Journal of Cleaner Production 346 (2022) 131169

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Journal of Cleaner Production

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

A sustainable approach for removing nitrate: Studying the nitrate

transformation and metabolic potential under different carbon source
by microorganism
Yimin You a, b, c, d, Shaohua Chu a, b, c, d, Muhammad Khalid a, b, c, d, Kashif Hayat a, b, c, d,
Xijia Yang a, b, c, d, Dan Zhang a, b, c, d, *, Pei Zhou a, b, c, d, **
a
School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China
b
Key Laboratory of Urban Agriculture, Ministry of Agriculture and Rural Affairs, 800 Dongchuan Rd., Shanghai, 200240, China
c
Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China
d
Shanghai Yangtze River Delta Eco-Environmental Change and Management Observation and Research Station, Ministry of Science and Technology, Ministry of
Education, 800 Dongchuan Rd, Shanghai, 200240, China

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

Handling Editor: Panos Seferlis Excessive nitrate is a critical concern for a long-term greenhouse production system and potentially pollutes
groundwater through leaching. Bacillus megaterium NCT-2 can be used to remove excessive nitrate. Enrichments
Keywords: were made from salinized soil, with and without strain NCT-2, to examine the influence of Bacillus megaterium
Excessive nitrate NCT-2 on the nitrate metabolic potential and microbial composition under various carbon sources using
Bacillus megaterium
detection of nitrogen content, high-throughput sequencing and quantitative real-time PCR. The results demon­
Carbon sources
strated that strain NCT-2 enhanced the ability of enriched microorganisms to convert nitrate to ammonium with
Microbial community
Metabolic potential some but not all carbon sources. The microbial composition and their functional potential had a significant
Sustainable approach relationship with the strain NCT-2 and carbon sources in the concentrates. Specifically, the enrichments with
strain NCT-2 significantly increased the relative abundance of Bacillus and Pseudomonas genera, while decreased
the relative abundance of Halomonas genus compared to enrichments without NCT-2 strain. Furthermore, in
terms of carbon sources, D-cellobiose promoted the enrichment of Enterobacter genus, methyl-glucoside favored
the enrichments of Photobacterium or Serratia genera, and D-mannitol favored the enrichments of Photobacterium,
Serratia, or Halomonas genera. In summary, the application of Bacillus megaterium NCT-2 will increase the po­
tential of microorganisms to remove nitrate from the environment. Moreover, the current study demonstrates
that different carbon sources can also increase the potential for excess nitrate removal by changing the microbial
community composition.

1. Introduction reduction to ammonium (DNRA), altering excess nitrate into a

The application of organic and chemical fertilizers to maximize crop
production can result in secondary salinization of the soil and the
accumulation of nitrate salt ions (Zhang et al., 2021). Nitrate is the main
nitrogen source for plant growth, but it can contaminate soil and water.
Therefore, there is an urgent need to find approach of sustainable
remediation of excess nitrate (Ma et al., 2020). Widely bacteria can
convert nitrate into ammonia and fix nitrogen by assimilation
(López-Gutiérrez et al., 2004). In microaerobic or anaerobic microsites,
some bacteria can convert nitrate to ammonia through dissimilative
soil-useable nitrogen source (Minick et al., 2016). Thus, microbial
remediation may be an environmentally friendly and sustainable
approach.
Generally, different carbon sources influences the microbial com­
munity, also influence the metabolic function (Zhalnina et al., 2018).
Previous research showed that the composition of the provided carbon
sources determined the collection of a microbial community (Goldford
et al., 2018). Microbial communities in grassland soils had different
substrate preferences, and changes in carbon supply are the key drivers
(Waldrop and Firestone, 2004). In addition, some studies have shown

* Corresponding author. School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China.
** Corresponding author. School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China.
E-mail address: peizhousjtu@163.com (P. Zhou).

