Bioresource Technology 315 (2020) 123802

Contents lists available at ScienceDirect

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

An oxic/anoxic-integrated and Fe/C micro-electrolysis-mediated vertical T

constructed wetland for decentralized low-carbon greywater treatment
⁎
Shihai Denga,d, Binghan Xieb, Qiang Kongc,d, , Shuai Penga, Hengchen Wange, Zhifeng Hua,
Desheng Lia
a
Department of Municipal and Environmental Engineering, School of Civil Engineering, Beijing Jiaotong University, Beijing 100044, PR China
b
School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai 264209, PR China
c
College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal
University, Jinan 250014, PR China
d
Centre for Water Research, Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, Singapore 117576, Singapore
e
China School of Environmental and Municipal Engineering, North China University of Water Resources and Electric Power, Zhengzhou 450046, PR China

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

A R T I C LE I N FO A B S T R A C T

Keywords: The treatment of decentralized low-carbon greywater in rural area, particularly in cold weather, remains a
Decentralized greywater challenge. Oxic/anoxic process and Fe/C micro-electrolysis were incorporated into vertical constructed wetland
Low organic carbon to develop ME-(O/A)CW for practical decentralized low-carbon greywater treatment. ME-(O/A)CW provided
Vertical constructed wetland NH4+-N, TN, TP and COD removal of 94.3%, 86.2%, 98.0% and 92.7%, respectively, at hydraulic loading rate of
Oxic/anoxic process
0.9 m3/(m2·d) under low ambient temperature of −11.5 to 8.0 °C. Effective nitrification, phosphorus-accu-
Iron-carbon micro-electrolysis
mulating and organic-degradation were proceeded in the aerobic layers and efficient H2-/Fe2+-mediated au-
totrophic denitrification and Fe3+-based phosphorus immobilization were developed in the anaerobic layers
through in-situ H2-/Fe2+-supply by Fe/C micro-electrolysis. AOB (e.g. Nitrosomonadales), NOB/PAOs (e.g.
Nitrospira), autotrophic denitrificans (e.g. Thiobacillus, Hydrogenophaga and Sulfurimonas), heterotrophic deni-
trificans (e.g. Denitratisoma) and Fe(II)-oxidizing bacteria (e.g. Ferritrophicum) dominated ME-(O/A)CW and
confirmed the reaction mechanisms. The developed ME-(O/A)CW presented significant potential in the practical
application for decentralized low-carbon greywater treatment under low ambient temperature.

⁎
Corresponding author at: College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of
Shandong, Shandong Normal University, Jinan 250014, PR China.
E-mail address: kongqiang@sdnu.edu.cn (Q. Kong).

