudate mimics, employing
three C/N stoichiometric ratios (CN6, CN10, and CN80) to explore the
underlying mechanisms involved in C mineralization. The input of root
exudates enhanced CO2 emission by 1.8--2.3-fold than that of the
control. Artificial root exudates with low C/N ratios (CN6 and CN10)
increased the metabolic quotient (qCO2) by 12\% over those obtained at
higher stoichiometric ratios (CN80 and C-only), suggesting a relatively
high energy demand for microorganisms to acquire organic N from SOM by
increasing N-hydrolase production. The stoichiometric ratios of enzymes
(\selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase) promoting organic
C degradation compared to those involved in organic N degradation showed
a significant positive correlation with qCO2; the stoichiometric ratios
of microbial biomass (MBC/MBN) were positively correlated with carbon
use efficiency. This suggests that root exudates with higher C/N ratios
entail an undersupply of N for microorganisms, triggering the release of
N-degrading extracellular enzymes. This in turn decreases SOM
mineralization, implying the C/N ratio of root exudates to be a
controlling factor. Our findings show that the C/N stoichiometry of root
exudates controls C mineralization by the specific response of the
microbial biomass through the release of C- and N-releasing
extracellular enzymes to adjust for the microbial C/N ratio.%
\end{abstract}%



\sloppy


\textbf{Different root exudates C/N ratios accelerate
CO\textsubscript{2} emission from paddy soil}

Guan Cai \textsuperscript{a,b}, Muhammad Shahbaz \textsuperscript{c},
Tida Ge \textsuperscript{a,b}, Yajun Hu \textsuperscript{a}, Baozhen Li
\textsuperscript{a}, Yuhuai Liu\textsuperscript{a,b}, Qiong Liu
\textsuperscript{a}, Olga Shibistova\textsuperscript{b,d}, Leopold
Sauheitl \textsuperscript{b}, Jinshui Wu\textsuperscript{a}, Georg
Guggenberger \textsuperscript{b}, Zhenke Zhu \textsuperscript{a*}

\emph{\textsuperscript{a}Key Laboratory of Agro-ecological Processes in
Subtropical Region \& Changsha Research Station for Agricultural and
Environmental Monitoring, Institute of Subtropical Agriculture, Chinese
Academy of Sciences, Hunan, 410125, China}

\selectlanguage{ngerman}\emph{\textsuperscript{b} Institute of Soil Science, Leibniz Universität
Hannover, Hannover 30419, Germany}

\emph{\textsuperscript{c} Centre for Environmental and Climate Research,
Lund University, 223 62 Lund, Sweden}

\emph{\textsuperscript{d} VN Sukachev Institute of Forest, Siberian
Branch of the Russian Academy of Sciences, Krasnoyarsk, 660036, Russian
Federation}

*Author for Correspondence: Zhenke Zhu; E-mail: zhuzhenke@isa.ac.cn



\textbf{ACKNOWLEDEMENTS}

This study was supported by the National Natural Science Foundation of
China (42177334, 42177330, 41877104), the Natural Science Foundation of
Hunan Province (2019JJ30028; 2020JJ4653), and the Youth Innovation
Promotion Association of the Chinese Academy of Sciences (2019357). The
grants or other support to Ge T. from the Alexander von Humboldt
Foundation of Germany are also acknowledged with gratitude.



\textbf{Abstract}

Root exudates can greatly modify microbial activity and soil organic
matter (SOM) mineralization. However, the mechanism of root exudation
and its stoichiometric ratio of C/N controlling upon paddy soil C
mineralization are poorly understand. In this study, we used a mixture
of glucose, oxalic acid, and alanine as root exudate mimics, employing
three C/N stoichiometric ratios (CN6, CN10, and CN80) to explore the
underlying mechanisms involved in C mineralization. The input of root
exudates enhanced CO\textsubscript{2} emission by 1.8--2.3-fold than
that of the control. Artificial root exudates with low C/N ratios (CN6
and CN10) increased the metabolic quotient (qCO\textsubscript{2}) by
12\% over those obtained at higher stoichiometric ratios (CN80 and
C-only), suggesting a relatively high energy demand for microorganisms
to acquire organic N from SOM by increasing N-hydrolase production. The
stoichiometric ratios of enzymes (\selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl
glucosaminidase) promoting organic C degradation compared to those
involved in organic N degradation showed a significant positive
correlation with qCO\textsubscript{2}; the stoichiometric ratios of
microbial biomass (MBC/MBN) were positively correlated with carbon use
efficiency. This suggests that root exudates with higher C/N ratios
entail an undersupply of N for microorganisms, triggering the release of
N-degrading extracellular enzymes. This in turn decreases SOM
mineralization, implying the C/N ratio of root exudates to be a
controlling factor. Our findings show that the C/N stoichiometry of root
exudates controls C mineralization by the specific response of the
microbial biomass through the release of C- and N-releasing
extracellular enzymes to adjust for the microbial C/N ratio.

