Article 1

Effect of Multi-Walled Carbon Nanotubes on the Carbon and 2

Nitrogen Cycling Processes in Saline Soil 3

Yutian Zuo 1,2, Chenchen Wei 3, Yue Hu 4, Wenzhi Zeng 1,5, Chang Ao 1* and Jiesheng Huang 1* 4

1 StateKey Laboratory of Water Resources Engineering and Management, Wuhan University, Wuhan, Hubei, 5
430072, China 6
2 Nanjing Hydraulic Research Institute, Nanjing, 210029, China 7
3Agricultural Water Conservancy Department，Changjiang River Scientific Research Institute, Wuhan 430010, 8
China 9
4Central-southern Safety＆environment Technology Institute Co.，LTD，Wuhan 430072, China. 10
5 College of Agricultural Science and Engineering, Hohai University, Nanjing, 210098, China 11
* Correspondence: [email protected] (C. Ao) [email protected] (J. Huang) 12

Abstract: Soil salinization is a pressing issue that needs to be addressed in current agricultural pro- 13
duction. In this study, we utilized the novel material, unfunctionalized multi-walled carbon nano- 14
tubes (MWCNT) and functionalized multi-walled carbon nanotubes (MWCNT-OH), to explored the 15
effects of soil carbon and nitrogen cycles in saline soil. We set up four treatments, which were ex- 16
posed to two exposure doses of 1g/kg and 1µ g/kg, and two MWCNT types of functionalized 17
MWCNT-OH and unfunctionalized MWCNT. Our results demonstrate that： Exposure of saline 18
soil to 1g/kg functionalized MWCNT-OH significantly increased the soil inorganic nitrogen (p﹤ 19
0.05), while also promoting the soil microbial biomass. It can potentially enhance greenhouse gas 20
emissions from saline soil. Moreover, exposure to MWCNTs significantly increased the proportion 21
of Actinobacteria and Proteobacteria, two dominant phyla (p﹤0.05), which in turn improved their 22
contribution to the carbon and nitrogen cycling processes within saline soil. High exposure dose 23
treatments (1 g/kg) significantly increased the abundance of functional genes associated with carbon 24
metabolism, carbon fixation, methane metabolism, and nitrogen cycling processes within saline soil. 25
In contrast, low exposure dose treatments (1 µ g/kg) had no significant effect on the abundance of 26
functional genes related to nitrogen cycling, but significantly increased the abundance of special 27
functional genes related to carbon cycling. Redundancy analysis revealed that the microbial com- 28
munity composition within saline soil was significantly impacted by the soil total carbon, total ni- 29
trogen, and nitrate nitrogen content. Furthermore, it was observed that over 80% of the carbon and 30
nitrogen cycling processes within the saline soil were contributed by the dominant phyla. In sum- 31
mary, our research confirm the potential applicability of MWCNTs within saline soil. Notably, ex- 32
posure of saline soil to 1g/kg functionalized MWCNT-OH exhibited the most significant promoting 33
effect on the carbon and nitrogen cycles. 34

Keywords: Functionalized multi-walled carbon nanotubes; Saline soil; Carbon and nitrogen cycling; 35
Metagenomics; Microbial community 36
37

1. Introduction 38
Soil salinization has become one of the main factors limiting agricultural productivity 39
and farmland use efficiency [1]. Currently, there are approximately 11 million square kil- 40
ometers of saline soil worldwide, and its total area is still expanding at a rate of 10% an- 41
nually [2]. Soil salinization is caused not only by climatic factors such as drought and in- 42
tense evaporation but also by human factors such as improper cultivation practices, lead- 43
ing to secondary soil salinization. Soil salinization suppresses normal crop growth, re- 44
duces crop yields, changes the structure and function of cell membranes, and ultimately 45

Agronomy 2023, 13, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/agronomy

Agronomy 2023, 13, x FOR PEER REVIEW 2 of 18

decreases soil microbial activity [3]. Furthermore, the decreased microbial activity within 46
saline soil inhibits the carbon and nitrogen cycling processes, which can lead to the accu- 47
mulation of salts such as nitrates and nitrites, exacerbating soil salinization. Current meth- 48
ods to address soil salinization, including irrigation and drainage, improving crop salt 49
tolerance, and applying chemical agents such as gypsum and calcium sulfite to reduce 50
soil salinity. While these methods can effectively improve the environment of saline soil 51
and promote nutrient cycles in farmland, they still have limitations such as high costs, low 52
efficiency, and potential damage to local ecosystems. Therefore, there is a pressing need 53
to identify efficient, cost-effective, and sustainable agricultural development approaches, 54
improving the living environment for soil microorganisms, and promoting carbon and 55
nitrogen cycles to alleviate the pressure of soil salinization in farmland. 56
In recent years, multi-walled carbon nanotubes (MWCNTs) have emerged as an effi- 57
cient and novel material with enormous development potential in many fields, owing to 58
their unique mechanical properties and thermal and chemical stability [4]. Prior research 59
has demonstrated the beneficial effects of MWCNTs on saline farmland. Specifically, ac- 60
cording to Carmen Martinez-Ballesta et al. [5], MWCNTs can enter plant cells, enhance 61
water absorption, mitigate salt stress-induced damage, and ultimately promote cauli- 62
flower growth in saline soil. Li et al. [6] have discovered that MWCNTs can enhance the 63
salt tolerance of grape seeds and seedlings. This novel approach not only significantly 64
increases the root length and seed germination rate of crops, but also maintains the anti- 65
oxidant capacity of grape seedlings under high salt stress. Chen and Wang [7] suggested 66
that MWCNTs can promote the growth of alfalfa in saline-alkaline soil while regulating 67
crop physiological characteristics such as photosynthesis and antioxidant systems. Ah- 68
madian et al. [8] have found that applying MWCNTs in agriculture has a more beneficial 69
effect than other plant growth regulators. MWCNTs not only directly change crop physi- 70
ological characteristics, but also alter microbial activity, thereby influencing the elemental 71
cycling process in soil systems. The effects of MWCNTs extend beyond immediate 72
changes to crop physiological characteristics, as it also alters microbial activity, thereby 73
influencing the elemental cycling process in soil systems. Soil carbon and nitrogen cycles 74
are crucial components of the global biogeochemical cycle, with vast implications for land 75
use and ecological balance on a worldwide scale. Therefore, a comprehensive understand- 76
ing of agricultural soil carbon and nitrogen cycling processes is of utmost importance for 77
improving crop productivity, increasing agricultural yields, and promoting sustainable 78
land management practices. Sekhon [9] indicates that compared to traditional fertilizers, 79
carbon nanotubes can enhance soil nutrients, promote soil carbon and nitrogen cycling 80
processes, and prevent nutrient loss caused by soil eutrophication or runoff. Furthermore, 81
alterations to soil carbon and nitrogen cycling processes have the potential to impact the 82
soil of greenhouse gas emissions, including carbon dioxide (CO 2), nitrous oxide (N2O), 83
and methane (CH4) [10]. However, the use of MWCNTs in agricultural production often 84
faces some challenges. Some researches have shown that exposure to single-walled carbon 85
nanotubes negatively affects soil nutrient cycling by affecting soil microbial communities 86
[11,12]. Wang et al. [13] indicated that the exposure of MWCNTs exposure doses of 1 or 87
10 mg/kg for 100 days led to a significant reduction in soil microbial diversity, altered soil 88
microbial community composition and metabolic pathways, and caused a decrease in spe- 89
cific microbial groups and functional genes. Ultimately, these changes significantly im- 90
pacted soil carbon and nitrogen cycling processes. Thus, the investigation of nutrient cy- 91
cling in soil systems is inextricably linked to the exploration of soil microbial communities. 92
The effects of carbon nanotube exposure on soil carbon and nitrogen cycling processes are 93
still a matter of debate. Specifically, future researches are required to evaluate the effect 94
of carbon nanotubes on carbon and nitrogen cycling processes in saline farmland, where 95
the soil environment is more intricate. 96
Furthermore, the effectiveness of carbon nanotubes is contingent upon multiple fac- 97
tors, including material type, exposure dose and time, functionalization type, and more 98
[14]. Previous research finds that the application of unfunctionalized carbon nanotubes in 99
Agronomy 2023, 13, x FOR PEER REVIEW 3 of 18