https://doi.org/10.1016/j.jclepro.2022.131169
Received 9 September 2021; Received in revised form 11 February 2022; Accepted 26 February 2022
Available online 28 February 2022
0959-6526/© 2022 Elsevier Ltd. All rights reserved.
Y. You et al.
that various carbon sources can enrich microbial subpopulations with
different carbon decomposition capabilities (Goldford et al., 2018).
Therefore, we hypothesized that some carbon sources may favor to
enrich microorganisms with nitrate metabolism.
Our previous studies had demonstrated that Bacillus megaterium NCT-
2 can effectively remediate excessive nitrate in secondary salinized soil
(Shi et al., 2014). Moreover, this strain has a genetic basis for metabo­
lizing nitrate through assimilation (Wang et al., 2020). Thus, the present
study focused on the excessive nitrate in the secondary salinized soil,
which found a sustainable approach for removing nitrate. The utiliza­
tion of carbon compounds by microbial communities was monitored in
parallel using 96-well plates, which have been frequently used as testing
the function of mixed microbial communities (Carlson et al., 2019).
These substrate arrays are often used to detect the metabolic potential of
microorganisms in the soil, water, and other environments (Medinets
et al., 2015). The current study focused on the microbial communities in
secondary salinized soil. This study used the substrate arrays as a
micro-bioreactor to analyze: the microorganisms and strain NCT-2
enriched cultures in secondary salinized soil metabolized the effi­
ciency of nitrate under various carbon sources; whether the community
composition is affected by strain NCT-2 and carbon composition; and the
functions of microbial communities enriched in different environments.
Therefore, the nitrate transformation and metabolic potential with
different carbon sources by microorganism were investigated. This
method of linked carbon sources with changes in the community
structure and functional genes of nitrate-metabolizing microorganisms.
Our goal is to extender the results to other systems and microbial
communities to understand how substrates and other factors affect
ecological functions and metabolic processes.
2. Materials and methods
2.1. Soil sample and preparation
Soil was collected by random sampling from greenhouse areas (idle
period, soil depth 0–20 cm) at six districts of Shanghai city in China. Soil
locations have shown in supplementary method 2.1. Twenty sub-
samples were randomly taken from greenhouse in idle period and
pooled together to form composite soils. Secondary salinized soils were
divided into different secondary salinization degrees by measuring ni­
trate content, and nitrate content was shown in Table 1. The nitrate
concentration of soil was extracted with 2 M KCl at a soil/extractant
ratio of 1:5 after shaking for 60 min at 250 rpm and 25 ◦ C and analyzed
on a Clever Chem ONE spectrophotometer (Alliance company, France).
Primary enriched cultures were different degrees of secondary salinized
soils.
In previous studies, Bacillus megaterium NCT-2 was screened and
stored in our laboratory. In this study, the strain was activated during
the preparation of microbial agent. The composition of inorganic salt
medium is as follows: KNO3, 1 g L− 1; KCl, 1 g L− 1; FeSO4⋅7H2O, 0.01 g
L− 1; MgSO4⋅7H2O, 0.5 g L− 1; CaCl2, 0.01 g L− 1; KH2PO4, 0.5 g L− 1;
glucose, 10 g L− 1. The pH of the medium was adjusted to 7. Thus the
strain was cultured in a medium where mineral N was supplied as KNO3.
After 24 h of cultivation at 35 ◦ C and 180 rpm, NCT-2 seed solution was
obtained. The NCT-2 strain was inoculated in different degrees of sec­
ondary salinization soils, and the viable count of NCT-2 strain in soils
reached more than 2 × 105 cells cfu g− 1. Three parallel tests were per­
formed for each treatment.
Table 1
Nitrate content of secondary salinized soils in six districts.
Distinct Chongming Qingpu
NO3¡ (mg kg− 1) 25.8 ± 2.1 160.2 ± 3.1
Data in the table are means ± standard deviation of the three samples.
Journal of Cleaner Production 346 (2022) 131169
2.2. Medium and cultivation conditions
Microbial enrichment cultures were respectively prepared by mixing
secondary salinized soil and soil+ NCT-2 strain mixture (~10 g) with
salinization chemically defined basal medium (50 mL total volume)
supplemented with 2 g L− 1 yeast extract as the primary organic carbon
sources and electron donor and 20 mM sodium nitrate as the primary
terminal electron acceptor and incubated for 48 h at 30 ◦ C. All chemicals
were from Sinopharm (Sinopharm Group, China). Basal medium was
consisting of: (NH4)2SO4 0.25 g L− 1 (1.89 mM), KH2PO4 1.0 g L− 1,
MnSO4 0.05 g L− 1, NaCl 30 g L− 1, microelement 2 mL L− 1. They were
inoculated into a medium with 20 mM Ca(NO3)2 and 30 g L− 1 NaCl to
enrich the nitrate-metabolizing microbial communities in secondary
salinized soil in high-salt environments. This experiment minimized
passaging of the enrichments in an effort to preserve as diverse a com­
munity as possible. After 10 times dilution twice, the concentrate was
stored in a fresh basic medium with nitrate but without yeast extract and
containing 25% glycerol.
2.3. Biolog plate culture
To test the impact of carbon sources on the products of the nitrate
metabolizing microorganisms, cryopreserved enrichments were recov­
ered in basal medium amended with 2 g L− 1 yeast extract and 20 mM Ca
(NO3)2. Cells from recovered enrichment cultures were pelleted at 4000
RCF. They were washed three times and then resuspended with 2 ×
concentrated basal medium lacking a carbon sources to an optical
density (OD600) of 0.09. Then, the cell suspension was transferred into
Biolog EcoPlate (Biolog, Inc., Hayward, CA), in which 31 carbon sources
and water controls were arrayed. These 31 carbon sources include six
categories: sugars, amino acids, carboxylic acids, amines, phenolic acids
and polymers. At this stage, we calculated the number of cells by plate
counting method and made sure the same number of cells are added to
each well. The inoculation was performed and incubated at 30 ◦ C and
700 rpm in an incubator. During experiment, cell growth was monitored
by optical density (OD600) using a Tecan M200 Pro microplate spec­
trophotometer (Tecan Austria GmbH, Salzburg, Austria). Finally, cul­
tures were harvested at 48 h for DNA sequencing and colorimetric assays
to test nitrogen cycle metabolic intermediates. When the cells were
harvested, the OD600 value was already constant, and most bacteria
were already in a stationary phase.
2.4. Colorimetric determined nitrogen metabolism intermediates
The procedure for preparing the reagent utilized in the colorimetric
determination is given in supplementary method 2.2. The colorimetric
method was used to determine the amounts of nitrate, nitrite, and
ammonium (Carlson et al., 2019). To measure nitrite, 2 μL of culture and
Griess reagent (20 μL) was added to assay plates. Then, it was kept at
30 ◦ C for 30 min prior to reading absorbance at 548 nm using a Tecan
M200 Pro microplate spectrophotometer (Rivett and Thomas, 2018).
For ammonium measurement, 4 μL of culture, the citrate reagent (4 μL),
salicylate/nitroprusside reagent (8 μL), and bleach reagent (4 μL) were
added to assay plates and were incubated at 30 ◦ C for 30 min. Absor­
bance was measured at 697 nm (Rivett and Thomas, 2018).
To quantify nitrates, all nitrates were first reduced to ammonium.
The nitrates was decompose to NO2− by 0.21M sulfamic acid (NH3SO3).
The samples were reduced with Devarda alloy and 0.2 M H2SO4. Plates
were sealed and incubated at 35 ◦ C until complete reduction achieved.
Jinshan Fengxian Jiading Minhang
202.9 ± 2.5 306.8 ± 3.9 384.2 ± 4.7 430.3 ± 2.8
2
Y. You et al. Journal of Cleaner Production 346 (2022) 131169

