Applied Soil Ecology 166 (2021) 103996

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Differentiated mechanisms of biochar- and straw-induced greenhouse gas

emissions in tobacco fields
Tiantian He 1, Fei Yun 1, Tian Liu, Jiawei Jin, Yang Yang, Yunpeng Fu *, Jing Wang *
The Key Lab of National Tobacco Cultivation; College of Tobacco Sciences, Henan Agricultural University, Zhengzhou 450002, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Addition of biochar and straw has constituted an effective way to improve soil fertility in tobacco fields.
Biochar However, little is known about the effects of these amendments on CH4 and N2O emissions and their potential
Organic amendment microbial mechanisms. Here, we conducted an experiment in tobacco-planted soil using four treatments: mineral
Nitrous oxide emissions
fertilizers only (F), biochar with mineral fertilizers (BF), wheat straw with mineral fertilizers (SF), and biochar +
Methane emissions
Denitrification
wheat straw with mineral fertilizers (BSF). Soil physical and chemical parameters, nitrous oxide (N2O) and
methane (CH4) emissions, and the related microorganisms and functional genes (pmoA, mcrA, amoA, nirS, nirK,
and nosZ) were analyzed. The results showed that biochar and straw addition significantly increased the uptake
of CH4 by 34.2%, 9.3%, and 22.1%, respectively, and significantly increased the relative abundance of pmoA.
Compared to the F treatment, the SF and BSF treatments significantly increased cumulative N2O emissions by
1.2- and 1.1-fold, respectively, while the BF treatment significantly decreased N2O emissions from the soil by
almost 30%. Correspondingly, decreased relative abundance of amoA-ammonia-oxidizing archaea (AOA), nirS,
nirK, and nosZ genes in response to biochar amendment were observed, whereas the relative abundance of amoA-
ammonia-oxidizing bacteria (AOB) increased. Conversely, straw addition increased the relative abundance of
amoA-AOA and nirS, nirK, and nosZ. Compared to SF, BSF significantly increased the relative abundance of nosZ
and decreased the number of denitrifying bacteria. A structural equation model indicated that the important
factors affecting N2O emissions were NH+ 4 -N, NO3 -N, pH, and amoA-AOA and nirK genes, while the important
−

factors affecting CH4 emissions were bulk density, pH, SMBC, and pmoA and mcrA genes. Biochar amendment
could significantly reduce CH4 and N2O emissions by regulating functional microorganisms and soil physico­
chemical properties. By contrast, returning straw to the field would increase N2O emissions by increasing the
relative abundance of denitrification-related genes.

1. Introduction Biochar, a carbon (C)-rich and C sequestration material, is produced

Nitrous oxide (N2O) and methane (CH4) are important greenhouse
gases (GHGs) that cause global warming effects (Smith et al., 2003;
Munoz et al., 2010). The main source of GHGs is thought to be agri­
cultural soils, producing approximately 66% of N2O and 50% of CH4
emissions (Thauer, 1998; Smith et al., 2003). With increasing demand
for food, further increases in these GHGs are expected in the future
(Xiang et al., 2015). To mitigate global warming, strategies are needed
to reduce these gas emissions from agricultural soils.
by the pyrolysis of crop residues under oxygen-limited conditions (Cao
et al., 2011). A large number of studies have shown that biochar can
significantly increase soil pH and cation exchange capacity (CEC), pro­
mote crop growth, etc., which has become a good soil improvement
material (Lehmann, 2007; Van Zwieten et al., 2010). Meanwhile, the
application of biochar has been considered one of the most promising
emission reduction measures in recent years, and its emission reduction
potential reaches 0.7 Gt Ceq yr− 1 (Smith, 2016). A meta-analysis showed
that the application of biochar could reduce more than half of

