Bioresource Technology 364 (2022) 128036

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Mixotrophic denitrification processes in basalt fiber bio-carriers drive

effective treatment of low carbon/nitrogen lithium slurry wastewater
Huicheng Ni a, Muhammad Arslan b, Zhishui Liang c, Chencheng Wang d, Zhijun Luo d,
Junchao Qian e, Zhiren Wu d, Mohamed Gamal El-Din b, d, *
a
School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang 212003, Jiangsu Province, PR China
b
Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
c
School of Civil Engineering, Southeast University, Nanjing 211189, Jiangsu Province, PR China
d
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu Province, PR China
e
Jiangsu Key Laboratory for Environment Functional Materials, Suzhou University of Science and Technology, SuZhou 215009, Jiangsu Province, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Efficacy of R-BF was studied for the

biological treatment of lithium slurry
wastewater.
• TN removal rate was 4.462 kg/m3/d in
R-BF while qmax in Monod was 0.32284
mg-N/mgVSS/d.
• R-BF could regulate diverse nitrogen
metabolic pathways to adapt to low C/N
ratios.
• HN-ADs, AOB, NOB and DGAOs were
the main functional groups in R-BF.
• (Aerobic)chemoheterotrophy, nitrogen
respiration, and nitrate reduction were
dominant processes.

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

Keywords: Lithium battery slurry wastewater was successfully treated by using basalt fiber (BF) bio-carriers in a biological
Basalt fiber contact oxidation reactor. This resulted in a significant reduction of COD (93.3 ± 0.5 %) and total nitrogen (77.4
Bio-carrier ± 1.0 %) at 12 h of HRT and dissolved oxygen (DO) of 0–1 mg/L. The modified Stover-Kincannon model
Carbon to nitrogen ratio (C/N)
indicated that the total nitrogen removal rate was 4.462 kg/m3/d in R-BF while the substrate maximum specific
Nitrogen removal
Simultaneous nitrification and denitrification
reaction rate (qmax) in the Monod model was 0.323 mg-N/mgVSS/d. A stable internal environment was estab­
(SND) lished within the bio-nest. Metataxonomic analysis revealed the presence of denitrification and decarbonization
bacteria, combined heterotrophic nitrification-aerobic denitrification bacteria, nitrite-oxidizing bacteria, and
ammonia-oxidizing bacteria. Functional analysis displayed changes related to (aerobic)chemoheterotrophy, ni­
trogen respiration, nitrate reduction, respiration/denitrification of nitrite, and nitrate in R-BF. The study pro­
poses a novel approach to achieve denitrification for the treatment of lithium slurry wastewater at low C/N
conditions.

* Corresponding author.
E-mail address: mgamalel-din@ualberta.ca (M. Gamal El-Din).

https://doi.org/10.1016/j.biortech.2022.128036
Received 29 July 2022; Received in revised form 19 September 2022; Accepted 23 September 2022
Available online 27 September 2022
0960-8524/© 2022 Elsevier Ltd. All rights reserved.
H. Ni et al.
1. Introduction
Biological treatment of lithium slurry wastewater under stringent
operating conditions is an emerging challenge faced by environmental
scientists (Kim et al., 2021). On the other hand, lithium battery industry
is developing rapidly, which produces large volumes of lithium slurry
wastewater containing low carbon to nitrogen (C/N) ratio. Lithium
slurry wastewater typically includes NMP (N-Methyl-2-pyrrolidone),
carbon powder, high suspended solids (SS), and esters (Frolov et al.,
2012). As of today, conventional treatment strategies have been
employed to treat lithium battery wastewater such as chemical oxida­
tion decomposition, activated carbon adsorption, and reverse osmosis
(Kim et al., 2017; Qiu et al., 2019; Zheng et al., 2016). Among them,
granular activated carbon (GAC) displayed successful treatment of
esters-rich wastewater obtained from a lithium-ion battery plant. Prin­
cipally, GAC allows the adsorption of esters containing organic matter,
which are later decomposed, and finally, GAC can be regenerated
through microwave radiation (Zheng et al., 2016). Accordingly, appli­
cation of rice-like iron oxide hollow structure, which was synthesized by
hydrothermal method using silk fiber template, also displayed successful
removal of adsorbents present in lithium-oxygen battery wastewater.
Here, C/Fe3O4 hollow particles improved the kinetics of oxygen reduc­
tion and precipitation, and enhanced the performance of adsorbing
organic pollutants from wastewater (Kim et al., 2017). Similarly, reverse
osmosis (RO) and electrodialysis (ED) were integrated into a deep con­
centration process of industrial lithium-containing wastewater to extract
lithium salt from industrial lithium-containing wastewater for industrial
production (Qiu et al., 2019). These technologies are often expensive,
difficult to operate, unsustainable, and prone to secondary pollution.
Contrarily, biofilm contact methods with simultaneous nitrification and
denitrification (SND) potential could offer alternative solutions to treat
large-scale lithium slurry wastewater. The efficiency of SND reaction
depends on the co-existence of general autotrophic nitrifying bacteria (i.
e. ammonia-oxidizing bacteria, nitrite-oxidizing bacteria) and hetero­
trophic denitrifying microorganisms, which is principally driven by
dissolved oxygen gradient in a particular micro-ecosystem (Liu et al.,
2019). The phenomenon has been previously studied in oxidation
ditches, sequencing batch reactors, and sequencing batch biofilm re­
actors (Liu et al., 2019). However, nothing is known about lithium slurry
wastewater remediation except that a few studies have reported reme­
diation via microbial inoculum in traditional reactors (Yoo et al., 2010).
The use of conventional bio-carriers such as combination filler,
elastic fibers, polyurethane sponge fillers, and non-woven fabric fillers
often results in poor treatment efficiency (Zhao et al., 2019). These
materials retain a low proportion of microbial biomass which is prone to
shedding, while the system cannot endure wastewater-derived toxicity
ultimately resulting in secondary pollution (Zhang et al., 2019b).
However, the application of basalt fiber (BF) as a bio-carrier could offer
several advantages such as large surface area, high chemical stability,
corrosion resistance, and no secondary pollution. The high performance
is attributed to the three-dimensional structure known as a biological
nest (bio-nest) which establishes a dissolved oxygen gradient, i.e. oxic at
the periphery and anoxic at the core. These micro-gradient environ­
ments provide niches to unique microbial communities including SND
bacteria in addition to other biogeochemical processes (Ni et al., 2018).
Nevertheless, in the case of lithium slurry wastewater, ions generated
from lithium batteries may inhibit bacterial activity which is worthwhile
investigating in the SND context (Yoo et al., 2010). The previous studies
on R-BF have shown that BF can be used as an excellent bio-carrier to
enhance the removal of pollutants in domestic wastewater, landfill
leachate, and printing and dyeing wastewater (Ni et al., 2021a; Ni et al.,
2021b; Zhang et al., 2019a).
The components in lithium battery slurry wastewater are not
monolithic; therefore, weaker metabolic functioning and gradual accu­
mulation of pollutants could disturb the reactor’s performance and
affect pollutant removal efficiency. This study reported for the first time
2
Bioresource Technology 364 (2022) 128036
the potential of biological contact oxidation reactor containing basalt
fibers (R-BF) bio-carrier for the treatment of lithium battery slurry
wastewater in a field pilot. The results were compared with a conven­
tional activated sludge reactor (R-AS) previously established at a
regional wastewater treatment plant with the primary aim to propose an
alternative and effective solution for lithium battery slurry wastewater
treatment. For a stable R-AS operation, glucose is regularly fed in the
reactor to enhance the C/N ratio and support heterotrophic denitrifying
bacterial communities. This problem could be solved by R-BF as the
system can be operated without any additional support. Thus, the ni­
trogen removal performance was investigated and microbial growth
kinetic models were applied to compute the removal efficiency. The
spatial distribution and structure of the bacterial community were
correlated, and the metabolic functions were putatively predicted.
2. Materials and methods
2.1. Lithium battery slurry wastewater
Lithium battery slurry wastewater was obtained from a high-tech
enterprise, Jiangsu Technology Co., ltd., Jiangsu, China (E
119.629891◦ , N 32.167067◦ ) engaged in the production and application
of carbon nanotubes and graphene. The wastewater was collected from
the sections receiving slurry wastewater, workshop floor and equipment
flushing water, domestic water, initial rainwater and catalyst waste­
water, and exhaust gas treatment drainage. The main contaminants in
the raw wastewater were N-Methylpyrrolidone (NMP) and toner, which
presented poor biochemical properties (C/N:1.57). The water quality
parameters of raw wastewater were as follows: pH – 6.6–7.3,
biochemical oxygen demand (BOD5) – 12.1–126.7 mg/L, chemical ox­
ygen demand (COD) – 350–395 mg/L, ammonia–nitrogen (NH4-N+) –
162.1–178 mg/L, total nitrogen (TN) – 231–245 mg/L, total phosphorus
(TP) – 4.510.1 mg/L, and SS – <60 mg/L.
2.2. Experimental setup
The experimental setup comprised a water collection tank, a contact
oxidation reactor equipped with an OHR aerator (Original Hydrody­
namic Reaction) and air pump, and a settling tank. The effective volume
of the contact oxidation reactor was 3.6 m3 (dimensions: 2.4 m × 1.0 m
× 1.5 m) (Fig. 1). A total of 360 bundles of basalt fiber carriers were
suspended in a reactor to build R-BF, which was supplied by Jiangsu
GMV New Material Science T&D Co., ltd., China. In the BF, auxiliary
material was a titanium wire wrapped in a Teflon tube while the top and
bottom ends of the packing were fixed to prevent the oscillation of the
BF bio-carrier. The pH and temperature of R-BF were maintained at 7.6
± 0.2 and 27.5 ± 1.5 ◦ C, respectively.
Seed sludge, the inoculum source, was taken from the sludge thick­
ener of a wastewater treatment plant in Zhenjiang, Jiangsu Province,
China (E 119.520822◦ , N 32.219372◦ ). The mixed liquid suspended
solids (MLSS) concentration was recorded as 20,000 ± 50 mg/L, and the
initial dosing rate was 1/3 of the effective reactor volume (3.6 m3). The
reactor was operated for a 30-day domestication period (activated
sludge domestication) in which domestic wastewater was used in the
first 10 days and then gradually replaced with lithium battery slurry
wastewater for the next 20 days until completely replaced. At the end of
the domestication period, BF formed the bio-nest of approximately 12
cm in diameter and the ratio of mixed liquid volatile suspended solids
(MLVSS) to MLSS was 0.53. After domestication, the reactor was oper­
ated at different HRT and DO concentrations having two phases. Phase I
comprised of initial 60 days in which HRT was fixed at 12 h while the
effect of different DO (0–1, 1–2, 2–3 mg/L) was studied. Phase II lasted
another 120 days in which the effect of varying HRT (6 h, 8 h, 10 h, 12 h,
14 h, 16 h) was investigated. The reactor conditions were stable and
efficient in both phases showing no abnormalities while a maximum
total nitrogen removal rate of 4.462 kg/m3/d was recorded.
H. Ni et al. Bioresource Technology 364 (2022) 128036

