Desalination and Water Treatment 318 (2024) 100300

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Desalination and Water Treatment

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Effect of electrode in UASB-MFC reactor for nitrogen removal under

anammox condition and its microbial community profile ]]
]]]]]]
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Asif Iqbala, Zulfiqar Ahmad Bhattib, , Farhana Maqboola, , Muhammad Faisal Siddiquia,
⁎ ⁎

Samia Zeba, Yang-Guo Zhaoc, Lina Xud, Sajjad Ahmada, Zakir Hussaine
a
Department of Microbiology, Hazara University Mansehra, 21300, Pakistan
b
Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan
c
College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China
d
College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266100, China
e
Department of Mathematics, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan

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

Keywords: Nitrogen removal through anammox is well-known for its low cost and less CO2 emissions, in this process
UASB-microbial fuel cell ammonium converted into nitrogen gas with the help of anammox. It is hypothesized that the presence of
Biokinetics electrodes in upflow anaerobic sludge blanket-microbial fuel cell (UASB-MFC) might increase nitrogen removal.
Microbial community In order to check this, two experimental setup were used to remove ammonium with electrode (UASB-MFC) and
Upflow anaerobic sludge blanket
without electrode (UASB), under anammox condition. Different parameters were analyzed including nitrate,
Nitrate
nitrite, total nitrogen and voltage. After 164 days, the NH4+ concentration in effluent was 3.20 mg/L and
4.23 mg/L, total inorganic nitrogen was 11.9 mg/L and 8.45 mg/L in UASB and UASB-MFC effluent respectively.
Bacterial growth rate was calculated through biokinetic modelling, indicating anammox growth rate 0.00294 g
VSS/m3.d in UASB-MFC with maximum voltage production of 774 mV. Metagenomic study was performed
through 16 S rRNA genome sequencing showed, Firmicutes phylum was high in both reactors, while
Planctomycetes, abundance 2.8% showing anammox growth achievement only in UASB-MFC. Lower con-
centration of nitrate, nitrite and total inorganic nitrogen were found in the UASB-MFC effluent which suggested
the higher conversion of ammonium into nitrogen gas. It is concluded that the presence of an electrode oxidizes
more nitrogen; hence total nitrogen was removed much more than in the absence of an electrode.

1. Introduction
High nitrogen content in the wastewater is an environmental con-
cern especially in developing countries as it has dangerous impact on
human well-being and water resources [1]. The eutrophication in
aquatic ecosystems is one of the direct and damaging impacts of ex-
cessive nitrogen in wastewater. It destroys aquatic ecosystems by in-
creasing algal blooms, oxygen reduction and hence damages the key
aquatic species. Additionally, bloom of blue green algae can harvest
natural toxins from water that pose risks to the human health [2].
Ammonium removal from wastewaters has been accomplished by using
both physicochemical and biological approaches [3]. In biological
methods, municipal and other industrial wastewater treatment facilities
frequently utilize the nitrification/denitrification process [4]. This is
costly process, causing greenhouse gas emission and insufficient to
meet standard value of EPA for effluent’s nitrate. Less carbon tech-
nology for ammonium removal evoking interest in anaerobic ammo-
nium oxidation processes (Anammox). Heterotrophic ammonium oxi-
dation is one of the most economical process to reduce the ammonium
nitrogen with limited oxygen and carbon source resulting nitrite for-
mation which further act as electron acceptor from ammonia to form
N2.
Biological wastewater treatment is achieved through different mi-
crobial community, due to the advancement in the field of high
throughput sequencing and researchers are able to find microbial di-
versity and physiology in different wastewater treatment systems in-
cluding anammox (anaerobic ammonium oxidation) [5]. Anammox is a
biological nitrogen removal process converting ammonium into ni-
trogen gas under anoxic conditions with nitrite and nitrous oxide be-
having as an electron acceptor [6]. Compared with conventional

⁎
Corresponding authors.
E-mail addresses: zabhatti@cuiatd.edu.pk (Z.A. Bhatti), drfarhana@hu.edu.pk (F. Maqbool).

