Journal of Cleaner Production xxx (2017) 1e10

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Journal of Cleaner Production

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Pollution control and in situ bioremediation for lake aquaculture using

an ecological dam
Zhifan Ni a, f, 1, Xiaogang Wu b, 1, Lingfang Li a, Zhe Lv a, Zhenjia Zhang a, Aimin Hao c,
Yasushi Iseri c, Takahiro Kuba d, Xiaojun Zhang b, Wei-Min Wu e, **, Chunjie Li a, *
a
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
b
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
c
College of Life and Environmental Science, Wenzhou University, Wenzhou, 325035, China
d
Graduate School of Engineering, Kyushu University, 744 Motooka, Nisi-ku, Fukuoka, 819-0395, Japan
e
Department of Civil & Environmental Engineering, Center for Sustainable Development and Global Competitiveness, William & Cloy Codiga Resource
Recovery Research Center, Stanford University, Stanford, CA, 94305-4020, USA
f
Shanghai Environmental Monitoring Center, Shanghai, 200235, China

a r t i c l e i n f o a b s t r a c t

Article history: Aquaculture has become one of the major sources of lake eutrophication, due to the lack of direct and
Received 1 December 2016 efficient technologies for pollution control and remediation. This study proposes a novel ecological dam
Received in revised form (Eco-dam) system, which consists of biofilter floating beds and plant floating beds that form an enclosure
22 November 2017
(the test zone) around the breeding area that allows lake water to pass through. A pilot-scale test was
Accepted 23 November 2017
Available online xxx
conducted to test pollution control and in situ bioremediation during breeding of Chinese mitten crab
(Eriocheir sinensis) in Yangcheng Lake, China. The results showed slight improvement of water quality in
the test zone compared with the breeding zone. The biofilm that formed on the biofilter played a major
Keywords:
Ecological dam
role in removal of organic pollutants and nitrogen (0.609 kg COD/(m2$d), 0.512 kg NHþ 2
4 -N/(m $d),
 2  2
Lake aquaculture 0.482 kg NO2 -N/(m $d), and 0.112 kg NO3 -N/(m $d)). Both proteins and volatile suspended solids (VSS)
Pollution control in the Eco-dam biofilm decreased from the water surface towards the lake bottom, especially below
Bioremediation 0.5 m depth. The average ratio of VSS to suspended solids in the biofilm was 0.337 ± 0.025 g/g. Analysis
Microbial activity with the Illumina MiSeq System confirmed the presence of a diverse microbe population in the biofilm,
Microbial community performing organic carbon removal and nitrification and denitrification, with limited photosynthesis
near the lake surface and methane oxidation near the lake bottom.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction growing demand for aquatic products, expansion of aquaculture

Aquaculture has been a rapidly growing food industry world-
wide, including China. In 2014, the total world production by all
fisheries was 158 Mt, 44.1% of which was production by aquaculture
(FAO, 2016). Almost 60% of these aquaculture activities occurred in
freshwater systems, and 88% of all aquaculture products were
produced in Asia, including 62% in China (Pauly and Zeller, 2017).
With the continuous increase of the world's population and its
* Corresponding author. 800 Dong Chuan Road, School of Environmental Science
and Engineering, Shanghai Jiao Tong University, Shanghai, China.
** Corresponding author. Department of Civil & Environmental Engineering,
Stanford University, 473 Via Ortega, Stanford, CA, 94305, USA.
E-mail addresses: billwu@stanford.edu (W.-M. Wu), cjli@sjtu.edu.cn (C. Li).
1
Zhifan Ni and Xiaogang Wu contributed equally to this work.
has been identified as critical to enhance the supply of food prod-
ucts in the future (Little et al., 2016). Rapid global expansion of the
aquaculture industry has caused many environmental issues, such
as water pollution (Edwards, 2015), ecosystem degradation
(Ottinger et al., 2016), and disease outbreaks (Liu et al., 2017),
suggesting that this expansion may not be sustainable (Li et al.,
2011). It is important to develop innovative technologies for
pollution control and remediation to allow resource recovery and
sustainable growth in aquaculture (Lin et al., 2015).
In China, lakes are the major water bodies used in freshwater
fisheries production, next to the pond aquaculture (Jia et al., 2013).
Pond aquaculture is a closed-containment system (Ayer and
Tyedmers, 2009) that needs a continuous supply of fresh water
from a river or other water source, while old water drains away
through a drainage gate to the sea or river. Lake aquaculture is