Abbreviations: AOA, ammonia-oxidizing archaea; AOB, ammonia-oxidizing bacteria; F, mineral fertilizer treatment; BF, biochar with mineral fertilizer treatment;
SF, wheat straw with mineral fertilizer treatment; BSF, biochar + wheat straw with mineral fertilizer treatment; GHG, greenhouse gas; CEC, cation exchange capacity;
BD, bulk density; TOC, total organic C; SMBC, soil microbial biomass C; SMBN, soil microbial biomass N.
* Corresponding authors.
E-mail addresses: yunpengfu@henau.edu.cn (Y. Fu), jingwang040922@henau.edu.cn (J. Wang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.apsoil.2021.103996
Received 4 October 2020; Received in revised form 12 March 2021; Accepted 14 March 2021
Available online 25 March 2021
0929-1393/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
T. He et al.
agricultural N2O emissions (Cayuela et al., 2014). However, some
studies have shown that the addition of biochar can increase N2O
emissions (Clough et al., 2010; Troy et al., 2013; Yoo et al., 2016). The
differing results may be related to the characteristics of biochar and soil
conditions. The large specific surface area and well-developed porous
structure of biochar can improve soil aeration and limit nitrogen (N)
utilization, inhibiting N2O emissions (Samad et al., 2016). However, the
labile C in biochar can also have a positive priming effect, stimulating
the activities of microorganisms and increasing the production of N2O
(Cross and Sohi, 2011). In addition to the physical and chemical char­
acteristics of soil, changes in N2O emissions caused by biochar may also
be related to the microbial pathway of N2O production. Microbial
nitrification and denitrification pathways are major contributors to soil
N2O emissions (Baggs, 2011). The process of ammonia oxidation and the
process of NO-2 reduction to NO are the rate limiting steps of nitrification
and denitrification, respectively. The amoA gene can be used as a mo­
lecular marker of ammonia-oxidizing bacteria (AOB) and ammonia-
oxidizing archaea (AOA) (Wang et al., 2016) and the nirK, nirS and
nosZ genes can been used as molecular markers for denitrification
(Zhang et al., 2016). Biochar contains a large number of soluble base
cations, which can be rapidly released into the soil, thus increasing the
soil pH. With increasing soil pH, the activity of N2O reductase can be
increased (McMillan et al., 2016), reducing N2O emissions. Cayuela
et al. (2013) found that the proportion of N2O / (N2 + N2O) decreased in
14 soils after biochar amendment, indicating that N2O was reduced to
N2, thus reducing N2O emissions. Similarly, some studies have reported
that biochar amendment reduces N2O emissions by increasing the
abundance of the nosZ gene (Harter et al., 2014; Van Zwieten et al.,
2014; Xu et al., 2014). However, Lin et al. (2017) found that the
increased N2O emissions stimulated by biochar amendment in paddy
soil were related to an increase in amoA genes, but not related to nosZ or
nirK genes. Therefore, the mechanism by which biochar affects N2O
emissions is still unclear, and further research is needed.
The production and consumption of CH4 are closely related to
methanogens and methanotrophs. Soil organic matter is decomposed by
various microorganisms, and is eventually used by methanogens to
produce CH4, which can be consumed by methane-oxidizing bacteria as
the only source of energy before being released into the atmosphere
(Bridgham et al., 2013). It was reported that the large surface area of
biochar can increase the adsorption of CH4, thereby reducing emissions
of CH4 (Yaghoubi et al., 2014). For example, Han et al. (2016) showed
that CH4 emissions from paddy soils amended with biochar decreased by
39.5%. However, the labile C component of biochar can also be used as a
substrate for methanogens in anoxic environments, thus promoting CH4
production (Wang et al., 2012). Some studies have reported that cu­
mulative CH4 emissions were negatively correlated with the gene
abundance of pmoA and positively correlated with mcrA in soil. Biochar
amendment could increase the abundance of the pmoA gene, thus
reducing CH4 emissions (Wu et al., 2018; Huang et al., 2019). However,
other studies have found that biochar amendment increased CH4 emis­
sions and had no significant effect on the pmoA gene (Wang et al., 2017).
At present, the effects of biochar addition on CH4 and N2O emissions
from farmland soil are not clear.
In China, returning straw to soil is a common agricultural practice to
improve soil physical properties and nutrients (Wang et al., 2019). Straw
application can not only increase soil organic C storage (Li et al., 2010;
Xia et al., 2014; Chaudhary et al., 2017) but also reduce environmental
pollution associated with straw burning (Romasanta et al., 2017).
However, studies on the effects of straw addition on GHGs are incon­
sistent in their findings (Chen et al., 2013). Some studies have shown
that straw addition increased CH4 emissions (Ma et al., 2009; Zhang
et al., 2015) and N2O emissions (Huang et al., 2019; Wang et al., 2019)
from farmland. For example, Wang et al. (2019) found that CH4 and N2O
emissions increased by 44–138 kg CH4-C ha-1 yr-1 and 0.68–1.49 kg N2O-
N ha-1 yr-1 after wheat and rice straw addition, respectively. However,
other studies reported the opposite results (Yao et al., 2009). The
2
Applied Soil Ecology 166 (2021) 103996
inconsistent results may be due to the different C/N ratio of straw; straw
with a high C/N ratio can inhibit the production of N2O (Toma and
Hatano, 2007). At present, there are few studies on the microbial
mechanism driving CH4 and N2O emissions following straw addition.
One study has shown that returning straw to soil increases N2O emis­
sions but has no significant effect on the abundance of amoA and nirK
genes (Wang et al., 2018). However, Hu et al. (2019) reported that straw
application had no significant effect on N2O emissions but significantly
increased mcrA gene abundance and CH4 emissions. The contradictory
results show that biochar and straw can affect the production and con­
sumption of N2O and CH4, and their effects on GHGs lie in the regulation
of various microbial functional genes.
As a cash crop, tobacco is planted worldwide. Biochar and straw
returning have been widely used in the tobacco planting industry in
recent years in China, but their effects on GHG emissions in tobacco
fields has received less attention. Therefore, it is necessary to study the
changes in N2O and CH4 emissions and the related microbial commu­
nities in tobacco fields after biochar and straw application and to un­
derstand the response mechanism of GHGs to such additions. We
hypothesized that biochar and straw addition alter GHG emissions in
tobacco-planted soils via changes to the microbial community and
functional genes. The purpose of this study was to explore the mecha­
nism by which biochar and straw amendment affect GHG emissions.
2. Materials and methods
2.1. Study site and materials
The test site was located in the Xuchang campus of Henan Agricul­
tural University (113◦ 85′ E, 34◦ 14′ N), which has a warm temperate
sub-humid monsoon climate, with a mean annual temperature and
precipitation of 15 ◦ C and 700 mm, respectively. The tested soil was
brown soil from the North Plain in China, which was classified as a
Luvisol (IUSS Working Group WRB). The basic physicochemical prop­
erties of the soil at 0–20 cm depth were as follows: organic matter, 19.09
g kg− 1; available N, 74.7 mg kg− 1; available phosphorus, 8.7 mg kg− 1;
available potassium, 114.5 mg kg− 1; and pH (soil: water = 1:5), 7.8. The
biochar used in the test was produced from peanut shells via high-
temperature pyrolysis (400–450 ◦ C) by Henan Biochar Technology En­
gineering Laboratory. It had a pH of 8 and a total C, N, and P concen­
tration of 49.68%, 1.21%, and 1.22%, respectively. The straw used in
the experiment was wheat straw. The decomposed straw had a pH of
7.86; a total C, N, P, and K concentration of 42.32%, 1.41%, 0.96%, and
0.97%, respectively; and a C/N of 30.01.
2.2. Experimental design
The long-term field experiment started on May 1st 2017 and
involved four treatments: (1) F, no addition of biochar or straw (con­
ventional fertilization); (2) BF, conventional fertilization +2.25 t hm− 2
biochar-C; (3) SF, conventional fertilization +2.25 t hm− 2 straw-C; and
(4) BSF, conventional fertilization +1.125 t hm− 2 biochar-C + 1.125 t
hm− 2 straw-C. Treatments 2, 3, and 4 had equal C contents. Three
replicate plots (0.02 hm− 2) were set up for each treatment. Biochar and
straw were applied to the test field two weeks before tobacco trans­
plantation and thoroughly mixed with the top 0–20 cm of the soil. The
conventional fertilization was in accordance with the fertilization
method commonly used in Henan tobacco growing areas, that is, the N
application amount was 37.5 kg hm− 2, and the N: P: K ratio was 1: 2: 5.
Tobacco-specific compound fertilizers (10% N, 10% P2O5, 10% K2O),
potassium sulfate (52% K2O), and superphosphate (12% P2O5) served as
base fertilizers, and potassium nitrate (13.5%N, 44.5% K2O) served as
the top-dressing fertilizer. Tobacco was transplanted around May 1st
every year, and all operations were performed in accordance with high-
quality tobacco production management practices.
T. He et al. Applied Soil Ecology 166 (2021) 103996