https://doi.org/10.1016/j.biortech.2020.123802
Received 24 May 2020; Received in revised form 2 July 2020; Accepted 4 July 2020
Available online 10 July 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
S. Deng, et al.
1. Introduction
Greywater has been recognized as domestic wastewater excluded
toilet wastewater (blackwater) (Abu Ghunmi et al., 2010; Boyjoo et al.,
2013). The separate collection and decentralized treatment of grey-
water and blackwater is being increasingly encouraged around the
world, e.g. the USA, Europe and Australia (Boyjoo et al., 2013), as well
as the development of rural virgin areas in China (Xu et al., 2019). It
reduced the treatment load and increased the possibility of reuse of
greywater in rural area (Boyjoo et al., 2013). The separation collection
also increased the pollutants concentrations of blackwater and reduced
its volumetric burden. Therefore, the efficiency of ammonia recovery
(e.g. by struvite precipitation and ammonium stripping) (Ye et al.,
2018) and phosphorus recovery (e.g. by anaerobic fermentation from
phosphorus-rich sludge) (Li et al., 2018) could be improved, and the
energy generation (e.g. by methanogenesis and microbial fuel cells)
could be strengthened (Kurade et al., 2019; Wang et al., 2020).
Due to the harmful effect of untreated greywater and increasingly
water resources scarcity, greywater treatment has been indispensable
(Boyjoo et al., 2013; Hernández Leal et al., 2010). Many treatment
methods, e.g. physicochemical (Thirugnanasambandham et al., 2014)
and biological technologies including rotating biological contactor
(Friedler & Hadari, 2006), sequencing batch reactor (SBR) (Tombola
et al., 2019), and membrane bioreactor (MBR) (Lesjean & Gnirss, 2006),
have been developed. Although the physicochemical technologies are
efficient, their costs are quite high for sewage treatment (Li et al., 2015;
Thirugnanasambandham et al., 2014). Biological technologies are
capable for the treatment with much lower costs. However, the complex
maintenance of SBR and MBR rendered them ineligible when serving
for one or several households (Boyjoo et al., 2013; Tombola et al.,
2019).
Constructed wetlands (CWs) is an ecological technology obtaining
the advantages of small-footprint, low-construction-cost, energy-effi-
cient and simple-maintenance (Wu et al., 2015). Hence, it is well-suited
for the construction of various scales. However, with the diversion of
greywater and blackwater, a significant reduce of COD and BOD5 in
greywater is usually obtained (Boyjoo et al., 2013; Wu et al., 2015),
which has affected the biological denitrification/dephosphorization
(Gong et al., 2019; Xie et al., 2020). Eilersen et al. (1994) demonstrated
efficient denitrification could be achieved when COD to TN ratio (C/N)
exceeded 6–11 and low-carbon sewage was defined by Sun et al. (2010)
when C/N ratio was below 8.0. The C/N ratio of greywater varied a lot
with area and season at the range of 0.9–26.4 (Boyjoo et al., 2013; Xu
et al., 2019), indicating that existing carbon source is not sufficient for
denitrification/dephosphorization. Additionally, denitrifying phos-
phate accumulation would be inhibited (Peng et al., 2020).
Iron-carbon (Fe/C) galvanic cells can be formed with the contact of
Fe(0) and activated carbon (AC) in solutions with AC-cathode (Eq. (1))
and Fe(0)-anode (Eq. (2)), being also known as Fe/C micro-electrolysis
(Fe/C-ME) (Deng et al., 2016; O'Brien, 1964). Fe/C-ME could initiated
effective redox reaction using Fe(0) as sole electron donor comparing to
natural Fe(0)-corrosion (Eq. (3)). Therefore, Fe/C-ME has been adopted
to accelerate [H]/H2 and Fe2+ production to initiate H2-/Fe2+-sup-
ported autotrophic denitrification processes (ADP) (Deng et al., 2020;
Hu et al., 2019; Li et al., 2015). Jia et al. (2020) introduced Fe/C-ME to
CW to achieved high-rate nitrate reduction in groundwater treatment.
Additionally, the production waste iron-scraps (ISs), obtaining < 1/3 of
the price of zero-valent iron, can be used as Fe(0)-source even with a
low purity and activity (Deng et al., 2016). Moreover, Fe3+ produced
by Fe(II)-oxidation is capable for the phosphorus immobilization (Deng
et al., 2017; Li et al., 2019). Peng et al. (2020) combined Fe/C-ME into
anoxic/anaerobic/oxic process and obtained efficient simultaneous ni-
trogen and phosphorus removal. Hence, incorporation of Fe/C-ME into
CW is potential to enhance both the denitrification and depho-
sphorization.
2
Bioresource Technology 315 (2020) 123802
2H+ + 2e− → H2, φθ(H+/[H]) = 0.00 V (1)
− θ
Fe → Fe
0 2+
+ 2e , φ (Fe 2+
/Fe) = −0.44 V (2)
−
Fe + 2H2O → H2/2[H] + Fe 2+
+ 2OH (3)
Another key issue for CWs is its operation in cold weather. For in-
stance, the ambient temperatures in northwest and northern China are
usually as low as –10 °C and the water temperature could be below 6 °C,
and the activity of microorganisms are reduced (Li et al., 2014). Meng
et al. (2019) reported the reducing of AOB, NOB and denitrificans ac-
tivities by over 20% when operating temperature decreased from 35 °C
to 15 °C. Zhang et al. (2017) claimed a markedly decrease of biological
organic degradation rate. Extremely cold environment also leads to the
decrease of the air permeability of soil and block oxygen supply (Zhang
et al., 2017). Guarantee of the efficiency of CWs in cold weather is still
lack of research, particularly for the low-carbon greywater treatment.
This study aimed to: 1) develop an oxic/anoxic process (O/A)-in-
tegrated and Fe/C-ME-mediated CW [ME-(O/A)CW] for decentralized
low-carbon greywater treatment under various environmental condi-
tions; 2) explore the efficiency and mechanism of ME-(O/A)CW through
comparative experiments with AC- and ISs-mediated (O/A)CW; 3) in-
vestigate the impact of operation parameters on ME-(O/A)CW; and 4)
finally develop an strategy for the decentralized low-carbon greywater
treatment under low temperature.
2. Materials and methods
2.1. The development of ME-(O/A)CW
In the lab, Fe/C-ME based biocarrier (Fe/C-biocarrier) was devel-
oped based on ISs, AC, catalysts, binders and foaming agents. Fe/C-
biocarrier obtained feasible physical characteristics (BET specific sur-
face area of 69.3 ± 1.9 m2/g and porosity of 54.3 ± 2.8%) comparing
to the common used biocarriers, i.e. the bio-ceramic (BET specific
surface area of 20.8 ± 5.9 m2/g and porosity of 38.1 ± 5.5%), bio-
zeolite (BET specific surface area of 188.0 ± 35.0 m2/g and porosity of
50.7 ± 10.2%) and quartz-sand (BET specific surface area of
12.8 ± 2.4 m2/g and porosity of 33.8 ± 8.7%), which also have been
used in this study. It was incorporated into CW as biocarriers and in-situ
electron donor producer in the anaerobic layer (Fig. 1). The schematic
diagram and proposed mechanism of the developed ME-(O/A)CW are
shown in Fig. 1(c)-(d), in which vegetation layer, soil layer (A, 40 cm,
mixture of local soil and quartz sand), oxic layer (B, 40 cm, bio-zeolite),
anoxic layer (C, 40 cm, Fe/C-biocarrier) and anaerobic layer (D, 40 cm,
bio-cerramic) are set from top to bottom. The quartz sand layer (10 cm)
was set to stop the dripping of soil and the pebble layer (10 cm) was set
as an aquifer of effluent. Hardy plants reed (50%) and calamus (50%)
were planted in the vegetation layer at growing density of 20 plants/m2
(Li et al., 2014). Water distributers were set at the top of layer A. Air
distributers were set at the depth of 30 cm of layer B to maintain an oxic
condition in layers A-B. The lower 10 cm of layer B was reserved to
control the dissolved oxygen concentration (DO) of layer C.
2.2. The presumptive designing reaction mechanisms
Fig. 1(d) also shows the presumptive designing reaction mechan-
isms in the developed ME-(O/A)CW system. In the vegetation layer,
nitrogen and phosphorus can be partly removed by the absorption of
plant roots for assimilation process of the plants, during which, nitrogen
is transformed into amino chemichass for protein synthesis, phosphorus
is utilized to synthesize adenosine triphosphate, nucleic acid, phos-
pholipid, etc (Wu et al., 2014). With the continuous supply of oxygen in
layers A and B, NH4 ± can be stepwise oxidized in to NO2−-N and
NO3−-N by the ammonia-oxidizing bacteria, i.e. AOB, and the nitrite-
oxidizing bacteria, i.e. NOB, respectively. Phosphorus can be absorbed
as poly-P by the phosphate-accumulating organisms (PAOs), e.g.
S. Deng, et al. Bioresource Technology 315 (2020) 123802

Fig. 1. Schematic diagram of the reaction systems and the reaction mechanisms of ME-(O/A)CW. Lab-scale reactors (ɸ8 cm × 200 cm) of (a) AC-(O/A)CW, (b) ISs-
(O/A)CW and (c) ME-(O/A)CW. Pilot-scale ME-(O/A)CW (0.6 m × 0.4 m × 2 m) filled with Fe/C-carriers and other bio-carriers.