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Please cite this article in press as: Ni, Z., et al., Pollution control and in situ bioremediation for lake aquaculture using an ecological dam, Journal
of Cleaner Production (2017), https://doi.org/10.1016/j.jclepro.2017.11.185
2 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10
performed in a large area, and is greatly influenced by the water
current. Lakes offers quality breeding conditions, but it may not be
practical to treat effluent from lake aquaculture using the land-
based wastewater treatment units or a fixed central plant. Lake
aquaculture has become an important source of eutrophication,
especially in the shallow lakes densely distributed along China's
eastern coast or in the middle and lower areas near the Yangtze
River, such as Taihu Lake (Cai et al., 2013).
Pen or enclosed aquaculture is the main form of aquaculture in
shallow lakes (Belle and Nash, 2009). Pen aquaculture shifted from
the natural-food based extensive culture of low-valued species
such as grass carp (Ctenopharyngodon idellus) in the 1980s and
1990s to a high-density, feed-based, intensive culture of relatively
higher-valued species, such as Eriocheir sinensis, commonly known
as Chinese mitten crab (Wang et al., 2015). Feeds enriched with
nitrogen and phosphorus were commonly used to enhance pro-
duction in intensive pen culture, generating a large quantity of
aquaculture wastes including residual solid feed, fish (or crab)
faeces, and soluble excretions (Wang et al., 2016). Excessive gen-
eration of solid wastes in pen enclosures might result in serious
oxygen depletion and anoxic conditions harmful to benthic biota,
owing to the oxygen-consuming decomposition of the organic
wastes by bacteria and other organisms (Li et al., 2011). Suspended
solid wastes and dissolved pollutants such as ammonia and phos-
phate derived from soluble excretions and organic decomposition,
could be flushed and dispersed from pens by natural water cur-
rents, leading to eutrophication due to excessive accumulation of
nutrients (Wang et al., 2016). Consequently, it is very important for
pen aquaculture to develop direct and efficient technologies for
pollution control and in situ bioremediation in the shallow lakes
(Zou and Huang, 2015).
To cope with increasing lake aquaculture pollution, the local
government has mainly focused on enforcement of pen aquaculture
management, and decrease of the culture area (Mungkung et al.,
2013). Several management methods have been applied to reduce
the water pollution caused by lake aquaculture (Samuel-Fitwi et al.,
2012). Feed management could be used to minimize the amount of
unconsumed feed (Cho and Bureau, 2002), optimizing the feed
conversion ratio (FCR; weight of feed offered/weight of fish pro-
duced) by adopting high-quality diets and efficient feeding
methods (Wu et al., 2015). Offshore culture was commonly used to
reduce potential pollutants from entering lake. However, these
methods did not directly control or remediate the pollutants
generated by lake aquaculture. Numerous physical, chemical and
biological methods have been proposed to reduce or control lake
eutrophication (Le et al., 2010), including phytoremediation (Li
et al., 2017), bio-manipulation (Gao et al., 2014), and microbial
remediation (Zhao et al., 2014). Bioremediation could improve
water quality and mitigate the environmental stress of lake aqua-
culture. However, there is still a lack of practicable in situ tech-
nology to prevent aquaculture pollution from endangering ambient
lake ecosystems in China. This study presents a design for a novel
ecological dam (Eco-dam) that can serve as a lake aquaculture
system with in-situ bioremediation.
2. The Eco-dam concept
The Eco-dam is composed of a set of functional units (Li et al.,
2012), each constructed with a submerged biofilter (SBF) and a
plant floating bed (PFB) as shown in Fig. 1. The SBF unit has a
floating plastic frame connected with elastic polypropylene (PP)
fibre roots, forming a brush-like biofilter for microbial attachment
(Li et al., 2014). The SBF unit is fixed by ropes to pilings set into the
lake bed. The PFB is composed of floating plastic bed units in which
aquatic plants (or vegetables) grow, and is placed within the
Please cite this article in press as: Ni, Z., et al., Pollution control and in situ bioremediation for lake aquaculture using an ecological dam, Journal
of Cleaner Production (2017), https://doi.org/10.1016/j.jclepro.2017.11.185
rectangular area formed by the SBF unit (Fig. 1). For field test pur-
poses, several Eco-dam units are combined to form an enclosure
(i.e., remediation zone or test zone) surrounded by the breeding
zone in which fishes and crabs were cultured (Fig. 1).
The Eco-dam allows lake water to pass between the test zone
and breeding zone. It reduces wave disturbance in the test zone and
serves as a physical barrier against the migration of solid wastes
and algae from this zone, reducing the risk of eutrophication.
The Eco-dam removes aquaculture pollutants through biodeg-
radation by diverse microbial populations, including bacteria, fungi,
protozoa, metazoa, and planktonic animals, and uptake by aquatic
plants in the plant floating bed and the biofilter. Organic pollutants
generated in the breeding zone, such as faeces and residual feed,
are deposited in the sediment and decomposed by microorganisms,
resulting in the release of soluble organic matter and ammonia and
phosphorus compounds (Lefebvre et al., 2001). Under aerobic
conditions, ammonia is oxidized by ammonia-oxidizing bacteria
(AOB) to nitrite and then to nitrate by nitrite-oxidizing bacteria
(NOB). When water flows through the Eco-dam, microbial activity
in the attached biofilm on SBF degrades soluble organic matter to
CO2 by heterotrophic microorganisms, and nitrifying bacteria
metabolizing ammonia to nitrite and nitrate, and then to nitrogen
gas. Aquatic plants and microorganisms uptake pollutants such as
phosphate and nitrate as nutrients. These pollutants serve as
feedstock in the ecosystem where biofilm is generated from the
biodegradation of pollutants by the microorganisms. Indirectly,
pollutants constitute a food source for fishes, aquatic insects,
shrimp, and spiral shells.
To test the Eco-dam concept, a pilot-scale Eco-dam was con-
structed for in situ bioremediation of an aquafarm zone. The mi-
crobial activities and communities in the functional biofilm of this
system were characterized to evaluate the metabolic performance
of the biotic system. The pilot-test results can be used to scale up to
the design of a full scale Eco-dam, and allow primary analysis of
capital costs and potential economic benefits.
3. Materials and methods
3.1. Pilot scale ecological dam
The pilot scale Eco-dam was constructed in an aquaculture farm
that has been breeding Chinese mitten crab since 2012 (Fig. 2). The
farm is located in Yangcheng Lake, Suzhou City, Jiangsu Province,
China (Fig. 2a); with a total area of 118.2 km2, an average depth of
1.61 m, and a storage capacity of 1.7  108 m3, this is the third
largest freshwater lake in the Taihu Lake Plain, and is well known
for the production of high-quality Chinese mitten crabs, which are
now sold at 45e55 RMB (6.5e7.9 USD) per crab. Intensive large-
scale pen culture had led to deterioration of water quality, reduc-
tion of submerged macrophytes, and eutrophication of the lake for
years; Total nitrogen (TN) and total phosphorus (TP) were the
primary pollutants (Song et al., 2010). Current pollutant levels were
below the TN ¼ 2.0 mg/L (Chinese state regulation limit for a
hypereutrophic lake (inferior Class V)), and were primarily due to
NHþ 4 -N; TP was 0.2 mg/L, still above the surface water quality
criteria of Class Ⅲ (TN of 1.0 mg/L; TP of 0.05 mg/L) for a drinking
water resource, according to the Environmental Quality Standards
for Surface Water (GB 3838-2002) (MEPPRC, 2002). The submerged
macrophytes, which provide natural shelter for exuviating crabs,
had been greatly reduced due to the low water transparency,
resulting in a negative impact on crab production. The excessive
addition of the aquaculture feeds has been a major source of pol-
lutants. To date, the total breeding area in Yangcheng Lake has been
reduced to 21.3 km2, and the local government restricted the pen
culture area to 13,340 m2 each farming in order to protect the lake
 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10 3

Fig. 1. Conceptual illustration of Eco-dam where the bioremediation zone was surrounded by the breeding zone.