https://doi.org/10.1016/j.dwt.2024.100300
Received 24 March 2024; Received in revised form 7 April 2024; Accepted 16 April 2024
1944-3986/© 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A. Iqbal, Z.A. Bhatti, F. Maqbool et al.
nitrification/denitrification process, in this process very limited oxygen
is required whereas CO2 and N2O emission reduced [1]. This process
take places inadequately, due to its limited dissolved oxygen require-
ment and prerequisite of low chemical oxygen demand (COD). Ana-
mmox is an autotrophic which converts ammonium to nitrogen gas
through anammox bacteria. As anammox are electroactive micro-
organism producing electron when ammonia is used as substrate.
Zekker et al. [7], has utilized the anammox biomass collected from pilot
scale plant and used in anodic chamber of MFC for energy production in
microbial fuel cells (MFC). One of the new finding suggests that ana-
mmox alone could be used for ammonium removal, while also produ-
cing energy in the form of an electric current or energy-rich hydrogen
gas in microbial electrolysis cells [8]. MFCs use microorganisms as
biocatalysts in anode chamber to decompose organic matter, electrons
are released, which transferred from the anode to the cathode through
an exterior circuit for oxygen reduction and to produce H2O, simulta-
neously protons are produced and transferred to the cathode to gen-
erate electricity [9]. Conventional MFC is two chambered system with 3
basic components, anaerobic anode, aerobic cathode and a proton ex-
change medium [PEM] [10]. Sometimes in MFC, cation exchange
membrane or salt bridge was used for the same purpose. These salt
bridge or PEM helps to separate the cathode and anode chamber hence
making the MFC a dual chamber reactor. Microbes which are present in
the anodic chamber are responsible for the oxidation of substrate pre-
sent in wastewater [11]. Previously, supercapacitor made up of com-
posite nanomaterial electrodes was used for the treatment of landfill
leachate [12] and single chambered MFC for the degradation of re-
calcitrant pyridine and azo dye which are nitrogen compound [13]. In
the application of widely use of anammox process, lack of the electron
acceptors in the anammox system is one of the hurdle [14]. The current
study designed a novel UASB-MFC system in which conditions were
forced to create multiple electron acceptor options, such as nitrite, ni-
trate and anode electrode during ammonia oxidation under anoxic
condition for nitrogen removal. Biokintics model was also used to see
the growth rate of anammox, ammonium oxidizers and nitrite oxidizing
bacteria (NOB) in the presence of anode electrode in UASB-AMFC and
without electrode in UASB.
2. Materials and Methods
2.1. UASB and UASB-MFC Construction
The UASB-MFC and UASB reactors were constructed with 2 mm
thick acrylic transparent sheet. The capacity of the both reactors was 4-
liter, half of the reactor filled with 2 Kg of biomass was collected from
the old domestic septic tank, which was partially digested and granu-
lated (Fig. 1A and B). The UASB had influent port at the bottom and
2
Desalination and Water Treatment 318 (2024) 100300
effluent port on the upper side. Water was filled in aerobic (cathodic)
chamber supplied with aeration. UASB-MFC reactor was consisted of
two parts: the UASB served as the anode, and aerobic chamber served as
cathode. Both chambers are connected with a salt bridge to pass protons
from the anode to the cathode [15]. UASB part containing carbon rods,
which were connected with wire, while aluminium (Al) hollow rods
were used in the cathode chamber. Other operating parameters are
given in Table 1. An agarose salt bridge of 33.6 cm2 area was prepared
to connect anode and cathode, and for better flow of charges and lower
resistance, its size was kept as low as possible [16].
2.2. UASB-MFC functioning
In the startup, both reactors were operated for 74 days with syn-
thetic wastewater without carbon source to washout the impurities
(lower down the chemical oxygen demand (COD) in the range 20 to
50 mg/ L). On 75th day, 40 mg/ L of ammonium containing synthetic
wastewater was fed with 2 days HRT into both reactors with the help of
a peristaltic pump. Dissolved oxygen (DO) was kept below 0.5 mg /L
from the influent through nitrogen sparging for anammox condition,
which was run further for 90 days. During these 164 days different
parameters were analyzed including pH, temperature, DO, COD, ni-
trate, nitrite, ammonium, voltage and current. In order to prepare
synthetic wastewater, different macro and micronutrients used in g/ L
of distilled water as described by [17] in Table 2a & b.
2.3. Analytical Parameters
For determination of DO, HANNA instrument HI 9147 was used.
Closed reflux method was used for COD analysis, in COD tubes reaction
mixture containing 1.5 mL of digestion solution, 3.5 mL sulfuric acid
and 2.5 mL of wastewater sample was mixed and placed in COD di-
gester (HACH-LTG 082.99.40001) at 150°C for 2 h. After 2 h′ readings
were taken through COD spectrophotometer [18].
For ammonium determination, 0.27 N NaOH solution, boric acid,
0.27 N H2SO4 solution and mixed indicator were prepared. Sample
25 mL was distilled by trapping ammonium in boric acid solution in a
distillation apparatus, titrated with 0.27 N H2SO4 solution till lavender
color developed [18].
Concentrated H2SO4 solution and stock nitrate solution were pre-
pared for the determination of nitrate. Water sample (25 mL) was taken
and filtered, then 0.5 mL of concentrated H2SO4 was added and shaken
well. Absorbance on spectrophotometer (T80 + Sr28) was measured at
wavelength of 250 nm [18].
For nitrite analysis, in 25 mL filtered water sample, color reagent
(including 100 mL sulphuric acid, 10 g sulfonamide, 1 g naphthylamine
diluted to 1 L) was added. Stock solution was also prepared with the
Fig. 1. Schematic diagram of (A) UASB-MFC
used in the experiment. (1) Inlet (2) UASB/
Anodic chamber (3) Electrogenic bacteria (4)
Anode rods (5) Gas bubble (6) Biogas outlet (7)
Cathode hollow rods (8) cathode wire (9) salt
bridge/Proton exchange membrane (10)
Aerobic/cathodic chamber (11) Open circuit
(12) voltmeter (13) outlet. (B) UASB reactor.
A. Iqbal, Z.A. Bhatti, F. Maqbool et al. Desalination and Water Treatment 318 (2024) 100300

