Water Research 135 (2018) 112e121

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Water Research
journal homepage: www.elsevier.com/locate/watres

Improving water quality using settleable microalga Ettlia sp. and the
bacterial community in freshwater recirculating aquaculture system of
Danio rerio
Seong-Jun Chun a, b, Yingshun Cui a, Chi-Yong Ahn a, b, **, Hee-Mock Oh a, b, *
a
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of
Korea
b
Department of Environmental Biotechnology, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-
gu, Daejeon 34113, Republic of Korea

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

Article history: A highly settleable microalga, Ettlia sp., was applied to a freshwater recirculating aquaculture system
Received 28 September 2017 (RAS) of Danio rerio to improve the treatment of nitrogenous compounds. The growth characteristics of
Received in revised form the microalgae, water quality parameters, and bacterial communities were monitored for 73 days. In the
29 December 2017
treatment RAS, the inoculated Ettlia sp. grew up to 1.26 g/L and dominated (>99%) throughout the
Accepted 5 February 2018
Available online 8 February 2018
experiment, whereas naturally occurring microalgae grew to 0.57 g/L in the control RAS. The nitrate,
nitrite, and ammonium concentrations in the treatment RAS were reduced by 50.1%, 73.3%, and 24.2%,
respectively, compared to the control RAS. A bacterial community analysis showed that Rhodospirillales,
Keywords:
Recirculating aquaculture systems
Phycisphaerae, Chlorobiales, and Burkholderiales were the major bacterial groups in the later phase of the
Settleable microalgae treatment RAS. Meanwhile, a network analysis among the Ettlia sp., bacterial groups, and environmental
Ettlia sp. parameters, revealed that the bacterial groups played key roles in both water quality improvement and
Water quality Ettlia sp. growth. In conclusion, the inoculation and growth of the Ettlia sp. and its associated bacteria in
Nitrate the RAS produced beneficial effects on the water quality by reducing the nitrogenous compounds and
Algae-bacteria interaction providing a favorable environment for certain bacterial groups to further improve water quality.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction nitrogenous compounds, phosphorus, and solids concentration

The estimated annual aquaculture production worldwide is 73.8
million tons and the contribution of aquaculture to the world's total
fish production is 44.1% (FAO, 2016). Recirculating aquaculture
systems (RAS) is generally designed to maintain clean water and
provide a suitable environment for aquatic organisms. However,
the operation of an RAS requires regular expert care and constant
water exchange (5e10%/d) to maintain the water quality, including
a tolerable range of dissolved oxygen, pH, carbon dioxide,
* Corresponding author. Cell Factory Research Center, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141,
Republic of Korea.
** Corresponding author. Cell Factory Research Center, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141,
Republic of Korea.
E-mail addresses: cyahn@kribb.re.kr (C.-Y. Ahn), heemock@kribb.re.kr
(H.-M. Oh).
(Badiola et al., 2012).
Among the various water quality parameters, excessive nitrog-
enous compounds (ammonium (NHþ 
4 ), nitrite (NO2 ), and nitrate
(NO3 )) need to be more carefully controlled for aquaculture sys-
tems. In nature, these compounds are controlled by photosynthetic
organisms and denitrifying bacteria. However, nitrogenous com-
pounds (mainly nitrate) tend to accumulate in aquaculture systems
(~500 mg NO 3 -N/L) (Pierce et al., 1993), due to high feed loads and
high fish densities. High concentrations of nitrate can reduce ani-
mal growth and decrease survival in aquacultures (Davidson et al.,
2014; Pierce et al., 1993). Further, even low levels of nitrate can lead
to chronic toxicity problems in sensitive aquatic organisms,
particularly during the early stages of certain freshwater in-
vertebrates, fish, and amphibians (Camargo et al., 2005). Tradi-
tionally, the nitrate concentration in aquaculture systems is
controlled by water exchange, phytoremediation, biofilter treat-
ment, and denitrification (Crab et al., 2007; Martins et al., 2010;
Yang et al., 2001).

https://doi.org/10.1016/j.watres.2018.02.007
0043-1354/© 2018 Elsevier Ltd. All rights reserved.