Nitrosospira (Xu et al., 2014) and Candidatus (De Vleeschauwer et al.,
2019). Simultaneously, organics is partly degraded into CO2 and H2O.
In layer C, Fe2+ and [H]/H2 can be continuously in-situ produced by
Fe/C-ME and raise H2-/Fe2+-supported autotrophic-denitrifying bac-
teria (ADB) for denitrification. At the same time, heterotrophic-deni-
trifying bacteria (HDB) conducts heterotrophic denitrification using the
residual organics as electron donor. Moreover, phosphorus is partly
released in layer C and D by PAOs, which could be efficiently pre-
cipitated with Fe3+ generated from the Fe2+-ADP (Deng et al., 2017; Li
et al., 2019). Phosphorus could also be partly removed by denitrifying
phosphate-accumulating organisms (DPAOs) during denitrification (Wu
et al., 2020). In cold weather, aeration can effectively enhance ni-
trification and organic degradation process. Coupling of Fe/C-ME into
CW could made up for the insufficient carbon source supply during
greywater treatment, and simultaneously enhance denitrification and
dephosphorization in cold weather by in-situ [H]/H2 and Fe2+ supply.
2.3. Lab-scale study: comparative study of AC-, ISs- and ME-(O/A)CW
To evaluate the performance and verify the reaction mechanism of
ME-(O/A)CW, AC-mediated (O/A)CW [AC-(O/A)CW] and ISs-mediated
(O/A)CW [ISs-(O/A)CW] were developed by packing AC and ISs in
layer C of (O/A)CW, respectively (Fig. 1(a) and (b)). Lab scale AC-(O/
A)CW, ISs-(O/A)CW and ME-(O/A)CW were set up with plexiglass
columns at the same size (8-cm inner diameter and 200-cm height) with
packing volume of 9.0 L obtaining water capacity of 4.2 ± 0.1 L after
packing biocarriers to the height of 180 cm (Fig. 1). Continuous op-
eration of the three reactors were initiated naturally without inocula-
tion. The same operation condition of aeration intensity 4:1 (given as
air to water volume ratio, A/W), hydraulic loading rate (HLR) 0.7 m3/
(m2·d) and water temperature 18–22 °C were maintained. After a start-
up, stable operation of over 20 days on the CWs was conducted. The
effluent and influent were sampled daily. Samples along the reactors
were collected on operational days 52–56.
2.4. Pilot-scale study: environmental factors affecting the ME-(O/A)CW
An outdoor pilot-scale system was set up in northern China to in-
vestigate the impact of operational conditions and ambient temperature
on ME-(O/A)CW. The system was set up with a burried cuboid stain-
less steel container with height of 2.0 m, sectional area of 0.6 × 0.4 m2
(Fig. 1(d)). The reactor obtained a packing volume of 432 L and water
capacity of 181.6 L after packing biocarriers to the height of 1.8 m.
During the start-up, biocarriers in the lab-scale ME-(O/A)CW were
mixed into the corresponding layers of the pilot-scale system for in-
oculation. Continuous operation of the pilot-scale ME-(O/A)CW totally
lasted 236 days including start-up process and 10 operational stages
(Table 1). Table 1 summarized the operation procedure, conditions and
duration of each stage. Reeds and calamus were harvested in October
and top of ME-(O/A)CW was covered with 30-cm thick insulated cotton
(Softwoll®, China) for heat preservation. The effluent and influent were
sampled daily.
2.5. Practical domestic greywater and analytical methods
Practical decentralized low-carbon greywater from a countryside of
northern China was treated in the lab-scale and pilot-scale study of this
research. An anaerobic regulation tank was set and a 24-h hydraulic
retention time was given to maintain a relatively stable influent quality
for the CWs: NH4+-N of 29.7–41.2 mg/L, total phosphorus of
2.8–4.9 mg/L, NO3−-/NO2−-N of < 0.1 mg/L (ignored during discus-
sion), COD of 88.7–204.1 mg/L and SS of 55.3–122.9 mg/L. The cal-
culated C/N ratio of the used practical domiectical greywater in this
study is 2.9–6.8 which showed a typical low-carbon character. Water
samples were daily analyzed, during which NO3−-N, NO2−-N, NH4+-N,
COD and TP were analyzed using an ultraviolet and visible

3
S. Deng, et al. Bioresource Technology 315 (2020) 123802

Table 1
Variation of operating conditions of the pilot-scale ME-(O/A)CW at different operational stages (Bold values indicate the change of operation parameters.).
Operational stage Duration (day) Month Aeration intensity (A/W) Hydraulic loading rate (m3/(m2·d)) Ambient temperature (°C) Water temperature in CW (°C)