For multilocation biochemical analyses, bio-nests were collected 2.4. Substrate removal/microbial growth kinetic model
from the reactor diagonally. The bio-nests were frozen and sliced into
two parts, while each part was further divided into six samples (a-f) from 2.4.1. Substrate removal kinetics
the core to the periphery (strip dimensions: 1.0 cm × 1.0 cm × 6.0 cm).
The size of each sample was 1.0 cm3 (length 1.0 cm × width 1.0 cm × a) Grau secondary substrate removal model
height 1.0 cm). The same samples from different bio-nests were pooled.
The samples were then stored at − 20 ◦ C until processed for content and Grau secondary substrate removal model was adopted to study the
composition analysis, bio-nest activity test, and microbial community reactor’s denitrification performance (Jin & Zheng, 2009). The model is
analyses (see supplementary material). During initial 60 days, pollutants shown in Eq. (1):
removal efficiency was studied at HRT of 12 h under different DO (0–1, ( )
dS Se
1–2, 2–3 mg/L). During remaining 120 days, removal efficiency was − = kX (1)
dt Si
studied under different HRT (6 h, 8 h, 10 h, 12 h, 14 h, 16 h). Finally, the
matrix removal model and matrix inhibition model in R-BF were Eq. (1) is integrated to obtain Eq. (2):
applied, and the activities at different depths in the bio-nest were
SiHRT Si
measured. = HRT + (2)
Si − Se kX
2.3. Comparison with activated sludge reactor (R-AS) In this equation, Si and Se are the TN influent and discharge con­
centrations (g/L), respectively; whereas, HRT is the hydraulic retention
The results of R-BF were compared with a reactor containing acti­ time (d), kX
Si
is the constant a, and Si S-i Se is the reciprocal of the TN
vated sludge (R-AS). The influent wastewater in R-AS was same as in R- removal rate E. Eq. (2) is modified as:
BF. R-AS is being operated as a sewage station at the enterprise that uses
HRT
the activated sludge method for wastewater treatment, i.e. primary = a + bHRT (3)
sedimentation tank-conventional activated sludge method- E
sedimentation tank. The wastewater station has an effective volume of In equation (3), b is a constant, k is Grau secondary substrate removal
900 m3, with an average daily treatment capacity of approximately 90 rate constant (1/d), and X is the concentration of denitrifying bacteria in
m3, a hydraulic retention time of 10 d, a MLSS concentration of the reactor (g/L) (Raja Priya et al., 2009).
2500–3000 mg/L, and a dissolved oxygen concentration of 3.5 ± 1.5
mg/L. R-AS was operated using activated sludge principles, therefore, b) Modified Stover-Kincannon model
samples were collected from the front, middle, and back of the tank
(Fig. 1b). The reactor operation further required addition of 100 kg of Stover-Kincannon model, originally used to predict the biological
glucose per day as a supplemental carbon source. performance of attached growth system, has been modified and widely
used to describe or predict bioreactor performance, and to describe the
kinetics of denitrification during the stabilization phase (Ni et al., 2010).
It has been shown that the modified Stover-Kincannon model is not

Fig. 1. Schematic representation of a) R-BF, b) R-AS, c) structural arrangement of basalt fiber bio-carriers, and d) bio-nests sampling developed under optimal
operating parameters.