2.3. Gas sampling and measurement
Gas samples were collected between 9 and 11 a.m. in 7-d intervals
from 1 May 2019 to 2 September 2019 (collected 2–3 times in one week
after fertilization and rainfall) using a static chamber during the whole
tobacco growing season. The plexiglass static chamber (0.4 m × 0.4 m)
was placed on a fixed plexiglass base (0.45 m × 0.45 m × 0.2 m) in each
plot at every collection time. An electric fan was fixed at the top of the
chamber to mix the gases, and a groove (0.05 m depth) along the top
edge of each base was filled with water to ensure an air-tight system. To
minimize air temperature changes inside the chambers during sampling,
the chambers were wrapped with a layer of porous thermal insulation
material (aluminum foil). For each collection, a 50-ml syringe was used
to collect gas samples at 0, 10, 20, and 30 min after chamber closure. The
concentrations of N2O and CH4 were analyzed within 24 h using a gas
chromatograph (Agilent 7890B, USA) equipped with an electron capture
detector (ECD) for N2O and a flame ionization detector (FID) for CH4
analysis. N2O and CH4 fluxes were calculated using linear regression
analysis. The equation is as follows:
ρ⋅h⋅dc⋅t⋅273
F= , (1)
dt⋅(273 + T)
where F is the CH4 or N2O flux (mg m− 2 h− 1), ρ is the density of CH4 or
N2O under standard atmospheric pressure, h is the height of the static
chamber (m), dc/dt is the change rate of the CH4 or N2O concentration,
T is the mean temperature inside the chamber (◦ C), and t is the time of
chamber closure (h).
CH4 and N2O cumulative emissions were calculated using Eq. (2) as
follows:
∑n
Fi + Fi+1
C= × 24 × D, (2)
2
i=1
samples were stored at − 80 ◦ C for subsequent analysis. The expression
level of genes was detected using a quantitative real-time polymerase
chain reaction (qPCR) device according to the instructions of a SYBR
Premix Ex Taq kit (TaKaRa, China). Gene-specific primers are listed in
Table 1. The qPCR reaction was performed in a 20-μL PCR mixture
containing 10 μL SYBR® Premix Ex Taq, 0.4 μL of each primer, 1 μL
template DNA, and 8.2 μL sterile water. The relative abundance of the
target gene were normalized to 16S rRNA and were calculated using the
2-ΔΔCt method.
2.6. Statistical analyses
Statistical analysis was performed using SPSS 22.0 (IBM Inc.
Armonk, NY, USA) and Microsoft 2010. One-way analysis of variance
(ANOVA) and the least significant difference (LSD) test (P < 0.05) were
used to analyze the differences in gas emissions, soil properties, micro­
bial quantities, and functional genes among the treatments. The re­
lationships among N2O and CH4 emissions, soil properties, and relative
abundance of microbial functional genes were analyzed using structural
equation modeling. All charts were created using Origin 2018.
3. Results
3.1. Soil physicochemical properties
Soil properties were significantly influenced by the addition of straw
and biochar. Straw addition alone (SF) significantly increased NH+ 4 -N by
8.5%, SMBC by 49.5%, and SMBN by 37.9% at day 100, but showed no
significant impact on NO−3 -N, in comparison with the control (F) (P <
0.05) (Table 2). When compared with SF, the pH significantly increased
by 0.1 and TOC by 31.3% after biochar amendment (BF) at day 100.
Similar trends of soil TOC and pH were also observed at day 30 and day