Start-up 1 ~ 33 Jun. ~ Oct. 4:1 0.7 16.5 ~ 37.5 14.2 ~ 24.3

I 34 ~ 50 8:1 0.7
II 51 ~ 66 12:1 0.7
III 67 ~ 82 8:1 0.9
IV 83 ~ 96 8:1 1.1
V 97 ~ 113 8:1 1.3
VI 114 ~ 138 8:1 1.1

VII 139 ~ 159 Oct. ~ Nov. 8:1 1.1 7.0 ~ 16.5 7.7 ~ 13.3
VIII 160 ~ 191 12:1 1.1

IX 192 ~ 217 Dec. ~ Jar. 12:1 1.1 –11.5 ~ 8.0 5.4 ~ 7.8
X 218 ~ 236 12:1 0.9

spectrophotometer (UV-1800PC, MAPADA Co., China) with the stan-
dard methods given by APHA (APHA, 2005). Total nitrogen (TN) was
calculated as the sum of concentrations of NO3−-N, NO2−-N and NH4+-
N. DO and water temperature were in-situ monitored by the DO-meter
(Cole-Parmer, USA) using DO probe (YSI 5739). Ambient temperature
was read from the daily local weather reports. The average value
(MEAN) and standard deviation (SD) were calculated and used for
discussion in this study.
2.6. Molecular microbiology analysis
Bio-samples of the lab-scale AC-, ISs- and ME-(O/A)CW were taken
on operational day 56 from the AC-, ISs- and Fe/C-layer (Fig. 1) namely
B-AC, B-ISs and B-Fe/C, respectively. Bio-samples at layers A, B, C and
D of the pilot-scale ME-(O/A)CW were collected and named L-A, L-B, L-
C and L-D, respectively. Each bio-sample was collected in triplicate and
mixed together as one sample for analysis. A 15-min centrifugation
process under 5000 rpm and 4 °C were conducted on the samples,
followed by genomic DNA extraction, which was conducted in triplicate
with Omega3® DNA Isolation Kits. Molecular biological analysis was
proceeded on the mixture of the extracts from each sample. The primer
pair of 338F/806R (5′-ACTCCTACGGGAGGCAGCAG-3′/5′-GGACTAC-
HVGGGTWTCTAAT-3′) was utilized in the 16S rRNA PCR amplification
at gene level. Illumina® MiSeq (Maj. Bio. Tech., Shanghai) high-
throughput sequencing (HTS) obtained valid sequences of 37,280,
24,613, 30,845, 23,188, 24,818, 35,242 and 26,408 were obtained
from B-AC, B-ISs, B-Fe/C, L-A, L-B, L-C and L-D, respectively, after
quality filtration. The diversity indices showed that 99.6%–99.7% of
the obtained sequences were valid, demonstrating that the sequencing
process is sufficient to characterize the microbial communities. OTUs
were clustered from the obtained sequences at similarity of over 97%
(UPARSE, Version7.0). Heat-map was generated with R-code on Ma-
jorbio Cloud Platform (http://www.majorbio.com) at the level of
genus.
3. Results and discussion
3.1. Comparison on the AC-(O/A)CW, ISs-(O/A)CW and ME-(O/A)CW
3.1.1. Performance
Continuous operation of lab-scale AC-, ISs- and ME-(O/A)CW lasted
56 days including microbial cultivation (36 days) and stable operation
(20 days). Their performances on nitrogen, phosphorus and COD re-
moval are shown in Fig. 2. As Fig. 2 shows, the start-up of the three CWs
lasted 25, 31 and 36 days, respectively. The AC obtained better ad-
sorption property than ISs and Fe/C-biocarrier, which benefited the
microbial cultivation of AC-(O/A)CW (Fig. 3(a1)-(c1)). However,
during the stable operation, the AC-(O/A)CW only obtained average TN
removal of 51.0% and surface denitrification load (SDL) of 11.7 g N/
(m2·d) with effluent NO3−-N, NO2−-N and NH4 ± -N concentrations of
12.4, 0.93 and 4.6 mg/L, respectively. Comparatively, ISs-(O/A)CW
and ME-(O/A)CW achieved higher TN removal of 57.9% and 82.6%,
respectively. The variation of COD along the CWs showed that COD
removal mainly happened in layers A and B but the TN removal of ME-
(O/A)CW mainly appeared in layer C. It was much higher than the TN
removal of AC-(O/A)CW. It was worth noting that NO2−-N accumula-
tion has been found in the AC- and ISs-(O/A)CW at effluent NO2−-N
concentration of 0.93 and 4.2 mg/L due to the insufficient electron
donor, comparing to the 0.29 mg/L in ME-(O/A)CW. The results de-
monstrated that the existing carbon source was not sufficient to support
HD process, and ISs and Fe/C-biocarrier performed as electron supplier.
Fe/C-biocarrier showed much higher capacity of [H]/H2 and Fe2+
supply than ISs, obtaining SDL of 20.7 g N/(m2·d) in ME-(O/A)CW
comparing to the 14.2 g N/(m2·d) in the ISs-(O/A)CW. Meanwhile, the
addition of ISs and Fe/C-biocarrier in CW significantly increased TP
removal from 37.1% to 82.2% and 94.8%, respectively. Meanwhile,
ME-(O/A)CW performed more effective in phosphorus immobilization
with effluent TP of 0.22 mg/L comparing to the 0.74 mg/L of ISs-(O/
A)CW and 2.5 mg/L of AC-(O/A)CW. Consequently, the incorporation
of Fe/C-ME is capable for the enhancement of greywater treatment by
CWs.
3.1.2. Nitrogen and phosphorus removal along the reactor
Fig. 3 illustrates the concentrations of NO3−-N, NO2−-N, NH4 ± -N,
and TP, as well as the corresponding TN and TP removal efficiency
along different layers of each reactor. As Fig. 3(a1)-(c1) shows, the CWs
obtained efficient nitrification in layers A and B under the enhancement
of aeration. The AC-, ISs- and ME-(O/A)CW achieved average ammonia
removal of 87.5%, 85.1% and 85.3% with average effluent NH4+-N of
4.6, 5.5 and 5.4 mg/L, respectively. The results demonstrated the suc-
cessful incorporation of O/A process for the enhancement of nitrifica-
tion in CW. Denitrification mainly proceeded in the layers C and D in
the CWs according to Fig. 3(a1)-(c1). In the AC- and ISs-(O/A)CW, the
TN removal efficiencies increased from 13.6% and 16.0% to 49.3% and
47.6% in layer C, respectively. Comparatively, the TN removal effi-
ciencies in ME-(O/A)CW increased from 14.3% to 58.2% in layer C, and
a continuous increase of TN removal to 82.6% in layer D of ME-(O/
A)CW was also observed. The variation of iron ions showed that Fe2+
and Fe3+ concentrations in layer C of ISs-(O/A)CW and ME-(O/A)CW
markedly increased comparing to that of AC-(O/A)CW, and that the
Fe2+ and Fe3+ concentrations in ME-(O/A)CW (3.3 mg/L and 3.7 mg/
L, respectively) were much higher than that of the AC-(O/A)CW
(0.17 mg/L and 0.06 mg/L, respectively) and ISs-(O/A)CW (1.1 mg/L
and 2.0 mg/L, respectively). The result demonstrated that the effective
[H]/H2 and Fe2+ production from Fe/C-ME supported denitrification in
layer C and the residual dissolved H2/Fe2+ continuously supported
denitrification in layer D. It was worth noting that ISs-(O/A)CW has
shown a significant NO2−-N accumulation of 5.4 and 4.2 mg/L, re-
spectively. The phenomenon accords with the previous study that
NO2−-N accumulated in Fe2+-ADP with limited electron donor supply