KEYWORDS: Root exudates -- Stoichiometric ratios -- Microbial biomass --
Extracellular enzyme -- Metabolic quotients -- Carbon use efficiency

\textbf{INTRODUCTION}

Plants can dramatically modify the soil environment through the
rhizosphere, either by the release of C from plant roots (e.g., root
exudates) or via the rapid uptake of their associated microorganisms by
the soil (Kuzyakov, 2002; Jones et al., 2009; Fisk et al., 2015; Liu et
al., 2019; Xiong et al., 2019). Across different plant species, around
1--10\% of photoassimilated C is released into the soil as root exudates
(Jones et al., 2004; Phillips et al., 2011; Qiao et al., 2014; Yin et
al., 2014), consisting primarily of sugars, while also containing
organic acids, amino acids, phenolics, and other secondary metabolites
(Haichar et al., 2014; Yuan et al., 2017). In addition to acting as
direct substrates for microorganisms and possess nutritional value, the
stoichiometric ratio of the C and N-containing are considered to have an
impact on their utilization by microorganisms (Wild et al., 2014; Liu et
al., 2020). Thus, elucidating the role and underlying mechanisms of
action of different exudate C/N ratios on microbial substrate
utilization is of key importance for understanding soil C and N cycling,
and likewise for soil C sink strength.

Several mechanisms have been proposed to explain the changes in the
microbial decomposition of soil organic matter (SOM) because of root
exudate addition, namely: (i) root exudates provide energy for the
stimulation of SOM decomposition and change the chemical and physical
properties of the soil environment (Qiao et al., 2016; Zhu et al., 2018;
Mehnaz et al., 2019; Du et al., 2020); (ii) labile C promotes microbial
growth, which in turn increases the N demand and likewise microbial N
mining from SOM (Manzoni et al., 2010; Dijkstra et al., 2013; Qiao et
al., 2016; Zhu et al., 2018); and (iii) microbial C and N demands cause
community shifts that alter microbial-mediated C decomposition (Phillips
et al., 2011; Wild et al., 2014; Yuan et al., 2017; Fang et al., 2020;
Wei, 2020). Moreover, impacts of nutrients on stoichiometry also need to
be considered (such as the addition of C substrates like glucose,
accompanied by several levels of N applications), which provide a
proportion of C that is incorporated into the microbial biomass at the
expense of CO\textsubscript{2} emission, and becomes stabilized as soil
organic carbon (SOC) (Creamer et al., 2014).

Recently, research on the stoichiometry of root exudate compounds
combined with mineral N {[}e.g.,
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4}{]} has shown
that higher C/N ratios increase CO\textsubscript{2} emissions due to
high microbial N demand (Du et al., 2020; Liu et al., 2020).
Subsequently, resource stoichiometry alters microbial community
composition to gain the required elements and increase SOM
mineralization (Zhu et al., 2018; Wei, 2020). Extracellular enzyme
activities, such as C- and N-acquiring enzymes, can also reflect the
resource demands of microbial communities (Schimel, 2003; Hill et al.,
2012). \selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase (BG) and \selectlanguage{greek}β-\selectlanguage{english}1,4-xylosidase (XYL) are the largest
contributors to the degradation of cellulose and hemicellulose,
respectively. Similarly, \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase (NAG) plays a
role in the degradation of chitin and is involved in the organic N pool.
The enzyme activities have similar stoichiometries, which could further
relate to the elemental stoichiometry of the microbial biomass for
microbial nutrient assimilation and growth (Sinsabaugh et al., 2008;
Sinsabaugh and Follstad Shah, 2012). To date, the understanding of the
influence of root exudates with different stoichiometric compositions on
microbial growth and activities is still in its infancy, especially with
respect to the interaction between microbial biomass stoichiometry and
extracellular enzyme stoichiometry depending on the root exudate
compositions.

In the present study, we applied different C:N stoichiometric ratios of
artificial root exudates to soil to investigate how they modify
microbial activities (including extracellular enzyme production and
microbial biomass stoichiometric ratio) and likewise influence SOM
mineralization. The stoichiometric ratios of artificial root exudates
were regulated by combining different amounts of glucose, oxalic acid,
and alanine, which represented low-molecular-weight organic compounds,
namely, sugars, organic acids, and amino acids, respectively. We
determined the activities of extracellular enzymes involved in C and N
decomposition, microbial biomass C (MBC) and N (MBN), along with the
associated CO\textsubscript{2} emissions after the addition of root
exudates. We hypothesized that (1) the application of C-only artificial
root exudates leads to an imbalance in resource stoichiometry, thereby
inhibiting microbial activity and SOM mineralization; and (2) the
inclusion of N-containing root exudates meets microbial stoichiometric
requirements, thus promoting microbial growth and increasing SOM
mineralization.

\textbf{MATERIALS AND METHODS}

\textbf{2.1 Study site and soil sampling}

Soil was collected from experimental rice fields located in Changsha,
Hunan, China (113\selectlanguage{ngerman}°19\selectlanguage{english}' 52\selectlanguage{english}" E, 28deg 33\selectlanguage{english}' 04\selectlanguage{english}" N). The site is characterized
by a subtropical climate, with a mean annual temperature of 16.7 degC and
mean annual precipitation of 1457 mm. Moist field soils were collected
from the plow layer (0--20 cm) using a stainless-steel drill (diameter:
5 cm) in September, which is after the growing season in summer. The
soil is derived from Quaternary Red Clay and classified as Haplic
Acrisol according to the IUSS Working Group WRB (WRB, 2015). The soil
received urea (80 kg ha\textsuperscript{-1} yr\textsuperscript{-1})
along with biological fertilizer (livestock manure) and rice plant straw
residues (6000 kg ha\textsuperscript{-1} yr\textsuperscript{-1}) within
a rice--rape rotation. Five soil cores were collected and thoroughly
mixed; they were immediately placed in a gas-permeable plastic bag, and
stored at 4 degC until analysis. A subset of soil samples was air-dried
and passed through a 2-mm sieve to remove fine roots and other plant
residues. The fresh soil samples were used to measure basic chemical
properties such as SOC (11.27 g kg\selectlanguage{english}\textsuperscript{-1}) and total N (TN)
(1.3 g kg\selectlanguage{english}\textsuperscript{-1}) using the potassium
dichromate-concentrated sulfuric acid-phenanthroline titration method
(Lu, 1999). Dissolved organic C (DOC) (69.84 mg kg\selectlanguage{english}\textsuperscript{-1})
was analyzed using a Shimadzu TOC-VWP analyzer (TOC-VWP, Shimadzu,
Japan). Soil pH (4.98) was measured using a Mettler Toledo 320 pH meter
at a soil-to-water ratio (w:v) of 1:2.5.

