Chemosphere 284 (2021) 131266

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Effective immobilization of Bacillus subtilis in chitosan-sodium alginate

composite carrier for ammonia removal from anaerobically digested
swine wastewater
Junyuan Guo *, Cheng Chen, Wenjing Chen, Jianying Jiang, Bozhi Chen, Fei Zheng
College of Resources and Environment, Chengdu University of Information Technology, Chengdu, Sichuan, 610225, China

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

Handling editor: Derek Muir. To overcome the easy loss of microorganism, the mass production of sludge and the consumption of aeration
energy during biological treatment of anaerobically digested swine wastewater, this study used chitosan–sodium
Keywords: alginate composite carrier to prepare immobilized bacteria pellets. The heterotrophic bacteria tolerant to high
Immobilized bacteria pellets concentrations of ammonia nitrogen were isolated and the conditions for immobilizing bacteria were optimized.
Chitosan–sodium alginate
The performance of immobilized bacteria pellets to remove ammonia nitrogen from ADSW was determined and
Ammonia nitrogen removal
the corresponding mechanism was investigated. Results showed that the isolated bacteria were Bacillus subtilis,
Anaerobically digested swine wastewater
Response surface methodology and the optimal conditions to prepare the immobilized bacteria pellets by response surface methodology tests
were sodium alginate of 0.84% (m/V), chitosan of 0.22% (m/V), embedding time of 32 min and embedding
amount of 15% (V/V). In ADSW treatment, at pH 6, 20 g/L of the immobilized bacteria pellets removed 96.5% of
ammonia nitrogen. Both adsorption and microbial action contributed to ammonia nitrogen removal, and their
contributions were 54.3% and 42.2%, respectively. Compared with the immobilized bacteria pellets using chi­
tosan–sodium alginate as carrier, the one using mono alginate as carrier had a weaker ability to remove ammonia
nitrogen, with a removal efficiency of 67.4%. The main mechanism was the formation of polyelectrolyte
membrane by the connection between amino groups of chitosan and carboxyl groups of sodium alginate, which
stabilized the immobilized bacteria pellets and prolonged their service life. To sum up, the immobilized bacteria
pellets using chitosan–sodium alginate as an embedding agent have a promising prospect in ammonia nitrogen
removal from wastewater.

1. Introduction
With the development of intensive livestock farming in China, more
and more swine wastewater containing high concentrations of organic
matter and nutrients has been produced, which caused serious envi­
ronmental pollution and toxicity to aquatic life (Wen et al., 2017; Zheng
et al., 2016). In order to reduce the above–mentioned hazards, several
treatment technologies have been proposed, such as constructed wet­
lands and activated sludge process (Ávila et al., 2015; Ben et al., 2014;
Yu et al., 2019). However, with the growth of China’s population and
the development of urbanization, the cost and availability of land have
become factors that limit the provision of satisfactory solutions for
constructed wetlands. Therefore, biological technologies are considered
to be the main promotion technology to treat swine wastewater and
swine manure, especially anaerobic digestion that can convert organic
matter into biogas (Chen et al., 2021). After anaerobic digestion treat­
ment, a large amount of ammonia nitrogen still remains in the effluent
(called “anaerobically digested swine wastewater, ADSW”), which re­
quires further treatment before discharge (Du et al., 2015; Sui et al.,
2014). Aerobic treatment technologies such as sequencing batch reactor,
biological contact oxidation reactor and biological aerated filter are
always applied to reduce ammonia nitrogen from ADSW (Calero et al.,
2018; Sun et al., 2015). Unfortunately, due to the low C/N ratio and poor
biodegradability of ADSW, the ammonia nitrogen removal efficiency in
actual projects still cannot meet people’s expectations (Sui et al., 2014).
In addition, there are disadvantages that cannot be ignored in the
application of biotechnologies, such as generating a large amount of
sludge and consuming a large amount of aeration energy (Zheng et al.,
2016).
Because of its strong resistance to pollutant impact and easy

* Corresponding author.
E-mail address: gjy@cuit.edu.cn (J. Guo).