agricultural farmland results in problems such as poor mixing with soil, as well as poten- 100
tial toxicity to crops and microorganisms, due to the hydrophobicity and unsatisfactory 101
biocompatibility [15].Thus, several studies have focused on the development of function- 102
alized carbon nanotubes to enhance their positive impact on crops and agricultural soil, 103
while minimizing their potential hazards [16,17]. Kerfahi et al. [18] shows that functional- 104
ized carbon nanotubes have a milder impact on soil microbial community structure com- 105
pared to unfunctionalized ones, with a smaller effect on microbial community diversity 106
as well. Su et al. [19] indicates that functionalized carbon nanotubes possess a larger num- 107
ber of hydrophilic groups on their surface, making them more easily utilized by microor- 108
ganisms via a lipid-assisted mechanism. Additionally, they exhibit improved dispersibil- 109
ity in water, which facilitates their application in agriculture. However, some researchers 110
have suggested that functionalized carbon nanomaterials, despite having stronger bio- 111
compatibility compared to unfunctionalized carbon nanomaterials, exhibit high toxicity 112
towards soil microorganisms at relatively low exposure levels, leading to a reduction in 113
soil microbial biomass [12]. Despite the potential benefits of functionalized MWCNTs in 114
agricultural soil, research on their application in saline farmland still needs to be com- 115
pleted. Given the potential negative impacts of MWCNTs on soil health, it is crucial to 116
explore the disparities in the efficacy of functionalized and unfunctionalized MWCNTs in 117
saline soil, with the aim of utilizing nanotechnology more effectively to mitigate salinity 118
stress in agricultural production. 119
Nanotechnology has emerged as a promising solution in agriculture, providing an 120
environmentally sustainable approach to preserving and managing soil and water re- 121
sources and simultaneously elevating the quality and quantity of global grain production. 122
Based on the research background, the objective of our study is to shed light on the effects 123
of distinct types and exposure doses of MWCNTs on the carbon and nitrogen cycling pro- 124
cess in saline soils. To accomplish this aim, we carried out experiments into the carbon- 125
nitrogen cycling process of saline soil that were subjected to two exposure doses (1 g/kg 126
and 1 µ g/kg) of both functionalized MWCNT-OH and unfunctionalized MWCNTs. The 127
objective of the study is (1) to investigate the suitability of MWCNTs in saline soils, (2) to 128
determine whether functionalized MWCNT-OH exhibit greater enhancement effects on 129
the carbon-nitrogen cycling process in saline soil than unfunctionalized MWCNTs, (3) to 130
investigate the effects of multi-walled carbon nanotube exposure on the structure and 131
function of microbial communities in saline soils by metagenomic. The study carries sig- 132
nificant scientific implications for enhancing soil quality, boosting crop productivity, and 133
promoting sustainable agricultural development in saline farmland. It provides a robust 134
theoretical basis for the future large-scale application of MWCNTs in saline farmland. 135

2. Materials and Methods 136

2.1. Test Materials 137
Experimental soil was collected from the autonomous county of Yanqi Hui, Bayingol 138
Mongol Autonomous Prefecture, Xinjiang, China, at a geographical location of approxi- 139
mately 41°45′N, 85°44′E. The experimental area experiences a climate that results in seri- 140
ous soil salinization. Soil samples were collected at the sampling site using a stratified 141
multi-point sampling method, and the undisturbed soil samples were placed in sealed 142
bags and stored for later use. Soil particle size characteristics were measured by a particle 143
size analyzer (S3500, Microtrac Inc., USA). The soil texture was determined to be sandy 144
loam, comprising 52.23% sand, 39.31% silt, and 8.46% clay. A conductivity meter (HQ40d, 145
HACH Co., USA) was used to measure the conductivity of the 1:5 soil-water extract was 146
841μS/cm, and the pH value was 8.02. 147
The selected unfunctionalized MWCNT was purchased from the Chengdu Organic 148
Chemical Co., Ltd., Chinese Academy of Sciences (http://www.cocc.cn/). The purity was 149
> 98%, the outer diameter was 5-15 nm, the inner diameter was 2-5 nm, the length was 0.5- 150
2 µ m, and the surface area was > 350 m2/g. And Functionalized MWCNT-OH purity was 151
Agronomy 2023, 13, x FOR PEER REVIEW 4 of 18

> 98%; the outer diameter was 5-15 nm, the inner diameter was 2-5 nm, the length was 0.5- 152
2 µ m, the surface area was > 380 m2/g, and the -OH content was 5.58%. Functionalization 153
treatment significantly increased the material’s surface area and -OH content. To prepare 154
a uniform suspension, both MWCNTs were soaked in sterile deionized water at 38℃ for 155
12 hours, then subjected to 15 hours of ultrasonic treatment in a high-frequency 40 kHz, 156
100W ultrasonic water bath. The resulting suspensions were mixed evenly with soil by 157
spraying, and the soil was adjusted to a uniform moisture content of 20%. 158

2.2. Experimental Scheme 159

The experiment was conducted in the microbial culture box of the Irrigation and 160
Drainage Test Field at the State Key Laboratory of Water Resources and Hydropower En- 161
gineering Science at Wuhan University. Prior to the experiment, the soil was air-dried, 162
crushed, and sieved through a 2 mm screen mesh. The soil samples were then layered and 163
compacted using the layering method based on a bulk density of 1.4 g/cm3. The soil was 164
then filled into 650 mL culture bottles with a filling volume of 200 mL. The filled soil col- 165
umns were placed in a constant temperature chamber at 25 ℃ and 50% humidity for 7 166
days to recover the soil condition for pre-incubation. During the experiment, the filled soil 167
columns were incubated for 56 days under the same constant temperature and humidity 168
conditions as during the pre-incubation. The samples were exposed to natural sunlight 169
for 12 hours and kept in darkness for 12 hours to simulate day-night variations. The cul- 170
ture bottles were weighed every 24 hours, and the soil moisture content was adjusted to 171
ensure that the moisture content was consistent across all treatments during the experi- 172
ment. Four treatments were set up in the experiment: MW1 treatment (soil exposed to 173
1g/kg of unfunctionalized MWCNTs), MW2 treatment (soil exposed to 1mg/kg of unfunc- 174
tionalized MWCNTs), HMW1 treatment (soil exposed to 1g/kg of functionalized 175
MWCNT-OH), and HMW2 treatment (soil exposed to 1mg/kg of functionalized MWCNT- 176
OH). In addition to the four treatments, a control group was set up and labeled as CK 177
treatment. Each treatment was replicated six times, resulting in a total of 30 culture bottles. 178