Table 1
Operating parameters of both reactors.

S# Parameters UASB UASB-MFC

1. Dissolved oxygen 0.3- 0.5 mg/L 0.3- 0.5 mg/L

2. COD 20 − 50 20-50
3. HRT 48 h 48 h
4. Electron donor Ammonium/Organic Compound Ammonium/Organic Compounds
5. Electron acceptor Nitrate/Nitrite Nitrate/Nitrite/Anode electrode

Table 2 2.4.3. Growth Rate of Anammox

a Composition of synthetic influent solution.
KpH , AN
Synthetic Influents Concentration (in 1 L)
µ = µ max
AN e
AN (T Tr )
KpH , AN 1 + 10 pHopt , AN | pH
MgCl2 1g STAN STN 02 K O, AN
XAN
KH2PO4 1g KTAN ,AN + STAN KTN 02, AN + STN 02 K O, AN + SO (3)
(NH4)2SO4 1g
NaHCO3 1g
Trace elements solution 1 mL
b Constituents of trace elements solution
2.5. Metagenomic Profiling
Components Concentration (in 1 L)
Na2 EDTA 5g DNA from the wastewater and sludge samples extracted through
FeCl2.4 H2O 3.58 g FastDNA™ Spin Kit (MP Biomedicals) for high throughput sequencing.
CoCl2.6 H2O 0.5 g
Wastewater sample 15 mL passed through 0.22 µ filter paper and
NaOH 11 g
MnCl2. H2O 2.5 g manufacturer procedure was followed. The DNA concentration and
(NH4)6Mo7O2.44 H2O 0.5 g quality were measured, based on spectroscopic absorbance ratios of
CaCl2.2 H2O 7.34 g 260/280 nm (∼1.8) and 260/230 nm (> 1.7). Polymerase Chain
ZnCl2 1.06 g Reaction was performed by using modified universal primer 515 MF
CuCl2.2 H2O 0.14 g
(GTGYCAGCMGCCGCGGTAA) 806 MR (GGACTACNVGGGTWTCT-
AAT) for bacteria and archaea identification [20]. The samples sent for
same procedure, by dissolving NaNO2 in the distilled water. At the end, next generation DNA sequencing. The amplicon sequencing data was
nitrite concentration was measured on spectrophotometer at wave- analyzed on Galaxy Pipeline work station and method was followed as
length of 543 nm [18]. provided in the manual of Deng Lab [21].