All the liquid was removed, and the absorbance was measured at 697 nm
according to the above ammonium determination procedure (Rivett and
Thomas, 2018). The concentration of nitrate, nitrite and ammonium
were calculated using the standard curve of calcium nitrate, sodium
nitrite, and ammonium chloride (Supplementary methods 2.3).
2.5. 16S rRNA sequencing and analysis
To understand the main factors affecting the microbial community,
enrichment cultures of secondary salinized soil at low, medium and high
degrees were selected according to the nitrate content (Table 1). The
secondary salinized soils of three degrees with and without strain NCT-2
were enriched in triplicate on three carbon sources and analyzed mi­
crobial community by 16S rRNA sequencing.
2.5.1. DNA extraction and PCR amplification
Microbial community genomic DNA was extracted from enrichment
samples using FastDNA® Spin Kit (MP Biomedicals, GA, U.S.) according
to manufacturer’s instructions. The quality of extracted DNA was
checked on 1% agarose gel, and DNA concentration and purity were
determined with NanoDrop 2000 UV–vis spectrophotometer (Thermo
Scientific, Wilmington, USA). The hypervariable region V3–V4 of the
bacterial 16S rRNA gene was amplified with primer pairs 338F (5′ -
ACTCCTACGGGAGGCAGCAG-3′ ) and 806R (5′ -GGAC­
TACHVGGGTWTCTAAT-3′ ) by an ABI GeneAmp® 9700 PCR thermo­
cycler (ABI, CA, USA). The PCR amplification of the 16S rRNA gene was
performed as supplementary method 2.4. The PCR product was extrac­
ted from 2% agarose gel and purified using the AxyPrep DNA Gel
Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to
manufacturer’s instructions and quantified using Quantus™ Fluorom­
eter (Promega, USA).
removed. The taxonomy of each OTU representative sequence was
analyzed by RDP Classifier version 2.2 (Wang, 2007) against the 16S
rRNA database (eg. Silva v138) using confidence threshold of 0.7.
According to the purpose of this study and the detection results of
nitrate and ammonium, three carbon sources were selected, which can
enrich the microbial communities that can efficiently metabolize nitrate
and produce ammonium, namely d-cellobiose, β- Methyl glucoside and
D-Mannitol. Under three carbon sources, the sample names of enriched
cultures in soils of different secondary salinization degrees were shown
in Table 2.
2.6. Quantitative real-time PCR (qPCR)
The FastDNA® Spin Kit (MP Biomedicals, GA, U.S.) was used to
extract DNA from enriched culture following the manufacturer’s rec­
ommendations. The nitrogen cycle genes (nasB nasD, glnA and narG)
were identified by qPCR in the StepOnePlus Real-time PCR System (Bio-
Rad, Hercules, CA, USA). Calibration curves for each gene were obtained
from serial dilutions of pMD18-T plasmid templates containing cloned
gene fragments of the respective genes. The primers and reaction con­
ditions of qPCR were shown in Table S1. Detailed experimental pro­
cedures and analysis were showed in supplementary method 2.5. The
copies of each standard plasmid were calculated according to the
equation described by Nathani et al. (2013).
The equations are as following:
6.02 × 1023 × C × 10− 9 × V
Gene ​ copy ​ number ​ (copies / μL) =
660 × L
Where C is the concentration of recombinant plasmids (ng mL− 1), V is
the total volume of target DNA (mL), L is the full length of recombinant
plasmid (bp).