Fig. 2. Pilot scale Eco-dam system in Yangcheng Lake, China. (a) Location of pilot scale facility; (b) Layout of the system with six different plants in PFB (A, B, C, and D: locations for
biofilm samples); (c) Site overview of test zone and breeding zone where crabs were reared; and (d) Detailed photos of Eco-dam units: (i) uninstalled submerged biofilter (SBF); (ii)
plant floating bed (PFB); (iii) in situ biofilter; and (iv) crab shells.

from further deterioration and eutrophication. Our test period
overlapped with the breeding cycle of Chinese mitten crabs from
March to October. In March and April, approximately 13,000 ju-
venile crabs were released into a breeding area of 13,340 m2,
together with 72 m3 of aquatic plants, including Hydrilla varticillata,
Elodea nattalli, and Vallisneria natans. The crabs experienced at least
5 episodes of exuviation prior to harvest. At the beginning of the
breeding cycle, 3500 kg of spiral shells were added, and commercial
feeds were added every three days, including 15 kg of corn (mainly
for the fish), 20e40 kg of small frozen fishes (for crabs). During the
peak breeding season from September to October, the feeding load
and frequency increased to 20 kg of corn and 240 kg of small fish
every other day, to meet the demand for rapid growth of the crabs.
The daily nutrient load to the breeding area was estimated as TN of
0.065 g/(m2$d) and TP of 0.0045 g/(m2$d), based on the mass of
residual feed, crab faeces, decay of aquatic weeds, and dissolved
chemicals released from the sediments during the breeding season
(Montanhini Neto and Ostrensky, 2015).
The Eco-dam system had 36 units (Fig. 2 b and c), each con-
sisting of one SBF (Fig. 2dei) and one PFB (Fig. 2deii), and was 2 m
long and 1 m wide. The floating plastic frame of an SBF was made of
polyvinyl chloride (PVC) tubes with a diameter of 16 cm. The 36
units were joined together to form a test zone or remediation zone
with an area of 18.48 m  9.92 m (183.32 m2). The total area
covered by the pilot Eco-dam system was 97.6 m2. The system was
surrounded by a purse seine with a pore size of 1 cm, to retard lake
water flow and keep the crabs inside the breeding zone. Each SBF
unit had 52 fibre roots (weighted at the end of each root) to form a
1.5-m deep biofilter (Fig. 2deiii). Each root had 7500 0.4-mm
diameter polypropylene (PP) fibres fixed on it. The length of each
fibre was 12.5 cm, for a total length of fibres of 937.5 m per root
(right insert picture Fig. 2deiii). In May 2013, six different aquatic

Please cite this article in press as: Ni, Z., et al., Pollution control and in situ bioremediation for lake aquaculture using an ecological dam, Journal
of Cleaner Production (2017), https://doi.org/10.1016/j.jclepro.2017.11.185
4 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10
plants d Lythrum salicaria, Thalia dealbata, Pontederia cordata, Iris
tectorum, Iris wilsonii, and Canna warscewiezii d were planted on
the floating bed (Fig. 2 b and c). The Eco-dam also provided places
for crab shelling (Fig. 2 d-iv). Tests were performed from March 14
to October 5, 2013, following formation of the biofilm on the SBF.
3.2. Water sampling and analyses
Water samples were collected from the breeding zone and the
test zone in the afternoon at 30 cm below the water surface every
15 d, preserved in cooler boxes and transported from the site to
Shanghai Jiao-tong University laboratory for analysis of chemical
oxygen demand (COD), ammonia, nitrite, and nitrate within 48 h.
Water temperature, pH, and dissolved oxygen (DO) were measured
on site with a Hach portable water quality analyser (HQ30d, USA).
Turbidity was measured using a Hach turbidity meter (2100Q, USA),
and chlorophyll a (Chl-a) was measured with the Chlorophyll
Fluorescence System (PHYTO-PAM, WALZ, Germany); both were
measured on site. Daily mean values of the data were recorded on
each sampling day. A portion of each water sample was filtered
through a mixed cellulose esters membrane (0.45 mm pore size) and
then analysed for NHþ 4 -N concentration by Nessler's reagent
colorimetric method, and for NO3 -N concentration by an ultraviolet
(UV) spectrophotometric method (NEPA, 2002). Unfiltered sub-
samples were analysed for TN by alkaline potassium persulfate
digestion-UV spectrophotometry, TP by ammonium molybdate
spectrophotometry, and COD, using Standard method (NEPA,
2002).
3.3. Biofilm sampling and analyses
3.3.1. Sampling sites
Biofilm samples were taken from the SBF for microbial activity
tests and bacterial community analysis in August 2013. The biofilm
samples were collected from four locations on the biofilter, denoted
by A, B, C, and D (Fig. 2 b). For each location, samples with the PP
fibres were taken from three different positions at three different
depths, representing the upper (30-cm depth), middle (below the
water surface, 70-cm depth), and lower (120-cm depth) portions of
the biofilter; sample B2, for example, referred to the biofilm sample
collected at the middle portion of biofilters from location B. All
samples were preserved at 4  C before analysis.
3.3.2. Microbial activity
Biofilm samples from the upper locations were mixed for mi-
crobial activity tests. The specific oxygen uptake rates (SOUR) of
heterotrophic bacteria and nitrifying bacteria, including ammonia
oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), were
analysed. The biofilm attached to the PP fibre filters was carefully
washed with deionized water and added to a 250-mL conical flask.
The flask was fully filled with pre-aerated nutrient solutions
(Table S1 in Supplementary Information) and sealed with a perfo-
rated silica gel plug. A dissolved oxygen probe (Hach HQ30d, USA)
and a 5-mL syringe were immediately inserted into the flask
through the plug, so that the flask was tightly sealed and no
headspace remained. All tests were conducted in a temperature-
controlled vibrating water bath (25  C, 120 rpm). DO concentra-
tion was recorded at intervals of 30 s, and oxygen uptake rates
(OURs) were determined by linear regression from the slope of DO
concentration versus time, and SOURs were obtained by dividing
OUR by the total amount of volatile suspended solids (VSS) deter-
mined by direct gravimetry; all rates were expressed as mg O2/(g
VSS$h).
The OURs of nitrifying bacteria, (OUR)N, were determined with
the three-phase method of Moussa et al. (2003). The endogenous
Please cite this article in press as: Ni, Z., et al., Pollution control and in situ bioremediation for lake aquaculture using an ecological dam, Journal
of Cleaner Production (2017), https://doi.org/10.1016/j.jclepro.2017.11.185
OUR (OUR1) was measured for 12 min with the nutrient solution.
The OURs of NOB, (OUR)NO2, were measured by injecting 1.0 mL of
NaNO2 (resulting in 0.2 mg/L of NO 2 N in solution) and recorded as
OUR2. The OUR of AOB, (OUR)NH4, was measured by injecting 1.0 mL
NH4Cl (resulting in 1 mg/L of NH4Cl in solution), and recorded as
OUR3. (OUR)NO2 was calculated from the difference between OUR2
and OUR1, while (OUR)NH4 was the difference between OUR3 and
OUR2. The OURs of heterotrophic bacteria, (OUR)H, were deter-
mined using a similar respirometry test, and calculated from the
difference between endogenous and exogenous respiration rates.
Allylthiourea (ATU) was used in the nutrient solution to inhibit
ammonia oxidation by AOB. (OUR)H was measured with 1 mL of
NaAc injected, resulting in 20 mg/L of CODCr.
The specific microbial activity of denitrifying bacteria in the
biofilm (specific denitrification rate, or SDNR), was determined by a
substrate utilization test, performed in a 500-mL conical flask
sealed by tinfoil in a constant-temperature vibrating water bath
(SHA-C, Guohua Electric Appliance Co. Ltd, China) at 25  C and
120 rpm. Substrate solution containing NO 3 -N at 1.0 mg/L and
CODCr at 20 mg/L (prepared with KNO3 and sodium acetate) was
poured into the flask, and the headspace was flushed with nitrogen
gas for 2e3 min to create an anoxic condition (DO
concentration < 0.5 mg/L). At pre-determined times (e.g., 30 min
and 60 min), 5 mL supernatant samples were collected with a
pipette for measurement of the NO 3 N concentration. After each
sample collection, the flask headspace was flushed immediately
with nitrogen gas for 30 s to maintain anoxic conditions. SDNR was
calculated by the decreasing slope of NO 3 -N concentration with
time and dividing by the total amount of VSS (Gong et al., 2012).
The total protein content of the biofilm at different depths was
measured to estimate the microbial biomass distribution. Seven
samples, P1 to P7, were collected from the top (just below the water
surface) to the bottom of the filter (effective depth of 1.5 m) at 0.25-
m intervals. The samples were stored at 20  C prior to analysis. To
extract the protein, the biofilm samples were centrifuged with
12,000  g for 15 min under 4  C after overnight treatment in a 1 N
NaOH solution for cell lysis (Bassin et al., 2012). The supernatant
was collected after filtration through a 0.22 mm membrane. Total
protein content was determined using the method of Lowry et al.
(1951).
3.3.3. Bacterial community analysis
The microbial community of the biofilm was analysed with the
Illumina MiSeq System (MiSeq System, Illumina Inc., USA). Deox-
yribonucleic acid (DNA) was extracted using the modified method
of Griffiths (Griffiths et al., 2000). The extracted metagenomic DNA
was used as the template to amplify the V3eV4 region of 16S rRNA
(ribosomal ribonucleic acid) genes. Polymerase chain reaction
(PCR), sequencing of the PCR amplicons and quality control of raw
data were performed with minor modification of the MiSeq system
protocols.
Both the forward and reverse ends of the same reads were cut at
the first base where the Q value was no more than 2. If the pair of
reads had a minimum of 50 bp-length overlap, they would be
merged into complete reads, which were kept unless they were
longer than 399 bp and the expected error was no more than 0.5
(Edgar, 2010). Quality-filtered reads were dereplicated into unique
sequences, which were sorted by decreasing abundance, and sin-
gletons were abandoned. Non-chimeric operational taxonomic unit
(OTU) representative sequences were picked by Uparse's default
(Edgar, 2013). Further detection of chimeras was performed using
the UCHIME method (Edgar et al., 2011). UCHIME is a new program
that detects chimeric sequences with two or more segments. The
name “UCHIME” indicates that these are “undetected chimeras”
when compared to the ribosomal database project (RDP) classifier
 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10 5