4
S. Deng, et al.
Fig. 2. Nitrogen, phosphorus, and COD removal of (a1 ~ a2) AC-(O/A)CW, (b1 ~ b2) ISs-(O/A)CW, and (c1 ~ c2) ME-(O/A)CW. a1, b1 and c1: Influent (Inf.) NH4+-
N, effluent (Eff.) NH4+-N, Eff. NO3−-N and Eff. NO2−-N, and surface denitrification load (SDL); a2, b2 and c2: Inf. TP, Eff. TP, and removal efficiencies (RE) of NH4+-
N, TN, TP and COD.
(Tian & Yu, 2020). Therefore, special attention on NO2−-N accumula-
tion is required when utilizing ISs as electron donor for denitrification.
In terms of TP removal in Fig. 3(a2)-(c2), PAOs showed effective
phosphorus absorption under oxic condition in layers A and B. Never-
theless, a slightly rise of TP was observed in layers C and D of AC-(O/
A)CW due to the phosphorus releasing of PAOs by the simultaneous
disintegrating of poly-P and glycogen under anoxic and anaerobic
conditions (De Vleeschauwer et al., 2019). However, continuous in-
crease of TP removal was observed with average TP removal of 83.5%
and 94.6% in ISs-(O/A)CW and ME-(O/A)CW, respectively. They are
higher than that in the AC-(O/A)CW (38.2%). The variation of iron ions
showed that Fe3+ concentration in layer C of ME-(O/A)CW was much
higher than those of the AC-(O/A)CW and ISs-(O/A)CW. The result
verified the effective phosphate immobilization with the Fe3+ gener-
ated from the Fe2+-ADP. The result demonstrated that the incorpora-
tion of ISs and Fe/C-ME in layer C effectively enhanced the TP removal
from greywater by CWs.
3.1.3. Microbial characteristics of the AC-, ISs- and Fe/C-layers
Abundant microorganisms into bacterial genus based on 16S rRNA
Illumina® MiSeq HTS in the AC-, ISs- and Fe/C-layers of the AC-, ISs-
and ME-(O/A)CW have been analyzed, respectively. Overall, the
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Bioresource Technology 315 (2020) 123802
microbial communities showed abundance in AOB, NOB, HDB, ADB,
PAOs and DPAOs. Abundant AOB of Nitrosomonadales (Maharjan et al.,
2020; Wang et al., 2018) and NOB of Nitrosospira (Peng et al., 2020; Xu
et al., 2014) were obtained in layer C of three reactors covering
0.7%–2.8% of the sequences due to the residual DO (0.2–0.5 mg/L).
HDB Denitratisoma (9.4%), Zoogloea (4.2%) and Pseudomonas (6.0%)
were found dominant in AC-(O/A)CW, but their abundance decreased
in ISs-(O/A)CW (7.0%, 3.5% and 0.3%, respectively) and ME-(O/A)CW
(4.9%, 3.7% and 0.2%, respectively) (Jiang et al., 2020; Mohseni-
Bandpi et al., 2013; Wu et al., 2020). However, a significant increase of
ADB (e.g. Thiobacillus, Hydrogenophaga and Hyphomicrobium)
(Karanasios et al., 2010; Kiskira et al., 2017; Vandekerckhove et al.,
2018) and ferrous iron-oxidizing bacteria (Fe(II)OB, e.g. Ferritrophicum)
(Deng et al., 2020; Kiskira et al., 2017) in ISs-(O/A)CW and ME-(O/
A)CW was observed. Genera Hydrogenophaga and Hyphomicrobium have
been identified as H2-supported ADB (Karanasios et al., 2010) and
Thiobacillus has been identified as sulfur-, H2- and Fe2+-supported ADB
(Karanasios et al., 2010; Vandekerckhove et al., 2018). The results
demonstrated that the incorporation Fe/C-ME shifted the microbial
community structure thereby affecting the functions. The higher
abundance of genera Hydrogenophaga and Ferritrophicum in ME-(O/
A)CW than ISs-(O/A)CW verified the higher [H]/H2 and Fe2+
S. Deng, et al.
Fig. 3. Nitrogen and phosphorus removal of (a1 ~ a2) AC-(O/A)CW, (b1 ~ b2) ISs-(O/A)CW and (c1 ~ c2) ME-(O/A)CW. a1, b1 and c1: Influent (Inf.) NH4+-N,
effluent (Eff.) NH4+-N, Eff. NO3−-N and Eff. NO2−-N, and removal efficiency (RE) of TN; a2, b2 and c2: Inf. TP, Eff. TP, and RE of TP.
production of Fe/C-ME than ISs.
3.2. Influence of aeration intensity and hydraulic loading rate on ME-(O/
A)CW
Using biocarriers from the lab-scale reactors for inoculation, 23 days
were taken for pilot-scale ME-(O/A)CW start-up (Fig. 4). Subsequently,
the influence of aeration intensity and HLR on ME-(O/A)CW was in-
vestigated in stages I-VI under ambient temperature of 18.0–37.5 °C
and corresponding water temperature of 14.5–24.3 °C, which have a
relative small impact comparing to the changes of DO and HLR. After
start-up, ME-(O/A)CW stabled at A/W ratio of 4:1 (DO of 1.9–2.4 mg/L
was detected in layer B) and HLR of 0.7 m3/(m2·d) obtaining average
NH4+-N, TN, TP and COD removal of 85.3%, 82.2%, 95.1% and 82.1%
with corresponding effluent concentrations of 5.4, 6.5, 0.22 and
25.6 mg/L, respectively. The effluent NH4+-N and COD were relatively
high and resulted in the high effluent TN concentration. A/W ratio was
6
Bioresource Technology 315 (2020) 123802
increased to 8:1 and 12:1 in stages I and II with detected DO of 3.1–3.6
and 3.7–4.4 mg/L in layer B, respectively, to enhance the nitrification
processes. TP removal continuously increased to 98.9% when in-
creasing the A/W ratio to 12:1 (Fig. 4), which was attributed to the
enhancement of aeration on phosphorus-accumulating by the PAOs (Xu
et al., 2014). As Fig. 4 shows, average effluent NH4+-N dropped to
0.96 mg/L at A/W of 8:1, and the corresponding TN and COD removal
increased to 93.8% and 94.4%, respectively. However, when increasing
A/W to 12:1, effluent NO3−-N shift to 2.6 mg/L and TN removal
dropped to 90.6%. This result indicated that the relatively high residual
DO (0.6–1.1 mg/L) in layer C slightly inhibited denitrification process.
When the A/W ratio was controlled at 8:1, HLR was increased from
0.7 to 0.9, 1.1 and 1.3 m3/(m2·d) in stages III, IV and V, respectively. A
slight drop in removal efficiencies of the contaminants are observed in