https://doi.org/10.1016/j.chemosphere.2021.131266
Received 24 November 2020; Received in revised form 6 May 2021; Accepted 15 June 2021
Available online 22 June 2021
0045-6535/© 2021 Elsevier Ltd. All rights reserved.
J. Guo et al.
operation, adsorption methods have been increasingly recognized and
promoted in the treatment of ammonia–polluted water. For example, the
modified zeolite could remove 73.4% of ammonia nitrogen from ADSW
(Guo et al., 2017); natural bentonite from Yemen could remove
approximately 30% of ammonia nitrogen from a 1000 mg/L–ammonia
solution (Alshameri et al., 2014). However, the potential adsorption
capacities of these adsorbents are limited due to the salinity and dirti­
ness filled in the micro–pores in their crystal framework, which is the
bottleneck for their application in actual treatment engineering
(Duangkamol et al., 2018).
In order to overcome the shortcomings of biological treatment
technologies and introduce the advantages of adsorption technology,
immobilized microorganism technology was developed. Especially, in
recent years, the immobilized microorganism technology that retains
the advantages of adsorption and microbial action attracts peoples’ in­
terests in the field of environmental pollution control. The microor­
ganisms are immobilized in a specific space or area using materialized
methods, so as to be highly enriched to meet the needs of wastewater
treatment (Liu et al., 2013). Compared with traditional biological
technologies, the advantages of immobilized microorganism technology
are efficient pollutant removal, easy solid–liquid separation and small
sludge output, which make it of extremely high application value
(Esawy and Nasser, 2015; Wu et al., 2016). So far, the immobilized
microorganism technology has been successfully applied on the labo­
ratory scale. For example, the immobilized Leucobacter sp. JW–1 was
capable to remove 99.9% of atrazine, 99.9% of ametryn and 97.8% of
propazine from wastewater (Liu et al., 2018); the immobilized Rhodo­
bacter Shaeroide S and NR–3 efficiently removed 74.2% and 58.2% of oil
from cooking oil wastewater, respectively (Takeno et al., 2005); the
strains (isolated from Zhejiang coast in China) immobilized in calcium
alginate–active carbon performed better in oil biodegrading than free
ones (Chen et al., 2017); the polycyclic aromatic hydrocarbons (PAH)–
degrading bacteria immobilized in sodium alginate reduced 72.6% of
pyrene in contaminated soil (Wang et al., 2019). The microbial cells
immobilized in the carrier greatly increase the concentration and purity
of the microorganisms in the reactor, thereby increasing the load to 3–7
times to that of the traditional activated sludge and further improving
the removal efficiency of pollutants (Esawy et al., 2015; Wu et al., 2016).
Therefore, immobilized microbial technology may be an effective
method to treat ADSW.
To date, several approaches for immobilizing microorganisms have
been proposed, including entrapment, embedding, cross–linking and
covalent bonding. Among them, embedding is the most commonly used
because of its simple operation (Paolo and Enrico, 2014). Sodium algi­
nate has become one of the most widely used embedding agents due to
its low price and high enrichment of microbial cells. However, poor
mechanical strength and short service life limit its industrial application
(Bleve et al., 2011; Dong et al., 2014). Chitosan is a natural polymer with
beneficial properties such as biocompatibility, non–toxicity, film for­
mation and adsorption, and is usually used as an adsorbent in biological
treatment (Islem and Marguerite, 2015; Randy et al., 2015). The amino
groups in the chitosan structure can combine with carboxyl groups in the
sodium alginate molecular chain to form a polyelectrolyte membrane,
thereby stabilizing the immobilized bacteria pellets and prolonging the
service life (Julie et al., 2015; Kanagaraj et al., 2015; Maria et al., 2011).
Therefore, chitosan–sodium alginate will become a promising carrier,
and microorganisms immobilized in it by embedding method will
become a potential solution for ADSW treatment, which is of academic
and practical significance.
This study established an immobilized microorganism technology
using a novel embedding agent, chitosan–sodium alginate to treat
ADSW. The main purpose is: (1) isolating heterotrophic bacteria that are
tolerant to high concentrations of ammonia nitrogen; (2) preparing
immobilized bacterial pellets and optimizing immobilization condition
by using a statistical technique, response surface methodology (RSM);
(3) investigating the removal of ammonia nitrogen by the immobilized
2
Chemosphere 284 (2021) 131266
bacteria pellets; and (4) discussing the mechanism of ammonia nitrogen
removal by the above–mentioned immobilized bacteria pellets. The
implementation of this research will help to provide a high–efficiency,
low–cost, easy–to–operate and manage technology for wastewater
treatment in pig farms, and provide a basis for the development of
immobilized microorganism technology.
2. Materials and methods
2.1. Chemical reagents and bacteria
All chemical reagents are of analytical grade and used without
further purification in this study. The basal liquid medium used in this
investigation was as follows: glucose 10 g/L, peptone 10 g/L, yeast ex­
tracts 10 g/L, KH2PO4 2 g/L, K2HPO4 4 g/L, NaCl 5 g/L, MgSO4 2 g/L,
NH4Cl 0.2–1 g/L. Agar with a concentration of 20 g/L was added for the