2.3. Research Methods 179

Soil samples were collected after 7 and 56 days of incubation, and soil characteristics 180
related to the carbon-nitrogen cycling process were measured. Soil organic carbon content 181
was measured using the K2Cr2O7 oxidation-spectrophotometric method (UV8000, Metash 182
Instruments Co, China). Soil microbial biomass carbon and nitrogen content were deter- 183
mined using the chloroform fumigation-K2SO4 extraction method (multi N/C 3100, Ana- 184
lytik Jena AG, Germany) [20]. Soil nitrate and ammonium content were measured using 185
an Ultraviolet-visible Spectrophotometer (UV8000, Metash Instruments Co, China). Soil 186
nutrient cycling indices for each treatment were calculated using the method proposed by 187
Delgado-Baquerizo et al. [21] to evaluate the soil nutrient cycling capacity. In addition, on 188
days 7, 9, 11, 14, 17, 21, 28, 35, 42, 49, and 56 of the experiment, gas-tight caps with sam- 189
pling ports were used to seal the culture bottles. At 0 and 45 minutes after sealing, 20 mL 190
of gas was extracted from the headspace of the bottles using a syringe with a rotating 191
three-way valve. The extracted gas was used to measure soil greenhouse gas flux and 192
calculate cumulative emissions. The collected gas samples were immediately analyzed for 193
greenhouse gas concentrations using a gas chromatograph (GC2010, Shimadzu Corpora- 194
tion, Japan) equipped with a thermal conductivity detector (TCD) and an electron capture 195
detector (ECD). Soil greenhouse gas emission flux, cumulative greenhouse gas emissions, 196
and the greenhouse gas emission potential index were calculated according to the meth- 197
ods described in Cai et al. [22]. In addition, the microbial metabolic quotient qCO2, devel- 198
oped by Anderson and Domsch [23], was also calculated to represent soil microbial effi- 199
ciency. A high qCO2 value indicates low microbial efficiency and can be used as an indi- 200
cator of microbial stress. 201

2.4. Metagenomic Analysis 202

Agronomy 2023, 13, x FOR PEER REVIEW 5 of 18

The differences in soil microorganisms among all treatments were analyzed by met- 203
agenomic sequencing and were detected immediately after rapid freezing of liquid nitro- 204
gen during sampling. Microbial community DNA was extracted from the soil (0.5 g). After 205
microbial community DNA extraction, the extracted genomic DNA was detected by 1% 206
agarose gel electrophoresis. High quality DNA was sheared into 400-bp fragments using 207
an ultrasonicator (M220, Covaris, America), and metagenomic shotgun sequencing pro- 208
ceeded on the NovaSeq Reagent Kits. Fifteen soil samples of DNA were analyzed in trip- 209
licate by whole-genome shotgun sequencing. The original sequencing data were obtained 210
using Fastp (https://github.com/OpenGene/fastp) and MEGAHIT 211
(https://github.com/voutcn/megahit) for quality control and assembly. The open reading 212
frames of spliced contigs were predicted using Prodigal (https://github.com/hy- 213
attpd/Prodigal), and a nonredundant gene catalog was constructed using CD-HIT 214
(http://weizhongli-lab.org/cd-hit/). Non-redundant gene sets were compared with the 215
Non-Redundant Protein Sequence (NR) Database 216
(https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/) using DIAMOND (http://ab.inf.uni- 217
tuebingen.de/software/diamond/). The non-redundant gene set sequences were com- 218
pared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.ge- 219
nome.jp/kegg), and the abundance of functional classes was calculated based on the sum 220
of gene abundance corresponding to Kegg Orthology (KO), Pathway, Read numbers, and 221
Module. 222

2.5. Data Analysis 223

All data processing, analysis, and plotting were performed using R. One-way 224
ANOVA was used to test for significant differences among the treatments, and the least 225
significant difference (LSD) test was used to compare the differences between different 226
treatments. The mean values and standard errors of all parameters were calculated from 227
at least three replicates. Bias-corrected 95% confidence interval were non-overlapping, 228
and p<0.05 were considered statistically significant. 229

3. Research Results 230

3.1. Soil Characteristics and Greenhouse Gas Emissions Related to Carbon and Nitrogen Cycles 231
in Saline Soil 232
Based on the data presented in Table 1, both short-term and long-term exposure to 233
MWCNTs significantly affect the characteristics of saline soil and greenhouse gas emis- 234
sions related to the carbon and nitrogen cycle in saline soil. 235

Table 1. Changes of soil characteristics and cumulative greenhouse gas emissions related to carbon 236
and nitrogen cycles in saline soil. 237

NO3- NH4+ CH4

Treatm TOC MBC Total MBN N 2O CO2 Exposure
Total C (mg/kg (mg/kg (kg/ha
ents (g/kg) (mg/kg) N (mg/kg) (kg/ha) (kg/ha) time
) ) )

4.64±0. 14.88±0 10.62±1. 23.71± 0.74±0.0 58.02±0 1.01±0.2

CK - - -
53a .28c 23b 0.15b 4a .32ab 2a

3.75±0. 26.69±1 20.56±1. 29.39± 2.36±0.9 56.47±1 1.00±0.0

HMW1 - - -
28a .59a 37a 0.60a 8a .23ab 7a
7day
3.76±0. 19.70±1 16.21±3. 23.75± 1.50±0.7 60.76±3 1.10±0.0
HMW2 - - -
20a .39b 72ab 0.78b 9a .80a 5a

5.37±1. 20.41±0 17.57±2. 24.44± 2.83±1.7 53.16±1 1.26±0.1

MW1 - - -
20a .76b 71ab 0.40b 1a .40b 6a
Agronomy 2023, 13, x FOR PEER REVIEW 6 of 18

3.85±0. 18.43±1 14.62±3. 23.26± 1.10±0.6 61.17±2 1.35±0.1

MW2 - - -
24a .40bc 45ab 0.36b 8a .32a 6a

5.08±0. 13.22±0 12.52±0. 25.03± 0.81±0.4 72.47±4 0.32±0.0 0.58±0. 757.94±27 1.18±0.
CK
27a .25b 48b 0.97b 2b .53b 5b 02a .82b 26ab

3.68±0. 17.20±0 15.67±0. 27.95± 2.60±0.4 86.73±3 0.48±0.0 0.56±0. 841.21±48 1.08±0.
HMW1
27b .23a 53a 0.61a 3a .72a 5a 01a .83a 08b

3.36±0. 16.15±0 12.08±2. 27.85± 1.39±0.7 90.33±3 0.53±0.0 0.56±0. 735.33±17 1.09±0.
HMW2 56day
33b .58b 46b 0.32a 6b .86a 7a 01a 6.75ab 21b

4.29±0. 15.88±0 15.33±1. 28.72± 2.15±0.9 87.25±0 0.55±0.0 0.58±0. 726.06±13 0.63±0.
MW1
41ab .55b 84ab 0.38a 9ab .96a 3a 00a 0.19ab 35b