where C is the amount of CH4 or N2O emissions (kg ha− 1), F is the gas 70 (Table 2), but biochar addition alone significantly decreased NH+ 4 -N

flux (mg m− 2 h− 1), n is the total number of gas measurements, i is the throughout the growth season, which might have been caused by the
sampling time, and D represents the days between times i and i + 1. adsorption of biochar. For the BSF treatment, there were no significant
differences in soil TOC and NO-3-N compared with BF, but soil SMBC and
2.4. Soil physicochemical properties and microbial biomass analyses SMBN increased by 19.5%–100% and 6.76%–23.45%, respectively
(Table 2). In short, compared with single straw application, the addition
Soil samples were collected from topsoil (0–15 cm) while gas sam­
pling was carried out. Samples were randomly collected from five Table 1
points, thoroughly mixed, and passed through a 2-mm sieve. Each mixed Primer pairs used for real-time quantitative PCR (qRT-PCR).
soil sample was divided into two parts. One part was used to determine Gene Primer Primer sequence (5′ → 3′ ) Reference
soil physical and chemical properties, the other part was used to name name
determine soil microorganisms and genes. NH+ 4 - N and NO3 -N were
−
STA ATG GTC TGG CTT AGA
amoA-F
measured in all soil samples. Soil bulk density (BD), total organic C amoA- CG
Francis et al. (2005)
(TOC), microbial biomass C (SMBC), microbial biomass N (SMBN), pH, AOA GCG GCC ATC CAT CTG TAT
amoA-R
GT
and colony number were measured at 30 d (cluster stage), 70 d (vigorous amoA1F GGG GTT TCT ACT GGTGGT
stage), and 100 d (mature stage) after transplanting. N2O and CH4 amoA- Rotthauwe et al.
CCC CTC KGS AAA GCC TTC
AOB amoA1R (1997)
related genes were measured at 3 d (after basal fertilizer application), 30 TTC
d (cluster stage), 70 d (vigorous stage), and 100 d (mature stage) after cd2aF
GTS AAC GTS AAG GAS ACS
Throbäck et al.
nirS GG
transplanting. Measurement of soil physicochemical properties and (2004)
R3cd GAS TTC GGR TGS GTC TTG A
various forms of N content were conducted according to “Agricultural nirKFlaCu ATC ATG GTS CTG CCG CG
Soil Analysis” by Bao (2007). The numbers of bacteria and fungi were Hallin and Lindgren
nirK GCC TCG ATC AGR TTG TGG
nirKR3Cu (1999)
measured by the coating plate method, and the numbers of nitrifying TT
and denitrifying bacteria were measured by the most probable number AGA ACG ACC AGC TGA TCG
nosZ-F
ACA
(MPN) method (Xu and Zheng, 1986). Although the MPN method is not nosZ Kloos et al. (2001)
TCC ATG GTG ACG CCG TGG
accurate enough to determine the absolute number of bacteria (David­ nosZ-R
TTG
son et al., 1985), all samples were measured in the same step, and the GGTGGT GTM GGA TTC ACA
mcrA-F
results were comparable. mcrA
CAR TAY GCW ACA GC
Barbier et al. (2012)
TTC ATT GCR TAG TTW GGR
mcrA-R
TAG TT
2.5. DNA extraction and relative quantitative PCR A189f GGN GAC TGG GAC TTC TGG Costello and
pmoA
mb661r CCG GMG CAA CGT CYT TAC C Lidstrom (1999)
A Power Soil DNA Isolation Kit (Mo Bio, Carlsbad, CA, United States) 515F GTG CCA GCM GCC GCG G
was used to extract the total DNA from 0.5 g of freeze-dried soil ac­ 16SrRNA CCG TCA ATT CMT TTR AGT Zhou et al. (2011)
907R
TT
cording to the manufacturer’s instructions. All the extracted soil DNA

3
T. He et al. Applied Soil Ecology 166 (2021) 103996

Table 2
Effects of biochar and straw on soil physical and chemical properties in different periods.
Transplant days Treatments TOCa (g⋅kg− 1) NH+
4 -N NO−3 -N pH BDb (g⋅cm− 2) SMBCc SMBNd
(mg⋅kg− 1) (mg⋅kg− 1) (mg⋅kg− 1⋅h− 1) (mg⋅kg− 1⋅h− 1)