3
H. Ni et al.
limited by the number of kinetic stages and can predict substrate
removal under any loading condition, making it more suitable for pre­
dicting the nitrogen removal performance of the reactor (Ma et al.,
2015). Precisely, it can predict the matrix removal under any load
condition, which makes it a suitable choice for predicting the denitrifi­
cation performance of the R-BF reactor. The original equation of the
model is shown as Eq. (4):
dS Umax QSA i
= (4)
dt KB + QSA i
3
Where dSdt is the substrate removal rate (kg/m /d), Umax is the maximum
3
substrate utilization constant (kg/m /d), KB is the saturation constant
(kg/m3/d), and A is the surface area of the reactor (m2).
In this model, A can be replaced by the reactor volume V. Then Eq.
(4) is modified as.
dS Umax QS i
= V
QSi
(5)
dt KB + V
Where, dS
dt was proportional to the substrate load at steady state, as
shown in Eq. (6):
dS Q
= × (Si − Se ) (6)
dt V
Eq. (6) was substituted into Eq. (5) to obtain Eq. (7):
V KB 1 The genomic DNA was quantified using Qubit 3.0 DNA Assay Kit. Full
= + (7)
Q(Si − Se ) Umax QS
V
i Umax
2.4.2. Monod’s microbial growth kinetic model
Monod kinetic model was also applied to describe the effect of sub­
strate concentration on the microbial growth rate, i.e. matrix inhibition
(Eq. (8)):
S data were exported by SMRT Link software. USEARCH software package
q = qmax (8)
Ks + S
Where q and qmax denote the substrate-specific reaction rate and the
substrate maximum specific reaction rate (mg-N/mgVSS/d), respec­
tively, Ks the half-saturation constant (mg/L), and S is the substrate
concentration (mg/L) (Zhang et al., 2017).
2.5. Bio-nest multi-location parameter analyses
A microelectrode system (Unisense, Denmark) was used to determine
dissolved oxygen (DO) concentration (Gundersen et al., 1998). The
gravimetric method was used to determine the amount of microbial
biomass attached to the BF. The biological activity of the bio-nest was
studied by using LIVE/DEAD™ BacLight™ Bacterial Viability Kit
(Invitrogen) staining and Confocal Laser Scanning Microscopy (CLSM)
(Leica TCS SP5 II, Germany). BacLight™ Bacterial Viability Kit com­
prises two nucleic acid dyes namely SYTO 9 and PI which have been
extensively used to stain alive and dead communities (de la Fuente-
Núñez et al., 2014). The samples were stained using both dyes in an
equal ratio (1:1), and stored in the dark for 15 min before CLSM scan­
ning. Excitation/emission wavelengths of SYTO 9 and propidium iodide
(PI) were set at 485⁄498 nm and 535⁄617 nm to perform dual-channel
imaging for green and red fluorescence, respectively. The images ob­
tained after the layer sweep were convoluted and calculated in 3D using
Leica LAS X software and the threshold segmentation method to obtain
the final bioactivity micrograph. The fluorescence signal of BF before
treatment was negligible indicating the absence of microbial coloniza­
tion on its surface. Bioactivity can be defined as the proportion of alive
or active microorganisms within the total count of microorganisms in
the bio-nest. The microscopic count was performed as per the threshold
segmentation method (Grishagin, 2015).
4
Bioresource Technology 364 (2022) 128036
Extracellular polymeric substance (EPS) is a polymer secreted by
microorganisms that comprise polysaccharides and proteins with small
amounts of humic substances, glyoxalate, and DNA. These substances
contain functional groups that affect the performance of activated
sludge flocculation, sedimentation, and dewatering (Ni et al., 2021a).
Thus, EPS acts as a buffer layer to protect the microorganisms from
harsh environmental conditions and provides a stable microenviron­
ment for the growth and metabolism of microbial communities. In this
study, EPS extraction was performed by using the “Formaldehyde –
NaOH” method as established previously for sludge samples (Liu &
Fang, 2002). To this end, BF and sludge samples from R-BF and R-AS
were immersed in a formaldehyde aqueous solution for 1 h. Then, 4 mL
of 1 N sodium hydroxide solution was added following incubation at
room temperature for 3 h. The mixture was then centrifuged for 20 min
at 20,000g following dialysis for 24 h. The resulting EPS fraction was
lyophilized at − 50 ◦ C for 48 h before further analysis. Finally, poly­
saccharide (PS) and protein (PN) contents in EPS were determined by
Anthrone Colorimetric method and the Coomassie Brilliant Blue
method, respectively (Liu & Fang, 2002).
2.6. Metataxonomic analyses
The biomass samples from bio-nest and R-AS were collected for
metataxonomic analyses, i.e. full length 16S amplicon sequencing as
explained above. DNA extraction was carried out using the NucleoSpin
96 Soil DNA Kit (MN, Germany) as per the manufacturer’s instructions.
length 16S primers such as 27F (5′ -AGRGTTTGATYNTGGCTCAG-3′ ) and
1492R (5′ -TASGGHTACCTTGTTASGACTT-3′ ) were used for library
preparation. The PCR products were then purified using 0.6X DNA
cleaning magnetic beads (VAHTSTM, Vazyme Biotech Co., ltd.) before
sequencing. The sequencing was performed on PacBio SMRT sequencing
technology according to the manufacturers’ instructions. The circular
consensus sequence reads generated from the raw PacBio sequencing
was used for clustering OTU and taxonomy assignments (Dubois et al.,
2010). The alpha diversity analysis was carried out to understand spe­
cies abundances and richness, whereas beta diversity analysis was car­
ried out to compare the magnitude of differences in species diversity
among the samples. The potential microbial functional profiles were
predicted by using a computational pipeline ‘Functional Annotation of
Prokaryotic Taxa (FAPROTAX)’ (Yang et al., 2021). The correlation
network diagram was analyzed by using the Spearman algorithm (both
positive and negative correlations) in the python environment. The
linear model redundancy analysis (RDA) was performed using
BMKCloud.
2.7. Analytical analyses
All samples were centrifuged and filtered through a 0.45 μm mem­
brane filter to remove impurities. COD, NH+4 -N, and TN were determined
as per the instructions of the potassium dichromate rapid digestion
method, Nessler’s reagent spectrophotometry, and alkaline potassium
persulfate digestion method combined with a spectrophotometer,
respectively (Ni et al., 2021c). The relevant standard curves are shown
in the supplementary material. DO, temperature, and pH was measured
using a Hach meter connected with specific probes (HQ40d, Hach).
3. Results and discussion
3.1. Performance of the R-BF
Fig. 2 (a,b,c) illustrates the removal of COD, NH+
4 -N, and TN by R-BF
and R-AS at HRT of 12 h and different DO conditions (0–1, 1–2, 2–3 mg/
L) for 60 days. For R-BF, largest reductions in COD, NH+ 4 -N and TN (i.e.
93.3 ± 0.5 %, 75.4 ± 1.2 % and 77.4 ± 1.0 %, respectively), which were
H. Ni et al.
Fig. 2. Removal trends for COD, NH+ 4 -N, and TN in R-BF and R-AS. Reactor operation during (a-c) initial 60 days at HRT of 12 h and DO of 0–1 mg/L, and (d-f)
remaining 120 days at DO of 0–1 mg/L at different HRT conditions (6 h, 8 h, 10 h, 12 h, 14 h, 16 h). At HRT of 12 h, the pollutant removal efficiency of R-BF was
significantly higher as compared to the other HRT conditions.
significantly higher than the removal in R-AS (88.1 ± 0.3 %, 65.4 ± 0.6
% and 62.4 ± 0.5 %). Importantly, dissolved oxygen in the bio-nest
successively decreased from the outside to the inside, i.e. 0.73 ± 0.02
mg/L, 0.64 ± 0.03 mg/L, 0.47 ± 0.02 mg/L, 0.36 ± 0.02 mg/L, 0.30 ±
0.04 mg/L and 0.13 ± 0.03 mg/L (Fig. 4). The maximum removal effi­
ciency could be attributed to the development of anaerobic/anoxic/
aerobic zones inside the R-BF. These conditions are known to facilitate
the degradation and transformation of pollutants in the wastewater (Ni
et al., 2021a).
At higher HRT of 12 h, the pollutant removal efficiency of R-BF was
significantly higher as compared to the other conditions. Further,
effluent quality was better than that of R-AS. At shorter HRTs, pollutants
and microorganisms do not get sufficient contact time, which in turn
affects the operational stability and treatment effect of R-BF. On the
other hand, when HRT is too long, the organic loading rate of R-BF is
reduced which increases the endogenous respiration of microorganisms.
This may affect the activity of sludge and the removal effect of pollut­
ants. Likewise, TN removal efficiency of R-BF (77.4 ± 1.0 %) was much
higher than that of R-AS (62.4 ± 0.5 %) when DO was 0–1 mg/L and
HRT was 12 h. This indicates that R-BF could save a large amount of
carbon input as well as reactor volume compared to the R-AS.
5
Bioresource Technology 364 (2022) 128036
3.2. Substrate removal/microbial growth kinetic model analysis
Results of Grau secondary substrate removal model for R-BF are
shown in Fig. 3(a). As per Eq. (3), fitting result of the model was HRT
E =
0.053 + 1.308HRT, where the correlation coefficient (R2) was
computed as 0.945 at a rate constant of 0.053. The removal efficiency
constant (k) of Grau secondary matrix in R-BF can be calculated ac­
cording to the corresponding constant a=kX Si
. When the influent con­
centration Si and HRT of the reactor are known, the model can be
substituted into the known fitting equation to predict the effluent con­
centration of R-BF, that was, the denitrification performance of the
reactor (Jin & Zheng, 2009). The modified Stover-Kincannon model was
not limited by the kinetic order and could predict the matrix removal
under any load condition. Therefore, it was more suitable for predicting
the denitrification performance of R-BF. The modified Stover-Kincannon
model for R-BF of correlation coefficient (R2) was computed to be 0.945
(Fig. 3b). The maximum substrate utilization constant (Umax) was 4.462
kg/m3/d, and the saturation constant (KB) was 5.835 kg/m3/d. KB refers
to the substrate concentration at which the substrate utilization constant
is maximum (Umax). The higher KB value indicates a higher system’s
H. Ni et al. Bioresource Technology 364 (2022) 128036