3.49±0. 15.73±1 12.28±3. 28.75± 1.69±0.4 81.49±2 0.60±0.0 0.57±0. 716.41±73 1.65±0.
MW2
15b .01b 36ab 0.82a 7b .11a 2a 01a .59b 28a
Note: Different lowercase letters represent significant differences between different treatments 238
(p<0.05). 239

After 7-day exposure to MWCNTs, the differences between the various treatments 240
and the CK treatment were relatively small, except the HMW1 treatment. Notably, the 241
HMW1, HMW2, and MW1 treatments significantly increased the total carbon content of 242
the saline soil (p<0.05). Furthermore, the HMW1 treatment greatly increased the MBC and 243
MBN content of the saline soil (p<0.05). Compared to the CK treatment, the MBC and 244
MBN content of the HMW1 treatments were increased by 93.60% and 218.92%, respec- 245
tively, with the CK treatment soil containing 10.62 mg/kg and 0.74 mg/kg of MBC and 246
MBN, respectively. Additionally, the HMW1 treatment significantly increased the total 247
nitrogen content of the saline soil (p<0.05). 248
After 56-day exposure to MWCNTs, significant changes in the saline soil character- 249
istics occurred, with increasing differences between the various treatments and the control 250
group. The exposure to MWCNTs significantly increased the total nitrogen content, ni- 251
trate nitrogen, and ammonium nitrogen in the soil and greatly promoted the nitrogen cy- 252
cling process in saline soil (p<0.05). With the increased exposure dose to MWCNTs, the 253
MBC content of the soil also significantly increased (p<0.05). The HMW1 and MW1 treat- 254
ments increased the soil MBC content compared to the HMW2 and MW2 treatments. No- 255
tably, the HMW1 treatment significantly increased the soil MBC and MBN content com- 256
pared to other treatments (p<0.05). Compared to the CK treatment, the MBC and MBN 257
content of the HMW1 treatments were increased by 25.16% and 220.99%, respectively, 258
with the CK treatment soil containing 12.52 mg/kg and 0.81 mg/kg of MBC and MBN, 259
respectively. However, the exposure to MWCNTs significantly decreased the organic car- 260
bon content of the saline soil (p<0.05). Furthermore, this research analyzed the changes of 261
soil particle characteristics and found that the exposure to MWCNTs had no significant 262
effect on the soil texture. Under conditions of high exposure doses, the proportion of clay 263
in the saline soil decreased, and overall soil particle size decreased as well. 264
The exposure to MWCNTs significantly reduced the soil MBC/MBN (p<0.05), with 265
the HMW1 treatment showing the smallest value. After 56 days of exposure to MWCNTs, 266
the MBC/MBN content of the CK treatment was 15.51, which was significantly reduced 267
by 61.19% in the HMW1 treatment as compared to the CK treatment. With the exposure 268
time increased, the MBC/MBN content of all treatments, except for the CK treatment, ex- 269
hibited a decline. This suggests that the promotion of nitrogen cycling by MWCNTs ex- 270
posure surpassed that of carbon cycling. In addition, exposure to MWCNTs significantly 271
reduced the loss rate of soil ammonium nitrogen (p<0.05) and promoted the increase of 272
nitrate nitrogen, demonstrating that exposure to MWCNTs can significantly increase the 273
soil mineralization rate (p<0.05). After 56 days of cultivation, CK treatment resulted in a 274
Agronomy 2023, 13, x FOR PEER REVIEW 7 of 18

decrease of 68.79% in soil ammonium nitrogen, while the other treatments, HMW1, 275
HMW2, MW1, and MW2, showed reductions of 52.24%, 51.71%, 56.32%, and 55.41%, re- 276
spectively. In addition, except for the CK treatment, all other treatments showed a de- 277
crease in soil organic carbon content compared to the 7-day exposure. Furthermore, it was 278
observed that higher soil microbial biomass was associated with higher soil organic car- 279
bon content. 280
As the exposure time of MWCNTs increased, the greenhouse gas emission flux from 281
saline soil gradually stabilized, and the changes in greenhouse gas emissions from each 282
treatment were small after 56 days of exposure. The exposure of MWCNTs did not have 283
a significant effect on the emission flux of N2O from saline soil but had a significant effect 284
on the emissions of CO2 and CH4. Among them, the exposure of functionalized MWCNT- 285
OH had a significantly greater effect on CO2 emissions than unfunctionalized MWCNT. 286
The HMW1 treatment significantly increased the CO2 emissions from saline soil (p<0.05), 287
and the cumulative CO2 emission flux from the CK treatment after 56 days was 757.94 288
kg/ha. Compared with the CK treatment, the HMW1 treatment increased by 10.99%. In 289
addition, the MW2 treatment significantly promoted the emission of CH 4 from saline soil 290
(p<0.05), while the other three treatments had no significant effect on CH 4 emissions. By 291
calculating the comprehensive greenhouse effect index of the soil, the HMW1 treatment 292
showed a substantial increase of 7.98% compared to the CK treatment (p<0.05). In contrast, 293
other treatments showed no significant difference compared to the CK treatment. 294

3.2. Changes in Microbial Community Structure in Saline Soil 295

The metagenomics sequencing generated a total of 246 million raw reads, with an 296
average of 49.11 million raw reads per treatment. Strict read and quality control measures 297
resulted in an average of 97.34% high-quality sequences, which were subsequently used 298
for more accurate downstream analyses. 299
The microbial community structure in saline soil was mainly annotated by compar- 300
ing against the NR database, and the soil samples included 5 domains, 12 kingdoms, 233 301
phyla, 440 classes, 847 orders, 1577 families, 4887 genera, and 29909 species. Figure 1(a) 302
displays the microbial community composition at the phylum level. Among them, the CK 303
treatment phylum level microbial community composition includes Actinobacteria 29%, 304
Proteobacteria 28%, Chloroflexi 10%, Gemmatimonadetes 9.6%, Acidobacteria 9.4%, and Others 305
14%. HMW1 treatment significantly increased the proportion of Actinobacteria and Pro- 306
teobacteria (p<0.05), especially Proteobacteria, which significantly increased by 11.14% 307
compared to CK treatment. The MW1 treatment significantly increased the proportion of 308
Actinobacteria (p<0.05) but had no significant impact on the proportion of other species 309
and had a smaller impact on the microbial community composition than the HMW1 treat- 310
ment. However, the effects of low exposure dose, including MW2 and HMW2 treatment, 311
on soil microbial community composition were insignificant. We further analyzed the top 312
30 relative abundances of microbial genera in different treatments, created a heatmap, and 313
conducted cluster analysis between different treatments (Figure 1(b)). Among them, the 314
exposure of MWCNTs significantly increased the proportion of Methylibium (p<0.05), and 315
compared to CK treatment, HMW1 treatment significantly increased by 343.78%. Accord- 316
ing to cluster analysis, the exposure dose of MWCNTs is the main factor affecting the soil 317
microbial community composition. HMW1 treatment and MW1 treatment are divided 318
into one group, while MW2 treatment and HMW2 treatment are divided into one group. 319
Furthermore, the dissimilarity in soil microbial community composition between the CK 320
treatment and the low exposure dose treatments is comparatively smaller than that ob- 321
served between the high exposure dose treatments. In addition, we analyzed the effects 322
of exposure to MWCNTs on microbial α-diversity in saline soil (Figure 1 (c)). Except for 323
the HMW1 treatment, there was no significant difference in soil microbial diversity be- 324
tween the CK treatment and the other three treatments. Among them, HMW1 treatment 325
significantly reduced the Simpson index (the smaller the Simpson index, the greater the 326
microbial diversity) and increased the soil Shannon index and Chao index (p<0.05). The 327
Agronomy 2023, 13, x FOR PEER REVIEW 8 of 18