2.6. Analysis of Power Generation

2.4. Growth Rate Model
With the help of different parameters including pH, temperature,
dissolved oxygen, VSS, ammonium, nitrite and nitrate concentration in
the effluent, growth rate of three different dominant groups of bacteria
were calculated by using biokinetics modelling as described by Veys
et al. [19]. The definition of each symbol used in Eqs. (1), (2), (3) and
the constant values are given in supplementary material Table S1 and
S2.
Voltage and current was measured by using voltmeter Uni-T55 in
millivolts (mV) and current in milli amperes (mA).
2.7. Statistical Analysis
MS excel ver. 2010 with Add-in XLSTAT, software was used for
descriptive statistics to calculate ranges and mean values.
3. Results and Discussion

2.4.1. Growth Rate of Ammonia Oxidizing Bacteria

3.1. Ammonium conversion into Nitrate and Nitrite
KpH , NH SO
µ = µ max
NH e NH(T Tr )
pHopt , NH pH Two reactors UASB and UASB-MFC were run separately for total 164
KpH , NH 1 + 10 K o,NH + SO
days with same operating parameters (Fig. 2A and B). Initially both
STAN
1+ 10 pH
reactors were fed with fresh water of COD less than 20 mg/L including
K e, NH
XNH other nutrients and trace elements (Table 2a & b) for 74 days. After 74
1+
STAN
+ K NH3NH , days ammonium nitrogen feeding was started and kept around 40 mg/L
10 pH
K e, NH (1) in influent for both rectors. Reactors were kept at room temperature
where average temperature was around 25 °C. A study indicated that
the nitrification level rises gradually with increased in temperature
from < 14 °C to 30 °C [22] and temperature can also affect the activity,
2.4.2. Growth Rate of Nitrite Oxidizers abundance and community structure of AOB which effect on ammo-
nium reduction. According to Zeng et al., better growth of AOB starts at
KpH , NO SO the 15 °C-25 °C and maximum treatment efficiency observed at 35 °C
µ = µ max
NO e
NO(T Tr )
KpH , NO 1 + 10 pHopt , NH pH K o, N0 + SO [23]. In UASB-MFC electrons were released during ammonium oxida-
STNO 2 tion, nitrate, nitrite and electrode possibly accept the electrons under
K e, NO
1+
10 pH
anoxic oxidation. In case of UASB, nitrate concentration in the effluent
STNO2
XN 0 was 12 mg/ L which decreased to 2.8 mg/L till 164 day, relatively ni-
+ KHNO2, NO
trite production was in the range of 17 to 5.9 mg/ L till day 73 to 164
K e, NO
1+
10 pH (2)
respectively.

3
A. Iqbal, Z.A. Bhatti, F. Maqbool et al. Desalination and Water Treatment 318 (2024) 100300

Fig. 2. A: Ammonium, nitrate and nitrite concentration during 162 days of treatment of ammonium containing synthetic wastewater in UASB.B: Ammonium, nitrate
and nitrite concentration in UASB-MFC during 162 days of treatment of ammonium containing synthetic wastewater.