Fig. 3. Substrate removal kinetic model and microbial growth kinetic model of R-BF. Grau secondary substrate removal model (a); Modified Stover-Kincannon model
(b); Monod model (c).

potential for nitrogen removal. Here, results indicated that the

maximum total nitrogen removal rate was 4.462 kg/m3/d in R-BF. In Table 1
Biochemical parameters of bio-nest at different locations under optimal oper­
this study, values of Umax and KB were higher than the long-term oper­
ating conditions.
ated UASB-anammox mixed culture process, which was previously
recorded up to 3.33 kg/m3/d and 4.03 kg/m3/d, respectively (Niu et al., Depth EPS Polysaccharide Protein Biomass Activity DO
(mg per (mg/g) (mg/g) (kg/m3) (%) (mg/
2016). For the treatment of synthetic wastewater, Umax and KB for
g-VS) L)
anammox non-woven membrane reactor (A NMR) were 8.98 kg/m3/
55 740.56 198.43 ± 9.23 277.12 25.7 ± 31.31 0.13
d and 7.89 kg/m3/d, respectively (Ni et al., 2010). Hence, R-BF could
mm ± 9.63 ± 9.78 0.1 ±
achieve more than half of the TN removal as compared to the A NMR, (a) 0.03
suggesting its efficient denitrification potential for lithium battery slurry 45 693.77 172.13 ± 7.89 246.12 24.3 ± 37.57 0.30
wastewater with a low carbon–nitrogen ratio (C/N: 1.57). mm ± 7.92 ± 8.67 0.1 ±
The Monod model was used to fit the microbial growth rate of R-BF (b) 0.04
35 649.25 156.72 ± 6.42 211.67 21.5 ± 41.49 0.36
under different NH+ 4 -N concentrations according to the effect of influent mm ± 8.36 ± 7.34 0.2 ±
matrix concentration on denitrification performance of the system (c) 0.02
(Zhang et al., 2017). The results are shown in Fig. 3(c). The correlation 25 582.10 142.18 ± 6.91 182.18 19.7 ± 41.65 0.47
coefficient (R2) of the fitting curve of microbial growth kinetic model mm ± 8.95 ± 8.11 0.3 ±
(d) 0.02
(Monod model) was 0.937, half-saturation constant (Ks) was 622.14
15 528.69 127.68 ± 5.82 157.10 17.8 ± 46.80 0.64
mg/L, and the substrate maximum specific reaction rate (qmax) was mm ± 7.67 ± 5.63 0.1 ±
0.323 (mg-N/mgVSS/d). This indicates that R-BF is highly adaptable to (e) 0.03
ammonia–nitrogen and could play a vital role in its removal from 5 mm 439.21 112.73 ± 6.17 142.16 15.9 ± 71.84 0.73
wastewater. (f) ± 6.29 ± 6.48 0.1 ±
0.02
R-AS 89.56 22.78 ± 1.18 28.98 2.75 ± 31.52 3.50
3.3. Microbial activity analysis (g) ± 0.95 ± 1.02 0.25 ±
1.50
1
The biological activity of the bio-nest, estimated by LIVE/DEAD® All results presented are means of duplicate experiments ± SD.
BacLightTM Bacterial Viability Kit, was established by comparing green
vs red fluorescence signals using CLSM (see supplementary material). In inner part of the bio-nest displayed the highest amount of EPS (740.56
R-AS, biological activity was recorded to be 31.52 %. However, for R-BF, ± 9.63 mg/g-VS) as compared to the outer part (439.21 ± 6.29 mg/g-
the biological activity of the samples was increased gradually from inner VS). Nevertheless, EPS content in R-AS was comparatively low, i.e.,
to outside of the bio-nest at optimal operating parameters, i.e., 31.31 %, 89.56 ± 0.95 mg/g-vS (Table 1). The high EPS content in R-BF could
37.57 %, 41.49 %, 41.65 %, 46.80 %, and 71.84 % (Table 1). The high have provided a better adsorption capacity and a stable internal envi­
microbial activity in R-BF was likely due to better impact resistance and ronment for microbial growth. The high EPS content is also known to
system stability. On the other hand, the weak green fluorescence signal facilitate flocculation and precipitation which could have contributed to
of R-AS indicated a low microbial activity (see supplementary material). the reduction of SS (Gorin & Mazurek, 1979). SS concentrations in the
Bio-nest provides a large specific surface area for microorganisms to effluents of R-BF and R-AS were < 12 mg/L and ~ 25 mg/L. Accord­
grow and multiply with a diversity of habitats including anaerobic/ ingly, floating carbon powder content in the effluent of R-BF was
anoxic/aerobic niches, which hosts unique microbial communities apparently low than R-AS (visual observations). The PS and PN in R-BF
accordingly (Ni et al., 2021b). In this study, diverse microbial commu­ were also higher than in the R-AS. Previously, it was found that PS and
nities could have allowed R-BF to be more active than R-AS in treating PN promote the formation of 3-dimensional bio-nest structure (Ni et al.,
lithium battery slurry wastewater. 2021c). The overall biomass in the bio-nest for all samples was higher
than the total biomass detected in R-AS, i.e., 25.7 ± 0.1, 24.3 ± 0.1, 21.5
± 0.2, 19.7 ± 0.3, 17.8 ± 0.1, and 15.9 ± 0.1 kg/m3, vs 2.75 ± 0.25 kg/
3.4. Bio-nest multi-location parameter analysis
m3.
During 180 days of continuous operation, low DO conditions likely
favored efficient attenuation of COD, NH+ 4 -N, and TN via anaerobic 3.5. Taxonomic composition and function of microbial communities
processes. DO concentration of the bio-nest gradually decreased from
the outside to the inside while the innermost part displayed 0.13 ± 0.03 3.5.1. Bacterial community structures
mg/L of DO (Fig. 4). Contrarily, EPS, PS, PN, and biomass contents were Full-length 16S rRNA sequencing was performed to study bacterial
gradually decreased from interior to exterior of the bio-nest (a-f). The community structures in R-BF and R-AS. Venn diagram illustration of