F 10.63 ± 0.39ce 6.08 ± 0.10b 8.35 ± 0.3a 7.98 ± 0.03ab 1.15 ± 0.01b 68.42 ± 4.34c 38.65 ± 0.69c
BF 13.88 ± 0.39a 6.80 ± 0.14a 8.02 ± 0.16ab 8.01 ± 0.01a 1.14 ± 0.01b 73.68 ± 2.55c 46.56 ± 1.92b
30
SF 12.40 ± 0.47b 6.14 ± 0.11b 7.38 ± 0.12c 7.94 ± 0.01b 1.18 ± 0.02a 105.26 ± 5.38b 50.34 ± 0.20a
BSF 14.50 ± 0.52a 6.28 ± 0.28b 7.84 ± 0.12b 7.89 ± 0.02c 1.14 ± 0.01b 147.37 ± 8.51a 49.71 ± 0.93a
F 11.89 ± 0.32d 6.89 ± 0.11a 7.81 ± 0.15b 7.87 ± 0.01c 1.23 ± 0.01a 294.74 ± 5.8d 42.31 ± 3.23b
BF 16.29 ± 0.29b 6.39 ± 0.08b 8.27 ± 0.16a 7.98 ± 0.01a 1.11 ± 0.01c 402.63 ± 7.99a 48.57 ± 6.64ab
70
SF 13.23 ± 0.20c 6.39 ± 0.16b 6.82 ± 0.16c 7.92 ± 0.01b 1.16 ± 0.02b 371.05 ± 4.18c 52.94 ± 6.70a
BSF 17.10 ± 0.45a 6.39 ± 0.22b 7.98 ± 0.05ab 7.91 ± 0.01b 1.11 ± 0.01c 389.47 ± 4.28b 55.90 ± 6.54a
F 11.06 ± 0.10c 4.30 ± 0.28b 8.52 ± 0.18a 7.89 ± 0.02c 1.23 ± 0.01a 239.47 ± 8.18c 41.66 ± 1.27b
BF 15.74 ± 0.28a 3.97 ± 0.55c 8.45 ± 0.10a 8.03 ± 0.03a 1.16 ± 0.02c 315.79 ± 7.03b 44.85 ± 1.22b
100
SF 11.99 ± 0.54b 4.66 ± 0.28a 7.84 ± 0.10a 7.94 ± 0.01b 1.20 ± 0.03b 357.89 ± 11.36a 57.46 ± 8.42a
BSF 16.05 ± 0.38a 4.36 ± 0.07b 7.91 ± 0.46a 7.98 ± 0.01b 1.17 ± 0.01bc 377.37 ± 9.28a 55.37 ± 2.83a
a
Total organic carbon.
b
Bulk density.
c
Microbial biomass carbon.
d
Microbial biomass nitrogen.
e
Different lowercase letters indicate significant differences among different treatments at p < 0.05.

of biochar alone and straw incorporation with biochar significantly
increased soil TOC, pH, and significantly reduce soil BD, while the straw
treatment greatly increased SMBN, SMBC, and NH+ 4 -N compared with
BF. (See Table 2.)
3.2. Soil functional colonies
The number of bacteria decreased over time in all treatments. The
addition of biochar and straw significantly increased the number of
bacteria, by 2.97–41.89%, 24.53–48.33%, and 9.24–112.02%, respec­
tively, compared with when only fertilizer was added (Fig. 1a). Similar

Fig. 1. Effects of biochar and straw on the number of soil microbial colonies in different periods. Different lowercase letters indicate significant differences among
different treatments at P < 0.05.

4
T. He et al.
trends for nitrifying bacteria and denitrifying bacteria were also found in
SF and BSF during the growing season (Fig. 1c–d), with maximum values
observed on day 30. The single application of biochar (BF) significantly
reduced the number of denitrifying bacteria on days 70 and 100, which
decreased by 33.02%–45.21% in comparison with the F treatment.
Meanwhile, the number of nitrifying bacteria decreased by 25% at day
30 in the BF treatment. In contrast, straw addition alone (SF) signifi­
cantly increased the number of nitrifying bacteria and denitrifying
bacteria, which were 36%–75% and 7%–764% higher than those in F,
respectively. The amount of fungi decreased slightly after 70 d of
transplantation, and then sharply increased in all treatments. The pro­
portion of fungi in BF was not significantly different from that in F after
70 d of transplantation, but then sharply increased on day 100, by
Fig. 2. Effects of biochar and straw on soil NH+
4 -N, NO3 -N, and N2O, and CH4 emissions Different lowercase letters indicate significant differences among different
−
treatments at P < 0.05.
5
Applied Soil Ecology 166 (2021) 103996
59.60%, which was substantially larger than the increase in the other
treatments. The patterns in the BSF treatment were opposite to those in
the BF treatment, where the proportion of fungi increased by 16.67%–
59.68% after 70 d of transplantation as compared with F. Straw addition
alone (SF) did not significantly affect the number of fungi (Fig. 1b).
3.3. N2O emissions from soils
The N2O emission flux of all treatments showed similar trends
throughout the growing season. High fluxes were usually recorded after
fertilization and continuous heavy rainfalls (Fig. 2), with a maximum
peak after adding base fertilizers (May 5). The fluxes quickly decreased
within a week, then peaked again within another week after two top
T. He et al. Applied Soil Ecology 166 (2021) 103996