\textbf{2.2 Artificial root exudate experiment}

The sieved and air-dried soil samples (3 kg dry weight) were
respectively placed in a 50-L plastic bucket and flooded with deionized
water to a depth of 2--3 cm. The soils were pre-incubated for two weeks
in the dark at 25 degC. The experiment consisted of five treatments with
four replicates in a completely randomized design. Afterwards, the soil
was mixed well, and subsamples of 50 g (equivalent to 30 g dry soil)
were placed in 500-mL incubation serum bottles. Deionized water was
added to each bottle to a level of 2--3 cm above the soil surface, and
the bottles were sealed using a rubber stopper to ensure anoxic
conditions. To imitate root exudates, which are mainly dominated by
sugars, amino acids, and organic acids (Lopez\selectlanguage{english}-Sangil et al., 2017; Xiong
et al., 2019), we chose different proportions of glucose
(C\textsubscript{6}H\textsubscript{12}O\textsubscript{6}), oxalic acid
(H\textsubscript{2}C\textsubscript{2}O\textsubscript{4}), and alanine
(C\textsubscript{3}H\textsubscript{7}NO\textsubscript{2}). We adjusted
three different C/N ratios (CN6-, CN10-, and CN80-treatments) by adding
different amounts of alanine, along with a treatment without alanine
(C-only). Treatment with only distilled H\textsubscript{2}O was used as
a control. The total amount of added C was maintained at 5\% SOC, about
0.6 mg C g\textsuperscript{-1} soil dry weight. The relative
contributions of sugar, amino acids, and organic acid-derived C were
within the ranges reported in the literature (Qiao et al., 2016; Liu et
al., 2020).

The solutions were prepared every five days by dissolving the
abovementioned proportions of glucose, oxalic acid, and alanine in 50 mL
of deionized water. The details are presented in Table 1 and Table S1.
To mimic a pattern of continuous exudate excretion, we manually injected
0.5 mL of the solution into the soil samples using a 10-mL syringe
daily. All samples were incubated at 25 degC in the dark for 45 days. Four
bottle samples were chosen randomly and destructively harvested at days
3, 12, and 45 after initial exudate addition. Before harvesting the soil
samples, four leachate water samples were collected in a 50-mL
centrifuge tube and the soil samples were then mixed thoroughly; both
leachate water and soil samples were stored at 4 degC for further
measurements.

\textbf{2.3 CO\textsubscript{2} efflux measurement}

Gas samples (15 mL) were collected from 500-mL anaerobic bottles every
day using a 20-mL syringe before exudate solutions were injected into
the soil samples. Subsequently, the serum bottles were flushed with
artificial air for another 10 min and then sealed using a rubber
stopper. CO\textsubscript{2} concentration was measured using a gas
chromatograph equipped with a thermal conductivity detector (Agilent
7890A, Agilent Technologies, Alto Palo, California, USA).

\textbf{2.4 Soil property analysis}

Soil MBC and MBN were determined using the chloroform fumigation
extraction method (Wu et al., 1990; Jenkinson et al., 2004). One
subsample (20 g) was directly extracted with
K\textsubscript{2}SO\textsubscript{4} solution (60 mL; 0.5 M); another
(also 20 g) was first fumigated with ethanol-free chloroform in the dark
for 24 h and then extracted with
K\textsubscript{2}SO\textsubscript{4}solution (60 mL; 0.5 M). The
extracted C in the fumigated and unfumigated samples was measured after
acidification using the Shimadzu TOC-VWP analyzer (Shimadzu), and
inorganic N (NO\textsubscript{3}\textsuperscript{-} and
NH\textsubscript{4}\textsuperscript{+}) was extracted with 2 M KCl and
analyzed using a continuous-flow analyzer (Fiastar 5000; Foss Tecator
AB, Hoganas, Sweden). The TN of the soil samples was determined using an
elemental analyzer (MACRO cube, Elementar, Germany).

MBC and MBN were calculated as the difference in C and N contents
between fumigated and non-fumigated sample extracts, adjusted by a
proportionality coefficient (kEC = 0.45, kEN = 0.54) to account for the
extraction efficiency. The 0.5 M
K\textsubscript{2}SO\textsubscript{4}-extractable C from the unfumigated
sample was considered as DOC (Vance et al., 1987).

The leaching water samples collected for
NO\textsubscript{3}\textsuperscript{-}(NO\textsubscript{3}\textsuperscript{-}-L)
and
NH\textsubscript{4}\textsuperscript{+}(NH\textsubscript{4}\textsuperscript{+}-L)
were analyzed using a continuous-flow analyzer (Fiastar 5000; Foss
Tecator AB, Hoganas, Sweden). Leaching water DOC (DOC-L) was measured
using the Shimadzu TOC-VWP analyzer (Shimadzu).