6
H. Ni et al. Bioresource Technology 364 (2022) 128036

Fig. 4. Parametric comparisons in the bio-nest of R-BF at different locations. DO in the bio-nest successively decreased from the outside to the inside likely affecting
biomass, bio-activity, polysaccharides, proteins and EPS content.

operational taxonomic units (OTUs) and alpha diversity indices indi­ abundance was 26.84 % inside the bio-nest, and 20.22 % to 20.57 % at
cated that R-BF harbored rich microbial species and diversity as the periphery. Its abundance in R-AS was 24.13 %. The members of
compared to the R-AS. Shannon Index (H) showed highest diversity and Bacteroidetes are mostly known as anaerobic bacteria, which play a key
richness at the core of the bio-nest (4.03), followed by outer parts in a role in the degradation of macromolecular organic matter (Tang et al.,
gradient manner (i.e., 3.79, 3.74, 3.60, 3.47, 3.30), which were higher 2017). Bacteroidetes also carry a hydrolytic effect on sludge flocs and
than the diversity in R-AS (3.08) (Table 2). Typically, a value of H > 3.5 may play an important role in the removal of COD, ammonia nitrogen,
suggests a stronger degree of biogeochemical cycles due to rich and total nitrogen, and TP from a variety of wastewater (Peng et al., 2015).
diverse microbial communities within a particular (micro)ecosystem The lower rank taxonomy (i.e. genus and species) was considered to
(Shade, 2015). Further, rarefaction analysis confirmed that sequencing interpret underlying biogeochemical processes in R-BF and R-AS
coverage in both R-BF and R-AS was sufficient to capture the majority of (Fig. 5). In R-BF, both SND and conventional nitrifica­
bacterial sequences (see supplementary material). tion–denitrification reactions were dominant; whereas, in R-AS, only
The bacterial community structure of all samples in R-BF and R-AS conventional nitrification–denitrification reactions were main meta­
was characterized from the phylum to species level ranks. The phylum- bolic processes.
level taxonomy indicated that Proteobacteria and Bacteroidetes domi­ Conventional denitrification: The bacterial community structures
nated the total bacterial community in both reactors (see supplementary indicated that both R-BF and R-AS were able to remove TN via con­
material). The highly abundant phylum was Proteobacteria, whose ventional denitrification mechanisms (Fig. 5). A denitrifying genus
relative proportion was 60.2 % within the bio-nest, and 58.3 % to 59.7 % Ottowia was prominent in R-BF (12.95 %, 16.54 %, 14.98 %, 12.69 %,
at the periphery; whereas the relative abundance in R-AS was 57.94 %. 18.86 %, and 6.12 % from inside to outside of the bio-nest), whereas its
Typically, members of Proteobacteria are known for a variety of meta­ abundance in R-AS was 3.97 % (Felföldi et al., 2010). Another deni­
bolic processes including aerobic, anaerobic, autotrophic, heterotro­ trifying genus Chujaibacter was detected up to 12.1 % inside the R-BF,
phic, and both phototrophic and chemotrophic pathways. The second while a comparable abundance was also observed in R-AS (g: 11.46 %)
most abundant phylum was identified as Bacteroidetes, whose relative (Senevirathna et al., 2022). Contrarily, a facultative bacterium namely
Rhodanobacter, capable of oxidizing nitrite to nitrate, was found 19.89 %
in R-AS whose abundance in R-BF was 0.6 % to 4.27 % (Han et al.,
Table 2 2019). The poor survival in R-BF might be due to low DO concentration
A comparison of Alpha diversity indices for R-BF and R-AS.
(0.13 ± 0.03 mg/L) in the bio-nest as well as competition with other
Sample ID Shannon ACE Chao1 Index Simpson Coverage microbes that could affect the growth and metabolism of nitrite-
55 mm (a) 4.0284 225.0435 215.1463 0.036 0.9842 oxidizing bacteria (NOB). The other denitrifying genera were also
45 mm (b) 3.5964 230.8254 234.2143 0.0736 0.9792 detected at relatively high percentages in R-BF (Fig. 5). For instance,
35 mm (c) 3.7471 184.624 187.5652 0.0478 0.9848 Acidovorax is capable of denitrification using various carbon sources (Di
25 mm (d) 3.7914 234.2525 243.0357 0.0505 0.9801
Pippo et al., 2020); Linebacker (4.67 %) could symbiotically grow with
15 mm (e) 3.4738 195.0181 196.4348 0.0713 0.9857
5 mm (f) 3.2971 189.7768 186 0.0782 0.9879 anaerobic ammonia-oxidizing bacteria during denitrification (Xing
R-AS (g) 3.0796 128.6483 122.375 0.096 0.9959 et al., 2020); Arcobacter (8.41 %) can carry out nitrate reduction (Huang