2.5.2. Illumina MiSeq sequencing 2.7. Data analysis

The above purified amplicons were pooled in equimolar and paired-
end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250
platform (Illumina, San Diego,USA) according to the standard protocols
by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw
reads were deposited into the NCBI Sequence Read Archive (SRA)
database (Accession Number: PRJNA703295). The detailed processing
of sequencing data was as follows.
The raw 16S rRNA gene sequencing reads were demultiplexed,
quality-filtered by fastp version 0.20.0 (Chen et al., 2018) and merged
by FLASH version 1.2.7 (Magoč and Salzberg, 2011) with the following
criteria: (i) the 300 bp reads were truncated at any site receiving an
average quality score of <20 over a 50 bp sliding window, and the
truncated reads shorter than 50 bp were discarded, reads containing
ambiguous characters were also discarded; (ii) only overlapping se­
quences longer than 10 bp were assembled according to their over­
lapped sequence. The maximum mismatch ratio of overlap region is 0.2.
Reads that could not be assembled were discarded; (iii) Samples were
distinguished according to the barcode and primers, and the sequence
direction was adjusted, exact barcode matching, 2 nucleotide mismatch
in primer matching.
Operational taxonomic units (OTUs) with 97% similarity cutoff
(Edgar, 2013; Stackebrandt and Goebel, 1994) were clustered using
UPARSE version 7.1(3), and chimeric sequences were identified and
All experiments were performed on three parallel samples. The dif­
ference significance was analyzed according to the data of different
samples. Analyses of variance (ANOVA) and the least significant dif­
ference (LSD) test were performed to test the significance (p < 0.05) of
the differences among the samples. The relationship between carbon
sources, functional gene, index and microorganism was analyzed by
Pearson correlation coefficient. All statistical analyses were performed
using SPSS version 22.0. All results were reported as the mean ± stan­
dard deviation.
3. Results
3.1. The potential of strain NCT-2 on the remediation of excess nitrate
with different carbon sources
Overall, strain NCT-2 enhanced the ability of the microbial com­
munity to metabolize nitrate with some substrates but not all while
having little effect in others (Fig. 1 and Figs. S1–5). It was determined
that some carbon sources had a consistent effect on the ammonium
production in an enriched culture of different district (Fig. 1 and
Figs. S1–5). For example, in medium degree of secondary salinized, D-
cellobiose, β-methyl-glucoside, and D-mannitol induced

Table 2
The names of enriched cultures of soils with different secondary salinized degrees under three carbon sources.
Secondary salinized degree/Carbon Low secondary salinized degree Medium secondary salinized degree High secondary salinized degree
sources
soil soil+ NCT-2 strain soil soil+ NCT-2 strain soil soil+ NCT-2 strain
cultures cultures cultures cultures cultures cultures

D-cellobiose CM_a CM_A JS_a JS_A MH_a MH_A

β-methyl-glucoside CM_b CM_B JS_b JS_B MH_b MH_B
D-mannitol CM_c CM_C JS_c JS_C MH_b MH_C