Simpson index of CK treatment was 0.026, which decreased by 6.62% compared to CK 328
treatment. HMW1 treatment significantly increased soil microbial community diversity 329
and richness. Finally, we investigated the differences between soil microbial communities 330
(Figure 1 (d)). The dissimilarity in soil microbial community follows a similar pattern to 331
the changes in community composition mentioned above, indicating that MWCNTs ex- 332
posure dose significantly impacts soil microbial diversity. Under low exposure doses, the 333
soil microbial community composition in the HMW2 and MW2 treatments showed little 334
difference compared to the CK treatment. There were no significant differences between 335
the two treatments. However, under high exposure doses, the soil microbial community 336
composition in the HMW1 and MW1 treatments showed significant differences compared 337
to the CK treatment. There were also noticeable differences between the two treatments. 338

(a) (b)

(c) (d)

339
Figure 1. Effects of MWCNTs exposure on the microbial community structure in saline soil. (a) Mi- 340
crobial community composition at the phylum level. (b) Microbial community composition at the 341
genus level (color depth represents the relative abundance of genera). (c) Microbial α-diversity 342
(Simpson index, Shannon index, Chao index). (d) Microbial β-diversity. (Purple arrow indicates in- 343
creasing MWCNTs exposure dose from low to high.). 344

3.3. Changes of Microbial Carbon and Nitrogen Cycles Function in Saline Soil 345
3.3.1. Changes of Functional Genes Related to the Carbon Cycles in Saline Soil 346
Given the intricate nature of carbon cycling processes in soil systems, this research 347
delved into the modules associated with three key carbon cycling processes, including 348
Agronomy 2023, 13, x FOR PEER REVIEW 9 of 18

carbon metabolism, carbon fixation, and methane metabolism in saline-alkaline soil, 349
based on the KEGG database. 350
From Figure 2 (a), the exposure of MWCNTs significantly affects the carbon metabo- 351
lism process in saline soil, and 14 related modules were significantly affected by the ex- 352
posure of MWCNTs. Overall, the exposure of MWCNTs significantly increased the num- 353
ber of functional genes related to carbon metabolism in saline soil, including M00001 (Gly- 354
colysis, glucose => pyruvate), M00002 (Glycolysis, core module involving three-carbon 355
compounds), M00003 (Gluconeogenesis, oxaloacetate => fructose-6P), M00004 (Pentose 356
phosphate pathway (Pentose phosphate cycle)), M00009 (Citrate cycle (TCA cycle, Krebs 357
cycle)), M00010 (Citrate cycle, first carbon oxidation, oxaloacetate => 2-oxoglutarate), 358
M00011 (Citrate cycle, second carbon oxidation, 2-oxoglutarate => oxaloacetate), M00012 359
(Glyoxylate cycle), M00307 (Pyruvate oxidation, pyruvate => acetyl-CoA), M00373 360
(Ethylmalonyl pathway), M00532 (Photorespiration), M00620 (Incomplete reductive cit- 361
rate cycle, acetyl-CoA => oxoglutarate), M00740 (Methylaspartate cycle), M00741 (Propa- 362
noyl-CoA metabolism, propionyl-CoA => succinyl-CoA) (p<0.05). The effectiveness of 363
HMW1 treatment is the most significant, and the high exposure dose of MW1 treatment 364
has a more robust promoting effectiveness than the low exposure dose of HMW2 treat- 365
ment and MW2 treatment. Therefore, exposure dose is one of the main factors affecting 366
the abundance of soil microbial carbon metabolism function genes. Specifically, the expo- 367
sure of MWCNTs promotes Glycolysis, Pentose phosphate cycle, Citrate cycle, Glyoxylate 368
cycle, Pyruvate oxidation, Incomplete reductive citrate cycle, Propanoyl-CoA metabolism 369
to promote carbon metabolism in saline soil. From Figure 2 (b), it can be seen that exposure 370
to MWCNTs significantly affects the carbon fixation process in saline soil, and six modules 371
were significantly affected by the MWCNTs. Among them, HMW1 treatment significantly 372
promoted the carbon fixation process of saline soil, including M00173 (Reductive citrate 373
cycle (Arnon-Buchanan cycle)), M00167 (Reductive pentose phosphate cycle, glyceralde- 374
hyde-3P => ribulose-5P), M00165 (Reductive pentose phosphate cycle (Calvin cycle)), 375
M00376 (3-Hydroxypropionate bi-cycle), M00375 (Hydroxypropionate-hydroxybutylate 376
cycle), M00374 (Dicarboxylate-hydroxybutyrate cycle) (p<0.05). The other three treat- 377
ments only significantly promoted the processes of M00173 and M00374 in saline soil 378
(p<0.05) and had no significant impact on the carbon fixation process in most soil systems. 379
Figure 2 (c) shows that exposure to MWCNTs significantly affects the methane metabo- 380
lism process in saline soil, and six modules were significantly affected by the MWCNTs. 381
The exposure of MWCNTs significantly promoted the M00346 and M00357 processes in 382
saline soil (p<0.05). In addition, high exposure dose treatments (HMW1 and MW1 treat- 383
ments) also significantly promoted saline soil M00563 (Methanogenesis, methylamine/di- 384
methylamine/trimethylamine => methane) and M00567 (Methanogenesis, CO2 => me- 385
thane) processes (p<0.05). 386
Agronomy 2023, 13, x FOR PEER REVIEW 10 of 18

387
Figure 2. Effects of MWCNTs exposure on carbon cycles-related functional genes in saline soil (as- 388
terisk indicates that there is a significant difference between this treatment and CK treatment, 389
p<0.05). 390

3.3.2. Changes of Functional Genes Related to the Nitrogen Cycle in Saline Soil 391
The nitrogen cycle in the soil system is an integral part of the global biogeochemistry 392
cycle. It can be seen from Figure 3 that exposure to MWCNTs significantly affects the ni- 393
trogen cycle process of saline soil. 394
Agronomy 2023, 13, x FOR PEER REVIEW 11 of 18

395
Figure 3. Effects of MWCNTs exposure on the nitrogen cycle-related functional genes in saline soil 396
(asterisk indicates that there is a significant difference between this treatment and CK treatment, 397
p<0.05). Notes: the digits in the figures show the relative content changes of nitrogen cycle functional 398
genes in MWCNTs exposure treatment and CK treatment, the arrows in different colors indicate the 399
process of the soil nitrogen cycle, and the asterisk indicates that the process in this treatment is 400
significantly different from that in CK treatment, p<0.05. 401