 S.-J. Chun et al. / Water Research 135 (2018) 112e121
Many studies have previously demonstrated the success of using
microalgae as a biological purifier for water treatment (Sutherland
et al., 2015). Microalgae are primary oxygen-releasing photosyn-
thetic microorganisms and have already been used in various ap-
plications. These microorganisms can improve the water quality by
reducing nitrogenous compounds and producing various beneficial
compounds (Richmond and Hu, 2013). Microalgae have also shown
numerous positive effects on aquatic organisms, such as improving
their stress response, physiological activity, starvation tolerance,
and disease resistance (Borowitzka, 1997). Moreover, harvested
microalgal biomass can be used as sustainable feed for aquaculture
(Hemaiswarya et al., 2010). Several well-known algal species, such
as Chaetoceros sp., Isochrysis sp., Chlorella sp., and Spirulina sp., have
been applied in aquaculture systems (Borowitzka, 1997; Lananan
et al., 2014; Sombatjinda et al., 2014). However, harvesting the
microalgal biomass from water remains a major problem for the
application of microalgae, due to their small cell size and float-
ability. Therefore, immobilization, periphyton, and bio-floc tech-
niques have all been developed to overcome the harvesting
problem of microalgae (Moreno-Garrido, 2008). In this study, an
Ettlia sp., which has been assigned to the family Chlorococcaceae,
was applied to a newly designed RAS to investigate its effects on
water quality and bacterial community structures. This green alga,
which is morphologically spherical and/or subspherical, was orig-
inally isolated from freshwater and later reported to also inhabit
terrestrial environments (Pegg et al., 2015; Yoo et al., 2013). Ettlia
sp. is known as a highly settleable microalga due to its auto-
flocculating property and is a suitable candidate for producing
biodiesel and high-value products (e.g. lutein, b-carotenoid, keto-
carotenoid, photoprotective reagent) (Lee et al., 2016; Salim et al.,
2014; Yoo et al., 2013).
Microorganisms form various ecological relationships, ranging
from mutualism to competition, that reshape microbial community
structures. Recently, association network techniques have
frequently been applied to microbial abundance data to detect
significant patterns of mutual exclusion between taxa and to
represent them as a network (Faust and Raes, 2012). It is generally
known that microalgae-bacteria exhibit relationships in the phy-
cosphere may range from mutualism to parasitism (Ramanan et al.,
2016). For example, microalgae can provide oxygen, microalgae-
derived substances, and a favorable environment for bacteria,
while bacteria can provide bacteria-derived substances and nutri-
ents for microalgae by decomposing organic polymers into small
molecules that can be used by microalgae. Therefore, when
applying microalgae to aquaculture systems, the effects of the
microalgae on the bacterial community also need to be considered.
Accordingly, this study investigated the water quality parame-
ters, microalgae growth characteristics, and bacterial communities
in a newly designed RAS to 1) uncover the effects of a highly
settleable microalga Ettlia sp. on the water quality parameters,
especially on nitrogenous compounds, 2) characterize the Ettlia sp.
growth in the newly designed microalgal tank, and 3) reveal the
effects of the Ettlia sp. on the bacterial communities in the RAS.
2. Materials and methods
2.1. System and experimental setup
This study used a newly designed RAS, which was composed of a
rearing tank (RT), biological sponge filters (BSFs), microalgal tank
(MT), gravel, and heater (Fig. 1). A total of four RASs, two as controls
and two for treatments, were established. The RT consisted of a
commercial glass tank (40  40  40 cm) with two BSFs (SP-L4,
Aquatech, Korea) connected to an air pump (SHD-60S, Shinhwa
Hitech Co, Korea). The water flow rate for the BSFs was
113
approximately 3 L/min. Commercial gravel (0.5e1 mm) was
washed 3 times with tap water and 2 times with autoclaved
distilled water and was then scattered to create an approximately
5 cm-deep layer at the bottom of the RT. To mimic a real aquacul-
ture system, 50 Danio rerio were added to each RAS after filling the
RT with approximately 50 L of tap water. The water temperature
was adjusted and maintained at 25  C using a heater (Sobo, Korea).
The MT was made of acrylic panels with two different colors: black
panels on the sides to block light from outside and a transparent
panel on the bottom to allow light to penetrate from a lighting
system. The MT capacity and bottom area were 4 L and 0.12 m2,
respectively. The lighting system (T5 8W  3) was attached to the
outside of the MT transparent panel. The light intensity was
measured using a quantum meter (LI-COR, USA), and set at
40e75 mmol/m2/s in the MT and 85 mmol/m2/s for the subsurface of
the RT (12:12 h light-dark cycle). A water pump (Hyubshin, Korea)
was used to circulate water between MT and RT. Flow rate between
the MT and RT was 1 L/min and the dilution rate in the MT was 0.25
min1. To compensate for water loss due to evaporation in the RAS,
distilled water was added using an automatic system. Commercial
fish feed, TetraBits® Complete (Tetra GmbH, Germany), was sup-
plied twice a day at 09:00 and 15:00 using an automatic feeder
(KWzone, Malaysia), that supplied the total feed at a rate of
0.88 ± 0.26 g/d.