The metabolic quotient (qCO\textsubscript{2}) and microbial carbon-use
efficiency (CUE) were calculated using previously published methods
(Anderson and Domsch, 1993; Sinsabaugh et al., 2013).
qCO\textsubscript{2} was defined as the ratio of C in respired
CO\textsubscript{2} and soil microbial biomass (qCO\textsubscript{2} =
CO\textsubscript{2}-C/MBC) and the CUE was defined as the amount of C in
the microbial biomass relative to the sum of the CO\textsubscript{2}-C
and MBC {[}CUE = MBC/(MBC + CO\textsubscript{2}-C){]}.

\textbf{2.5 Enzyme activity measurement}

Extracellular enzyme activity was determined using a previously
established method (Marx et al., 2001). Fluorogenic
methylumbelliferone-based artificial substrates were used to estimate
the activity levels of BG, XYL, and NAG, which are involved in soil
organic C (BG, XYL) and N (NAG) degradation (Sinsabaugh and Follstad
Shah, 2012). The fresh soil (1 g) was prepared by dissolving in 125 mL
of 50 mM sodium acetate buffer (pH 5) using low-energy sonication (50 J
s\selectlanguage{english}\textsuperscript{-1}) for 1 min. A 200-\selectlanguage{greek}μ\selectlanguage{english}L aliquot of the slurry was
incubated with 50 \selectlanguage{greek}μ\selectlanguage{english}L of the corresponding substrate at 20 \selectlanguage{ngerman}°C for 4 h in
the dark. Ten microliters of 1 M NaOH solution was added to each plate
to terminate the reaction before measurement; the fluorescence values
were then determined with an automated fluorometric plate reader
(Victor3 1420--050 Multi-label Counter; PerkinElmer, Waltham, MA, USA)
with an excitation wavelength of 365 nm and an emission wavelength of
450 nm. Enzyme activities were expressed as nM
g\textsuperscript{-1}h\textsuperscript{-1}.

\textbf{2.6 Calculations and statistical analysis}

Statistical analysis was performed with R Studio (R version 3.5.2, CDN,
Global) using one-way ANOVA, and significant differences between single
treatments were analyzed using Tukey's post-hoc test (p \textless{}
0.05). Structural equation modeling (SEM) was performed using the Amos
17.0 software package (Smallwaters Corporation, Chicago, IL, USA) to
test the significance of the hypothesized causal relationships among
total C, MBC/MBN, BG/NAG, XYL/NAG,
DOC/NH\textsubscript{4}\textsuperscript{+},
DOC/NO\textsubscript{3}\textsuperscript{-}, and
CO\textsubscript{2}emissions. The log-transformed CO\textsubscript{2},
SOC, DOC/NH\textsubscript{4}\textsuperscript{+},
DOC/NO\textsubscript{3}\textsuperscript{-}, MBC/MBN, BG/NAG, and XYL/NAG
were used to perform SEM on the data statistics and conceptual
assumptions (Hu et al., 2014). The best-fit model was determined using a
chi-square test (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}), \emph{P} -values,
goodness-of-fit index (GEI), root mean square error of approximation
(RESEA), and Akaike information criteria (Chen et al., 2016).

\textbf{RESULTS}

\textbf{3.1 SOM mineralization response to artificial root exudates}

The temporal patterns of CO\textsubscript{2} emissions from all
artificial root exudate--treated soils were similar. The
CO\textsubscript{2} efflux was highest at the beginning of the
incubation (days 1--4), and exponentially decreased until day 15.
Furthermore, it reached a stable level with only minor fluctuations
until the end of the chasing period (Fig. 1a). Compared with the soil
without additions (Control), the input of artificial root exudates led
to an increase in cumulative CO\textsubscript{2} emissions by 25\%,
25\%, 20\%, and 19\%, as CN6 \textgreater{} CN10 \textgreater{} CN80
\textgreater{} C-only, respectively (Fig 1b).

The input of root exudates with low C/N ratios (CN6 and CN10)
significantly increased qCO\textsubscript{2} on day 3. However, the
qCO\textsubscript{2} decreased from day 3 to day 45 in all treatments in
the following order: CN6 \textgreater{} CN10 \textgreater{} CN80
\textgreater{} C-only \textgreater{} Control (Fig. 2a). In contrast, the
CUE increased from day 3 to day 45 (Fig. 2b). The addition of artificial
root exudates likewise decreased the CUE value in the following order:
control \textgreater{} C-only \textgreater{} CN80 \textgreater{} CN10
\textgreater{} CN6.

\textbf{3.2 Responses of enzymes activity and microbial biomass to
artificial root exudates}

Compared with the control, the activities of the three enzymes measured
significantly increased. The BG and XYL enzymes increased in the order
C-only \textgreater{} CN80 \textgreater{} CN10 \textgreater{} CN6
\textgreater{} Control (Fig. 3). Conversely, NAG activity decreased with
decreasing N content in the simulated root exudates on day 3, following
the order of CN 6 \textgreater{} CN10 \textgreater{} CN80 \textgreater{}
C-only. Increasing the BG/NAG ratio increased
qCO\textsubscript{2}(R\textsuperscript{2} = 0.41, \emph{P} = 0.01) and
suppressed the CUE (R\textsuperscript{2} = 0.36, \emph{P} = 0.02) (Fig.
4a). Compared with the control, MBC and MBN remarkably increased (except
in CN6 and C-only). However, there was a minor rise from day 3 to day 45
in all treatments (Table S2). C/N ratios of the microbial biomass ranged
from 3.2 to 7.8 (Fig. 4c). The MBC/MBN ratio was negatively correlated
with qCO\textsubscript{2} (R\textsuperscript{2} = 0.29, \emph{P} = 0.01)
and CUE (R\textsuperscript{2} = 0.36, \emph{P} = 0.02) (Fig. 4b).