In UASB-MFC, nitrate concentration in effluent on day 73 was Table 3

16.3 mg/L which was decreased till day 86 to 95th day with minimum Comparative efficiencies of UASB and UASB-MFC in different forms of nitrogen
concentration of 3 mg/L and 0.38 mg/L respectively and then with removal.
some fluctuation nitrate concentration remained below 2 mg/L till the S.No. Different forms of N (mg/L) UASB UASB-MFC
end. Nitrite production was around 1–5 mg/L between 74 to 162 days
and 1.2 mg/L was achieved in the last day. In order to maintain ana- 1. Influent ammonium 40 40
2. Effluent ammonium 3.20 4.23
mmox condition and to produce more nitrite, the system was operated
3. Nitrite 5.9 3.02
at DO below 0.5 to 0.3 mg/L. Study indicated that DO less than 0.5 mg/ 4. Nitrate 2.80 1.2
L efficiently inhibit the development of nitrite oxidizer bacteria (NOB) 5. Total inorganic N in effluent 11.9 8.45
and affect nitrite accumulation [24].
Nitrite under heterotrophic condition acts as electron acceptor from
ammonium to produce N2. In UASB-MFC effluent, total inorganic
nitrogen,NO2 and NO3were 8.45, 3 and 1.2 mg/L respectively, lower electrode by electron shuttle of extracellular polymeric substances,
than in UASB effluent, whereas in UASB effluent ammonium con- present in the outer membrane of microbes 2) direct connection by
centration was 3.2 mg/L slightly lower than 4.23 mg/L of UASB-MFC attached extracellular polymeric substance with electrode which is an
effluent (Table 3). Although ammonium to nitrite and nitrate conver- ideal in electron transfer to electrode [14]. Anammox has special cy-
sion were higher in UASB, but less concentration of both in the UASB- toplasmic membrane, enable electron transit from the membrane to the
MFC effluent suggested the higher conversion of ammonium into ni- acceptor electrode. Continuous availability of electron acceptor such as
trogen gas, as depicted by more total nitrogen removal (Table 3). electrode, nitrite and nitrate are responsible to reduce more nitrogen
In UASB-MFC, bioelectrochemical treatment system depends upon under anoxic condition. Therefore it may support our assumption that
shift of electrons between electrode (anode) and biofilm formed on the presence of electrode helped in total nitrogen reduction. Another
electrode as attached growth or around the electrode as suspended study used anammox fluidized bed-MFC reactor, they found better
growth. Possibly two extracellular electron transfer mechanisms might ammonium and nitrite removal, as a result of bioelectricity production
have been taken place, 1) indirect process through electron shift to in MFC [25].

4
A. Iqbal, Z.A. Bhatti, F. Maqbool et al.
Fig. 3. A: Biokinetics study of the growth rate of anammox, nitrite and ammonia oxidizer of UASB reactor.B: Biokinetics study of the growth rate of anammox, nitrite
and ammonia oxidizer of UASB-MFC reactor.
3.2. Bio-kinetic study for the growth rate comparison between UASB-MFC
and UASB
Bio-kinetic growth model was applied on UASB and UASB-MFC to see
the effects on growth rate of anammox, ammonia oxidizing bacteria (AOB)
and nitrite oxidizing bacteria (NOB) under anammox condition of low DO
(0.5 mg/L) and unavailability of carbon source. In UASB, anammox growth
rate was increasing after 106 day and reached to its maximum value of
0.00213 g VSS/m3.d. till day 150 (Fig. 3A). Nitrite oxidizer growth rate was
start increasing after 74 days and gradual increase was observed till the end
and reached to its maximum value of 4.39 g VSS/m3.d. Ammonia oxidizer
was found in steady increased from 74 to 162 day with growth rate of 1.02 g
VSS/m3.d and 1.95 g VSS/m3.d.
In case of UASB-MFC, low growth rate of anammox 0.0005 g VSS/
m3.d was observed on day 77, which increased gradually with very slow
pace and reached to 0.0007 g VSS/m3.d on day 92 and maximum value
was observed as 0.0027 g VSS/m3.d on day 118. Later on insignificant
increased 0.00294 g VSS/m3.d was observed in the system, which might
be due to the low ammonium concentration in synthetic wastewater
[26,27]. The growth of AOB was 2 gVSS/m3.d on day 75 which was
gradually decreased and reached to minimum value of 1.03 g VSS/m3.d
5
Desalination and Water Treatment 318 (2024) 100300
on day 123. After day 135 it was increasing again and reached to above
2.21 g VSS/m3.d till 155 day. In UASB-MFC system AOB and NOB both
growth rate were observed in similar pattern (Fig. 3B). The growth rate
of NOB was increased from 0.397 g VSS/m3.d on day 123 to 0.95 g VSS/
m3.d on day 154. Average ammonium to nitrite ratio was 7:1 and later
on it was remain 1:1 which is good for anammox growth [5,19]. This
model predicts that AOB growth rate was higher than NOB throughout
the experiment and AOB was responsible to decrease ammonium con-
centration in UASB-MFC effluent [28].
Anammox growth appeared after 108 days in UASB whereas in
UASB-MFC anammox growth started after day 81 days. Nitrite oxidizer
may be supporting the growth of anammox bacteria by electron ac-
cepting mechanisms of the electrode. But in UASB nitrite production
found more than UASB-MFC which means ammonia oxidizing bacteria
working faster to oxidize ammonium to nitrite and due to lack of
electron acceptor less nitrate was produced.
In UASB, nitritation NH4 to NO2 conversion (40 mg/L to 5.9 mg/L)
was greater than the UASB-MFC (40 mg/L to 3.02 mg/L). Similarly,
nitratation in UASB (5.9 mg/L to 2.80 mg/L) was also greater than
UASB-MFC (3 to 1.2 mg/ L) (Table 3). This shows that AOB and NOB
was dominating in UASB than UASB-MFC (Table 4).
A. Iqbal, Z.A. Bhatti, F. Maqbool et al. Desalination and Water Treatment 318 (2024) 100300