Fig. 4 when increasing HLR to 0.9 and 1.1 m3/(m2·d). At HLR of 1.1 m3/
(m2·d), the removal efficiencies of NH4+-N, TN, TP and COD are 93.8%,
85.8%, 97.3% and 91.8% with corresponding effluent concentrations of
S. Deng, et al.
Fig. 4. Influence of aeration intensity (A/W) and hydraulic loading rate (HLR) on nitrogen, phosphorus, and COD removal of ME-(O/A)CW. (a): Influent (Inf.) NH4+-
N, effluent (Eff.) NH4+-N, Eff. NO3−-N and Eff. NO2−-N and surface denitrification load (SDL); (b): Inf. TP, Eff. TP, and removal efficiencies (RE) of NH4+-N, TN, TP
and COD. The system was operated from June to October at ambient temperature of 16.5 ~ 37.5 °C and the corresponding water temperature of in ME-(O/A)CW was
detected at 14.2 ~ 24.3 °C.
1.7, 4.9, 0.12 and 12.3 mg/L, respectively. However, with the con-
tinuous increase of HLR to 1.3 m3/(m2·d), the effluent NH4+-N, TN, TP
and COD shifted to 6.2, 13.4, 0.41 and 32.7 mg/L. Consequently, the
optimal A/W ratio of 8:1 and HLR of 1.1 m3/(m2·d) are concluded
under ambient temperature of 18.0–37.5 °C. Under this condition, ME-
(O/A)CW obtained TN, TP and COD removal loads of 35.2, 4.7 and
158.4 g/(m2·d), respectively, which were dramatically higher than the
8.2, 1.9 and 101.0 g/(m2·d) calculated from the vertical flow CW re-
ported by Dong et al. (2012) at HLR of 0.76 g/(m2·d), respectively, and
the 9.2, 0.70 and 68.9 g/(m2·d) calculated from the vertical flow CW
reported by Foladori et al. (2013) under HLR of 0.17 m3/(m2·d), re-
spectively. The results indicated that the incorporating O/A process and
Fe/C-ME effectively increased the treatment capacity of CWs.
3.3. Influence of ambient temperature on ME-(O/A)CW
Cold weather in northern China usually starts in October with sig-
nificant drop of ambient temperature, which started at the end of op-
eration stage VI (Fig. 5). According to the change of temperature and
performance of ME-(O/A)CW, the operation in cold weather was di-
vided into two phases in this study. Phase 1 was in October and No-
vember under ambient temperature of 7.0–16.5 °C and corresponding
detected water temperature of 7.7–13.3 °C (Stages VII-VIII in Fig. 5);
Phase 2 was in December and January of the coming year under am-
bient temperature of −11.5 to 8.0 °C and corresponding detected water
temperature of 5.4–7.8 °C (Stages IX-X in Fig. 5). The performance of
7
Bioresource Technology 315 (2020) 123802
ME-(O/A)CW at phase 1 and phase 2 were compared to operational
stage VI (in September and October at ambient temperature of
16.5–27.5 °C and corresponding detected water temperature of
14.2–20.7 °C).
As Fig. 5 shows, ME-(O/A)CW operated at the same A/W ratio and
HLR in stage VI and VII, but the water temperature dropped from
14.2–20.7 °C to 7.7–13.3 °C with the drop of ambient temperature. With
this drop of temperature, effluent concentrations of NO3−-N, NO2−-N
and NH4+-N remained relatively stable at 1.2–2.4, 0.12–0.66 and
0.05–0.21 mg/L, respectively. However, average concentrations of
NH4+-N and COD in the effluent increased to 5.7 and 25.7 mg/L, re-
spectively. The results indicated that the drop of ambient temperature
first affected the upper layer (A and B) of CWs in which nitrification and
organics degradation proceeded. Therefore, A/W ratio was increased to
12:1 in stage VIII to enhance the NH4+-N and COD removal. As is
shown in Fig. 5, the average effluent NH4+-N and COD was reduced to
1.4 and 16.4 mg/L, respectively. Consequently, optimal operation
conditions of A/W ratio 12:1 and HLR 1.1 m3/(m2·d) were obtained,
and removal efficiencies of 96.2, 87.1%, 97.8% and 93.7% on NH4+-N,
TN, TP and COD were achieved by ME-(O/A)CW at the ambient tem-
perature of 7.0–16.5 °C, respectively.
In stage IX, the water temperature decreased to 5.4–7.8 °C with
continuously drop of ambient temperature to –11.5–8.0 °C (Fig. 5). As is
observed in Fig. 5, the effluent NO3−-N, NO2−-N, NH4+-N, and TP rose
to 6.6, 1.5, 4.9 and 0.41 mg/L, respectively, and the TN, TP and COD
removal efficiencies decreased to 64.7%, 90.8% and 85.9%,
S. Deng, et al.
Fig. 5. Influence of ambient temperature (AT) and water temperature (WT) on nitrogen, phosphorus, and COD removal of ME-(O/A)CW. (a): Influent (Inf.) NH4+-N,
effluent (Eff.) NH4+-N, Eff. NO3−-N and Eff. NO2−-N and surface denitrification load (SDL); (b): Inf. TP, Eff. TP, and removal efficiencies (RE) of NH4+-N, TN, TP and
COD.
respectively. This results demonstrated that the extremely low ambient
temperature affected all the bio-reactions in the CW including ni-
trification, denitrification, dephorsphorization and organic degrada-
tion. Therefore, the HLR was reduced to 0.9 m3/(m2·d) to maintain the
removal efficiency of the contaminants. As is illustrated in stage X of
Fig. 5, the performance of ME-(O/A)CW recovered in 7 days after HLR
adjusting, with average effluent NO3−-N, NO2−-N, NH4+-N, and TP of
2.4, 0.58, 2.1 and 0.09 mg/L, and NH4+-N, TN, TP and COD removal of
94.3%, 86.2%, 98.0% and 92.7%, respectively. Therefore, optimal op-
eration condition of A/W ratio 12:1 and HLR 0.9 m3/(m2·d) was ob-
tained at ambient temperature of –11.5–8.0 °C. Consequently, under the
enhancement of aeration and Fe/C-ME the CW obtained efficient pol-
lutants removal under low temperature.
3.4. Distribution of functional microbes in different layers of ME-(O/A)CW
The principal component analysis (PCA) result was explained by
PC1 of 68.19% and PC2 of 21.71%. The communities of L-A/L-B and L-