training database (RDP database version 2.10, Michigan State Uni-
versity, USA) (Cole et al., 2014). The OTU table was finalized by
mapping quality-filtered sequences to the remained OTUs with the
Usearch method (Edgar, 2010) and a global alignment algorithm at
97% cutoff. All analyses were performed based on the quantitative
insights into microbial ecology (QIIME) platform (Caporaso et al.,
2010). Representative sequences for each OTU were submitted to
an RDP classifier to determine the phylogeny (RDP database version
2.10, Michigan State University, USA).
4. Results and discussion
removal. A significant difference between Chl-a concentrations was
observed after July. The differences between the two zones have
been less significant for other parameters (TP, TN, NO þ
3 -N, and NH4 -
N). It was likely that establishment of the pilot-scale Eco-dam in an
open lake where strong water currents caused water mixing be-
tween the test zone and breeding zone led to masking the effects of
bioreactions. In an open system, it is almost impossible to deter-
mine pollutant removal efficiencies and metabolic rates in terms of
COD removal, DO consumption, or nitrogen removal, due to the
rapid and dynamic water flow. In this case, the potential of the Eco-
dam for biological degradation of pollutants could be evaluated by
estimating the microbial activity of the SBF biofilm.

4.1. Operational performance of Eco-dam

4.2. Biofilm microbial activity
The water quality of the test zone and breeding zone was
monitored weekly during the crab breeding period from March 14 Laboratory batch assays were performed to determine the mi-
to October 5, 2013. Bimonthly average results for change of tem- crobial activities of SBF biofilms to indirectly evaluate the biore-
perature, pH, dissolved oxygen, turbidity, Chl-a, COD, NHþ 
4 -N, NO3 - mediation performance of the Eco-dam. The specific activities of
N, TN, and TP were summarized in Table 1. The lake water tem- heterotrophic bacteria, NOB, and AOB in terms of SOUR, and deni-
perature was 16e17  C in March and April, and reached approxi- trifying bacteria in terms of SDNR, were shown in Fig. 3 and
mately 28  C in July and August. The concentrations of DO, COD, TP, compared with the activities reported for various wastewater
TN, NO þ
3 -N, and NH4 -N were maintained within acceptable ranges treatment systems in Table 2.
for crab growth (Table 1). No algal bloom was observed during the The SBF biofilm showed all the necessary microbial activities for
test period, suggesting that the Eco-dam performed properly. Based
on the profiles of temperature, pH, DO, and electrical conductivity
(EC), the changes in temperature (29.37 ± 0.05  C), pH (8.03 ± 0.04)
and EC (491.67 ± 24.92 mS/cm) were insignificant for the water
depth in the test zones during the peak breeding period (from
September to October 2013).
The DO concentrations were as high as 8e10 mg/L below the
water surface and 5e7 mg/L near the bottom of the lake, indicating
no significant DO depletion. A slightly lower DO was observed in
the test zone (Table 1), possibly due to oxygen consumption by the
SBF biofilms. The average values of turbidity, COD, TP, TN, NO 3 -N,
NHþ 4 -N, and Chl-a were slightly lower in the test zone than the
breeding zone, indicating the removal of suspended solids and
nutrients and decreased algal density in the test zone. The differ-
ence in the concentrations between the two zones became signif- Fig. 3. Specific activities of heterotrophic bacteria, NOB, and AOB in terms of SOUR,
and denitrifying bacteria in terms of SDNR. ((SOUR)H: specific oxygen uptake rate of
icant at a 0.1 level (confidence level: 90%, p < 0.1) after May, when heterotrophic organic-degrading bacteria; (SOUR)NO2: specific oxygen uptake rate of
temperature exceeded 24  C (Table 1). This indicated that the Eco- nitrite-oxidizing bacteria; (SOUR)NH4; specific oxygen uptake rate of ammonia-
dam had performed effectively, especially for organic pollutant oxidizing bacteria; SDNR: specific denitrification rate of denitrifying bacteria).