plate medium. The final pH of the medium was adjusted to 7 by using 1
M NaOH or HCl solution. The medium were autoclaved at 121 ◦ C for 30
min.
The bacteria in the study were isolated from biological sludge in the
aerobic tank of a wastewater treatment plant in Shuangliu city, China by
the dilute coating method. The wastewater treatment plant treating 6.5
× 104 m3 of domestic sewage every day by A2/O biotechnology. 1 mL of
the biological sludge was diluted to 106 folds with distilled water and 1
mL of the dilution was inoculated to the plate medium with 0.2 g/
L–NH4Cl. And then, the inoculated sample was cultured in a constant
temperature incubator (SPX–150B–Z, Boxun Industry and commerce
Co., Ltd. Medical Equipment) at 25 ◦ C until the microorganisms
multiply. The large and viscous colonies were streaked into 0.4 g/
L–NH4Cl plate medium and incubated under the same temperature.
Subsequently, the concentration of NH4Cl in the plate medium was
gradually increased to 0.6, 0.8 and 1.0 g/L in order, and the finally
obtained microorganisms was donated as heterotrophic bacteria that
could tolerant to high concentrations of ammonia nitrogen. The bacte­
rial colonies were round and opalescent. Surface of the bacteria was
smooth on the agar medium and under the microscope (Fig. S1). Phys­
iological and biochemical characterization indicated that the bacteria
are glycolysis positive, nitrate reduction positive, gelatin positive, cit­
rate positive and V–P negative, facultative aerobe and immobile. The
16S rDNA gene of the bacteria was sequenced and the homology with
Bacillus subtilis reached 99.6%. The phylogenetic tree constructed with
the Neighbor–Joining method was shown in Fig. S2. According to the
characteristics of the bacteria and the 16S rDNA gene, it was identified
as Bacillus subtilis.
2.2. Immobilization of the bacteria
The bacteria were immobilized according to the following steps.
Firstly, the isolated bacteria were inoculated in 100 mL of liquid me­
dium in 250 mL Erlenmeyer flask and incubated on a reciprocal shaker
(SHA–A) at 25 ◦ C and 200 r/min. After cultivation for 48 h, the bacteria
concentration in the resulting bacterial solution was counted as 1.8 ×
107 CFU/mL. The component of the liquid medium was glucose 10 g/L,
peptone 10 g/L, yeast extract 10 g/L, KH2PO4 2 g/L, K2HPO4 4 g/L, NaCl
5 g/L, MgSO4 2 g/L and NH4Cl 1 g/L. Secondly, a certain amount of
sodium alginate was dissolved in 50 mL of distilled water to obtain the
sodium alginate solution. At the same time, 100 mL of bacterial solution
was centrifuged at 3500 r/min for 5 min to obtain bacteria (0.173 g),
which was mixed with the above sodium alginate solution. The mixture
was called “microbial–sodium alginate solution”. Thirdly, a certain
amount of chitosan was dissolved in 0.6% acetic acid solution (100 mL),
and 2 g of CaCl2 was added into the chitosan–acetic acid solution and
dissolved sufficiently. The obtained solution was neutralized by 0.5 M
NaHCO3 to get an embedding solution. The immobilized bacteria pellets
were formed by mixing the microbial–sodium alginate and embedding
solutions. Finally, the obtained immobilized bacteria pellets were
J. Guo et al.
washed by physiological saline and dried to a constant weight at 25 ◦ C.
The dimension of the finally obtained immobilized bacteria pellets was
2 ± 0.5 mm. All operations were performed under sterile condition.
In the preparation of immobilized bacteria pellets, this experimental
set–up comprising preliminary (one–factor–at–a–time approach) and
optimization (Response Surface Methodology, RSM) tests. The
one–factor–at–a–time approach was used to determine the range of
conditions for the immobilization of bacteria. RSM using central com­
posite design (CCD) was used to evaluate the optimal conditions, which
could offer the influences of individual factors and their interaction.
RSM is a statistical technique used to establish multivariable equations
and evaluate the optimal values of variables (Su et al., 2017).
In preparation of the immobilized bacteria pellets by
one–factor–at–a–time approach, sodium alginate dosage, chitosan
dosage, embedding time and embedding bacteria amount were selected
as the influencing factors. The value range of these parameters were set
to 0.4%–1.6% (m/V), 0.1%–0.8% (m/V), 20–60 min and 5%–25% (V/
V). By determining the physical and chemical properties of the immo­
bilized bacterial pellets and the removal efficiency of ammonia nitrogen,
the range of conditions of the above–mentioned various factors for the
immobilization of bacteria were evaluated. Based on the results of the
preliminary tests in preparation of the immobilized bacteria pellets,
RSM was used to evaluate the optimal preparation conditions. Sodium
alginate dosage (A), chitosan dosage (B) and embedding time (C) were
setup as the effective variables, and the removal efficiency of ammonia
nitrogen was selected as the response variable. RSM experiments were
designed by Design–Expert (version 8.06) software and given in
Table S1. The response variable was fitted with a full quadratic model as
Eq. (1):
∑ ∑ ∑
Y = β0 + βi α2i + βii α2i + βij αi αj (1)
where Y is the response variable; αi and αj are coded independent vari­
ables; β0, βi, βii and βij are coefficients of intercept, linear, square and
interaction effects.
Meanwhile, the control pellets (without embedding bacteria) were
prepared under the same procedure and optimal conditions.