dressings (June 1, June 10, and June 25). No obvious variation was
found in the following growth stages, except for the second largest
emission peak flux on August 13. Generally, the biochar addition alone
(BF) increased the N2O emission flux at 20 d after transplantation, and
then significantly decreased, compared with F. However, the N2O
emission fluxes of the SF and BSF treatments were significantly higher
than those of the F treatment during the whole growth period, with the
largest increase in the SF treatment.
The cumulative N2O emissions from SF (0.56 kg ha− 1) and BSF
(0.499 kg ha− 1) were significantly higher than those from the F treat­
ment (0.458 kg ha− 1) (P < 0.05), which increased 1.23- and 1.09-fold,
respectively (Fig. 2). However, the cumulative N2O emissions from the
BF treatment (0.322 kg ha− 1) were significantly lower than those from
the F treatment (P < 0.05), which decreased by 29.59%.
June 25, when the CH4 fluxes of the BF, SF, and BSF treatments reached
− 62.29 μg m− 2 h− 1, − 76.27 μg m− 2 h− 1, and − 63.48 μg m− 2 h− 1,
respectively, which was significantly lower by 75.36%, 114.7%, and
78.71% compared with the F treatment.
In short, the addition of biochar and straw increased the uptake of
CH4 in tobacco-planted soil, compared with soil only fertilized (− 0.69
kg ha− 1). The cumulative emissions of CH4 from BF (− 0.92 kg ha− 1) was
lowest and significantly lower, by 22.78%, as compared with SF (− 0.75
kg ha− 1). Meanwhile, straw incorporation with biochar (− 0.84 kg ha− 1)
greatly decreased the cumulative CH4 emissions as compared to SF, but
significantly increased the emissions as compared to BF.
3.5. Relative abundance of N2O-related functional genes

The relative abundance of amoA-ammonia-oxidizing archea (AOA)

3.4. CH4 emissions from soils
Upland soil is a sink of CH4, the CH4 emission flux showed remark­
able differences in all treatments and varied over the tobacco growing
season (Fig. 2). The maximum absorption peak of CH4 was observed on
decreased by 0.15–0.28-fold in several periods in the BF treatment, but
there was no significant difference compared with F (except for 100
d after transplanting). In addition, no significant variation in AOA gene
quantity was observed between the BSF and F treatments during the
entire growth period. In contrast, the relative abundance of amoA-AOA

Fig. 3. Effects of biochar and straw on the relative abundance of N2O- and CH4-related genes. The relative abundance of functional genes (amoA, nirK, nirS, nosZ,
pmoA, and mcrA) related to N2O and CH4 emission were quantified using real-time quantitative PCR (qPCR). And the relative abundance of all functional genes was 1
for the “F” treatment. Error bars present standard deviations of means (n = 3). Different lowercase letters indicate significant difference among different treatments at
P < 0.05.