Table 4 3.3. Metagenomic profile of UASB-MFC

Biokinetics model for the growth rate of anammox, AOB and NOB (gVSS/m3.d)
of UASB and UASB-MFC. Total nine bacterial phyla were identified through sequence ana-
S# Parameters UASB UASB-MFC lysis. By comparing different bacterial phylum of initial day, last day
and control samples, initially Firmicutes phylum was higher in both
1. Anammox growth rate 0.00213 0.00294 reactors. In UASB the level decreased from 51.5% to 18% while in
2. AOB 1.59 1.17
UASB-MFC, initially it was 35.7% which decreased to 20% in last day
3. NOB 2.86 0.464
sample (Fig. 4A and 4B). Firmicutes are anaerobic Gram positive bac-
AOB: Ammonium oxidizing bacteria growth rate, NOB: Nitrite oxidizing teria, mostly spore forming and involved in decomposition of organic
bacteria growth rate matter therefore found in higher level in UASB. They play important
role in phytoremediation, plant growth promotion and in microbial fuel
Table 5 cell power generation attached on the anode rods [29,30].
Alpha diversity indices results of 16 S rRNA sequencing from first and last day While Proteobacteria abundance increased from 23% to 34% in
samples of two reactors. UASB-MFC and 34.6% to 37% in UASB from initial to last day sample.
Shannon Observed Pielou Initially very low abundance of Actinobacteria (8.8%) (biofilm forming
diversity richness evenness bacteria) was found in UASB as compared to UASB-MFC (35%) abun-
dance, later on a drastic increased 31% was observed in the last day
UASBi 4.08 958 0.59
sample, while small increased 37% found in UASB-MFC but remained
UASBf 3.90 821 0.58
UASB-MFCi 3.68 842 0.55 very low in control sample of raw sludge 2.4% (Fig. 5A). One important
UASB-MFCf 3.24 664 0.50 phyla belongs to anammox bacteria named as Planctomycetes, its 2.8%
Control 3.86 970 0.56 abundance was found in the last day sample, which was initially absent,
showing anammox growth achievement in UASB-MFC, while its

Fig. 4. Percentage of relative abundance of different phylum in (A) UASB (B) UASB-MFC in which UASBi and UASB-MFCi = initial day, UASBf and UASB-
MFCf= last day and Control= raw sludge sample.