2.3. Ammonia nitrogen removal by the immobilized bacteria pellets
The ADSW used for the ammonia nitrogen removal tests was ob­
tained in July from a pig farm at Santai city, China. The pig farm had
10,000 pigs and produces approximately about 800 m3 of swine
wastewater every day, which mainly contained pig urine and pig house
washing water. The swine wastewater was treated with Up–flow
Anaerobic Sludge Bed (UASB) and Sequencing Batch Reactor (SBR).
After treated by UASB, the ADSW was obtained, whose properties were:
pH value of 6.8 ± 0.1, COD concentration of 1347.2 ± 0.5 mg/L,
ammonia nitrogen concentration of 685.4 ± 0.5 mg/L. The ADSW was
autoclaved at 121 ◦ C for 30 min before the ammonia nitrogen removal
experiments by the immobilized bacteria pellets. After the autoclave
treatment, pH value of the ADSW was 6.8 ± 0.1, COD concentration of
the ADSW was 1292.6 ± 0.5 mg/L, ammonia nitrogen concentration of
the ADSW was 626.2 ± 0.5 mg/L. From the changes of COD and
ammonia nitrogen concentrations before and after autoclave treatment
of the ADSW, it can be concluded that autoclave treatment had a certain
effect on COD and ammonia nitrogen concentrations. COD and ammonia
nitrogen concentrations decreased by about 4.1% and 8.6%, respec­
tively, both of which were less than 10%. In addition, ADSW would not
be sterilized in practical processing before treated by the immobilized
bacteria pellets. Therefore, for calculating the ammonia nitrogen
removal efficiency, 685.4 ± 0.5 mg/L was selected as the initial con­
centration of ammonia nitrogen.
The ammonia nitrogen removal experiments by the immobilized
bacteria pellets were carried out at room temperature (25 ± 0.5 ◦ C). The
effects of the immobilized bacteria pellets dosage, ADSW pH and
3
Chemosphere 284 (2021) 131266
reaction time on the removal of ammonia nitrogen were discussed. 1 L of
ADSW sample was poured in a beaker, whose pH was adjusted using 1 M
NaOH or HCl if necessary. The immobilized bacteria pellets were then
added and the mixture was stirred at 200 r/min for 6 days. Ammonia
nitrogen concentration was measured according to the method of
Nessler’s reagent spectrophotometry (National Standard of China, HJ
535–2009) using UV–vis spectrometer (UV–3802, China). Ammonia
nitrogen removal efficiency was calculated with the following formula:
( )
Cn
Ammonia nitrogen removal efficiency = 1 − × 100% (2)
C0
where C0 and Cn are initial and final concentrations of ammonia nitro­
gen (mg/L).
Literature reported that carrier could be used as an adsorbent to
remove pollutants in wastewater (Nadia et al., 2015). In order to
determine the contribution of microbial action and adsorption ability of
the carrier itself in the removal of ammonia nitrogen, during the 6–day
ADSW treatment, performance of the control and the immobilized
bacteria pellets under the same conditions to remove ammonia nitrogen
was discussed.
2.4. Statistical analysis
Statistical analysis was performed using Origin 8.0 and data pre­
sented as mean ± SD of three replicates. Pearson correlation analysis
was carried out to identify the relationship between the variables, and
statistical significance was determined at 95% confidence interval at p
< 0.05.
3. Results and discussion
3.1. Optimization of bacteria immobilization condition
3.1.1. Determine the range of conditions by preliminary tests
In the presence of 2.0% (m/V) of calcium chloride, 0.4%, 0.8%, 1.2%
and 1.6% (m/V) of sodium alginate were applied to immobilize the
bacteria. Table 1 depicts that when the sodium alginate dosage was
0.4% (m/V), the immobilize bacteria pellets were transparent, soft, thin,
easily broken and had a poor elasticity; when the sodium alginate
dosage was 0.8% (m/V), the immobilize bacteria pellets were slightly
loose and had good elasticity, transparency and hardness; when the
sodium alginate dosage was 1.2% and 1.6% (m/V), the immobilize
bacteria pellets were relatively compact, opaque, hard and trailed. These
differences were due to the gelatin properties of sodium alginate at
different concentration (Dong et al., 2014). Fig. 1a depicts the removal
of ammonia nitrogen from ADSW by using the immobilized bacteria
pellets prepared with different dosages of sodium alginate. Within 6
days of ADSW treatment, the immobilized bacteria pellets showed a
similar ammonia nitrogen removal trend. In the first 3 days, ammonia
nitrogen removal was relatively poor, which increased rapidly and
reached a peak in the following days. At sodium alginate dosage of 0.8%
(m/V), the immobilized bacteria pellets removed 62.8% of ammonia
nitrogen at day 4, which was higher than that by the bacteria immobi­
lized in other dosages of sodium alginate. Excessive sodium alginate
prevented the transfer of ammonia nitrogen and inhibited the growth of
bacteria, thus reducing the reactivity and the removal of ammonia ni­
trogen (Soo et al., 2017). There was a statistically significant linear
correlation between ammonia nitrogen removal efficiency and the
excessive sodium alginate dosage (>0.8%). Using day 4 as an example,
ammonia nitrogen removal efficiency = (− 3312.5× sodium alginate
dosage + 88.98)% (R2 = 0.99829, p = 0.02634 < 0.05). The insufficient
sodium alginate caused the immobilized bacteria pellets to be broken
easily, which declined the ammonia nitrogen removal as well (Orrego
et al., 2018; Dong et al., 2014). Dong et al. (2017) also reported similar
findings.
J. Guo et al. Chemosphere 284 (2021) 131266