C/L-D were closely clustered, respectively, which were in accordance
with the oxic condition in layers A/B and anoxic condition in layers C/
D. At the level of phylum (Fig. 6(a)), Proteobacteria accounted for the
highest OTU sequences of 60.5%-77.5%. At the level of class, the α-, β-
and γ-Proteobacteria totally covered 52.5%, 58.6%, 69.5% and 70.9% of
the sequences in L-A, L-B, L-C and L-D, respectively (Fig. 6(b)), in which
a number of AOB, NOB, HDB, ADB, PAOs and DPAOs have been
identified. Moreover, over 14 species of ADB have been identified in
8
Bioresource Technology 315 (2020) 123802
bacterial class α-, β- and γ-Proteobacteria (Deng et al., 2020; Karanasios
et al., 2010).
Fig. 6(c) illustrated the heat-map at level of genus. A significant
enrichments of genera Nitrospira, Nitrosomonadales and Nitrosomonas
were observed in L-A and L-B. Genera Nitrosomonadales and Ni-
trosomonas have been verified as abundant AOB in the nitrification
systems (Maharjan et al., 2020; Wang et al., 2018). Many species pos-
sessed by genus Nitrospira have been recognized as NOB, e.g. spiece
Nitrospira defluvii (NC_014355.1) in NCBI Nucleotide (Peng et al., 2020;
Xu et al., 2014). Genus Nitrospira has also showed function of phosphate
accumulating as PAOs in TP removal (Xu et al., 2014). The increase in
the abundance of Nitrospira (from 6.5% to 8.9%), Nitrosomonadales
(from 2.5% to 9.2%) and Nitrosomonas (from 10.1% to 10.8%) de-
monstrated the function of nitrification and phosphorus immobilization
in layers A and B. Additionally, the dominant genus Algoriphagus in
layers A and B (13.2% and 1.7%, respectively) is functional in organic
degradation under aeration (Ahmad et al., 2019).
The abundance of genera Nitrospira, Nitrosomonadales, Nitrosomonas
and Algoriphagus significant decreased under the anoxic condition in
layers C and D, obtaining abundances of 1.1%, 2.8%, 0.0% and 0.0%,
respectively, in layer D. However, markedly increase in the abundances
of HDB/DPAOs, e.g. Denitratisoma (5.8%) (Peng et al., 2019; Wu et al.,
2020), Thauera (7.1%) (Deng et al., 2019; Li et al., 2017) and Zoogloea
(4.0%) (Mohseni-Bandpi et al., 2013), and ADB, e.g. Thiobacillus
(7.7%), Hydrogenophaga (7.3%), Sulfurimonas (2.0%) and Hyphomicro-
bium (3.2%), in layer C have been observed, which is quite similar to
S. Deng, et al.
Fig. 6. Distribution of microbes into (a) bacterial phyla and (b) bacterial classes, and (c) heat-map at the genus level based on Illumina® MiSeq sequencing of 16S
rRNA. The relative abundances of the genera in the heat-map are indicated by the color intensity, representing the percentages (%) of the OTU-reads of targeted
genera in the total reads. The communities accounted for ≥1.0% of the total OTU reads are shown.
the sample B-Fe/C (Section 3.1.3). The above ADB have been identified
as gram-negative bacterium, among which Thiobacillus and Hydro-
genophaga belong to class β-proteobacteria, Sulfurimonas belong to class
ε-proteobacteria and Hyphomicrobium belong to class α-proteobacteria.
Genus Thiobacillus has been found abundant in the sulfur-, H2- and Fe
(II)-mediated autotrophic denitrification systems (Deng et al., 2020;
Kiskira et al., 2017; Vandekerckhove et al., 2018). A number of pure
cultures of the functional species comprised by Thiobacillus have been
cultivated, e.g. Thiobacillus_denitrificans (NR025358.1) and Thioba-
cillus_thioparus (HM535225.1), and some of them have been facilitated
in engineering applications as obligate chemolithotrophs for nitrate
removal (Deng et al., 2020; Vandekerckhove et al., 2018). Genera Hy-
drogenophaga and Hyphomicrobium have been demonstrated as the main
functional bacteria in many H2-mediated and Fe(0)-corrosion-based
denitrification systems (Deng et al., 2016; Karanasios et al., 2010; Peng
et al., 2019). The autotrophic denitrifying ability of Sulfurimonas has
been verified in the sulfur-, pyrite- and other sulfide-mediated deni-
trification systems (Vandekerckhove et al., 2018). Dominance of ADB in
layer C demonstrated that the [H]/H2 and Fe2+ supply of Fe/C-ME has
supported effective autotrophic denitrification, which made up for the
low nitrogen removal due to lack of organic carbon source. The genera
Thiobacillus, Hydrogenophaga, Sulfurimonas and Hyphomicrobium also
obtained relatively high abundancces of 15.9%, 4.7%, 3.2% and 3.3%,
respectively. This result confirmed the continuous increase of TN re-
moval observed in layer D of ME-(O/A)CW in Section 3.1.2. Ad-
ditionally, the Fe(II)OB genera Ferritrophicum (Deng et al., 2020; Kiskira
et al., 2017) also obtained high abundances of 10.5% and 6.9% in layer
C and D, respectively, which verified the in-situ Fe3+ generation process
9
Bioresource Technology 315 (2020) 123802
for phosphorus immobilization. The above results have indicated that
the Fe/C-ME in ME-(O/A)CW is effective to supply [H]/H2 and Fe2+ in
layer C and simultaneously maintained residual dissolved H2 and Fe2+
for continuous denitrification and phosphorus immobilization in layer
D to further improve TN and TP removal for the decentralized low-
carbon greywater.
Consequently, the incorporation of O/A process and catalytic Fe/C-
ME into CW significantly enhanced the NH4+-N, TN, TP and COD re-
moval of decentralized low-carbon greywater. AOB, NOB and PAOs
were cultured in the oxic layers A and B under aeration, which pro-
ceeded effective nitrification, phosphorus accumulating and organic
degradation. Fe/C-ME was coupled in the anoxic layer C as an efficient