Table 1
Water quality results from the test zone and breeding zone, and statistical analysis using ManneWhitney U test (non-parametric t-test, confidence level: 90%) for discrim-
ination between the test and breeding zones (March 14 to October 5, 2013). (p: positive constant).

Temperature pH DO (mg/L) Turbidity Chl-a (mg/L) COD (mg/L) TP (mg/L) TN (mg/L) NO3--N (mg/ NH4-N (mg/
( C) (NTU) L) L)

Mar. to Test zone 16.98 ± 7.05 8.52 ± 0.19 10.99 ± 0.53 6.67 ± 1.18 9.99 ± 2.79 29.17 ± 2.33 0.06 ± 0.02 1.12 ± 0.14 0.95 ± 0.16 0.10 ± 0.01
Apr. Breeding 16.20 ± 6.06 8.62 ± 0.27 11.54 ± 0.84 7.06 ± 1.00 8.36 ± 2.07 31.75 ± 3.47 0.07 ± 0.03 1.16 ± 0.07 0.99 ± 0.08 0.11 ± 0.01
zone
p-value e 0.7381 0.4127 0.8016 0.3095 0.1508 0.7381 0.5238 0.4127 0.246
May. to Test zone 25.42 ± 1.60 8.62 ± 0.18 10.84 ± 1.12 4.23 ± 1.15 7.10 ± 2.83 34.36 ± 2.55 0.09 ± 0.03 0.86 ± 0.15 0.71 ± 0.12 0.07 ± 0.02
Jun. Breeding 24.74 ± 2.34 8.90 ± 0.17 11.33 ± 1.13 7.57 ± 1.57 7.76 ± 1.86 40.19 ± 3.20 0.13 ± 0.04 0.97 ± 0.31 0.89 ± 0.14 0.10 ± 0.02
zone
p-value e 0.2 0.9 0.0952 0.9444 0.0556 0.0952 0.0556 0.0556 0.1429
(p < 0.1) (p < 0.1) (p < 0.1) (p < 0.1) (p < 0.1)
Jul. to Aug. Test zone 28.28 ± 3.14 8.31 ± 0.41 10.30 ± 1.86 7.99 ± 4.22 4.88 ± 0.64 36.23 ± 2.00 0.10 ± 0.05 0.91 ± 0.18 0.59 ± 0.12 0.07 ± 0.03
Breeding 28.16 ± 2.59 8.39 ± 0.36 11.26 ± 1.50 11.48 ± 3.38 6.23 ± 0.83 42.45 ± 4.47 0.16 ± 0.05 0.74 ± 0.24 0.63 ± 0.17 0.11 ± 0.05
zone
p-value e 0.8 0.4 0.5317 0.0317 0.0159 0.1143 0.4524 0.6746 0.2
(p < 0.1) (p < 0.1)
Sept. to Test zone 27.12 ± 4.79 8.25 ± 0.41 10.20 ± 1.93 10.12 ± 2.18 3.70 ± 1.65 35.91 ± 2.44 0.10 ± 0.05 0.96 ± 0.19 0.52 ± 0.14 0.07 ± 0.04
Oct. Breeding 26.92 ± 4.53 8.42 ± 0.39 11.30 ± 1.44 12.07 ± 3.31 5.48 ± 0.69 42.59 ± 4.38 0.16 ± 0.05 0.81 ± 0.22 0.52 ± 0.13 0.12 ± 0.06
zone
p-value e 0.3333 0.6571 0.4127 0.0952 0.0571 0.7 0.4 0.9 0.9
(p < 0.1) (p < 0.1)

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6 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10

the removal of organic matter and nitrogen. The heterotrophic

bacterial activity (SOUR)H for degrading organic carbon was
7.34 ± 1.91 mg O2/(g VSS$h). For nitrogen removal via nitrification,
the activity of AOB was measured based on oxygen consumption by
ammonia i.e. (SOUR)NH4 at 19.89 ± 4.18 mg O2/(g VSS$h); the NOB
activity, or (SOUR)NO2, was slightly lower at 6.12 ± 1.22 mg O2/(g
VSS$h). The specific substrate utilization rate was also used to
describe the activities of AOB and NOB through the specific
ammonium utilization rate (SAUR) and specific nitrite utilization
rate (SNUR), which were estimated from (SOUR)NO2 and (SOUR)NH4,
based on the stoichiometry of nitrification (0.95 mg NO 2 -N/mg O2
and 0.31 mg NHþ 4 -N/mg O2) (Bassin et al., 2012). SNUR and SAUR
were estimated as 5.81 ± 1.16 mg NO 2 -N/(gVSS$h) and
6.17 ± 1.30 mg NHþ 4 -N/(gVSS$h). SDNR (1.35 ± 0.32 mg NO3 -N/