3
Y. You et al.
Fig. 1. The enrichment culture of secondary salinized soil in Jinshan district metabolized nitrate to produce nitrite and ammonium under 31 carbon sources. Content
of ammonium (a), nitrite (b) and nitrate (c) in enrichment cultures. Blue represents the enriched culture of secondary salinized soils. Orange represents the enriched
culture of secondary salinized soil containing strain NCT-2.
microorganisms to utilize nitrate and produce more ammonium through
the intermediate metabolite nitrite (Fig. 1). Similar results were
observed in other five districts (Figs. S1–5). It was observed that organic
acids and sugars were distributed in the range of ammonium concen­
tration, and the concentrations of all carbon sources were the same
(Fig. 1 and Figs. S1–5). Therefore, the significant difference of ammo­
nium production demonstrated the selective influence of the carbon
sources on the nitrate reduction process (Fig. 1 and Figs. S1–5).
The nitrate-metabolizing ability of strain NCT-2 in different carbon
sources was clearly different (Fig. 2). The carbon sources were D-
cellobiose, β-methyl-glucoside or D-mannitol, which produced more
ammonium (Fig. 2). Under three carbon sources, the soil enriched cul­
ture containing strain NCT-2 had a stronger tendency of nitrate meta­
bolism and ammonium production than the original enriched culture
(Fig. 1 and Figs. S1–5). To better understand the process, the present
study aimed to focus on those three carbon sources which metabolized
nitrate and produced more ammonium. Meanwhile, we speculated the
difference in ammonium production between enrichment cultures
recovered on different carbon sources, which could be attributed to
differences in the composition and gene content of the nitrate-
metabolizing microbial communities selectively enriched on each car­
bon sources.
3.2. The main factors influenced the microbial composition
This study found that microbial community composition was
significantly influenced by the degree of soil secondary salinization,
carbon sources and strain NCT-2 in the enrichment cultures (Fig. 3).
Especially, the degree of soil secondary salinization was the main factor
affecting the microbial community (p < 0.01) (Fig. 3a). Moreover, in
4
Journal of Cleaner Production 346 (2022) 131169
enriched cultures of low-degree secondary salinized soil, carbon sources
(p < 0.01) had a more significant impact on the community than strain
NCT-2 (p < 0.05) (Fig. 3b). Strain NCT-2 had a greater impact on the
community than carbon sources in medium and high-degree secondary
salinized soils, but the effects of both factors were highly significant (p <
0.01) (Fig. 3c and d).
3.3. Changes in microbial community composition
To know the influence of strain NCT-2 on microbial composition and
the preferences of carbon sources enriched nitrate-metabolizing micro­
bial community in different secondary salinization levels. Primarily, the
different degrees of secondary salinization resulted in differences in the
initial soil enrichment culture’s community structure. In enriched cul­
tures of low-degree secondary salinized soil, Photobacterium was more
significantly enriched, followed by Enterobacter, Exiguobacterium and
Pseudomonas (Fig. 4). However, Enterobacter was mainly enriched, fol­
lowed by Serratia, Pseudomonas and Arachidicoccus in medium-degree
secondary salinized soil (Fig. 4). Enterobacter was mainly enriched, fol­
lowed by Halomonas, Bacillus and Pseudomonas in high-degree secondary
salinized soil (Fig. 4).
Secondly, this study investigated the impact of strain NCT-2 on
enriched microbial composition in different carbon sources. The addi­
tion of strain NCT-2 changed the relative abundance of microorganisms,
but the preference of carbon sources enriched microbial subpopulation
was altered (i.e., the dominant genera contained in the enriched culture
did not change) (Fig. 4). For example, in enrichment cultures of low-
degree secondary salinization, the addition of strain NCT-2 signifi­
cantly increased the abundance of Photobacterium, Exiguobacterium,
Glutamicibacter and Acinetobacter genera, and decreased the abundance
Y. You et al.
Fig. 2. NCT-2 strain metabolizes nitrate to produce nitrite and ammonium under different carbon source. After enrichment culture, content of ammonium (a), nitrite
(b) and nitrate (c). Blue represents the enriched culture of secondary salinized soils. Orange represents the enriched culture of secondary salinized soil remediated
with NCT-2 strain.
of Enterobacter and Microbacterium genera in D-cellobiose (p < 0.05)
(Fig. 4). In β-methyl-glucoside, this strain significantly increased the
Enterobacter genus and decreased Photobacterium and Exiguobacterium
genera (p < 0.05) (Fig. 4). In D-mannitol, the abundance of Enterobacter,
Pseudomonas and Exiguobacterium genera was significantly increased,
while the abundance of Photobacterium genus was significantly
decreased (p < 0.05) (Fig. 4). Strain NCT-2 had a more significant in­
fluence on microbial community composition in the enriched culture of
medium and high-degree secondary salinized soil (Fig. 3). In enrichment
culture of medium-degree secondary salinized soil by three carbon
sources, the addition of strain NCT-2 significantly increased the abun­
dance of Serratia and Pseudomonas genera, and decreased the abundance
of Enterobacter and Arachidicoccus genera (p < 0.05) (Fig. 4). In the high-
degree secondary salinization, the abundance of Enterobacter, Bacillus
and Pseudomonas genera was significantly increased, and the abundance
of Halomonas genera was significantly reduced (p < 0.05) (Fig. 4).
Thirdly, the carbon sources had preference for the enrichment of
microbial sub-community. In enrichment culture of low-degree sec­