Table 1
Properties of the immobilized bacteria pellets prepared under different sodium alginate dosages, chitosan dosages, embedding time and embedding bacteria amount.
Properties Sodium alginate dosage (%, m/V) Chitosan dosage (%, m/V) Embedding time (min) Embedding bacteria amount (%, V/V)

0.4 0.8 1.2 1.6 0.1 0.2 0.4 0.6 0.8 20 30 40 50 60 5 10 15 20 25

Tailing – – ++ + – – ++ ++ + – – – – – – – – – –
Collapse + – – – – – – – – – – – – – – – – – –
Hardness – + + + + + + + + – + + ++ ++ + + + + +
Transparency ++ + – – + ++ – – – ++ + + + + + + + – –
Sphericity + + – – + + – – – + + + + + + + + +

Note.
Tailing: -, No; +, Yes; ++, Obvious. Collapse: -, No; +, Yes. Hardness: -, Soft; +, Moderate; ++, Hard.
Transparency: -, Opaque; +, Yes, ++, Very. Sphericity: -, No; +, Yes.

Fig. 1. Effects of sodium alginate dosage (a), chitosan dosage (b), embedding time (c) and embedding bacteria amount (d) on ammonia nitrogen removal efficiency
by the immobilized bacteria pellets. (Ammonia nitrogen concentration: 685.4 ± 0.5 mg/L).