6
A. Iqbal, Z.A. Bhatti, F. Maqbool et al.
Fig. 5. Microbial community genera abundance (%) in (A) UASB (B) UASB-MFC. Different colors show relative abundance percentage. 1 = initial sample, 2 = last
day sample, 3 = raw sludge sample.
abundance was negligible in UASB. Kinetic growth model for anammox
growth rate also shows the limited growth rate. Previous study showed
that, although anammox exist in very small proportion of total bacteria,
they perform efficient role in ammonium and nitrite removal [31]. Si-
milarly a study by Sotres et al. [32] also identified Firmicutes, Proteo-
bacteria, Bacteroidetes, Actinobacteria and Tenericutes in MFCs fed with
synthetic wastewater. A study by Zhi et al. [33] listed that around 50
bacteria belonging to three phyla Proteobacteria, Firmicutes, and Acid-
obacteria have been identified as exoelectrogenic.
16 S rRNA sequencing at genera level study showed that the com-
positions of the microbial community in wastewater were changed
significantly during treatment (Fig. 5B). Genera Nocardioides which
belongs to Actinobacteria, its abundance increased at the end of treat-
ment in both reactors, Nocardioides are nitrate reducing electrogenic
7
Desalination and Water Treatment 318 (2024) 100300
bacteria [34]. Genera Clostridium, Aneurinibacillus and Lysinibacillus of
Firmicutes phylum abundance decreased in the last day sample in both
reactors compared with control. In UASB-MFC, phylum level classifi-
cation showed increased in Planctomycetes abundance in last day
sample but genera level classification has not identified any anammox
genera, while unclassified genera increased from 4% to 22% in the first
to last day sample respectively. Among this high amount of unclassified
genera might be some anammox genera, which have not been identified
through modified universal primers of Walters et al., [19] used in this
study.
Shannon diversity index, shows more diverse, rich and even bacterial
community was present initially in both reactors and in control samples,
while in the end under specific UASB and UASB-MFC conditions less diverse
and more specific dominant genera were found (Table 4).
A. Iqbal, Z.A. Bhatti, F. Maqbool et al.
Fig. 6. Voltage production variation in UASB-MFC.
3.4. Bioelectricity generation in UASB-MFC
In the UASB-MFC, during initial days the voltage production was
low (102 mV) then increased up to 774 mV on day 124 (Fig. 6) and
remained high till 150 day. It was also noticed that during this time
anammox growth rate was also at its peak value (Fig. 3B), which means
that ammonium oxidation resulted in higher voltage production. Si-
milarly, He et al., (2009) also found that, the addition of ammonium
alone or with nitrate or nitrite has increased bioelectricity production
[35]. Another study explored the effect of nitrogen removal on elec-
tricity generation; they found that the significantly higher nitrogen
removal of 92% was associated with higher voltage production [25].
Voltage production decreased till 162 day and fluctuation in the voltage
was observed till the end, with 433.7 mV an average voltage produc-
tion. Prolonged voltage production decreases the fuel cell life, therefore
it is well-known that the voltage production in MFCs drops with time
[36], due to decline in electrode working efficiency.
4. Conclusions
It is concluded that the presence of anode as an electron acceptor has
increased the total nitrogen removal under anammox condition in UASB-
MFC with high voltage production. Lower concentration of nitrate, nitrite
and total inorganic nitrogen were found in the UASB-MFC effluent, which
suggested the higher conversion of ammonium into nitrogen gas. Biokinetics
model also shows that AOB and NOB was dominating in UASB than UASB-
MFC but Planctomycetes (anammox) abundance increased significantly in
the last day sample only in UASB-MFC which shows better anammox
conditions developed in this reactor. Maximum voltage production was
achieved when ammonium oxidizers were high which related with higher
nitrogen removal in the UASB-MFC.
Funding
This research was funded by Higher Education Commission of
Pakistan NRPU No. 8985.
CRediT authorship contribution statement
Muhammad Faisal Siddiqui: Formal analysis. Samia Zeb: Writing
– review & editing. Yang-Guo Zhao: Methodology. Xu Lina:
Visualization, Software. Sajjad Ahmad: Investigation. Zakir Hussain:
Validation, Software. Asif Iqbal: Formal analysis, Data curation.
Zulfiqar Ahmad Bhatti: Funding acquisition, Conceptualization.
Farhana Maqbool: Writing – original draft, Supervision, Project ad-
ministration, Conceptualization.
8
Desalination and Water Treatment 318 (2024) 100300
Data Availability
Data will be made available on request.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Authors would like to thanks to Professor Deng Ye from Research
Centre of Eco-environmental Sciences CAS institute, Beijing for their
corporation in next generation DNA sequencing.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.dwt.2024.100300.
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