(gVSS$h)) was obtained during a 540 min substrate utilization test
under anoxic conditions.
These results indicated that the nitrification activity, especially
the AOB activity of the biofilm, was higher than the NOB and SDNR.
This could be due to high DO in the lake water in general. The
slightly low heterotrophic bacterial activity ((SOUR)H) was likely
due to relatively low COD concentrations (30e40 mg/L) in the lake
water.
Microbial activities, i.e. the specific activities of heterotrophic
bacteria, nitrifying bacteria, and denitrifying bacteria in the SBF
biofilm, were compared to the results obtained from various
wastewater treatment systems (Table 2), including aerobic biofilms,
activated sludge, and aerobic granular sludge systems. The het-
erotrophic organic degradation and all nitrogen-metabolism
related activities measured in this study were within the ranges
reported for the wastewater treatment systems. The exception was
that the denitrifying activity (SDNR) of the SBF appeared lower
(1.35 mg NO
3 -N/(gVSS$h)) than that of the biofilm used for
wastewater treatment by Chu and Wang (2011) (12.7 mg NO
3 -N/
(gVSS$h)) and Gong et al. (2012) (4.0e5.0 mg NO
3 -N/(gVSS$h)). The
low SDNR could be due to the high DO concentration (8e10 mg/L)
in the test zone, which depressed the activity of anoxic denitrifiers.
The results suggested that the Eco-dam possessed a stronger
capability for organic matter oxidation and nitrification, reflected
by the decreased COD and NHþ
4 -N in the test zone.
4.3. Biomass profile of SBF
The biofilm distribution profile (from water surface to the water
bottom) was characterized by the contents of proteins and VSS per
cm of fibre material, as shown in Fig. 4. Both proteins and VSS
contents showed similar trends, exhibiting a gradual decrease from
the water surface towards the bottom of the fibers. Most biomass
(68.2% of the total biomass) attached at the upper portions within
Fig. 4. Profiles of VSS and proteins on biofilter PP fibres.
0.5 m of the water surface, with 1.45e5.33 mg VSS per cm of fibre
and 0.56e2.14 mg protein per cm of fibre. The VSS and protein
contents dropped significantly at depths below 0.5 m. The higher
biomass concentration above 0.5 m might be due to higher near-
surface turbulence of lake water, and therefore the higher mass-
transfer rates of substrates (nitrate, ammonia, and COD) from the
breeding zone to the biofilm in the test zone, which could enhance
reaction rates and thus the biomass growth rate. Photosynthetic
microorganisms might also contribute to the higher biomass con-
tent, because sunlight can easily penetrate to depths of 50 cm from
the water surface.
The average ratio of volatile suspended solids to suspended solid
(VSS/SS, g/g) in the SBF biofilm was 0.337 ± 0.025, indicating that
the biofilm contained a large fraction of inert materials, such as soil
or clay, and that the biofilm contained fewer active microbes than
the activated sludges and biofilms of biofilters in the municipal
wastewater treatment plants (VSS/SS ratio of 0.7 or higher). The
average ratio of protein to VSS (g/g) was 0.41 ± 0.05, which was also
slightly lower than that found in the wastewater treatment plants
(0.5e0.75, g/g).
The total biomass on each 1.5-m filter fibre root included
approximately 180.2 g VSS and 73.9 g protein. Based on the mi-
crobial activities determined in Section 4.2, the maximum micro-
bial activities of the organic-degrading bacteria (ODB), AOB, NOB,
and DNB could reach 0.609 kg COD/(m2$d), 0.512 kg NHþ 2
4 -N/(m $d),
 2  2
0.482 kg NO2 -N/(m $d), and 0.112 kg NO3 -N/(m $d), respectively,
with the Eco-dam system (Note: in the dimensions of the above
parameters, m2 refers to the water area covered by the Eco-dam
system). In this study, these parameters were used for the pri-
mary scale-up design of the Eco-dam system for lake-aquaculture
pollution control and in situ bioremediation and economic esti-
mation. Considering mass transfer and kinetic limitations, the

Table 2
Comparison of microbial activities of SBF biofilms obtained in this study and those of various wastewater treatment systems reported in the literature.

Type of samples (SOUR)H (SOUR)NH4 (SOUR)NO2 SDNR References

mg O2/(gVSS$h) mg O2/(gVSS$h) mg O2/(gVSS$h) mg NO
3 -N/(gVSS$h)

Biofilm in MBBR 10.3 29.0 NR 4.0e5.0 (Gong et al., 2012)

Biofilm in UBAF NR 15.2 4.5 NR (Fdz-Polanco et al., 2000)
Activated sludge 6.4e10.6 NR NR NR (Clouzot et al., 2011)
Activated sludge 4.9 12.6 4.0 NR (Raszka et al., 2011)
Aerobic granules NR 20.1e27.7 1.2e10.5 NR (Winkler et al., 2011)
Biofilm on biopolymers NR NR NR 12.7 (Chu and Wang, 2011)
Sludge with glucose NR NR NR 2.33
Suspended sludge NR NR NR 0.75
Biofilm in Eco-dam 7.34 19.89 6.12 1.35 This study

((SOUR)H: specific oxygen uptake rate of heterotrophic organic-degrading bacteria; (SOUR)NO2: specific oxygen uptake rate of nitrite-oxidizing bacteria; (SOUR)NH4: specific
oxygen uptake rate of ammonia-oxidizing bacteria; SDNR: specific denitrification rate of denitrifying bacteria. NR: not reported; MBBR: moving-bed biofilm reactor; UBAF:
upflow biological aerated filter.).

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 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10 7

actual pollutant removal rates would be lower than the rates esti- present in very small numbers (0.1e0.2%). Methanogens have been
mated above. found in aerobic activated sludge, due the presence of anaerobic
nuclei within the sludge (Wu et al., 1987). This study confirmed the
presence of an anaerobic layer with methanogens in the SBF bio-
4.4. Microbial community analysis
films, even at DO concentrations up to 8e10 mg/L or greater in
ambient water. The role of methanogens in the carbon cycle in the
Microbial community analysis was performed for the 12 sam-
Eco-dam system might be limited or negligible, due to their
ples of SBF collected from four locations at three different depths
extremely low abundance. Aerobic Methylocystis sp. was present in
(surface, middle, and bottom), in order to verify the roles of the
abundance in the biofilm, with its relative abundance increasing
microbes in removal of organic carbons and nutrients, especially
from top (1.8%) to bottom (5.2%). Methylocystis sp. was typical Type
nitrogen compounds (Fig. 5). A total of 146,064 of 16S rRNA se-
II methane-oxidizing bacteria, with accumulation of poly-
quences were selected for classification. The phylum and genus-
hydroxobutyrate (PHB) inside the cell (Belova et al., 2011). The
level distributions presented were based on the 80% similarity
abundance distribution of Methylocystis sp. was likely related to the
clusters of OTUs. Other low abundance bacteria represent the set of
consumption of methane released from the lake-bottom sediment,
genera for which the average relative abundances were less than
rather than methane from the anaerobic layer of the SBF biofilm.
1%. The series numbers of bacteria names correspond to the order
Hyphomicrobium was also a high-abundance genus in the SBF
of bars from top to bottom. Based on the 97% similarity, a total of
biofilms (1.7e5.25%), and its presence was correlated with the
2119 OTUs derived from the sequences were detected. The
metabolism of methanol for denitrification (Timmermans and
sequencing analysis showed that the most abundant bacteria
Haute, 1983). The role of these bacteria could be related to
among the 12 samples, at phylum level, were Proteobacteria, Acti-
methane metabolism or biodegradation of organic matter. Several
nobacteria, Cyanobacteria, Firmicutes, Planctomycetes, Bacteroidetes
types of denitrifying bacteria detected in the SBF biofilms,
and Nitrospirae (Fig. 5a). The predominant bacterial genera (Fig. 5b)
including. Bacillus, Pseudomonas, Paracoccus and Rhizobium
showed similar microbial structure at the genus level in the upper,
(Table 3), were also considered to be organic-carbon degraders
middle, and bottom samples at all four different locations.
(Tiedje, 1988).
Based on the known metabolic properties of microbial genera
Nitrogen-cycle groups included nitrifying, denitrifying, and
and species, the trophic groups of the functional microorganisms
anaerobic ammonium oxidation (anammox) microorganisms. Un-
on the SBF were summarized as average abundances at different
classified Nitrosospira and Unclassified Nitrosococcus as AOB (Bock
depths in Table 3, and presented in detail in Table S2 (Supple-
et al., 1989) were found at different positions around the Eco-
mentary Information). Carbon-metabolic microbes included
dam. Nitrosospira was evenly distributed with a relatively low
mainly various heterotrophic aerobic microorganisms and a limited
abundance (0.42e0.56%), while Nitrosococcus was present at a
number of anaerobic microbes. Aerobic heterotrophic bacteria
much lower abundance. The main nitrite-oxidizing bacteria (NOB)
belonging to Steroidobacter sp. represented one abundant genus
was Nitrospira (Ehrich et al., 1995). Its relative abundance in the
(4.3% at the upper but declining to 2.28% at bottom), and could
upper, middle, and lower portions was 1.28, 1.71 and 7.68%,
contribute to the degradation of organic matter in the lake water.
respectively. NOB catalyses the conversion from nitrite to nitrate
Steroidobacter sp. were also considered to be steroid degraders
during nitrification, which is an important process in the biogeo-
(Wang et al., 2013), but it was not known if the lake water contained
chemical nitrogen cycle. Relative abundance of NOB in the lower
steroids. Strictly anaerobic bacteria, including methanogens (e.g.
positions was higher than the upper position. Although NOB such
Methanobacterium sp. which belongs to Archaebacteria), were