2.2. Microalgae preparation, sampling, and analysis
The microalgae strain used in this study was Ettlia sp. KCTC
12109BP, which was obtained from the Korean Collection for Type
Cultures (KCTC). The initial seed was cultivated using 4-L photo-
bioreactors with a BG11 medium (Stanier et al., 1971). The har-
vested microalgae were washed 10 times with distilled water to
remove any remaining medium, cell detritus, etc., and added to the
treatment MT. After Ettlia sp. flocculated and stacked at the bottom
of the MT, the pump was started at a low speed and gradually
increased to a final dilution rate of 0.25/min.
To determine the growth characteristics of the microalgae in the
MT, the dry cell weight (DCW) and chlorophyll-a (chl-a) concen-
trations were measured in triplicate every 10e15 days using the
following procedures. The water circulation was turned off, and
following manual mixing, 50 ml of the microalgal suspension was
collected from the MT. For comparison, the control microalgal
biomass was collected using a scraper to detach the microalgal
biofilm. The DCW was measured by filtering an aliquot of the
harvested microalgal suspension through pre-weighed GF/C filters
(Whatman, United Kingdom). After rinsing with distilled water, the
filters were dried at 105  C for 24 h and reweighed. Chl-a was
extracted using a chloroform and methanol mixture (2:1, v/v), and
measured using a fluorometer (Turner 450, Barnstead/Thermolyne,
Dubuque, IA). The biomass productivity was calculated based on
the DCW and expressed as [mg DCW/L/d]. Furthermore, the par-
ticulate nitrogen (PN) in the MT was measured using a commercial
kit (C-mac, Korea) after sonication at a resonance of 10 kHz for
5 min (Chisti and Moo-Young, 1986).
2.3. Water quality analysis
The water quality parameters (temperature, pH, and dissolved
oxygen (DO)) in the RT were measured using a portable instrument
(Multi 3410, WTW GmbH, Germany). Nitrite, nitrate, and phos-
phate were determined using an ion-exchange chromatograph
with suppressed conductivity (ICS 1600, Dionex, USA) and equip-
ped with an IonPac AS22-HC (Dionex, USA) guard and analytical
columns. Total ammonium nitrogen (TAN) was measured using a
kit (C-mac, Korea). The turbidity was measured using a turbidity
114 S.-J. Chun et al. / Water Research 135 (2018) 112e121
Fig. 1. (A) Drawing of recirculating aquaculture system (RAS) used in this study, including description of system components. Photographs of microalgae on the surface of MT at day
24: (B) Control; (C) Treatment. Black arrows indicate water circulation direction.
meter (Lutron, Taiwan). The water quality analysis was performed
in triplicate every 2e3 days.
2.4. DNA preparation and 16S rRNA gene amplification
For the bacterial community analysis, samples were collected
from the RT, BSFs, and MT at days 0, 29, and 60. One-liter water
samples were collected from the RT and filtered using a sterilized
0.22 mm polycarbonate membrane filter (Millipore Corporation,
USA). To analyze the BSF bacterial communities, a quarter of the
sponges were detached from the BSFs and squeezed 10 times in
autoclaved distilled water (approximately 500 ml); then, 50 ml was
filtered to analyze the attached bacteria. After sampling, the
sponges were re-attached to the BSFs. Ten milliliters of the
microalgal suspension was collected from the MT and filtered as
described above. All the membrane filters were stored at 80  C in
a deep freezer until DNA extraction.
Genomic DNA was extracted using a ChargeSwitch® Forensic
DNA Purification Kit (Invitrogen, USA) according to the manufac-
turer's instructions. The bacterial 16S rRNA gene was amplified
using a universal bacterial primer set, 341F/805R (341F:
CCTACGGGNGGCWGCAG; 805R: GACTACHVGGGTATCTAATCC),
which targets the V3-V4 regions (Herlemann et al., 2011). The
amplicons obtained were then purified using Agencourt AMPure
XP beads (Beckman Coulter, USA) according to the manufacturer's
instructions. Quantification of the DNA concentrations was per-
formed using a Quant-iT dsDNA HS Assay Kit (Thermo Fisher Sci-
entific, USA), then the purified amplicons were pooled in equimolar
concentrations, and then sequenced using high-throughput paired-
end Illumina sequencing (MiSeq, 2  250 bp reads) by the Macro-
gen Corporation (Seoul, South Korea).
2.5. Sequence analysis procedure
The resulting sequences were processed using mothur (Schloss
et al., 2009) according to the MiSeq standard operating procedure
(http://www.mothur.org/wiki/MiSeq_SOP) (Kozich et al., 2013).