In general, the high exposure dose treatments (HMW1 treatment and MW1 treat- 402
ment) significantly promoted the nitrogen cycle process of saline soil, while the low expo- 403
sure dose treatment (HMW2 treatment and MW2 treatment) had no significant effect. 404
Moreover, the improvement effects of different types of MWCNTs were also significantly 405
different, and the promotion effect of HMW1 treatment on the nitrogen cycle of saline soil 406
was more substantial than that of MW1 treatment. HMW1 treatment significantly pro- 407
moted the transformation of NO3- to NO2- during denitrification, nitrogen fixation, NH4+ 408
to NH2OH, and NO2- to NO3- during nitrification in saline soil, and significantly promoted 409
the mutual transformation of organic nitrogen and ammonium nitrogen in the soil 410
(p<0.05). In addition, MW1 treatment also considerably affected the nitrogen cycle process 411
of saline soil, significantly promoted the transformation of NO 2- to NO3- during nitrifica- 412
tion, NO3- to NO2- during assistant nitrate reduction, and NO3- to NO2- during soil denitri- 413
fication, and also significantly promoted the mutual transformation of organic nitrogen 414
and ammonium nitrogen in the soil (p<0.05). However, the effect of MW1 treatment on 415
the nitrogen cycle of saline soil was less than that of HMW1 treatment. 416

3.3.3. Responses of Biotic and Abiotic Factors to Carbon and Nitrogen Cycles in Saline 417
Soil 418
The exposure of MWCNTs significantly affects the functional contribution of soil mi- 419
croorganisms in saline soil. Figure 4 (a) shows the functional contribution of dominant 420
phyla, including carbon metabolism, carbon fixation, methane metabolism, and nitrogen 421
Agronomy 2023, 13, x FOR PEER REVIEW 12 of 18

metabolism in saline soil. Our results show that more than 80% of the carbon and nitrogen 422
cycling processes in saline soil can be contributed by four dominant phyla, Actinobacteria, 423
Proteobacteria, Chloroflexi, and Gemmatimonadetes. Among them, Actinobacteria and Proteo- 424
bacteria contributed significantly more to the carbon and nitrogen cycles of saline soil than 425
Chloroflexi and Gemmatimonadetes. Under the exposure of MWCNTs, the proportion of Ac- 426
tinobacteria and Proteobacteria in saline soil is the highest, thereby making the most signif- 427
icant contribution to the biological function of saline soil. In addition, the exposure of 428
MWCNTs can promote the contribution of dominant phyla to the carbon and nitrogen 429
cycles in saline soil. Actinobacteria contribution to the nitrogen cycle of saline soil was sig- 430
nificantly higher than that to the carbon cycle. At the same time, Proteobacteria’s contrib- 431
utes to the carbon cycle of saline soil was considerably higher than that of the nitrogen 432
cycle. HMW1 treatment significantly increased the proportion of Actinobacteria and Prote- 433
obateria (Figure 1) and further promoted the carbon and nitrogen cycling processes in sa- 434
line soil. 435

436
Figure 4. (a) The functional contribution of dominant phyla (%); (b) RDA analysis of the relationship 437
between soil microbial community and environmental factors (RDA, redundancy analysis). 438

Exposure of MWCNTs significantly affects the response relationship between soil 439
microbial community and environmental factors. Figure 4 (b) shows the response rela- 440
tionship between dominant phyla and saline soil characteristics and greenhouse gas emis- 441
sions based on RDA analysis. The cumulative contributions of the first and second axes 442
reach 89.75%, so the response mechanisms of most microorganisms to environmental fac- 443
tors can be determined through RDA1 (81.96%) (p<0.05). RDA analysis showed that soil 444
total carbon (p=0.024), total nitrogen (p=0.012), and nitrate nitrogen (p=0.046) significantly 445
affected the microbial community composition in saline soil (p<0.05). There is no signifi- 446
cant response relationship between other soil characteristics and the microbial community 447
composition in saline soil, while there is a strong correlation between different saline soil 448
characteristics. Apart from the direct effects of MWCNTs in saline soil microorganisms, 449
the difference in soil environment caused by the diverse exposure doses and material 450
types of MWCNTs also indirectly influences the carbon and nitrogen cycles in saline soil 451
(p<0.05). Furthermore, a significant positive correlation exists between the exposure dose 452
of MWCNTs and the dominant phyla within saline soil (p<0.05). 453

4. Discussion 454
Both macroscopic soil characteristic indicators and microscopic soil microbial indica- 455
tors indicate that exposure to MWCNTs significantly affects the carbon and nitrogen cy- 456
cling processes in saline soil. 457
Agronomy 2023, 13, x FOR PEER REVIEW 13 of 18

4.1. Effects of MWCNTs Exposure on Carbon and Nitrogen Cycles in Saline Soil 458
The HMW1 treatment significantly increased the microbial biomass (MBC and MBN) 459
in saline soil (p<0.05), and the positive effect of the HMW1 treatment increased gradually 460
with exposure time (Table 1). Compared to the CK treatment, the HMW1 treatment 461
demonstrated a notable 9.03% increase in metagenomic read length, suggesting a substan- 462
tial enhancement in the soil microbial biomass. Furthermore, the HMW1 treatment signif- 463
icantly increased the microbial community diversity in saline soil (p<0.05). The enhance- 464
ment of soil microbial biomass and diversity often promotes soil microbial metabolism, 465
producing organic acids and changing soil aggregate structure, which can effectively mit- 466
igate soil pH and salinity [24]. The significant reduction of soil MBC/MBN by HMW1 467
treatment (p<0.05) indicates that exposure to functionalized MWCNT-OH increases bac- 468
terial community and decreases fungal community in saline soil, representing a more ro- 469
bust ability of soil to resist adverse factors [25]. The HMW1 treatment significantly en- 470
hances the total carbon and total nitrogen content in the soil by promoting the carbon 471
fixation and nitrogen fixation processes (p<0.05). This can effectively mitigate nutrient 472
leaching losses in the soil during crop growth [25]. Furthermore, the HMW1 treatment 473
significantly increases the ammonium nitrogen and nitrate nitrogen content in saline soil 474
(p<0.05), increasing soil nutrient content and reducing the reliance on traditional fertiliz- 475
ers used in saline farmland to a certain extent. In addition, the soil nutrient cycling index, 476
as calculated by Delgado-Baquerizo et al. [21], indicates that applying MWCNTs signifi- 477
cantly enhances the carbon and nitrogen cycling processes in saline soil, with the HMW1 478
treatment demonstrating the most notable improvement effect. The soil nutrient cycling 479
process of the high exposure dose treatments (HMW1 and MW1 treatments) is more ro- 480
bust than that of the low exposure dose treatments (HMW2 and MW2 treatments). In con- 481
trast, the low exposure dose has no significant effect on most of the soil carbon and nitro- 482
gen cycling processes in saline farmland. Furthermore, Dharni et al. [26] indicated that as 483
the exposure dose of carbon nanomaterials increases, inhibited soil microbial activity, re- 484
duced soil microbial diversity, and the development of some fungi and bacteria may be 485
hindered. Jin et al. [27] indicated that the exposure dose of single-walled carbon nanotubes 486
is negatively correlated with soil microbial biomass, and the exposure of MWCNTs has a 487
particular impact on the soil microbial community composition. The divergent conclu- 488
sions of these studies from the present study may be attributed to variations in the appli- 489
cation mode of carbon nanomaterials and soil environmental conditions. Moreover, dis- 490
crepancies in the definition of high and low exposure doses of carbon nanomaterials 491
across different studies may also contribute to these differences. The exposure of 492
MWCNTs also significantly affects greenhouse gas emissions in saline soil (p<0.05), with 493
CO2 emission flux often positively proportional to soil microbial activity. This finding also 494
indicates that the HMW1 treatment can effectively increase microbial activity in saline 495
soil. Moreover, the exposure of MWCNTs reduces qCO2, with the HMW1 treatment sig- 496
nificantly increasing qCO2 by 11.33% compared to the CK treatment, considerably enhanc- 497
ing the functional biological efficiency of saline soil (p<0.05). The microbial community 498
composition and functional structure drive the most critical soil element cycling pro- 499
cesses. And the utilization of metagenomic offers a powerful means of elucidating the 500
underlying mechanisms by which exposure to MWCNTs impacts carbon and nitrogen 501
cycling processes in saline soil. 502
Several studies have shown that the major phyla in the soil ecosystems have higher 503
competitive potential and growth rates and are significantly correlated with soil biological 504
function. In contrast, species with lower proportions contribute less to soil biological func- 505
tion [28]. In this study, the dominant bacterial phyla Actinobacteria and Proteobacteria in 506
saline soil also contribute the most to soil biological function (Figure 4). Therefore, the 507
HMW1 treatment in this study significantly increases the number of dominant phyla in 508
saline soil, thereby considerably enhancing the biological function of saline soil (p<0.05). 509
Moreover, from the perspective of soil microbial functional genes, the exposure of 510
MWCNTs significantly increases the abundance of functional genes related to soil carbon 511
Agronomy 2023, 13, x FOR PEER REVIEW 14 of 18