Fig. 5. Composition of the bacterial community composition attached in SBF biofilms. (a) Phylum-level community structure at different sampling sites; (b) Genus-level bacterial
community structure analysis.

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8 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10

Table 3
Relative abundance (%) of functional bacteria at different sampling depths in the Eco-dam.

Function Description Classification Upper portion Middle portion Lower portion

Nitrogen cycle AOB Unclassified Nitrosospira 0.42 0.46 0.56

Unclassified Nitrosococcus UD 0.01 0.02
NOB Nitrospira 1.28 1.71 7.68
Anammox Unclassified Candidatus Brocadia 0.01 0.01 UD
Unclassified Candidatus Anammoxoglobus 0.02 0.02 0.01
DNB/ODB Neisseriaceae (family) 0.62 0.07 0.02
Rhodospirillaceae (family) 0.27 0.87 0.22
Bacillaceae 1 (family) 0.03 0.10 0.11
Cytophagaceae (family) 0.08 0.02 0.15
Rhizobiaceae (family) 0.40 0.61 0.51
Bacillus 0.01 0.03 0.04
Pseudomonas 0.12 0.13 0.04
Paracoccus 0.03 0.02 0.03
Rhizobium 0.14 0.20 0.19
Carbon cycle Methanogenic Methanobacterium UD 0.02 0.02
Methane oxidizing Methylocystis 1.79 2.11 5.25
ODB Hyphomicrobium 2.09 2.72 4.24
ODB Steroidobacter 4.26 3.69 2.28
Photosynthetic Photosynthetic Cyanobacteria (phylum) 14.39 10.84 5.45
Photosynthetic Rhodobacter 0.58 0.34 0.21
Photosynthetic Unclassified Rhodobacter 4.65 3.74 3.08
Sulphur cycle SRB Desulfobacterium 0.04 0.04 0.05

(AOB: ammonia-oxidizing bacteria; ODB: organic-degrading bacteria; NOB: nitrite-oxidizing bacteria; DNB: denitrifying bacteria; SRB: sulphate-reducing bacteria. UD: under
detection limit.).

as Nitrospira were abundant in the lower samples, the absolute
biomass at the lower portion was not high because the lower
biomass was attached in the lower portion (Fig. 4).
Anammox bacteria were present the biofilm at very low levels
(0.1e0.2%). They were mainly the Unclassified Candidatus Brocadia
and Unclassified Candidatus Anammoxoglobus (Kartal et al., 2008),
indicating that the anammox process was present, but not the
major nitrogen removal pathway in the Eco-dam. The main deni-
trifying bacteria, at family level, could be Neisseriaceae, Rhodospir-
illaceae, Bacillaceae 1, Cytophagaceae, and Rhizobiaceae (Tiedje,
1988). The main genera of aerobic denitrifying bacteria could
contain the families Bacillus, Pseudomonas, Paracoccus, and Rhizo-
bium (Tiedje, 1988).
Photosynthetic bacteria and algae were found in the SBF biofilm,
especially the upper portion. Cyanobacteria were one of the high
abundant phyla, and their distribution depended on the water
depth. Their relative abundance decreased from the upper samples
(14.39%) to the deeper samples (5.45%). Cyanobacteria were a group
of photosynthetic, nitrogen-fixing bacteria that could occur as
planktonic cells or could form phototrophic biofilms. Cyanobacteria
could fix atmospheric nitrogen under aerobic conditions by means
of specialized cells called heterocysts, and were known for exten-
sive and highly visible blooms that could form in both freshwater
and marine environments (Oren, 2004). The growth of these bac-
teria in biofilms due to photosynthesis could have a negative
impact due to nitrogen fixation, which would increase the nitrogen
content of the lake water. No algal blooms occurred in the Eco-dam
system during this study. Rhodobacter was a genus of Rhodo-
bacteraceae, a model organism used to study bacterial photosyn-
thesis (Imhoff et al., 1984). Rhodobacter and unclassified
Rhodobacter were common in the SBF biofilms. The extent of any
negative impact due to photosynthetic microorganisms should be
studied in detail in the future.
The predominant sulphate-reducing bacteria (SRB) attached on
the filters were Desulfobacterium, which reduce sulphate to sul-
phide. In environments with little or no sulphate, they might also
reduce sulphite or elemental sulphur to sulphide. In rare cases,
nitrate might also be used as an electron acceptor, and would be
reduced to ammonia (Hao et al., 1996). Their low abundance
(<0.05%, Table 3) suggested that the role of SRB in the Eco-dam
system was almost negligible.
4.5. Estimation of the potential benefits of Eco-dam
The Eco-dam concept is to achieve flexible and suitable in situ
bioremediation and pollution control for lake aquaculture, through
simple infrastructure offering ease of operation and minimal
maintenance costs. The pilot-scale Eco-dam test in Yangcheng Lake
showed promising results for breeding high-value species such as
Chinese mitten crabs. Four aspects are significant for estimation of
the performance potential of the Eco-dam system, and for deter-
mining the parameters for optimization in further study.
The first is the determination of the pollutant (organic materials,
nitrogen, and phosphorus) loading rate for the Eco-dam system.
The pollutant input could be based on aquaculture acreage of
10,000 m2 with 10,000 crabs. The organic material input mainly
originated from the commercial crab feed, including a one-time
input of 2625 kg of spiral shells in March, and 11.25 kg of corn
(for lake fish) and 15e30 kg of small frozen fish every three days.
During the peak breeding season (from September to October),
feeding frequency increased to 15 kg of corn and 180 kg of fish
every other day. Approximately 54 m3 of aquatic plants, including
Hydrilla varticillata, Elodea nattalli, and Vallisneria natans were
placed in the breeding area to provide shelter for crabs at the
beginning of the breeding cycle. Considering that more than 95% of
the feed added could be utilized by crabs and lake fish, the organic
pollutants were eventually transferred to nutrients, and N and P
loads would be the key factors in the design of the Eco-dam system.
The TN and TP loads originated from the residual feed, faeces of
crabs and fish, and dissolved pollutants released from sediments
during crab-breeding season. According to the mass balance
method described in the literature (Montanhini Neto and
Ostrensky, 2015), the TN and TP loads were preliminarily esti-
mated as 0.065 g/(m2$d) and 0.0045 g/(m2$d) (note: m2 refers to
the breeding area). These values might be overestimated because
pollutants could continuously move out of aquaculture area due to
lake water currents, which made it difficult to provide a conclusive
model of an ecological dam using existing models of lake