in-situ supplier of inorganic electron donors (i.e. [H]/H2 and Fe2+).
Subsequently, autotrophic denitrification and Fe3+-based phosphorus
immobilization were raised in layers C and D, which made up for the
lack of organic carbon source and achieved high TN and TP removal
efficiencies in the decentralized low-carbon greywater treatment.
3.5. Implications to environmental application
Generally, hydraulic loading of the treatment system has to be sla-
shed to achieve higher nitrogen and phosphorus removal due to the
reducing of microbial activity during wastewater treatment at low
temperature (Meng et al., 2019; Zhang et al., 2017). Moreover, in-
sulation measure (e.g. constructing the treatment plants indoor) need to
be taken and the chemical dosages need to be increased to achieve high
denitrification and dephosphorization rates (Meng et al., 2019). These
measures would significantly increase both the capital investment and
S. Deng, et al.
operational cost. Additionally, the addition of external electron donors
has been required in the low-carbon wastewater treatment (Tian & Yu,
2020). In the present study, ME-(O/A)CW was developed, in which O/A
and Fe/C-ME processes were incorporated to simultaneously enhance
the nitrification, denitrification and dephosphorization under low
temperature. As the results shows, nitrification effectively proceeded in
the aerobic layers, and efficient denitrification and dephosphorization
were initiated in the anaerobic layers. Under the low temperature of
−11.5 to 8.0 °C, the ME-(O/A)CW achieved high TN, TP and COD re-
moval loads of 35.2, 4.7 and 158.4 g/(m2·d) with high HLR of 0.9 m3/
(m2·d), respectively, comparing to the results of Dong et al. (2012) and
Foladori et al. (2013) utilizing traditional CW. The effluent NH4+-N
(2.1 mg/L), TN (5.1 mg/L), TP (0.09 mg/L) were significantly lower
than the level A of National Discharge Standard of Pollutants for Mu-
nicipal Wastewater Treatment Plant of China (GB18918-2002) (NH4+-
N ≤ 5.0 mg/L, TN ≤ 15.0 mg/L, TP ≤ 0.5 mg/L). Meanwhile, the
effluent concentrations were lower than the limitation of water reuse to
toilet-flushing and supplement of landscape water specified by the
Chinese Design Specification for Sewage Reuse (GB/T 18920-2002).
The results demonstrated the successful development and operation of
ME-(O/A)CW for the low-carbon greywater treatment under low tem-
perature.
In the developed ME-(O/A)CW, the low-cost production waste ISs
has been used to initiate micro-electrolysis, which is potential to reduce
the cost. According to the purchase price of the raw materials and the
energy consumption ($0.05/kg) in the preparation in the lab, the pro-
duction cost of the Fe/C-biocarrier has been calculated as $0.20/kg
with the ISs purchasing price of $0.11/kg (Deng et al., 2019). Con-
sidering that mass fraction of ISs in the developed Fe/C-biocarrier was
90.0%, average mass fraction of Fe(0) in ISs was 94%, and 5 M of Fe(0)
was required to 3 M nitrogen, the cost of the Fe/C-biocarrier based
nitrogen removal was calculated as $1.56/kg TN. This cost has been
competitive with the other AD processes ($9.60/kg TN for the Fe2+-
supported denitrification (Kiskira et al., 2017), $7.59/kg TN for the
ZVI-supported denitrification (Deng et al., 2019; Peng et al., 2019),
$1.20/kg TN for the H2-supported denitrification without counting
energy consumption (Karanasios et al., 2010) and $1.43 ~ 2.83/kg for
the sulfur-/sulfur-compounds-supported denitrification (Peng et al.,
2019). Hence, the ME-(O/A)CW obtained competitive contaminants
removal as well as low cost. Moreover, only aeration is required during
the operation of ME-(O/A)CW. Consequently, it is potential to scaled up
ME-(O/A)CW for practical engineering application in decentralized
low-carbon greywater treatment.
4. Conclusions
Oxic/anoxic (O/A) process Fe/C micro-electrolysis (Fe/C-ME) were
incorporated into constructed wetland (ME-(O/A)CW). Effective ni-
trification, phosphorus-accumulating and organic-degradation were
proceeded in the aerobic layers and efficient H2-/Fe2+-autotrophic
denitrification and Fe3+-based phosphorus immobilization were de-
veloped in the anaerobic layers by the in-situ H2/Fe2+ supply of Fe/C-
ME. ME-(O/A)CW achieved NH4+-N, TN, TP and COD removal of
94.3%, 86.2%, 98.0% and 92.7%, respectively, under low ambient
temperature of −11.5 to 8.0 °C. AOB, NOB, autotrophic denitrificans
and Fe(II)-oxidizing bacteria dominated ME-(O/A)CW. The results
confirmed the functional microorganisms and successful operation of
the recommended ME-(O/A)CW at low temperature.
CRediT authorship contribution statement
Shihai Deng: Conceptualization, Methodology, Data curation,
Writing - original draft. Binghan Xie: Methodology, Formal analysis,
Data curation. Qiang Kong: Funding acquisition, Supervision,
Conceptualization, Writing - review & editing. Shuai Peng:
Methodology, Formal analysis, Data curation. Hengchen Wang:
10
Bioresource Technology 315 (2020) 123802
Methodology, Formal analysis, Data curation. Zhifeng Hu:
Methodology, Formal analysis, Data curation. Desheng Li: Funding
acquisition, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests/personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the Key Research and Development
Program of Shandong Province, PR China (No. 2019GSF109103), and
the National Natural Science Foundation of China (No. 51778040).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2020.123802.
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