In the presence of 2.0% (m/V) of calcium chloride and 0.8% (m/V) of
sodium alginate, 0.1%, 0.2%, 0.4%, 0.6% and 0.8% (m/V) of chitosan
were applied to immobilize the bacteria. Table 1 depicts that when the
chitosan dosage was changed from low to high, except for tailing, the
immobilized bacteria pellets were not only loose, but also had good
elasticity and hardness. When the chitosan dosage exceeded 0.4% (m/
V), the immobilized bacteria pellets significantly trailing due to the high
gel viscosity at high chitosan concentration (Mini et al., 2011). Fig. 1b
depicts an obvious effect of the chitosan on ammonia nitrogen removal
by the immobilized bacteria pellets. In the process of preparing the
immobilized bacteria pellets using chitosan–sodium alginate as
embedding agent, when chitosan dosage was 0.2% (m/V), the immo­
bilized bacteria pellets removed 83.9% of ammonia nitrogen on the 4th
day. Lower or higher chitosan dosage decreased the removal efficiency
of ammonia nitrogen due to the decreasing diffusion of chitosan mole­
cules and film–forming reaction (Thai et al., 2016). As the dosage of
chitosan increasing, the driving force of the chitosan molecules deep­
ened the diffusion of the chitosan into the immobilized bacteria pellets,
which increased the formation degree and thickness of the film, thus
immobilizing more bacteria (because chitosan has a good bio­
–compatibility) and promoting the removal of ammonia nitrogen.
Meanwhile, the increase in the dosage of chitosan increased the chance
of the combination of chitosan and sodium alginate through the reaction
between amino and carboxyl groups (due to the increase of the number
of amino groups), which made the immobilized bacteria pellets more
stable and prolonged their service life. However, the highly viscosity of
the excessive chitosan was not conducive to the transfer of ammonia
nitrogen and the growth of bacteria, thus significantly reducing the
removal of ammonia nitrogen (Mitra et al., 2014; Wang et al., 2018).
The thick film formed by excessive chitosan may be another reason for
increasing the diffusion resistance of ammonia nitrogen and products
(Soon et al., 2017). There was a statistically significant linear correlation
between ammonia nitrogen removal efficiency and the excessive chi­
tosan dosage (>0.4%). Using day 4 as an example, ammonia nitrogen
removal efficiency = (− 6320.0 × chitosan dosage + 94.35)% (R2 =
0.96923, p = 0.01551 < 0.05). In addition, compared with the bacteria
immobilized in single sodium alginate, the bacteria immobilized in
chitosan–sodium alginate showed better ammonia nitrogen removal
efficiency because of the good bio–compatibility of chitosan (Duan et al.,
2015).
In the presence of 2.0% (m/V) of calcium chloride, 0.8% (m/V) of
sodium alginate and 0.2% (m/V) of chitosan, 20, 30, 40, 50 and 60 min
of the embedding time were applied to immobilize the bacteria. Table 1
depicts that when the embedding time was 20 min, the immobilized

4
J. Guo et al. Chemosphere 284 (2021) 131266

bacteria pellets were obviously transparent and soft, relatively thin and
easily broken, whereas at embedding time longer than 30 min, the Y = 91.29 + 4.46A + 4.89B + 0.053C + 4.54AB + 10.3AC − 1.9BC
immobilized bacteria pellets had better transparency and hardness. − 16.72A2 − 17.92B2 − 4.67C2 (3)
Fig. 1c depicts that the immobilized bacteria pellets removed more than
The analysis of variance (ANOVA) result in Table 2 indicates that the
75% of ammonia nitrogen on the 4th day, especially the immobilized
model was significant at 97.7% confidence level, with ‘Prob > F’ less
bacteria pellets prepared when the embedding time was 30 min, the
than 0.05 and Fstatistic greater than 2.57. The p–value for the lack of fit
removal efficiency of ammonia nitrogen reached 86.9%. Too short or too
(LOF) test less than 0.0001 indicates that there was almost no systematic
long embedding time decreased the removal efficiency of ammonia ni­
variation unaccounted in the model (only 0.01%). The determination
trogen, because the embedding time affected the mechanical strength,
coefficient R2 of 0.9768 indicates that there was a good agreement be­
the chitosan film thickness of and the embedding bacteria amount of the
tween the predicted and the experimental values. In addition, the
immobilized bacteria pellets (Zou et al., 2018). There was a statistically
neglected difference between the adjusted R2 (0.9559) and the deter­
significant linear correlation between ammonia nitrogen removal effi­
mination coefficient R2 (0.9768) indicates that almost all of the signif­
ciency and the embedding time (>30 min). Using day 4 as an example,
icant terms were included in the model. The adequate precision (AP)
ammonia nitrogen removal efficiency = (− 0.47 × embedding time +
value of 17.484 that higher than 4 indicates that the model was
101.0)% (R2 = 0.99513, p = 0.00244 < 0.05). If the embedding time was
desirable.
too short, the chitosan film was thin, the embedding amount of bacteria
Results of the significance tests in Table 3 depicts that in linear terms,
was low and the immobilized bacteria pellets were fragile, resulting in
sodium alginate and chitosan dosages were significant. Among higher
poor ammonia nitrogen removal; if the embedding time was too long,
order effects, quadratic terms of sodium alginate and chitosan dosages
the chitosan film would be aging, which decreased the ammonia nitro­
were the significant factors, demonstrating that sodium alginate and
gen removal by the immobilized bacteria pellets (Dong et al., 2014).
chitosan dosages played a decisive role in immobilizing bacteria and the
Appropriate embedding time could make sodium alginate and calcium
corresponding removal of ammonia nitrogen. Proper sodium alginate
chloride fully react, and the formed immobilized bacteria pellets with
produced proper pellets strength, and proper chitosan produced proper
good mechanical strength could embed bacteria to the greatest extent,
bio–compatibility, making bacteria to grow well (Wang et al., 2016).
thereby effectively removing ammonia nitrogen (Dong et al., 2014; Liu
Clear peaks appeared in the response plot in Fig. 2 indicate that the
et al., 2016). A similar trend was observed when the immobilized Rho­
optimal immobilization conditions were determined by sodium alginate
dococcus Shaeroide JX–2 pellets were used to remove 17β–estradiol (Liu
dosage, chitosan dosage and embedding time inside the design bound­
et al., 2016).
ary. The interactions of sodium alginate and chitosan dosages, sodium
In the presence of 2.0% (m/V) of calcium chloride, 0.8% (m/V) of
alginate dosage and embedding time were significant. Fig. 2a depicts
sodium alginate, 0.2% (m/V) of chitosan and 30 min of embedding time,
that when sodium alginate dosage was at its central value of 0.8%
5%, 10%, 15%, 20% and 25% (V/V) of embedding amount of bacteria
(m/V), the addition of chitosan improved ammonia nitrogen removal by
were applied to immobilize the bacteria. Table 1 depicts that the amount
the immobilized bacteria pellets. This finding was consistent with the
of bacteria embedded almost had no significant effect on the physical
conclusion in single–factor experiment in section 3.1.1. Notably, when
properties of the immobilized bacteria pellets. Only when the embedded
sodium alginate dosage was at a high level, the improvement of
bacteria amount was higher than 15% (V/V), the transparency of the
ammonia nitrogen removal by the increasing chitosan dosage was not
immobilized bacteria pellets was relatively worse. Fig. 1d depicts that
satisfactory, similar to the conclusion reported by Mahesh et al. (2017).
the removal efficiency of ammonia nitrogen increased from 61.2% to
The response on the sodium alginate dosage–embedding time surface in
89.4% as the embedding bacteria amount embedded increased from 5%
Fig. 2b depicts that when sodium alginate dosage was at its central value
to 15% (V/V) during the immobilization process. There was a statisti­
of 0.8% (m/V), the embedding time significantly affected the formation
cally significant linear correlation between ammonia nitrogen removal
of the immobilized bacteria pellets and the corresponding removal of
efficiency and the embedding bacteria amount (5%–15%). Using day 4
ammonia nitrogen. In addition, the center of the under–surfaces in Fig. 2
as an example, ammonia nitrogen removal efficiency = (2820.0 ×
represents the optimal conditions of the immobilized bacteria pellets
embedding bacteria amount + 46.97)% (R2 = 0.99973, p = 0.01043 <
preparation and the best ammonia nitrogen removal. Moving away from
0.05). This may be due to the increase in microbial action caused by the
these centers, a decline in ammonia nitrogen removal efficiency was
increase in the number of bacteria (Dong et al., 2014). With continuous
observed.
increasing the amount of embedded bacteria (>15%), too much bacteria
According to the target values of the response, ammonia nitrogen
occupied the pores on the surface of the immobilized bacteria pellets,
removal efficiency of 100%, the optimal bacteria immobilization con­
which increased the mass transfer resistance and ultimately decreased
ditions calculated according to the regression equations were: sodium
the ammonia nitrogen removal efficiency (Wang et al., 2014).