\textbf{3.3 Structural equation modeling analysis on CO2 efflux}

To quantify the effects of stoichiometry of root exudates on
CO\textsubscript{2} efflux as a proxy of SOM mineralization, a SEM was
constructed based on biotic parameters (microbial biomass and enzyme
activity) and products of biological activity (DOC and
NH\textsubscript{4}\textsuperscript{+}). The SEM showed a reasonable fit
to our hypothesized causal relationships (GEI = 0.98, Chi/DF= 1.00,
RESEA \textless{} 0.01. Fig. 5) and explained 58\% of the variance in
CO\textsubscript{2} emissions using all available biotic parameters and
the products of biological predictors. The increase in C/N ratios had a
negative effect on CO\textsubscript{2} emissions (-0.29,
\emph{P}\textless{} 0.05, Fig. 6). Extracellular enzyme stoichiometry
(BG/NAG and XYL/NAG ratios) exerted a positive direct effect (+0.39,
\emph{P}\textless{} 0.001), whereas the
DOC/NH\textsubscript{4}\textsuperscript{+} ratio showed a negative
direct effect (-0.30, \emph{P} \textless{} 0.001).

\textbf{DISCUSSION}

\textbf{4.1 Microbial response to the addition of root exudates}

Artificial root exudates were designed by using three low-molecular
organic compounds in four different C/N ratios, which are easily
degradable and accessible by the microbial community (de Graaff et al.,
2010; Bastida et al., 2013). When C and N are sufficient, microorganisms
prefer to easily utilize compounds from root exudates than from native
SOM (Shahbaz et al., 2017; Wei et al., 2019). We found that the addition
of root exudates stimulated microbial activity, as indicated by large
accumulative CO\textsubscript{2} emissions. These emissions responded
differently to various stoichiometric ratios of root exudates with the
same amount of C input, indicating that microbial activity is modified
by N demand. Higher activity of the N-acquiring extracellular enzyme
(NAG) was observed in C-rich soil (N limit at high C/N ratio). Thus, a
continuous supply of low C/N ratio artificial root exudates increased N
availability and decreased N-acquiring enzymes (Fig. 3). This is
consistent with previous studies that have applied stoichiometric root
exudates exacerbate CO\textsubscript{2} emission due to microbial N
demand in paddy soils (Liu et al., 2020). However, in contrast to the
observation of Liu et al. (2020), we found that a higher proportion of N
in artificial root exudates stimulated more CO\textsubscript{2} emission
than at high C/N ratios (CN6 [?] CN10 \textgreater{} CN80 [?] C-only) (Fig.
1a, b), and the C-acquiring enzyme activity (BG and XYL) was increased
by a higher C/N ratio of artificial root exudates. These differences due
to the different forms of N nutrients
{[}(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4} vs.
alanine{]} added to paddy soils.

Furthermore, unlike labile inorganic forms of N
(NH\textsubscript{4}\textsuperscript{+} and
NO\textsubscript{3}\textsuperscript{-}), N-containing low-molecular
organic compounds (alanine in this study) can decrease microbial
nutrient limitation from C/N ratios: carbohydrate hydrolase has been
shown to increase with increasing C/N stoichiometric ratios of root
exudates due to unbalanced nutrient addition (Allison and Vitousek,
2005; Sinsabaugh et al., 2008). On the other hand, higher N-rich
artificial root exudates (CN6) showed higher qCO\textsubscript{2} and
lower CUE than other treatments at day 3 and day 12, whereas C-only
caused low CUE (Fig. 2a, b). A higher proportion of organic N-containing
root exudates may have promoted microbial catabolism relative to
anabolism, as there is a higher energy demand for microorganisms in
acquiring organic N compared to inorganic N (N\selectlanguage{ngerman}äsholm and Persson, 2001;
Czaban et al., 2016). Moreover, the positive relationship between BG/NAG
ratio and qCO\textsubscript{2} suggested that microbial catabolism of
root exudates attributed to microorganisms enhancing C- and N-acquiring
enzymes to obtain available C and N from both root exudates and SOM.
Labile C from root exudates can be quickly utilized by microorganisms
for nutrients acquiring to enhance microbial consumption of energy and
released as CO\textsubscript{2} (Zhu et al., 2018).

Along with the increase in CO\textsubscript{2} emission, the continuous
supply of artificial root exudates caused lower microbial CUE in all
treated soils than in the control. This indicated that the addition of
root exudates caused substrate and nutrient uptake by microorganisms
(Blagodatskaya et al., 2014; Chen et al., 2020). Simultaneously, the
higher CUE corresponded to a higher C/N stoichiometric ratio, except in
C-only. This indicated that N-containing stimulates affected microbial
catabolic activity, and that low N-containing formations of root
exudates (i.e., with high C/N ratios) increased the CUE for SOC
accumulation. A positive relationship between the MBC/MBN and CUE (Fig.
4d) suggested that the stoichiometry of microbial biomass played a key
regulator in promoted microorganism utilize input labile C source and
increased the soil C accumulation (Sinsabaugh et al., 2016; Soares and
Rousk, 2019). These findings thus supported our hypothesis that the
addition of a higher C/N ratio with decreasing N content increased
microbial biomass with lower CO\textsubscript{2} emissions and
stimulated soil C sequestration.