7
H. Ni et al.
Fig. 5. Heatmaps illustrating bacterial community structures at genus and species levels in R-BF and R-AS.
et al., 2021); Ignavibacterium uses NO–2-N as an electron acceptor for
denitrification; and Ellin6067 can oxidize ammonium to nitrite being
ammonia-oxidizing bacteria (Chen et al., 2020).
Heterotrophic nitrification and aerobic denitrification: R-BF was
also capable of attenuating nitrogen species via combined heterotrophic
nitrification and aerobic denitrification (HN-AD) mechanism. This is
because Hydrogenophaga, whose members are capable of HN-AD pro­
cesses, was highly abundant at the periphery of the bio-nest (20.05 %)
(Xing et al., 2017). Biochemically, Hydrogenophaga members can
perform oxidative sugar metabolism with oxygen as a terminal electron
acceptor, which is likely the reason for its decrease in the interior of the
bio-nest where oxygen availability was low (Xing et al., 2017). A few
other HN-AD genera namely Dokdonella, Thauera, and Terrimonas were
also detected in R-BF (Fig. 5). Members of Dokdonella can perform
nitrification and denitrification in different DO concentrations using
oxygen and NO–3 as electron acceptors under oxic and anoxic conditions
(Jiang et al., 2018a). Thauera, whose abundance was 2.45 % in R-BF and
0.2 % in R-AS, is also known for denitrification (Si et al., 2020).
Organics degradation: In R-BF, some bacteria likely supported TN
removal indirectly by providing electron donors via organics degrada­
tion. For instance, Tolumonas (species: T. osonensis), an anaerobic
fermentative bacterium commonly found in hydrolytic acidification
reactors, was identified at the periphery of R-BF. It can degrade organics
and provide electron donors for denitrification. Anaerobic fermenters
such as Macellibacteroides, mainly detected in R-BF (~1.16 % vs 0.02 %
in R-AS), could convert granular carbon sources into dissolved carbon
sources (lactate, acetic acid, n-butyric acid, and isobutyric acid) that
may also support the denitrification mechanism (Guo et al., 2019).
Lentimicrobium, a hydrolytic fermentative genus, was relatively high in
R-BF (a: 1.58 %, f: 2.13 %) than R-AS (0.02 %) likely have performed a
similar function (González-Cortés et al., 2021). Stenotrophobacter,
capable of degrading a variety of organics using a variety of growth
substrates such as PN, was also detected in smaller fractions in R-BF
(0.91 %, 0.31 %, 1.04 %, 0.51 %, and 0.17 %) but absent in R-AS (Ryu
et al., 2021). On the other hand, Candidatus Saccharimonas, a sugar-
fermenting bacterium mainly abundant in R-AS (6.74 %), was
enriched due to glucose addition during the reactor’s operation.
Other processes: Acidovorax, Comamonas, Thauera, and Ferrugini­
bacter were mainly identified in R-BF and their abundance in R-AS was
8
Bioresource Technology 364 (2022) 128036
relatively low (Fig. 5). These genera could synthesize and secrete EPS
effectively (Di Pippo et al., 2020). The synthetically secreted EPS of
these genera could have allowed better adhesion and bridging to the
mid-term of the bio-nest in R-BF (Si et al., 2020). Similarly, an abun­
dance of Novosphingobium in R-BF was higher than in R-AS. The presence
of Novosphingobium could reduce COD as they can degrade a variety of
lignin and cyclic compounds. Also, Novosphingobium performs phos­
phate solubilizing activity, which can convert insoluble phosphates in
lithium battery slurry wastewater into dissolved and available phos­
phates, thus supplementing the deficiency of elemental phosphorus in
the wastewater and promoting the removal of pollutants (Coll et al.,
2020).
3.5.2. Putative function prediction
The metabolic functions of identified bacterial taxa were computa­
tionally assessed by using the FAPROTAX pipeline (see supplementary
materials). R-BF displayed potential for variety of functions including
chemoheterotrophy (20.8 %), aerobic chemoheterotrophy (19.0 %),
nitrogen respiration (6.19 %), nitrate reduction (6.04 %), nitrate
respiration (5.99 %), nitrite respiration (5.78 %), nitrate denitrification
(4.33 %), nitrite denitrification (4.33 %), nitrous oxide denitrification
(4.33 %), denitrification (4.33 %), nitrification and aerobic ammonia
oxidation processes (4.33 %). On the other hand, only chemohetero­
trophy and aerobic chemoheterotrophy appeared as dominating meta­
bolic functions in R-AS, i.e. 42.6 % and 42.3 % respectively (84.9 %).
3.5.3. Redundancy analysis (RDA)
RDA was performed at the genus level to study the relationship be­
tween bacterial abundance and environmental factors. The environ­
mental factors influence on bacterial communities was as follows: DO >
PN > PS > Biomass > EPS > Activity. Precisely, ‘DO’ had a stronger
influence on bacterial community structure, whereas ‘Activity’ had the
weakest (Fig. 6). ‘DO’ was negatively correlated with ‘EPS’, ‘PS’, ‘PN’
and ‘Biomass’, whereas the ‘Activity’ was not correlated with ‘EPS’,
‘PN’, ‘PS’ and ‘Biomass’. The large variability in bacterial community
structure between sample a-e and sample f was mainly due to the DO
gradient created during the oxygen transfer inside the bio-nest. This
made the DO inside lower (0.73 ± 0.02 mg/L) than the outside con­
centration. The high variability in bacterial community structure
H. Ni et al.
Fig. 6. Linear model redundancy analysis (RDA) represents the relationship between genus ratio and environmental factors.
between sample g of R-AS and sample a-f of R-BF is in accordance with
the high efficiency and stability of the R-BF system in treating
contaminants.
Hydrogenophaga was positively correlated with ‘Activity’, and the
activity at sample f was 71.84 %, while the species abundance of
Hydrogenophaga was as high as 20.05 %, indicating that the increase in
Activity favored the enrichment of Hydrogenophaga as a genus of HN-AD.
Terrimonas, Dokdonella, and Thauera were positively correlated with
‘EPS’, ‘PN’, ‘PS’ and ‘Biomass’, and were negatively correlated with
‘DO’. This indicates that the abundance of these three HN-AD genera
was influenced by EPS content and composition. Previously, it was re­
ported that the growth of HN-AD genera could be promoted by regu­
lating DO and EPS content, which may facilitate simultaneous
nitrification and denitrification reaction (Tan et al., 2021). Acidovorax,
Thauera, and Ferruginibacter were also positively correlated with ‘EPS’,
‘PN’, ‘PS’, and ‘Biomass’ whereas Comamonas was positively correlated
with DO. This suggests that EPS secretion by Acidovorax, Ferruginibacter,
Thauera, and Comamonas was likely influenced by ‘EPS’, ‘Biomass’, and
‘DO’. Ottowia, Limnobacter, Ferruginibacter, Acidovorax, and Alicycliphilus
were also positively correlated with ‘EPS’, ‘PN’, ‘PS’, and Biomass, as
well as with Ellin6067 (Ammonia Oxidizing Bacteria) and positively
correlated with Fusibacter and Macellibacteroides (anaerobic fermenta­
tive genera). Singulisphaera, Comamonas, Curvibacter, Castellaniella, and
Chujaibacter were positively correlated with DO and with Rhodanobacter