ondary salinized soil, D-cellobiose was more favor to be enriched in
Enterobacter, Pseudomonas, Microbacterium and Glutamicibacter genera
(Fig. 4). β-Methyl-glucoside was similar to D-mannitol, which was
preferred to enrich Photobacterium and Exiguobacterium (Fig. 4).
Enterobacter was mainly enriched in cultures of medium-degree sec­
ondary salinization enriched with three carbon sources (Fig. 4). Then,
β-methyl-glucoside and D-mannitol were preferred to be enriched in
Serratia (Fig. 4). In enriched culture of high-degree secondary salinized
soil, D-cellobiose and β-methyl-glucoside both were favored to enrich
Enterobacter, while D-mannitol was preferred to enrich Halomonas
(Fig. 4).
5
Journal of Cleaner Production 346 (2022) 131169
3.4. The potential and preference for metabolizing nitrate
To explore the potential and preference of the microbial community
to metabolize nitrate, the key enzyme genes of the nitrate assimilation
(nitrate reductase gene nasB, nitrite reductase gene nasD and glutamine
reduction enzyme gene glnA) and DNRA (nitrate reductase gene, narG)
were examined. Overall, the addition of strain NCT-2 increased the copy
number of nitrate-metabolizing genes in microorganisms of secondary
salinized soil enriched with the three carbon sources (Fig. 5). Especially
in high-degree secondary salinization, strain NCT-2 had a more signifi­
cant influence on the copy number of key genes in assimilation (Fig. 5),
indicating that this strain could enhance the metabolic potential of ni­
trate in the microbial community. The results were consistent with the
decrease of nitrate content (Fig. 1 and Figs. S1–5).
The effect of carbon sources on nitrate-metabolizing potential of
microbial community was also analyzed. It was observed that D-cello­
biose was positively correlated with the copy number of nasD, glnA and
narG genes, and the correlation with narG gene was more significant (p
< 0.01) (Fig. 6a). D-mannitol was positively correlated with nasD gene,
and was significantly negatively correlated with narG gene (p < 0.05).
β-Methyl-glucoside was negatively correlated with nasD and glnA genes
(Fig. 6a). These results indicated that there were difference in the effect
of different carbon sources on the nitrate-metabolizing potential of mi­
crobial community.
The correlation between the microorganisms and nitrate metabolism
genes were compared to analyze the nitrate-metabolizing potential of
microbial communities. The results showed that the Bacillus was
significantly positively correlated with the nasB, nasD and glnA genes of
assimilation (p < 0.01) (Fig. 6b). Enterobacter genus was significantly
positively correlated with nasD and glnA (p < 0.05). On the other hand,
Y. You et al.
Arachidicoccus was significantly negatively correlated with the nasB
gene (p < 0.05) (Fig. 6b). Exiguobacterium, Glutamicibacter and Micro­
bacterium genera were significantly positively correlated with narG gene
(p < 0.01) (Fig. 6b).
6
Journal of Cleaner Production 346 (2022) 131169
Fig. 3. Biplot of RDA showing the difference
and relationship of microbial communities
and environmental factors among different
samples. Relationship between secondary
salinization degree (0-NO3-), carbon sources,
strain NCT-2 and microbial community in
enriched cultures of different degree sec­
ondary salinized soil (a). Relationship be­
tween carbon sources, strain NCT-2 and
microbial community of enrichment culture
in low-degree secondary salinized soil (b),
medium-degree secondary salinized soil (c)
and high-degree secondary salinized soil (d),
respectively. Arrows represent environ­
mental factors, correlation between samples
and environmental factors were reflected by
the length and the angle between arrows,
environmental factors. CM_a, CM_b and
CM_c; JS_a, JS_b and JS_c; MH_a, MH_b and
MH_c; CM_A, CM_B and CM_C; JS_A, JS_B
and JS_C; MH_A, MH_B and MH_C expressed
different secondary salinized soil (with and
without strain NCT-2) enriched in different
carbon sources, respectively. The meaning of
each treatment was showed in Table 2.
Fig. 4. Microbial community composition of sec­
ondary salinized soil enriched by D-cellobiose,
β-methyl-glucoside and D-mannitol carbon sources
(genus level). The colors difference indicated the
microorganism of different genus. CM_a, CM_b and
CM_c; JS_a, JS_b and JS_c; MH_a, MH_b and MH_c;
CM_A, CM_B and CM_C; JS_A, JS_B and JS_C; MH_A,
MH_B and MH_C expressed different secondary sali­
nized soil (with and without strain NCT-2) enriched
in different carbon sources, respectively. The mean­
ing of each treatment was showed in Table 2.
3.5. Relationship between microbial community composition and
environmental factors
The strain NCT-2 was significantly positively correlated with the
relative abundance of Bacillus and Pseudomonas genera (p < 0.05), and
negatively correlated with Halomonas genus (p < 0.01) (Fig. 7). The
residual nitrate content in the medium was significantly positively
correlated with Halomonas genus, and significantly negatively
Y. You et al.
correlated with Serratia and Pseudomonas genera (p < 0.01) (Fig. 7). The
content of nitrite produced was significantly positively correlated with
Bacillus and Pseudomonas genera, and was significantly negatively
correlated with Arachidicoccus and Microbacterium genera (p < 0.05)
(Fig. 7). Meanwhile, Bacillus and Pseudomonas genera were significantly
positively correlated with ammonium production (p < 0.01), Arach­
idicoccus genus was significantly negatively correlated with the pro­
duction of ammonium (p < 0.05) (Fig. 7).
4. Discussion
The current study found that microbial remediation enhanced
environmental conditions while also changing the microbial composi­
tion and functional potential. According to the correlation between