\textbf{4.2 Combining microbial C and N metabolism in the SEM}

Root exudates are ubiquitous and quantitatively important drivers of SOM
turnover in flooded paddy soil ecosystems. The stoichiometric ratios of
resources that are important as energy and nutrients are dominant
drivers for the biogeochemical cycles of soil C (McGroddy et al., 2004;
Anderson et al., 2005).

There are two stoichiometric processes that combine microbial C and N
metabolism of root exudates in paddy soils (Figs. 5, 6, and 7). First,
the continuous supply of root exudates caused a C-rich condition, and
thus increased microbial activity via overflow respiration of root
exudates. Different C/N stoichiometric ratios of root exudates affected
the elemental stoichiometry demands for microorganisms to maintain the
microbial biomass C/N stoichiometric balance (Haichar et al., 2014; Liu
et al., 2020; Du et al., 2020; Zhu et al., 2021). Higher energy demand
for microorganisms to acquire organic N from amino acids can further
increase respiration. In the present study, the stoichiometric ratios of
root exudates (i.e., C-only) for microbe-regulated C mineralization did
not dependent on nutrient (N-containing compounds) limitation, as the
lack of N content did not affect the allocation of energy through C
mineralization. Second, a combination of exogenous C and N from root
exudates is required for the adaptation of microbe catabolic activity by
the enhancement of C- and N-acquiring enzyme activity and by causing
catabolism of soil C (Liu et al., 2020; Mori et al., 2021). This was
confirmed by the results of this study, which identified a significant
positive relationship between CO\textsubscript{2} emissions and enzyme
stoichiometry. The stoichiometric ratios of root exudates provide a
perspective on microbial stoichiometric requirement of nutrients and
leads us to further understand the root exudate--soil--microbe
interactions in the rhizosphere soil.

\textbf{CONCLUSIONS}

In this study, we used artificial root exudates with different C/N
ratios to clarify the association of elemental stoichiometric ratios in
microbial biomass and extracellular enzyme activities with soil C
metabolism. We found that the addition of C-only decreased
CO\textsubscript{2} emissions attribute to the N limitation.
Simultaneously, all three different C/N ratios of root exudates (CN6,
CN10, and CN80) increased microbial activities and catabolism to meet
the microbial biomass ratios, thus promoting SOM mineralization.
Microorganisms preferred to use easily available low-molecular compounds
that lead to increased CO\textsubscript{2} emissions, corresponding with
increased stoichiometric ratios of C- and N- hydrolases to meet
microbial nutrient demands. It is, therefore, the input of root exudates
with low C/N stoichiometric ratio stimulate soil C mineralization, while
the inputs with high C/N stoichiometric ratio benefit soil C
accumulation. These results highlight the fact that the stoichiometric
of root exudates represent important derivers for understanding C
cycling in plant-soil system.

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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Table 1. Amounts of individual substrates added (mg incubation
flask\textsuperscript{-1} day\textsuperscript{-1}) to the paddy soil as
artificial root exudates in the different treatments.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Treatment\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glucose (C)
(C\textsubscript{6}H\textsubscript{12}O\textsubscript{6})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Oxalic acid (C)
(H\textsubscript{2}C\textsubscript{2}O\textsubscript{4})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Alanine (C)
(C\textsubscript{3}H\textsubscript{7}NO\textsubscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Alanine (N)
(C\textsubscript{3}H\textsubscript{7}NO\textsubscript{2})\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Control\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.14\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.09\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.17\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.07\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN10\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.22\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.07\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.11\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.04\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN80\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.34\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.05\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.01\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.01\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
C-only\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.35\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.05\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figures}

Figure 1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 4\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 5\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 6\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 7\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}



\textbf{Figure legends}

Fig. 1. CO\textsubscript{2} efflux rates (a) and cumulative
CO\textsubscript{2} (b) over the 45-day incubation period. The control
represents no addition of artificial root exudates to soil; C-only
represents addition of C substrates glucose and oxalic acid only; CN6,
CN10, and CN80 represent addition of C substrates glucose and oxalic
acid as well as the N substrate alanine at different C/N stoichiometries
of CN6, CN10, and CN80, respectively. Different letters indicate
significant differences among stoichiometric ratios at the end of
cumulative CO\textsubscript{2} emission (one-way ANOVA and LSD
test;\emph{P} \textless{} 0.05 level). Error bars show standard errors
(n = 4).

Fig. 2. Metabolic quotient of soil microbial biomass
(qCO\textsubscript{2}) (a) and carbon use efficiency (CUE) (b) at days
3, 12, and 45 during the 45-day incubation period. C-only represents
addition of C substrates glucose and oxalic acid only; CN6, CN10, and
CN80 represent addition of C substrates glucose and oxalic acid as well
as the N substrate alanine at different C/N stoichiometries of CN6,
CN10, and CN80, respectively. Different letters and capital letters
indicate significant differences among stoichiometric ratios and
sampling periods, respectively (one-way ANOVA and LSD test;
\emph{P}\textless{} 0.05 level). Error bars show standard errors (n =
4).

Fig. 3. Activity of extracellular enzymes \selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase (BG),
\selectlanguage{greek}β-\selectlanguage{english}1,4-xylosidase (XYL), and \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl-glucosaminidase (NAG) at days
3, 12, and 45 during the 45-day incubation period. C-only represents
addition of C substrates glucose and oxalic acid only; CN6, CN10, and
CN80 represent addition of C substrates glucose and oxalic acid as well
as the N substrate alanine at different C/N stoichiometries of CN6,
CN10, and CN80, respectively. Different letters and capital letters
indicate significant differences among stoichiometric ratios and
sampling periods, respectively (one-way ANOVA and LSD test;
\emph{P}\textless{} 0.05 level). Error bars show standard errors (n =
4).