3.1.2. Optimize the preparation condition by RSM tests

20 experimental observations were taken at random orders to opti­ Table 3
Significance of quadratic model coefficient for the response.
mize the preparation condition by using RSM (Table S2). The experi­
mental results were analyzed through central composite design to obtain Response Independent variables Prob > F
an empirical model for the best response (Guo and Chen, 2017). The Ammonia nitrogen removal efficiency (%) A 0.00062
final quadratic equation obtained to explain the mathematical rela­ B 0.0035
tionship between the independent parameters and the dependent AB 0.0226
AC 0.0001
response of ammonia nitrogen removal efficiency (Y) was presented as A2 <0.0001
follow: B2 <0.0001

Table 2
ANOVA results for the response.
Response F–value Prob > F P–value R2 Adjusted R2 AP

Ammonia nitrogen removal efficiency (%) Model 46.73 <0.0001

Lack of fit 45.45 <0.0001 0.9768 0.9559 17.484

5
J. Guo et al.
Fig. 2. Surface graphs of ammonia nitrogen removal showing variable effect of sodium alginate dosage and the chitosan dosage (a); sodium alginate dosage and
embedding time (b). (Ammonia nitrogen concentration: 685.4 ± 0.5 mg/L).
alginate dosage 0.84% (m/V), chitosan dosage 0.22% (m/V) and
embedding time 32 min. Under this optimal condition, the immobilized
bacteria pellets removed 96.5% of ammonia nitrogen from the ADSW.
From this standpoint, sodium alginate–chitosan could be a potential
carrier for immobilizing bacteria to remove ammonia nitrogen.
3.2. Application of the immobilized bacteria pellets in ADSW treatment
The immobilized bacteria pellets obtained under the optimal con­
dition was used to remove ammonia nitrogen from ADSW. Fig. 3 depicts
that regardless of the dosage, the immobilized bacterial pellets per­
formed well in the pH range of 5–8. Dong et al. (2017) reported that the
strong acidic and strong alkaline conditions could destroy the immobi­
lized bacterial pellets, thereby decreasing the microbial action. In fact,
the large amount of H+ in a strong acidic environment affected the
electrical changes on the surface of bacteria, while nucleic acid and
protein were always denatured in a strong alkaline environment, both of
which would cause cell death. In addition, as the acidity increasing, the
–COO− groups of the immobilized bacterial pellets tended to transform
Fig. 3. Effects of immobilized bacteria pellets dosage and solution pH on
ammonia nitrogen removal. (Ammonia nitrogen concentration: 685.4 ± 0.5
mg/L).
6
Chemosphere 284 (2021) 131266
into –COOH groups. Conversely, as the alkalinity increasing, the –COOH
groups tended to ionize, which would corrode the immobilized bacterial
pellets. Therefore, an appropriate pH value could ensure that the
immobilized bacterial pellets had better microbial activity, thereby
effectively removing ammonia nitrogen. Fig. 3 also depicts the changes
of ammonia nitrogen removal efficiency under different dosages of
immobilized bacteria pellets. It clearly shows that as the dosage of
immobilized bacteria pellets increased to 20 g/L, the ammonia nitrogen
removal efficiency significantly increased, while the further increasing
immobilized bacteria pellets dosage was not helpful to improve
ammonia nitrogen removal efficiency. At pH 6, after treatment with 20
g/L pellets for 4 days, 96.5% of ammonia nitrogen was removed. There
was a statistically significant linear correlation between ammonia ni­
trogen removal efficiency and the dosage of the immobilized bacteria
pellets (5–20 g/L). Using pH 6 as an example, ammonia nitrogen
removal efficiency = (2.574 × immobilized bacteria pellets dosage +
47.45)% (R2 = 0.96849, p = 0.01588 < 0.05). As a comparison, the
ammonia nitrogen removal efficiency of the immobilized ammonia­
–oxidizing bacteria by polyvinyl alcohol and sodium alginate reached
90.3% under its optimal immobilization conditions (Dong et al., 2017);
the ammonia nitrogen removal efficiency of the immobilized anammox
bacteria by polyvinyl alcohol and sodium alginate reached approxi­
mately 70% (Tuyen et al., 2020); the highest ammonia nitrogen removal
efficiency of the immobilized nitrifying and denitrifying bacteria by
construction waste was 78.8% (Li et al., 2019). In the ammonia nitrogen
removal tests, a blank group without adding immobilized bacteria pel­
lets was also performed, under the experimental condition (200 r/min
for 4 d), the efficiency of the original microorganisms to remove
ammonia nitrogen ranged from 7.8% to 26.7%. Notably, if the sterilized
ADSW was stirred at a speed of 200 r/min for 4 d, the removal efficiency
of ammonia nitrogen was 3.8%–6.2%, which might be caused by vola­
tilization. These results indicate that the immobilized bacterial pellets
improved the removal of ammonia nitrogen by increasing the concen­
tration of microorganisms and the adsorption of the carrier itself (Esawy
et al., 2015; Liu et al., 2013; Wu et al., 2016).
The performance of the control pellets (without embedding bacteria)
to remove ammonia nitrogen was also discussed. Fig. 4 depicts that for
both of the control and the immobilized bacteria pellets, with the
extension of the treatment time, ammonia nitrogen removal efficiency
increased firstly and then reached equilibrium. At day 4, the immobi­
lized bacteria pellets removed 96.5% of ammonia nitrogen while the
control pellets removed 54.3%. This information indicates that both
adsorption and microbial action contributed to the removal of ammonia
nitrogen, with their contributions being 54.3% and 42.2%, respectively.
J. Guo et al.
Fig. 4. Comparison of the control and immobilized bacteria pellets in ammonia
nitrogen removal. (Ammonia nitrogen concentration: 685.4 ± 0.5 mg/L; the
pellets concentration: 20 g/L).
In order to investigate the superiority of chitosan, the immobilized
bacteria pellets with single alginate as embedding agent was prepared
under the optimal condition of the immobilization of bacteria, and
performances of the bacteria immobilized in chitosan–sodium alginate
and single alginate toward to ammonia nitrogen removal were
compared. Results show that under the same treatment conditions, the
removal efficiency of ammonia nitrogen by the bacteria immobilized in
single alginate was 67.4%, which was lower than that when chito­
san–sodium was used as the embedding agent, indicating that chitosan
was beneficial for the immobilized bacteria pellets to remove ammonia
nitrogen.
3.3. Mechanism of immobilized bacteria pellets to remove ammonia
nitrogen
3.3.1. FT–IR spectra analysis
Curves (a) and (b) in Fig. 5 depict the FT–IR spectra of the control
Fig. 5. FT–IR spectra of the control and the immobilized bacteria pellets before
(a, b) and after (c, d) ADSW treatment.
7
Chemosphere 284 (2021) 131266
and the immobilized bacteria pellets before ADSW treatment. The new
significant peak appeared in both curves (a) and (b) near 1020 cm− 1 was
attributed to amid bonds developed from the cross–linking reaction
between amino group of chitosan and carboxyl group of sodium alginate