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 Z. Ni et al. / Journal of Cleaner Production xxx (2017) 1e10
aquaculture (Lefebvre et al., 2001).
The second aspect is how to accurately estimate the bioreme-
diation capability of the Eco-dam system. In this study, the
maximum pollutant removal rates of 0.609 kg COD/(m2$d), 0.512 kg
NHþ 2  2
4 -N/(m $d), 0.482 kg NO2 -N/(m $d), and 0.112 kg NO3 -N/

2
(m $d) were based on batch assays of microbial activities. These
rates could not be achieved in situ, due to seasonal dynamics of
biomass content and microbial structure in SBF biofilms, the effect
of temperature on microbial activities, and mass transfer of pol-
lutants from the ambient water to the biofilm. As analysed in sec-
tion 4.3, photosynthetic microorganisms or algae might also have
additional impact. Future work should address these issues.
The third aspect is how to determine the nutrient removal,
especially phosphorus removal, by plants in the PFB. This could be
estimated using batch assays of typical aquaculture plants in the
laboratory. For example, Ni et al. (2016) found that water spinach
(Ipomoea aquatica) removed nitrogen at a rate of 4 mg NHþ 4 -N/
(m2$d) and phosphorus at a rate of 0.48 mg TP/(m2$d), using
simulated aquaculture water. Future studies will focus on estima-
tion of removal of N and P via harvest of plant biomass from PFB
under lake conditions.
The fourth aspect is to perform a reliable economic analysis of
the Eco-dam system. Variables that should be considered include
the costs of Eco-dam fabrication, installation, and maintenance, and
the potential benefit from the increased crab productivity and
harvest of vegetable products, as well as environmental and other
benefits.
Based on the results of this pilot study, a preliminary economic
analysis was conducted for a full-scale aquaculture operation of
10,000 m2 with 10,000 juvenile crabs, considering capital depre-
ciation over 8 y. The total capital investment for fabrication and
installation of the Eco-dam was estimated to be 280,000 RMB
(~41,500 USD), based on an area of 1400 m2 for the Eco-dam, or 14%
of the total breeding acreage, with the capital cost assumed to be
reduced by at least 30% by selecting proper materials and econo-
mies of scale. The Eco-dam could reduce net costs through the
increase in crab productivity and harvest of vegetables on FPB, as
well as cost saving from reduction of adding aquatic plantings and
the reduction in crab feed due to possible use of the biofilm by
crabs. A preliminary cost estimate was provided in Table S3 in the
Supplementary Information. More accurate estimates will be
developed in the future studies using more than one pilot system.
In this study, microbial community structures were only
investigated in August, when the lake experienced elevated tem-
peratures and accelerated feeding. The results showed that the Eco-
dam appeared to function well without algal blooms or deteriora-
tion of lake water quality. However, the information obtained was
still limited, and future studies will focus on investigation of the
microbial activities of respective functional microorganisms and
microbial community dynamics throughout the crab breeding
period, by collecting more detailed information to help scale up the
Eco-dam design and management of operation.
5. Conclusion
A pilot scale Eco-dam system comprising submerged SBF and
PFB units was successfully tested for in situ bioremediation and
pollution control for the breeding of Chinese mitten crab in an
aquaculture farm in Yangcheng Lake, China. The water quality in
the test zone was slightly improved compared with the breeding
zone although this was likely affected by lake water currents. The
SBF biofilm had high microbial activities relevant to pollution
control and bioremediation of lake aquaculture. The estimated
maximum bioremediation capability of the tested Eco-dam was
0.609 kg COD/(m2$d), 0.512 kg NHþ 2 
4 -N/(m $d), 0.482 kg NO2 -N/
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9
(m2$d), and 0.112 kg NO 2
3 -N/(m $d) for the removal of organic
carbon and nitrogen compounds during the breeding period. Both
proteins and VSS distributions in the SBF biofilms decreased from
the water surface towards the lake bottom, especially below 0.5 m
depth. The average ratios of VSS/TSS and protein/VSS were
0.337 ± 0.025 and 0.41 ± 0.05 g/g, respectively. The most abundant
bacteria, at the phylum level, were Proteobacteria, Actinobacteria,
Cyanobacteria, Firmicutes, Planctomycetes, Bacteroidetes and Nitro-
spirae. Other functional microbial genera identified in the biofilms
included trophic groups for the nitrogen cycle (AOB, NOB, DNB,
Anammox) and organic carbon degradation, as well as methano-
genic and methane-oxidizing bacteria, with limited sulphate-
reducing bacteria. Preliminary cost analysis suggested potential
benefits from use of the Eco-dam system, including increased
productivity of crabs.
Acknowledgments
This research project was funded by National Natural Science
Foundation Council of China (No. 51378305, 2014e2017). Dr.
Chunjie Li was financially supported by China Scholarship Council
during the research period (No. 201308310275, April 2014 to April
2015) as a Visiting Scholar at Center for Sustainable Development &
Global Competitiveness at Stanford University. We appreciate help
and suggestions from Drs. James O. Leckie, Craig Criddle, Jie Wang,
Colin Ong, and Sandy Robertson, Department of Civil & Environ-
mental Engineering, Stanford University, USA; Dr. Pei Xu, Civil En-
gineering Department, New Mexico State University, USA; Dr.
Weiwei Mo, Department of Civil and Environmental Engineering,
University of New Hampshire, USA; Dr. Salma Tabassum, School of
Environmental Science and Engineering, Shanghai Jiao Tong Uni-
versity, China; and Dr. Chew-Tin Lee, Faculty of Chemical & Energy
Engineering, University Technology Malaysia, Malaysia.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.jclepro.2017.11.185.
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Please cite this article in press as: Ni, Z., et al., Pollution control and in situ bioremediation for lake aquaculture using an ecological dam, Journal
of Cleaner Production (2017), https://doi.org/10.1016/j.jclepro.2017.11.185