Fig. 4. Relationships between metabolic quotient (qCO\textsubscript{2})
and soil enzyme stoichiometry (BG/NAG) (a), qCO\textsubscript{2} with
microbial biomass stoichiometry (MBC/ MBN) (c); carbon use efficiency
(CUE) with soil enzyme stoichiometry (BG/NAG) (b), CUE with microbial
biomass stoichiometry (MBC/ MBN) (d). Abbreviations are BG,
\selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase; NAG, \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase. The dots
represent data from all treatments in the 45-day incubation period.

Fig. 5. Structural equation model of the multivariate effects of C/N
ratios, leaching water sample
DOC-L/NO\textsubscript{3}\textsuperscript{-}-L ratios and
DOC-L/NH\textsubscript{4}\textsuperscript{+}-L ratios, soil sample
DOC/NO\textsubscript{3}\textsuperscript{-} ratios and
DOC/NH\textsubscript{4}\textsuperscript{+} ratios, microbial biomass
stoichiometry, and enzyme stoichiometry on the
CO\textsubscript{2}emission. Microbial biomass stoichiometry represents
the ratios of microbial biomass C to microbial biomass N; enzyme
stoichiometry represents the average ratios of the activity of
\selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase and the activity of
\selectlanguage{greek}β-\selectlanguage{english}1,4-xylosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase. The solid lines
indicate positive path coefficients; dashed lines, negative.
R\textsuperscript{2} values indicate the proportion of variance
explained by each variable contribution to the CO\textsubscript{2}
emission from soil application with substrates. The numbers and the
width of the arrows indicate the standardized path coefficients.

Fig. 6. Standardized effect of stoichiometric C/N ratios, leaching water
sample DOC-L/NO\textsubscript{3}\textsuperscript{-}-L ratios and
DOC-L/NH\textsubscript{4}\textsuperscript{+}-L ratios, soil sample
DOC/NO\textsubscript{3}\textsuperscript{-} ratios and
DOC/NH\textsubscript{4}\textsuperscript{+} ratios, enzyme stoichiometry,
and microbial biomass stoichiometry on CO\textsubscript{2} emission.

Fig. 7. Schematic diagram emphasizing the importance of the
stoichiometric control of plants by their release of root exudates of
different N contents in the flooded rice-paddy soil system. C and N
circles represent artificial root exudates; DOC-W represents dissolved
organic C from water solution; SOM represents the soil organic matter;
microbial biomass stoichiometry represents the C to N ratios of
microbial biomass; enzyme stoichiometry represents the ratios of the
activity of \selectlanguage{greek}β-\selectlanguage{english}1,4-glucosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase and the
activity of \selectlanguage{greek}β-\selectlanguage{english}1,4-xylosidase to \selectlanguage{greek}β-\selectlanguage{english}1,4-N-acetyl glucosaminidase. The
solid lines represent the transportation of microbial active pathways
from stoichiometric controls of root exudates.