(Emine and Alper, 2016). The similar patterns of peaks associated with
–OH and –COOH around 3400 and 1635 cm− 1 in both curves (a) and (b)
were favorable for ammonia adsorption. The new significant peak
appeared in curve (b) near 1411 cm− 1 was attributed to the CH3C–H
bond formed by the acetylation reaction between chitosan and amino
group of the immobilized bacteria pellets (Muhammad et al., 2017).
Curves (c) and (d) in Fig. 5 depict the FT–IR spectra of the control and
immobilized bacteria pellets after ADSW treatment. Obviously, the
characteristic peak of –COOH of the immobilized bacteria pellets was
slipped from 1635 to 1650 cm− 1, while in the control pellets was
changed to 1643 cm− 1. The characteristic peak of –OH at 3400 cm− 1 was
changed to 3448 cm− 1 for the immobilized bacteria pellets and 3440
cm− 1 for the control pellets. The characteristic peak of CH3C–H of the
immobilized bacteria pellets at 1411 cm− 1 was changed to 1419 cm− 1.
The changes of these groups from the FT–IR spectra indicate that these
groups played an important role in the absorption of ammonia nitrogen.
Compared with the FT–IR spectra before ADSW treatment, the peak
intensity of amid group increased after treatment. Meanwhile, this peak
changed from 1020 cm− 1 to 1033 cm− 1 for the control pellets and 1041
cm− 1 for the immobilized bacteria pellets, which was caused by the
reaction between ammonia nitrogen and the –COOH. In addition, in the
immobilized bacteria pellets, the change trend of bonds was more
obvious than that of the control pellets, which was attributed to the
appearance of bacteria (Muhammad et al., 2017).
3.3.2. Scanning electron microscopy (SEM) analysis
Fig. 6a depicts the internal morphology and photograph of the con­
trol pellets before ADSW treatment. The control pellets were spherical
and had good elasticity, permeability and mechanical strength. The in­
ternal shape of the control pellets showed an irregular honeycombed
and macro–porous structure, which provided a larger surface area for
bacteria to load, or ammonia nitrogen adsorption. In other words, the
diffusion of ammonia nitrogen from ADSW to the control pellets phase
and the leakage of cells from the control pellets were mainly affected by
the inter–spaces structure (Ahmad et al., 2012). Fig. 6b depicts the in­
ternal morphology and photos of the immobilized bacteria pellets before
ADSW treatment, which clearly showed that a large number of bacterial
cells adhered to the surface of the pellets. Before ADSW treatment, the
bacteria in the immobilized bacteria pellets were spherical and clustered
together in an orderly manner (Liu et al., 2013). The presence of bacteria
facilitated the removal of ammonia nitrogen. Gentili et al. (2006) found
that chitosan promoted the attachment and growth of bacteria due to
electrostatic bonding between cells and chitosan during the process of
immobilizing bacteria. This provided evidences that chitosan with high
molecular weight had low antibacterial activity and was a natural
embedding agent suitable for cell immobilization. Fig. 6c depicts that
after ADSW treatment, the inter–spaces structure of the control pellets
became tight which was due to the adhesion of ammonia nitrogen (Ni
et al., 2012). Fig. 6d depicts that after ADSW treatment, the number of
bacteria increased significantly and was widely distributed throughout
the interior of the immobilized bacteria pellets. This was due to the
growth of bacteria that used ammonia nitrogen and organics in ADSW as
nutrients (Su et al., 2017).
Overall, the results of this study show that the immobilized micro­
organism technology was favorable to treat ADSW. On the one hand,
sodium alginate led to higher mechanical strength of the immobilized
bacteria pellets, due to its high porosity and high molecular weight,
thereby decreasing the transfer resistance of ammonia nitrogen in the
immobilized bacteria phase. On the other hand, the bio–compatibility of
chitosan enabled bacteria to adapt to the environment in a relatively
short period of time and increased the density of bacteria in the pellets.
These two reasons improved the removal of ammonia nitrogen from
J. Guo et al.
Fig. 6. SEM images of the control and the immobilized bacteria pellets before (a, b) and after (c, d) ADSW treatment.
ADSW (Chen et al., 2014; Ganesh et al., 2015; Lv et al., 2013).
4. Conclusions
Biological technologies has been considered as the most critical
method for treating anaerobically digested swine wastewater (ADSW),
but the application of biotechnologies always produce a large amount of
sludge and consume a large amount of aeration energy. In addition,
because microorganisms are easily lost in the biological treatment pro­
cess, the removal efficiency of ammonia nitrogen in actual projects still
does not meet people’s expectations. Therefore, to improve ammonia
nitrogen removal, chitosan–sodium alginate was used as a carrier and
Bacillus subtilis was used as the target bacteria to prepare immobilized
bacteria pellets for ADSW treatment. Results demonstrated that the
immobilized bacteria pellets prepared under the optimal conditions of
sodium alginate of 0.84% (m/V), chitosan of 0.22% (m/V), embedding
time of 32 min and embedding amount of 15% (V/V) significantly
improved the removal efficiency of ammonia nitrogen to 96.5%. Mi­
crobial action and adsorption were the two main ways for ammonia
nitrogen removal by the immobilized bacteria pellets, accounting for
54.3% and 42.2%, respectively. The SEM image shows a relatively good
pore structure of the immobilized bacteria pellets. The FT–IR spectrum
curve confirms that the immobilized bacteria pellets have abundant
surface functional groups. All of which are beneficial to the removal of
ammonia nitrogen. Although the immobilized bacteria pellets with
chitosan–sodium alginate as a carrier has shown good performances in
the removal of ammonia nitrogen in this study, there are still some
limitations that need to be studied in the future, such as the effect of the
number of recycling times on the ammonia nitrogen removal ability of
the immobilized Bacillus subtilis pellets, and the bacterial activity, cell
density and metabolite products during ammonia nitrogen removal. The
in–depth study of these issues will help to carry out actual engineering,
evaluate reasonable cost–benefits and formulate or perfect environ­
mental control policies.
8
Chemosphere 284 (2021) 131266
Credit author statement
Junyuan Guo: Supervision; Revision. Cheng Chen: Investigation;
Writing. Wenjing Chen: Revision. Jianying Jiang: Editing; Review. Bozhi
Chen: Investigation; Revision. Fei Zheng: Editing; Review.
Declaration of competing interest
All of the authors declare that they have no conflict of interest.
Acknowledgments
The authors would like to acknowledge funding support by Program
of Chengdu Science and Technology Board (2018–YF05–00146–SN).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2021.131266.
Ethical approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
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