(NOB) and Candidatus Saccharimonas (genus of anaerobic fermenting
bacteria) were positively correlated with DO. This indicated that the
growth of most denitrifying bacteria was positively correlated with DO,
EPS, and Biomass, which, combined with the functions of various
9
Bioresource Technology 364 (2022) 128036
genera, was consistent with the interplay of different functional genera
in water treatment systems. The results of the RDA analysis were
consistent with the results of the correlation network diagram (see
supplementary material).
3.5.4. Practical implications
This study confirms the coexistence of nitrification–denitrification
and simultaneous nitrification and denitrification in R-BF, which
resulted in a stable and efficient removal of pollutants from the low
carbon to nitrogen ratio lithium battery slurry wastewater. By sus­
pending BF bio-carriers into the reactor, bio-nest with high biomass,
high EPS, and a rich microbial community were formed. The high
biomass and EPS content likely enhanced flocculation and sedimenta­
tion of SS in lithium battery slurry wastewater. The rich microbial
community and anaerobic/anoxic/aerobic microenvironments could
have further facilitated the denitrification process in R-BF. It can be
argued that denitrification process using NMP as an electron donor were
occurring in R-BF, and 79.2 % of the electron donor produced by NMP
could have been used for the reduction of exogenous nitrate-nitrogen
(see supplementary material). This approach appeared to be an effec­
tive, sustainable, and cost-effective solution to lithium slurry waste­
water treatment. Compared to the conventional biological treatment
processes (activated sludge method), R-BF requires less aeration power
input and no additional organic carbon source for denitrification during
treatment, which largely reduced CO2 emissions. Furthermore, in line
with previous studies, R-BF in this study had a longer sludge retention
time (SRT) of 90 d for the R-BF and 6 d for the R-AS, which was mainly
related to its internal anaerobic microenvironment and long food chain.
H. Ni et al.
On the contrary, shorter SRT could lead to the loss of potential nitrifying
bacteria. The reduction in residual sludge production contributed to the
lower operating costs of R-BF. Theoretically, with the rapid develop­
ment of new energy batteries, R-BF seems to be a valuable technology
for lithium battery wastewater treatment.
4. Conclusions
BF improved the treatment capacity of R-BF by successfully attenu­
ating COD, ammonia, and TN for low C/N lithium battery slurry
wastewater. Modified Stover-Kincannon model reflected that R-BF was
particularly efficient for denitrification. The higher biological activity,
EPS, PS, PN, and microbial biomass maintained a stable internal envi­
ronment within the bio-nest, which also promoted bacterial richness and
diversity. Bacterial communities involved in the nitrogen cycle, carbon
cycle, hydrolysis, and fermentation-related processes were more abun­
dant in R-BF. The multidimensional structure of the bio-nest could solve
the problem of R-AS, mainly the high microbial exfoliation and main­
tenance in terms of glucose addition.
CRediT authorship contribution statement
Huicheng Ni: Writing – original draft, Formal analysis. Muhammad
Arslan: Supervision, Validation, Writing – review & editing. Zhishui
Liang: Formal analysis. Chencheng Wang: Supervision, Writing – re­
view & editing. Zhijun Luo: Formal analysis. Junchao Qian: Formal
analysis, Writing – review & editing. Zhiren Wu: Funding acquisition,
Writing – review & editing, Conceptualization. Mohamed Gamal El-
Din: Writing – review & editing, 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.
Data availability
Data will be made available on request.
Acknowledgments
The authors wish to thank the National Key R&D Program of China
(2016YFE0126400) and the Natural Science Foundation of Jiangsu
Province (Grants No. BK20180971).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2022.128036.
References
Chen, Y., Shao, Z., Kong, Z., Gu, L., Fang, J., Chai, H., 2020. Study of pyrite based
autotrophic denitrification system for low-carbon source stormwater treatment.
J. Water Process. Eng. 37, 101414.
Coll, C., Bier, R., Li, Z., Langenheder, S., Gorokhova, E., Sobek, A., 2020. Association
between Aquatic Micropollutant Dissipation and River Sediment Bacterial
Communities. Environ. Sci. Technol. 54 (22), 14380–14392.
Di Pippo, F., Venezia, C., Sighicelli, M., Pietrelli, L., Di Vito, S., Nuglio, S., Rossetti, S.,
2020. Microplastic-associated biofilms in lentic Italian ecosystems. Water Res. 187,
116429.
Dubois, P.C.A., Trynka, G., Franke, L., Hunt, K.A., Romanos, J., Curtotti, A.,
Zhernakova, A., Heap, G.A.R., Ádány, R., Aromaa, A., Bardella, M.T., van den
Berg, L.H., Bockett, N.A., de la Concha, E.G., Dema, B., Fehrmann, R.S.N.,
Fernández-Arquero, M., Fiatal, S., Grandone, E., Green, P.M., Groen, H.J.M.,
Gwilliam, R., Houwen, R.H.J., Hunt, S.E., Kaukinen, K., Kelleher, D., Korponay-
Szabo, I., Kurppa, K., MacMathuna, P., Mäki, M., Mazzilli, M.C., McCann, O.T.,
Mearin, M.L., Mein, C.A., Mirza, M.M., Mistry, V., Mora, B., Morley, K.I., Mulder, C.
J., Murray, J.A., Núñez, C., Oosterom, E., Ophoff, R.A., Polanco, I., Peltonen, L.,
10
Bioresource Technology 364 (2022) 128036
Platteel, M., Rybak, A., Salomaa, V., Schweizer, J.J., Sperandeo, M.P., Tack, G.J.,
Turner, G., Veldink, J.H., Verbeek, W.H.M., Weersma, R.K., Wolters, V.M.,
Urcelay, E., Cukrowska, B., Greco, L., Neuhausen, S.L., McManus, R., Barisani, D.,
Deloukas, P., Barrett, J.C., Saavalainen, P., Wijmenga, C., van Heel, D.A., 2010.
Multiple common variants for celiac disease influencing immune gene expression.
Nat. Genet. 42 (4), 295–302.
Felföldi, T., Székely, A.J., Gorál, R., Barkács, K., Scheirich, G., András, J., Rácz, A.,
Márialigeti, K., 2010. Polyphasic bacterial community analysis of an aerobic
activated sludge removing phenols and thiocyanate from coke plant effluent.
Bioresour. Technol. 101 (10), 3406–3414.
Frolov, A.I., Arif, R.N., Kolar, M., Romanova, A.O., Fedorov, M.V., Rozhin, A.G., 2012.
Molecular mechanisms of salt effects on carbon nanotube dispersions in an organic
solvent (N-methyl-2-pyrrolidone). Chem. Sci. 3 (2), 541–548.
González-Cortés, J.J., Almenglo, F., Ramírez, M., Cantero, D., 2021. Simultaneous
removal of ammonium from landfill leachate and hydrogen sulfide from biogas using
a novel two-stage oxic-anoxic system. Sci. Total Environ. 750, 141664.
Gorin, P.A.J., Mazurek, M., 1979. Structure-dependent, molecular-motion parameters of
branched-chain polysaccharides. Carbohydrate Research 72, C1–C5.
Grishagin, I.V., 2015. Automatic cell counting with ImageJ. Anal. Biochem. 473, 63–65.
Gundersen, J.K., Ramsing, N.B., Glud, R.N., 1998. Predicting the signal of O2
microsensors from physical dimensions, temperature, salinity, and O2 concentration.
Limnol. Oceanogr 43 (8), 1932–1937.
Guo, N., Ma, X., Ren, S., Wang, S., Wang, Y., 2019. Mechanisms of metabolic
performance enhancement during electrically assisted anaerobic treatment of