6
T. He et al.
in the SF treatment was significantly higher than that in BF and BSF in
multiple periods (P < 0.05; Fig. 3). The relative abundance of amoA-
ammonia-oxidizing bacteria (AOB) in the SF and BSF treatments was not
significantly different from that of F (except for day 3 after trans­
planting). However, the single application of biochar (BF) significantly
increased the relative abundance of amoA-AOB by 0.25–1.04-fold,
compared with F. Generally, the single application of biochar (BF)
significantly decreased the relative abundance of amoA-AOA, and
significantly increased the relative abundance of amoA-AOB, when
compared with F. However, the addition of straw alone (SF) increased
the relative abundance of amoA-AOA but had no effect on AOB. The BSF
treatment had no significant effect on the relative abundance of amoA
genes in AOA and AOB.
Compared with CK, the application of biochar alone (BF) signifi­
cantly reduced the relative abundance of nirS and nirK genes, except for
a significant increase on day 3 after transplantation. However, straw
addition alone (SF) and biochar incorporation with straw (BSF) had little
effect on the relative abundance of nirS and nirK genes, except for a
significant decrease (0.47-fold) in the SF treatment on day 3 (P < 0.05)
compared with F (Fig. 3). Similarly, the addition of biochar alone
significantly reduced the relative abundance of nosZ genes, except for a
significant increase on day 3. In contrast, biochar incorporation with
straw (BSF) significantly increased the relative abundance of the nosZ
gene over time. However, straw addition alone (SF) had little effect on
the nosZ gene, except for a significant increase of 88.3% on day 100
(Fig. 3, P < 0.05).
3.6. Relative abundance of CH4-related functional genes
The relative abundance of the mcrA gene increased with SF and
showed statistically significant differences on days 30 and 100
compared with F (Fig. 3, P < 0.05). Interestingly, the relative abundance
of mcrA in BF showed no significant difference to that in F, except for a
significant decrease on day 70 (Fig. 3, P < 0.05). Similarly, biochar
incorporation with straw (BSF) had little effect on the relative abun­
dance of the mcrA gene, except for a significant decrease (0.44–0.60-
fold) on days 3 and 70 (P < 0.05) compared with F (Fig. 3).
Compared with the F treatment, SF and BSF significantly promoted
the relative abundance of the pmoA gene, except for day 100 (Fig. 3).
Moreover, the BF resulted in a visible increase in pmoA gene quantity on
days 70 and 100, which increased 2.01–3.05-fold compared with the
Fig. 4. Structural equation model of the effects of soil properties and N2O-related genes on N2O emissions from tobacco fields. The blue and red arrows indicate
positive and negative effects, respectively, and the adjacent number is the standardized path coefficient. *** P < 0.001, ** P < 0.01, * P < 0.05.
7
Applied Soil Ecology 166 (2021) 103996
BSF treatment. However, there was no significant difference between BF
and F during the initial 30 d after transplantation. In short, the addition
of straw and biochar both increased the relative abundance of the pmoA
gene, and the positive effect of the biochar treatment on the pmoA gene
was largest in the late growth stage, while the straw treatments (SF and
BSF) showed the opposite pattern.
3.7. Structural equation models explaining N2Oand CH4 emissions
The structural equation model showed that soil pH, NH+ 4 -N, NO3 -N,
−
amoA-AOA, and nirK were the main factors leading to N2O emissions
(Fig. 4). The contents of NH+ 4 -N and NO3 -N affected soil N2O emissions
−
via their effect on amoA-AOA and nirK genes. Although pH had no sig­
nificant effect on N2O-related genes, it directly affected N2O emissions,
and its path coefficient was − 0.37. Based on the above results, we found
that biochar could inhibit N2O emissions by reducing the content of
NH+ 4 -N, amoA-AOA, and nirK genes. On the contrary, straw addition
increased NH+ 4 -N and amoA-AOA, which promoted N2O emissions.
The structural equation model showed that soil pH, SMBC, pmoA,
and mcrA were the main factors directly leading to CH4 flux changes
(Fig. 5). Among them, soil pH and pmoA genes had significant negative
effects on CH4 emission, while mcrA genes had significant positive ef­
fects on CH4 emission. pH and SMBC not only directly affected CH4
emission, but also indirectly affected CH4 emission by affecting the
relative abundance of pmoA and mcrA genes. BD and fungi indirectly
affected CH4 emission by affecting pmoA and mcrA genes, respectively.
Adding biochar and straw could promote CH4 absorption by reducing
BD and increasing soil pH and the relative abundance of pmoA.
4. Discussion
4.1. Effects of straw addition and biochar incorporation on N2O emissions
N2O is an intermediate product of the nitrification and denitrifica­
tion processes. Various factors control the release of N2O from soil,
including an appropriate soil redox status and soil temperature as well as
N substrates and C sources for nitrifying and denitrifying bacteria (Lin
et al., 2017). In this study, N2O emission peaks occurred after fertiliza­
tion and continuous heavy rainfall (from August 1 to August 10) in all
treatments, and the largest peak appeared after the application of base
fertilizer (Fig. 2). A possible reason for these patterns is that an increase
T. He et al.
Fig. 5. Structural equation model of the effects of soil properties and CH4-related genes on CH4 emissions from tobacco fields. The blue and red arrows indicate
positive and negative effects, respectively, and the adjacent number is the standardized path coefficient. *** P < 0.001, ** P < 0.01, * P < 0.05.
in N substrates and C sources after fertilization and the strong fluctua­
tions in the soil redox status caused by frequent drying and wetting al­
ternations in August favored both denitrifiers and nitrifiers, thus,
promoting N2O emission (Laville et al., 2011). In this study, the single
straw amendment significantly increased N2O emissions during the
growth season (Fig. 2), which was inconsistent with Yao et al. (2009).
The inconsistent results may be due to the various C/N ratios of the
straw (Toma and Hatano, 2007). Incorporating straw with a higher C/N
ratio (> 40) can enhance the microbial N fixation capacity, thereby
reducing the N utilization by nitrification and denitrification (Toma and
Hatano, 2007; Rizhiya et al., 2011). In contrast, lower straw C/N ratios
would provide nitrifiers and denitrifiers with higher amounts of avail­
able N, contributing to increased N2O emissions (Baruah et al., 2016). In
this study, significantly higher N2O emissions in the single-straw
amendment were observed as well as higher numbers of nitrifying and
denitrifying bacteria (Fig. 1). The wheat straw C/N ratio of about 30
used in this experiment can well explain the significant increase in N2O
emissions after straw amendment. In addition, the decomposition of
straw could increase the contents of NH+ 4 -N and NO3 -N in soils (Kissel
−
et al., 1988; Lu et al., 2012), which is in line with the results of our study
(Table 2). The structural equation showed that the main factors affecting
N2O emissions were amoA-AOA and nirK genes, as well as soil NH+ 4 -N