and nitrogen cycling (p<0.05), indicating the carbon and nitrogen cycling process in saline 512
soil. According to previous studies, a rich set of functional genes often leads to higher soil 513
microbial activity. However, Yang et al. [29] showed that the abundance of functional 514
genes in soil microorganisms is not significantly correlated with soil microbial activity but 515
rather represents the primary energy flow and material cycling processes in the soil eco- 516
systems. Consistent with the findings of this study, the HMW1 treatment has a rich mi- 517
crobial community structure and functional gene abundance, representing a more fre- 518
quent carbon and nitrogen cycling process within the soil system and greater exchange 519
with the external environment. Furthermore, the exposure of MWCNTs not only increases 520
soil nutrients in saline soil but also provides fresh organic matter to soil microorganisms, 521
thereby accelerating the decomposition of existing soil organic matter. This has the poten- 522
tial to reduce the consumption of traditional fertilizers used in saline farmland, further 523
mitigating the increase of soil salinity and promoting sustainable agricultural develop- 524
ment. Furthermore, studies indicated that soil microbial communities could optimize soil 525
system function by regulating their structure and functional consistency under appropri- 526
ate exposure dose to soil amendments. However, beyond a certain threshold, they exhibit 527
toxicity in certain critical carbon and nitrogen cycling processes [30,31]. This research 528
found that the toxicity threshold of MWCNTs was increased under low exposure doses 529
or in saline soil. Specifically, the highest exposure dose of 1g/kg of MWCNTs did not ex- 530
hibit toxicity in saline soil. 531

4.2. Applicability Analysis of MWCNTs in Saline Soil 532

In previous studies, MWCNTs have often exhibited toxicity in soil. However, the 533
main reasons for MWCNTs showing predominantly positive effects in saline soil in the 534
present research might be explained in the following four aspects: 535
Firstly, the electrical charge strength of the soil can affect the surface charge of 536
MWCNTs, which in turn controls the degree of cellular absorption of carbon nanotubes 537
[32]. Therefore, the high electrical charge and high pH soil conditions modulated the bio- 538
compatibility of MWCNTs, enhancing their effectiveness. Secondly, along with soil nitri- 539
fication, hydrogen ions are produced, which weaken the alkalinity of saline soil. There- 540
fore, the exposure of MWCNTs promotes the nitrification process in saline soil, thereby 541
mitigating the detrimental effects of saline soil. Thirdly, the exposure of saline-alkali soil 542
to acidic carbon nanotubes can neutralize the alkalinity of the soil, lower the soil pH, and 543
improve the soil environment. Fourthly, in saline-alkali soil environments, along with in- 544
sufficient soil aeration and other conditions, the activity of nitrobacteria is inhibited, lead- 545
ing to an increase in the accumulation of nitrate and nitrite. The accumulation of nitrate 546
and nitrite in the soil will produce toxic effects on soil microorganisms (inhibiting micro- 547
bial activity and even death), crops (burning seeds, rotten buds, rotten roots, and seedling 548
death), and even human bodies (carcinogen). Promoting the nitrogen cycle of saline soil 549
will help alleviate the toxic effects of salt accumulation in saline soil [33]. The exposure of 550
MWCNTs has also been found to mitigate the toxic effects of salt accumulation in saline 551
soil on soil microorganisms by promoting the nitrogen cycle and reducing the rate of am- 552
monium nitrogen loss. 553

4.3. Advantages of Functionalized MWCNT-OH in Saline Soil 554

The direct effects of carbon nanomaterials on soil microorganisms and crops are pri- 555
marily attributed to their inherent chemical structures and biological properties [1]. This 556
study suggests that functionalized MWCNT-OH has a more significant promoting effect 557
on the carbon and nitrogen cycling processes in saline soil than unfunctionalized 558
MWCNT. The main reasons are as follows: 559
At first, the electronegative functional groups present in functionalized MWCNT- 560
OH can provide negative charges to moderately active nutrient ions and biological mac- 561
romolecules, thereby improving soil properties and enhancing the effectiveness of soil 562
Agronomy 2023, 13, x FOR PEER REVIEW 15 of 18