\textbf{Supporting information}

\textbf{Table S1.} Amounts of individual substrates added (mg incubation
flask-1) to the paddy soil as artificial root exudates in the different
treatments.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Treatment\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glucose (C)
(C\textsubscript{6}H\textsubscript{12}O\textsubscript{6})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Oxalic acid (C)
(H\textsubscript{2}C\textsubscript{2}O\textsubscript{4})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Alanine (C)
(C\textsubscript{3}H\textsubscript{7}NO\textsubscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Alanine (N)
(C\textsubscript{3}H\textsubscript{7}NO\textsubscript{2})\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Control\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN 6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6.17\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4.11\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7.71\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.00\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN 10\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9.82\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.27\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4.91\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.91\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CN 80\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
15.10\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.32\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.58\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.23\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
C-only\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
15.60\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.40\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S2} . Soil C/N ratios, microbial biomass C (MBC),
microbial biomass N (MBN), DOC, and
NH\textsubscript{4}\textsuperscript{+} at days 3, 12, and 45 during the
45-day incubation period. C-only represents addition of C substrates
glucose and oxalic acid only; CN6, CN10, and CN80 represent addition of
C substrates glucose and oxalic acid as well as the N substrate alanine
at different C/N stoichiometries of CN6, CN10, and CN80, respectively.
Different letters indicate significant differences among stoichiometric
ratios at the end of cumulative CO\textsubscript{2} emission (one-way
ANOVA and LSD test; \emph{P}\textless{} 0.05 level). Values represent
means \emph{+} standard errors (n = 4).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Treatment} & \textbf{3 day} & \textbf{3 day} & \textbf{3 day} &
\textbf{3 day} & \textbf{3 day}\tabularnewline
& C/N ratios & MBC (mg/kg) & MBN (mg/kg) & DOC (mg/kg) &
NH\textsubscript{4}\textsuperscript{+} (mg/kg)\tabularnewline
Control & 8.71\emph{+}0.13\textsuperscript{Bab} &
101.48\emph{+}11.00\textsuperscript{Aab} &
13.80\emph{+}1.97\textsuperscript{Ac} &
140.02\emph{+}5.84\textsuperscript{Aa} &
84.69\emph{+}1.49\textsuperscript{Abc}\tabularnewline
CN6 & 8.44\emph{+}0.49\textsuperscript{Ad} &
92.50\emph{+}20.09\textsuperscript{Bb} &
24.25\emph{+}1.46\textsuperscript{Ab} &
153.91\emph{+}10.06\textsuperscript{Aa} &
90.47\emph{+}2.00\textsuperscript{Aa}\tabularnewline
CN10 & 8.65\emph{+}0.08\textsuperscript{Bc} &
117.23\emph{+}7.10\textsuperscript{Aa} &
22.37\emph{+}1.66\textsuperscript{Aa} &
142.71\emph{+}4.82\textsuperscript{Aa} &
87.03\emph{+}2.22\textsuperscript{Aab}\tabularnewline
CN80 & 8.88\emph{+}0.09\textsuperscript{Cab} &
111.94\emph{+}34.90\textsuperscript{Bab} &
25.91\emph{+}0.68\textsuperscript{Bb} &
146.17\emph{+}13.46\textsuperscript{Aa} &
82.78\emph{+}2.59\textsuperscript{Ac}\tabularnewline
C-only & 9.18\emph{+}0.11\textsuperscript{Ba} &
61.10\emph{+}15.49\textsuperscript{Cc} &
15.70\emph{+}1.55\textsuperscript{Bc} &
152.34\emph{+}4.51\textsuperscript{Aa} &
83.83\emph{+}1.06\textsuperscript{Abc}\tabularnewline
\textbf{Treatment} & \textbf{12 day} & \textbf{12 day} & \textbf{12 day}
& \textbf{12 day} & \textbf{12 day}\tabularnewline
& C/N ratios & MBC (mg/kg) & MBN (mg/kg) & DOC (mg/kg) &
NH\textsubscript{4}\textsuperscript{+} (mg/kg)\tabularnewline
Control & 9.40\emph{+}0.23\textsuperscript{Aa} &
74.92\emph{+}16.25\textsuperscript{Cc} &
13.12\emph{+}1.10\textsuperscript{Ac} &
118.26\emph{+}6.55\textsuperscript{Ba} &
87.35\emph{+}10.92\textsuperscript{Bc}\tabularnewline
CN6 & 8.84\emph{+}0.22\textsuperscript{Ad} &
95.72\emph{+}23.31\textsuperscript{Bb} &
24.25\emph{+}1.46\textsuperscript{Ab} &
107.00\emph{+}3.72\textsuperscript{Ba} &
75.87\emph{+}7.78\textsuperscript{Aa}\tabularnewline
CN10 & 8.87\emph{+}0.22\textsuperscript{Bc} &
105.86\emph{+}21.85\textsuperscript{Ba} &
22.37\emph{+}1.66\textsuperscript{Bb} &
113.92\emph{+}5.44\textsuperscript{Ba} &
65.09\emph{+}4.97\textsuperscript{Bab}\tabularnewline
CN80 & 9.18\emph{+}0.08\textsuperscript{Bab} &
100.75\emph{+}11.83\textsuperscript{Cb} &
31.09\emph{+}2.61\textsuperscript{Aa} &
113.92\emph{+}5.44\textsuperscript{Ba} &
65.09\emph{+}4.97\textsuperscript{Bd}\tabularnewline
C-only & 9.01\emph{+}0.20\textsuperscript{Bab} &
104.17\emph{+}34.14\textsuperscript{Aa} &
21.85\emph{+}1.61\textsuperscript{Ab} &
114.93\emph{+}12.59\textsuperscript{Ba} &
76.36\emph{+}6.54\textsuperscript{Bab}\tabularnewline
\textbf{Treatment} & \textbf{45 day} & \textbf{45 day} & \textbf{45 day}
& \textbf{45 day} & \textbf{45 day}\tabularnewline
& C/N ratios & MBC (mg/kg) & MBN (mg/kg) & DOC (mg/kg) &
NH\textsubscript{4}\textsuperscript{+} (mg/kg)\tabularnewline
Control & 9.59\emph{+}0.46\textsuperscript{Aa} &
82.98\emph{+}3.31\textsuperscript{ABd} &
15.50\emph{+}0.47\textsuperscript{Ad} &
73.94\emph{+}9.39\textsuperscript{Ca} &
19.07\emph{+}1.15\textsuperscript{Cd}\tabularnewline
CN6 & 8.88\emph{+}0.26\textsuperscript{Ab} &
159.45\emph{+}2.39\textsuperscript{Aa} &
20.24\emph{+}1.11\textsuperscript{Bc} &
68.52\emph{+}4.92\textsuperscript{Ca} &
95.74\emph{+}3.36\textsuperscript{Aa}\tabularnewline
CN10 & 9.43\emph{+}0.08\textsuperscript{Aa} &
102.82\emph{+}0.83\textsuperscript{Cc} &
22.66\emph{+}0.87\textsuperscript{Bb} &
72.76\emph{+}2.58\textsuperscript{Ca} &
73.22\emph{+}5.31\textsuperscript{Bb}\tabularnewline
CN80 & 9.65\emph{+}0.15\textsuperscript{Aa} &
121.17\emph{+}11.54\textsuperscript{Ab} &
26.41\emph{+}1.22\textsuperscript{Ba} &
66.49\emph{+}9.79\textsuperscript{Ca} &
36.72\emph{+}0.97\textsuperscript{Cc}\tabularnewline
C-only & 9.65\emph{+}0.08\textsuperscript{Aa} &
99.66\emph{+}13.99\textsuperscript{Bc} &
22.25\emph{+}0.61\textsuperscript{Ab} &
74.29\emph{+}2.78\textsuperscript{Ca} &
37.45\emph{+}1.21\textsuperscript{Cc}\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
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\end{document}