strain NCT-2 and microbial subpopulation, the strain had the ability to
promote nitrate metabolism and reduce the salinity in the environment.
Thus, this altered the living environment of other microorganisms,
resulting in a shift in community composition (Zhang et al., 2018). The
microbial community composition and functional activity may change
with the variation of their living environment (Chen et al., 2022). This
study analyzed the relationship between environmental factors and the
relative abundance of microorganisms. The results found that the higher
the nitrate content in the secondary salinized soil, the higher the relative
abundance of Enterobacter, Bacillus and Halomonas genera in the
enriched culture, while the lower the relative abundance of Exiguo­
bacterium, Glutamicibacter, Microbacterium, Photobacterium and Pseudo­
monas genera. These results shown that compared with other
7
Journal of Cleaner Production 346 (2022) 131169
Fig. 5. The copy number of nitrogen meta­
bolism genes in cultures of secondary sali­
nized soil enriched by three carbon sources.
(a) Nitrate reductase gene, NasB, (b) nitrite
reductase gene, NasD, (c) glutamine synthe­
tase gene, GlnA and (d) nitrate reductase
gene, NarG. CM_a, CM_b and CM_c; JS_a,
JS_b and JS_c; MH_a, MH_b and MH_c; CM_A,
CM_B and CM_C; JS_A, JS_B and JS_C; MH_A,
MH_B and MH_C expressed different sec­
ondary salinized soil (with and without
strain NCT-2) enriched in different carbon
sources, respectively. The meaning of each
treatment was showed in Table 2.
microorganisms, Enterobacter, Bacillus and Halomonas genera are more
tolerant to salt and can survive well in high salt environment. Our
findings are consistent with previous studies showing that salinity
significantly promoted the growth of Halomonas genus (Oueriaghli et al.,
2013).
In case of microbial nitrate metabolism, carbon concentration is said
to be a key control factor (Wang et al., 2019). However, the effects of
specific substrates on microbial community composition and function
are still poorly understood. In the present study, all cultures were
adjusted with high concentrations of carbon, having significant differ­
ences in nitrate metabolism and ammonium production. Compared to an
open environment like agricultural soils, carbon sources modifiers may
have a high metabolizing ability in less complex and less dispersal mi­
crobial communities, such as enrichments or industrial reactors (Kraft
et al., 2014). Therefore, specific carbon sources can enrich different
microbial communities with special functions, which leads to the dif­
ferences in nitrate metabolism (Cruz-Garcia et al., 2015).
It was hypothesized that given the preferential enrichment of mi­
crobial communities by carbon sources, various carbon sources will
selectively prefer distinct microbial subpopulation with different func­
tions during strain NCT-2 remediation. For instance, D-cellobiose
favored to enrich Enterobacter and Pseudomonas genera in the enrich­
ment of low secondary salinized soil. Enterobacter genus has been re­
ported with the potential to dissolve insoluble phosphorus and
hydrolyze organic phosphorus, which promote plant growth in the soil
(Jha et al., 2011). Pseudomonas genus can promotes plant growth and
have the capability to remediate the contaminated soils (David, 2018).
Y. You et al.
Fig. 6. Nitrate metabolic potential. In cultures of secondary salinized soil enriched by three carbon sources, the relationship between function genes of nitrate
metabolism and carbon sources (a) and the relationship between function genes of nitrate metabolism and microbial community (b). * means p < 0.05, ** means p <
0.01, *** means p < 0.001.
Fig. 7. The relationship between the microbial genus of secondary salinized
soil enriched by different carbon sources and the initial secondary salinization
degree (0-NO3-), strain NCT-2, residual nitrate (NO3− ), nitrite (NO2− ) and
ammonium production (NH4+). * means p < 0.05, ** means p < 0.01, ***
means p < 0.001.
β-Methyl-glucoside and D-mannitol prefer to enrich Photobacterium and
Exiguobacterium genera. Photobacterium genus can remediate soil pollu­
tion caused by pesticides (Ravi et al., 2015). Exiguobacterium genus has
been reported in playing an important role in dissolving phosphorus
(Collavino et al., 2010). In addition to Enterobacter genus,
8
Journal of Cleaner Production 346 (2022) 131169
β-methyl-glucoside, and D-mannitol also preferred to enrich Serratia
genus in the microbial community with medium secondary salinization.
Serratia genus dissolves inorganic phosphates and fixes nitrogen, which
is a valuable resource for plant growth (Li et al., 2008). In enriched
cultures of high secondary saline soil, D-mannitol prefers to enrich
Halomonas genus. It mainly grows in high salt environment and has the
denitrification function, which produces gas and causes loss of nitrogen
in the soil (Oueriaghli et al., 2013). The degree of secondary salinization
had a great impact on primitive microorganisms, so the preference of
microorganisms from different degrees of secondary salinized soils to
carbon sources was not completely consistent. Overall, Enterobacter
genus showed a preference for D-cellobiose in all saline environments,
and Serratia and Arachidicoccus genera showed a preference for
β-Methyl-glucoside. Microorganisms have no specificity for D-mannitol
in different environments, but they have a certain preference in specific
salinity environments. For example, Halomonas genus was preference
for D-Mannitol in high salinity environments. Our findings are consis­
tent with previous studies showing that a given carbon source will
temporarily enrich different subgroups with different functions in any