nutrients [34]. Secondly, heteroaggregation may occur due to the interaction between pos- 563
itively charged sites on soil components and negatively charged nanomaterials [35]. Un- 564
der alkaline soil conditions, unfunctionalized MWCNTs with strong negative charges 565
tend to have a stronger aggregation effect with soil compared to functionalized MWCNT- 566
OH, which can weaken their bioavailability. Moreover, functionalized MWCNT-OH has 567
a higher positive charge, which can better interact with negatively charged soil microor- 568
ganisms and improve the utilization efficiency of MWCNTs [36,37]. Then, the nanoscale 569
structure and richer hydrophilic functional groups of functionalized MWCNT-OH make 570
it a better transport carrier for providing water and nutrients to soil microorganisms and 571
crop cells [38]. Next, soil nutrient availability is crucial in determining soil microbial bio- 572
mass. Under conditions of nutrient deficiency, promoting microbial nutrient retention can 573
lead to more available nutrients being stored within the microorganisms, thereby increas- 574
ing nutrient effectiveness [39,40]. Compared to unfunctionalized MWCNT, functionalized 575
MWCNT-OH has a higher surface area structure, which can retain more nutrients in con- 576
ditions of soil nutrient deficiency, alter the habitat of soil microorganisms, regulate the 577
soil microbial communities structure, and enhance the effectiveness of soil carbon and 578
nitrogen elements [37]. Furthermore, the high surface area of functionalized MWCNT-OH 579
can effectively reduce the availability of unstable organic carbon and the decomposition 580
of existing soil organic carbon by adsorbing available organic carbon, thereby further 581
strengthening the limiting effect of carbon on nitrogen denitrification. Additionally, as a 582
carbon source, MWCNTs have a high carbon-nitrogen ratio, which can further inhibit mi- 583
crobial denitrification [41]. Eventually, functionalized MWCNT-OH altered its hydropho- 584
bicity, making it more dispersible in suspension and soil and increasing its chances of 585
contact with crops and microorganisms, thereby enhancing the bioavailability of 586
MWCNTs [19]. 587
Although the above results have demonstrated the positive effects of MWCNTs ex- 588
posure on the carbon and nitrogen cycles in saline soil, there are still many issues that 589
need to be addressed: 590
Firstly, exposure to MWCNTs cause changes in soil microbial communities and 591
higher microbial diversity, which pose risks to ecological stability and weaken the soil to 592
resist adverse effects. Subsequently, the longest exposure time set in this study was 56 593
days. After 56 days of exposure, the greenhouse gas emission fluxes from each treatment 594
showed only marginal change, indicating the effects of the MWCNTs on soil microbial 595
activity and nutrient cycling had reached a relatively stable state. However, Ge et al. [42] 596
suggested that soil exposed to MWCNTs and graphene can still alter the soil microbial 597
communities structure after one year. Therefore, future research should explore the long- 598
term effects of carbon nanotubes exposure in the saline soil and select appropriate appli- 599
cation modes to the peculiarities of the local soil environment. At last, exposure to func- 600
tionalized MWCNT-OH (HMW1 treatment) significantly increased the microbial biomass 601
in saline soil and greenhouse gas emissions (p<0.05), exacerbating global greenhouse ef- 602
fects. Nevertheless, this should not be considered a limitation of carbon nanotube materi- 603
als since nearly all fertilizers utilized in agricultural production may yield comparable 604
adverse consequences. Therefore, in future research, we intend to contrast the dissimilar- 605
ities between the impacts of MWCNT and conventional fertilizers on greenhouse gas 606
emissions within saline soil. 607
Based on this study, the exposure to MWCNTs positively impacts the carbon and 608
nitrogen cycling processes in saline soil. MWCNTs can improve the quality and produc- 609
tivity of saline farmland. Moreover, functionalized MWCNT-OH is more suitable for sa- 610
line soil as it has a more pronounced promoting effect on the carbon and nitrogen cycling 611
processes in saline soil than unfunctionalized MWCNT (p<0.05). This article highlights 612
the potential of MWCNTs as a novel material for employment in saline soil. Controlling 613
the exposure time and dose is necessary for the large-scale application of MWCNTs in 614
saline farmland in the future. However, it is crucial to regulate the exposure time and dose 615
Agronomy 2023, 13, x FOR PEER REVIEW 16 of 18

as prerequisites for the widespread application of MWCNTs in saline farmland in the fu- 616
ture. 617

5. Conclusion 618
In this research, the effects of exposure to unfunctionalized and functionalized 619
MWCNTs on the carbon and nitrogen cycling processes in saline soil were investigated 620
from macroscopic and microscopic perspectives. Based on the results obtained, the fol- 621
lowing conclusions are as follows: 622
Firstly, short-term exposure of saline soil to MWCNTs, and HMW1 treatment signif- 623
icantly increased the total carbon and nitrogen content of the soil (p<0.05). In contrast, 624
other treatments did not have significant effects on the saline soil characteristics. Under 625
long-term exposure conditions, MWCNTs significantly increased saline soil's total nitro- 626
gen content, nitrate nitrogen content, and ammonium nitrogen content (p<0.05), promot- 627
ing the nitrogen cycling process in saline soil. Moreover, the HMW1 treatment signifi- 628
cantly increased the MBC and MBN in saline soil, promoting greenhouse gas emissions 629
from saline soil (p<0.05). Secondly, high exposure doses of MWCNTs significantly altered 630
the microbial community structure in saline soil. The HMW1 treatment significantly in- 631
creased the proportion of Actinobacteria and Proteobacteria (p<0.05). It increased the micro- 632
bial diversity in saline soil, while other treatments did not have a significant impact on 633
microbial diversity. The MW1 treatment significantly increased the proportion of Actino- 634
bacteria (p<0.05) while not having a significant impact on the proportion of other phyla. 635
Low exposure dose of MWCNTs did not significantly impact the microbial community 636
composition in saline soil. The exposure dose and material type of MWCNTs are im- 637
portant factors affecting the microbial community structure in saline soil. The microbial 638
community structure in saline soil is significantly influenced by the type and exposure 639
dose of MWCNTs. Then, exposure to MWCNTs significantly affects the quantity of func- 640
tional genes in saline soil. High exposure doses of MWCNTs increase the abundance of 641
functional genes related to carbon metabolism, carbon fixation, methane metabolism, and 642
nitrogen cycling processes in saline soil. The HMW1 treatment was observed to be more 643
efficacious in stimulating the aforementioned promotion effects compared to the MW1 644
treatment. However, low exposure doses of MWCNTs had no significant impact on the 645
quantity of most nitrogen cycling related functional genes in saline soil. Next, more than 646
80% of the carbon and nitrogen cycling processes are contributed by four dominant phyla, 647
including Actinobacteria, Proteobacteria, Chloroflexi, and Gemmatimonadetes in saline soil. 648
Furthermore, exposure of MWCNTs can increase the contribution of these dominant 649
phyla to the carbon and nitrogen cycling processes in saline soil. Within the dominant 650
phyla, Actinobacteria play a more prominent role in nitrogen cycling as compared to car- 651
bon cycling, whereas Proteobacteria exhibit a greater contribution to carbon cycling than 652
nitrogen cycling within saline soil. Eventually, the RDA analysis revealed that the micro- 653
bial community composition in saline soil is significantly influenced by the total carbon, 654
total nitrogen, and nitrate nitrogen content of the soil (p<0.05), while other soil character- 655
istics do not exert a significant impact. Furthermore, a significant positive correlation 656
(p<0.05) was observed between the exposure dose of MWCNTs and the proportions of 657
dominant phyla within saline soil. 658
In conclusion, applying MWCNTs in saline soil exhibits immense potential for future 659
development. Functionalized MWCNT-OH has a more pronounced promoting impact on 660
the carbon and nitrogen cycling processes in saline soil as compared to unfunctionalized 661
MWCNT. The exposure dose and material type significantly influence the effectiveness of 662
MWCNTs in saline soil, and therefore, precise control of the exposure dose of different 663
MWCNTs under varying environmental conditions is imperative. Notably, Exposure of 664
saline soil to 1g/kg functionalized MWCNT-OH exhibited the most significant promoting 665
effect on the carbon and nitrogen cycles. 666
Agronomy 2023, 13, x FOR PEER REVIEW 17 of 18

Acknowledgments: We are grateful for the financial support from the Program of the National Nat- 667
ural Science Foundation of China (NSFC) (Grant Nos. 52179039 and 52009093), the National Key 668
Research and Development Program of China (Grant No. 2021YFD1900805-03) and the Fundamen- 669
tal Research Funds for the Central Universities (Grant No. 2042023kf0158). 670

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