and pH (Fig. 4). Moreover, NH+ 4 -N significantly affected the expression
of amoA-AOA, with a path coefficient of 0.87. Straw addition signifi­
cantly increased the level of NH+ 4 -N, thus resulting in increased N2O
emissions.
Compared with the control, adding biochar significantly reduced the
cumulative emissions of N2O from the soil, with a decrease of approxi­
mately 30% (Fig. 2). Similarly, some previous studies also observed
significant reductions in soil N2O emissions after biochar application
(Liu et al., 2012; Hu et al., 2014; Zhang et al., 2017; Huang et al., 2019),
while others reported a significant increase in N2O emissions after bio­
char inputs (Yoo et al., 2016; Liu et al., 2019). The different effects on
N2O emissions may be related to the effect of biochar on soil nitrification
and denitrification. In this experiment, biochar significantly changed the
relative expressions of N2O-related genes by changing soil physical and
chemical properties (Table 2 and Fig. 3). Biochar can reduce N2O pro­
duction by adsorbing NH+ 4 -N (Case et al., 2012; He et al., 2016). This
study found that biochar treatment significantly reduced the content of
NH+ 4 -N, and through correlation analysis, it was found that there was a
highly significant positive correlation between NH+ 4 -N and the amoA-
AOA gene (Fig. 4). Moreover, the contribution of AOA to N2O emissions
reached 0.76 (Fig. 4). Biochar can decrease the relative expression of the
amoA-AOA gene by reducing the level of NH+ 4 -N; thus, reducing N2O
emissions during nitrification. Studies have shown that the abundance
and activity of the nirS, nirK, and nosZ genes are the main factors
affecting N2O production and consumption in denitrification (Thomson
8
Applied Soil Ecology 166 (2021) 103996
et al., 2012). Our data indicate that the biochar amendment significantly
reduced nirS and nirK gene abundances (Fig. 3), and that the nirK gene
significantly affects N2O emissions (Fig. 4). This is consistent with the
results of Wang et al. (2013), potentially because the biochar amend­
ment increased the soil organic C content and C/N ratio. A high soil C/N
ratio promotes N assimilation; thus, more N was consumed by nitrifiers
and denitrifiers (Huang et al., 2004). Li et al. (2016) also found that a
reduction in N2O emissions was due to an increase in the nosZ gene
following biochar amendment. In contrast, the current results show that
the nosZ gene was significantly reduced. This may be because biochar
reduced the expressions of the nirS and nirK genes. It is well known that
the reduction of NO−2 to NO is the rate-limiting step in denitrification,
which is mediated by the nitrite reductase genes nirS and nirK (Braker
et al., 2000). The addition of biochar reduced the conversion of NO−2 to
NO; therefore, no overexpression of the nosZ gene is required to reduce
N2O emissions.
4.2. Effect of straw addition and biochar incorporation on CH4 emissions
The single-biochar amendment significantly decreased the CH4
emissions during the whole growing season in this study, compared with
the control treatment (Fig. 2). Although some studies reported that
biochar application increased CH4 emissions (Wang et al., 2017), most
previous studies have shown that biochar amendment can effectively
reduce CH4 emissions (Karhu et al., 2011; Feng et al., 2012; Chen et al.,
2018). The reduction in CH4 emissions in the above-mentioned studies
was due to an improvement in soil aeration under biochar amendment,
thus, increasing CH4 oxidation by promoting the growth of methano­
trophs, an aerobic proteobacterial group utilizing CH4 as the sole C
source (Van Zwieten et al., 2009; Qin et al., 2016). It was found that the
application of biochar alone significantly reduced soil BD (Table 2). The
structural equation showed that there was a significant negative corre­
lation between BD and the pmoA gene, and the pmoA gene had a sig­
nificant negative effect on CH4 emissions, with a contribution of 31%.
This suggests that biochar addition can reduce CH4 emissions, poten­
tially due to the reduction in soil BD and the increase in the relative
abundance of methanotrophs. In addition, biochar can absorb CH4,
given its large surface area (Schimmelpfennig and Glaser, 2012). The
high surface area of biochar might be another factor leading to lower
CH4 emissions. At the same time, biochar incorporation with straw
significantly decreased the CH4 emissions in this study, which is in line
with Wang et al. (2018), probably due to a significant pH increase.
CH4 emissions have been found to be negatively correlated with
pmoA gene copies and positively correlated with mcrA gene copies (Wu
et al., 2018). Additionally, Hu et al. (2019) attributed a significant in­
crease in CH4 emissions to an increase in mcrA gene abundance, which is
inconsistent with the results of our study. This study found that straw
T. He et al.
addition increased the relative abundance of the mcrA gene, but also
increased the relative abundance of the pmoA gene, and the contribution
of the pmoA gene to CH4 emissions (0.31) was greater than that of the
mcrA gene (0.26), resulting in methane absorption (Fig. 5). Meanwhile,
compared with the biochar treatment, the straw treatment absorbed less
CH4. This may due to the decomposition of straw consuming oxygen,
providing anaerobic conditions and energy for the survival of metha­
nogens (Wang et al., 2018). Ma et al. (2007) attributed the increase in
CH4 emissions to the anaerobic decomposition of straw, which provided
additional substrates for methanogens. Our study also found that the
relative abundance of the mcrA gene in the straw treatment increased
significantly compared with the biochar incorporation treatments
(Fig. 3). Therefore, although the addition of straw also increased the
uptake of CH4 in upland soils, this uptake was far less than that in the
treatment with biochar alone, so that the methane absorption potential
in the straw treatment was lowest.
5. Conclusion
This three-year field experiment demonstrated that the biochar and
straw amendment could significantly increase the uptake of CH4 in soil,
while the absorption effect of the biochar amendment was most signif­
icant. This result could be explained by changes in straw- and biochar-
supplied C and N substrates and soil conditions, and changes in the
relative abundance of the mcrA and pmoA genes related to CH4 pro­
duction and consumption could well explain the changes in CH4 emis­
sions in all treatments. The single-straw and straw combined with
biochar treatments caused a significant increase in N2O emissions, while
the single-biochar amendment reduced N2O emissions from the soil.
This is closely related to the adsorption of NH+
4 -N, the retention of C, and
the inhibition of key gene expressions during nitrification and denitri­
fication after biochar addition. Straw increased N2O emissions by
increasing the number of nitrifying and denitrifying bacteria and pro­
moting the relative abundance of amoA-AOA genes. Our long-term
experiment showed that biochar plays a significant role in reducing
the risk of CH4 and N2O emissions in tobacco field.
Declaration of competing 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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