chloramphenicol wastewater. Water Res. 156, 199–207.
Han, Z., Dong, J., Shen, Z., Mou, R., Zhou, Y., Chen, X., Fu, X., Yang, C., 2019. Nitrogen
removal of anaerobically digested swine wastewater by pilot-scale tidal flow
constructed wetland based on in-situ biological regeneration of zeolite.
Chemosphere 217, 364–373.
Huang, C., Liu, Q., Li, Z.-L., Ma, X.-D., Hou, Y.-N., Ren, N.-Q., Wang, A.-J., 2021.
Relationship between functional bacteria in a denitrification desulfurization system
under autotrophic, heterotrophic, and mixotrophic conditions. Water Res. 188,
116526.
Jiang, X., Shen, J., Xu, K., Chen, D., Mu, Y., Sun, X., Han, W., Li, J., Wang, L., 2018a.
Substantial enhancement of anaerobic pyridine bio-mineralization by electrical
stimulation. Water Res. 130, 291–299.
Jin, R.-C., Zheng, P., 2009. Kinetics of nitrogen removal in high rate anammox upflow
filter. J. Hazard. Mater. 170 (2), 652–656.
Kim, S., Bang, J., Yoo, J., Shin, Y., Bae, J., Jeong, J., Kim, K., Dong, P., Kwon, K., 2021.
A comprehensive review on the pretreatment process in lithium-ion battery
recycling. J. Cleaner Prod. 294, 126329.
Kim, T.-S., Lee, G.-H., Lee, S., Choi, Y.-S., Kim, J.-C., Song, H.J., Kim, D.-W., 2017.
Carbon-decorated iron oxide hollow granules formed using a silk fibrous template:
lithium-oxygen battery and wastewater treatment applications. NPG Asia Mater. 9
(11), e450–e.
Liu, H., Fang, H.H.P., 2002. Extraction of extracellular polymeric substances (EPS) of
sludges. J. Biotechnol. 95 (3), 249–256.
Ma, R., Yan, J., Fang, L., Tao, H., Liu, Z., Zhou, Y., 2015. Kinetics of removal of chemical
oxygen demand in municipal sewage sludge by supercritical water oxidation.
J. Shenzhen University Sci. Eng. 32, 617–624.
Ni, H., Zhou, X., Zhang, X., Xiao, X., Liu, J.F., Huan, H., Luo, Z., Wu, Z., 2018. Feasibility
of using basalt fiber as biofilm Carrier to construct bio-nest for wastewater
treatment. Chemosphere 212, 768–776.
Ni, H., Arslan, M., Qian, J., Wang, Y., Liu, Z., Luo, Z., Cai, R., Gamal El-Din, M., Wu, Z.,
2021a. Application of basalt fibers in a biological contact oxidation reactor for the
treatment of landfill leachate. J. Cleaner Prod. 297, 126648.
Ni, H., Arslan, M., Wei, J., Dai, J., Luo, Z., Cai, R., Zhao, S., Gamal El-Din, M., Wu, Z.,
2021b. Treatment of printing and dyeing wastewater in biological contact oxidation
reactors comprising basalt fibers and combination fillers as bio-carriers: Elucidation
of bacterial communities and underlying mechanisms. Sci. Total Environ. 785.
Ni, S.-Q., Lee, P.-H., Sung, S., 2010. The kinetics of nitrogen removal and biogas
production in an anammox non-woven membrane reactor. Bioresour. Technol. 101
(15), 5767–5773.
Ni, H., Qian, J., Arslan, M., Zhou, X., Luo, Z., Wei, J., Gamal El-Din, M., Wu, Z., 2021c.
Treatment of high-load organic wastewater by novel basalt fiber carrier media. Sci.
Total Environ. 758, 143760.
Niu, Q., Zhang, Y., Ma, H., He, S., Li, Y.-Y., 2016. Reactor kinetics evaluation and
performance investigation of a long-term operated UASB-anammox mixed culture
process. Int. Biodeterior. Biodegrad. 108, 24–33.
Peng, L., Ni, B.-J., Ye, L., Yuan, Z., 2015. The combined effect of dissolved oxygen and
nitrite on N2O production by ammonia oxidizing bacteria in an enriched nitrifying
sludge. Water Res. 73, 29–36.
Qiu, Y., Ruan, H., Tang, C., Yao, L., Shen, J., Sotto, A., 2019. Study on Recovering High-
Concentration Lithium Salt from Lithium-Containing Wastewater Using a Hybrid
Reverse Osmosis (RO)–Electrodialysis (ED) Process. ACS Sustainable Chem. Eng. 7
(15), 13481–13490.
Raja Priya, K., Sandhya, S., Swaminathan, K., 2009. Kinetic analysis of treatment of
formaldehyde containing wastewater in UAFB reactor. Chem. Eng. J. 148 (2),
212–216.
Ryu, J., Jung, J., Park, K., Song, W., Choi, B., Kweon, J., 2021. Humic acid removal and
microbial community function in membrane bioreactor. J. Hazard. Mater. 417,
126088.
Senevirathna, S.T.M.L.D., Krishna, K.C.B., Mahinroosta, R., Sathasivan, A., 2022.
Comparative characterization of microbial communities that inhabit PFAS-rich
contaminated sites: A case-control study. J. Hazard. Mater. 423, 126941.
H. Ni et al.
Si, Z., Song, X., Wang, Y., Cao, X., Wang, Y., Zhao, Y., Ge, X., Sand, W., 2020. Untangling
the nitrate removal pathways for a constructed wetland- sponge iron coupled system
and the impacts of sponge iron on a wetland ecosystem. J. Hazard. Mater. 393.
Tan, X.u., Yang, Y.-L., Li, X., Gao, Y.-X., Fan, X.-Y., 2021. Multi-metabolism regulation
insights into nutrients removal performance with adding heterotrophic nitrification-
aerobic denitrification bacteria in tidal flow constructed wetlands. Sci. Total
Environ. 796.
Tang, B., Song, H., Bin, L., Huang, S., Zhang, W., Fu, F., Zhao, Y., Chen, Q., 2017.
Determination of the profile of DO and its mass transferring coefficient in a biofilm
reactor packed with semi-suspended bio-carriers. Bioresour. Technol. 241, 54–62.
Xing, W., Zhang, W., Li, D., Li, J., Jia, F., Cui, Y., Ren, F., 2017. An integrated O/A two-
stage packed-bed reactor (INT-PBR) for total nitrogen removal from low organic
carbon wastewater. Chem. Eng. J. 328, 894–903.
Xing, W., Wang, Y., Hao, T., He, Z., Jia, F., Yao, H., 2020. pH control and microbial
community analysis with HCl or CO2 addition in H2-based autotrophic
denitrification. Water Res. 168, 115200.
Yang, N., Zhan, G., Luo, H., Xiong, X., Li, D., 2021. Integrated simultaneous nitrification/
denitrification and comammox consortia as efficient biocatalysts enhance treatment
of domestic wastewater in different up-flow bioelectrochemical reactors. Bioresour.
Technol. 339.
11
Bioresource Technology 364 (2022) 128036
Yoo, K., Shin, S.-M., Yang, D.-H., Sohn, J.-S., 2010. Biological treatment of wastewater
produced during recycling of spent lithium primary battery. Miner. Eng. 23 (3),
219–224.
Zhang, Q., Liang, X., Wei, J., Li, S., Xiao, X., Liu, Z., Rong, X., Liang, Z., Wu, Z., 2019a.
Effects of size and spacing of basalt fiber carrier media on performance, extracellular
polymeric substances and microbial community of hybrid biological reactors.
Environ. Sci.: Water Res. Technol. 5 (7), 1253–1261.
Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Ishii, S., Okabe, S., 2017. Microbial
competition among anammox bacteria in nitrite-limited bioreactors. Water Res. 125,
249–258.
Zhang, X., Zhou, X., Xie, Y., Rong, X., Liu, Z., Xiao, X., Liang, Z., Jiang, S., Wei, J., Wu, Z.,
2019b. A sustainable bio-carrier medium for wastewater treatment: Modified basalt
fiber. J. Cleaner Prod. 225, 472–480.
Zhao, Y., Liu, D., Huang, W., Yang, Y., Ji, M., Nghiem, L.D., Trinh, Q.T., Tran, N.H.,
2019. Insights into biofilm carriers for biological wastewater treatment processes:
Current state-of-the-art, challenges, and opportunities. Bioresour. Technol. 288.
Zheng, T., Wang, Q., Shi, Z., Zhang, Z., Ma, Y., 2016. Microwave regeneration of spent
activated carbon for the treatment of ester-containing wastewater. RSC Adv. 6 (65),
60815–60825.