environment (Flynn et al., 2017). In summary, certain carbon sources
favored enriched microorganisms not only for enhancing nitrate meta­
bolism, but also for promoting growth. However, other carbon sources
may cause microorganisms to release more greenhouse gases. Therefore,
it is crucial to understand the preferences and functions of enriched
microorganisms under different carbon sources.
Analyzing the functional preferences of microbial communities
Y. You et al.
through functional genes and substance content may increase under­
standing of the functions and potential of microbial ecosystems (Torsvik
and Øvreås, 2002). Current findings showed that, the enriched microbial
communities can metabolize nitrate through assimilation and DNRA,
and the metabolic ability was enhanced by the addition of strain NCT-2.
According to the results of the correlation analysis between microor­
ganisms and NCT-2 strains, the addition of NCT-2 strains significantly
increased the abundance of Bacillus and Pseudomonas genera, which
were all significantly positively correlated with nitrate metabolism or
ammonium production. The reason could be that strain NCT-2 itself has
the ability to metabolize nitrate, which increases the metabolism of ni­
trate (You et al., 2021). In addition, the addition of the strain also
changed the microbial community composition, which may increase the
relative abundance of microorganisms with the ability to metabolize
nitrate. The result of the potential and preference for metabolizing ni­
trate indicated that Bacillus and Enterobacter could be related to the
assimilation, while Exiguobacterium, Glutamicibacter and Microbacterium
genera could be related to the DNRA potential. These results demon­
strated that the change in microbial composition may be the reason for
the increased conversion of nitrate to ammonium.
Microbes convert nitrate to ammonia through assimilation and
DNRA, which is considered to be a key mechanism for soil nitrogen
retention (Myrold and Posavatz, 2007). Nitrogen sequestered in mi­
crobial biomass can become available upon re-mineralization in the
subsequent growing season (Romero et al., 2015). Thus, it is important
to study the pathway of nitrate-metabolizing microbial community.
Meanwhile, this study demonstrated that the metabolic nitrate potential
of microbial communities has major implications for environmental
conservation.
To accurately predict the impact of selective factors on complex
microbial communities with diverse gene content and characteristics,
better functional annotations for genes and strains will be required.
Although the composition of the microbial community can be analyzed
by high-throughput sequencing, but laboratory simulations are also of
prime importance to understand how changing conditions influence
microbial community composition. Investigating the composition and
functions of low-complexity microbial communities through high-
throughput and qPCR will improve the ability to accurately predict
the effects of complex and variable environmental conditions on
microbial-mediated processes. Here, we understood the composition
and functional preference of microbial communities in different degrees
of secondary salinized soil, which will provide sustainable approach for
the improvement of excessive nitrate in the environment.
5. Conclusion
The present study investigated the nitrate transformation and
metabolic potential under different carbon sources by microorganism.
The results demonstrated that strain NCT-2 promoted enriched micro­
organisms to convert nitrate to ammonium in some carbon sources.
Furthermore, carbon sources clearly influenced the nitrate metabolism
potential of strain NCT-2 and secondary salinized soil concentrates. The
microbial composition and functional potential showed significant
relationship with the strain NCT-2 and carbon sources in the concen­
trates. Particularly, the strain NCT-2 significantly increased the relative
abundance of Bacillus and Pseudomonas genera, while reduced the
relative abundance of Halomonas genus. Additionally, in carbon sources,
D-cellobiose preferred to enrich Enterobacter genus, β-methyl-glucoside
favored to enrich Photobacterium or Serratia genera, and D-mannitol
preferred to enrich Photobacterium, Serratia or Halomonas genera. The
current research not only reveals the nitrate metabolic potential of mi­
crobial communities, but also provides a comprehensive understanding
of the impact of carbon composition on nitrate metabolism. Moreover, it
also provides a sustainable approach for the removal of excess nitrate.
9
Journal of Cleaner Production 346 (2022) 131169
CRediT authorship contribution statement
Yimin You: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Project administration, Resources, Soft­
ware, Validation, Visualization, Writing – original draft, Writing – re­
view & editing. Shaohua Chu: Funding acquisition, Project
administration, Resources, and, Supervision. Kashif Hayat: Revising.
Xijia Yang: Kokub. Dan Zhang: Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology,
Project administration, Supervision. Pei Zhou: Conceptualization, Data
curation, Formal analysis, Funding acquisition, Investigation, Method­
ology, Project administration, Supervision.
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.
Acknowledgements
Shanghai “Science and Technology Innovation Action Plan” Social
Development Science and Technology Project (20dz1204804); the Na­
tional Natural Science Foundation of China of China (31902105,
31702003); National Key Research and Development Program
2016YFD0800807; China Postdoctoral science Foundation
(2019M651505).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jclepro.2022.131169.
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