The Silva database (release 123) was used to align and classify the
sequences. All statistical analyses were performed using the R
package (version 3.4.0). Briefly, low-quality sequences were
removed from the analysis if they contained ambiguous characters,
contained more than two mismatches to the forward primer or one
 S.-J. Chun et al. / Water Research 135 (2018) 112e121
mismatch to the barcode, or were less than 300 bp or more than
500 bp in length. After removing doubletons, the pre-cluster
method was applied to further reduce any sequencing errors pro-
duced by the Miseq Illumina sequencing platform (Huse et al.,
2010). Chimeras were identified and removed using chimer-
a.uchime (Edgar et al., 2011). The average read length was
approximately 400 bp after trimming the barcode and primer se-
quences. A similarity cutoff of >99% was used to assign the same
OTUs. The bacterial 16S rRNA gene sequences and accompanying
metadata have already been deposited in the Sequence Read
Archive (SRA) of NCBI under the project number PRJNA406893.
2.6. Diversity indices, association network and statistical analysis
For a visual assessment of the differences between the controls
and the treatment tanks, non-metric multidimensional scaling
(NMDS) was performed using the R software (package: vegan)
(Oksanen et al., 2007). The top 100 most abundant OTUs were
selected to calculate the Bray-Curtis dissimilarities. The richness
(Chao1 and ACE) and diversity (Shannon's and Simpson's indices)
indices were calculated after subsampling using the mothur soft-
ware package (Schloss et al., 2009).
To examine the relationship among the microalgae, water
quality parameters (temperature, pH, DO, ammonium, nitrite, ni-
trate, and phosphate), and bacteria, an association network was
constructed based on Spearman's rank correlation coefficient (r),
which was calculated using the R software (package: Hmisc)
(Harrell et al., 2007). This study used the relative abundance of
distinct bacterial orders with median values above 0.01%. The as-
sociation network was constructed using highly correlated subsets
of variables with significant correlations (jrj  0.7, P < 0.05). The
network was visualized using open source software Cytoscape 3.5.1
(Shannon et al., 2003).
The statistical significance of the clustering was tested using
ANOSIM with the vegan package. Microalgal growth characteristics
(DCW and chl-a) and water quality data (TAN, nitrite, nitrate,
phosphate, temperature, pH, and DO) were analyzed by Student's t-
test for comparison between the control and the treatment using
Sigmaplot 12.0. Differences were considered significant at a P-value
< 0.05.
3. Results
3.1. Growth characteristics of microalgae in microalgal tank (MT)
The RASs were operated for a total of 73 days. The nutrient
loading in the RAS was completely dependent on the daily added
feed, where the feed supply was 0.88 ± 0.26 g/d that included
approximately 46.6 ± 13.8 mg/d of nitrogen and 12.5 ± 3.7 mg/d of
phosphorus. The inoculated Ettlia sp. became flocculated and
stacked at the bottom of the MT (Fig. 1C). In the control, the
naturally occurring microalgae (mainly diatoms) attached tightly
and formed a microalgal biofilm on the bottom panel of the MT
(Fig. 1B). The growth characteristics of the microalgae in both the
control and treatment MT are shown in Fig. 2. In the treatment MT,
the biomass (DCW) and chl-a concentration increased to 1.26 g/L
and 13.85 mg/L, respectively, by the end of the experiment. The
DCW and chlorophyll-a concentration in the control were 0.52 g/L
and 5.46 mg/L, respectively, both of which were lower than those in
the treatment MT. The biomass productivity of Ettlia sp. in the MT
was 19.83 mg DCW/L/d.
The chl-a concentration and biomass showed a significant cor-
relation (r ¼ 0.99, P < 0.01), suggesting that almost all the DCW
consisted of microalgal species. The microscopic observations
revealed that the Ettlia sp. was the dominant microalgae species
115
Fig. 2. Growth characteristics of microalgae in the MT: DCW and Chl-a. Asterisks
indicate significant differences in the control and the treatment.
(>99%) in the treatment MT throughout the experiment (Fig. S1B),
while diatoms dominated in the control MT (Fig. S1A). At the end of
the experiment (day 73), the PN contents were 1.7 ± 0.1 mg-N/g
DCW and 22.6 ± 1.4 mg-N/g DCW in the control and treatment MT,
respectively.
3.2. Changes in water quality
The water temperature, pH, and DO were all monitored during
the experiment (Fig. S2). The water temperature was regulated at
24e26  C. The DO and pH ranged from 7 to 8 mg/L and 8.0e8.3,
respectively. The turbidity in the rearing tanks remained under 0.1
NTU throughout the experiment for both the control and treatment
systems.
The concentrations of inorganic compounds in both systems
varied largely over the course of the experiment (Fig. 3). Therefore,
the experimental data were separated into three different phases
based on the fluctuations: phase 1 (0e29 days), phase 2 (30e60
days), and phase 3 (61e73 days). During phase 1, the ammonium
concentrations increased rapidly within two days and reached
maximum values of 0.44 mg/L and 0.32 mg/L in the control and
treatment systems, respectively (Fig. 3A). These ammonium con-
centrations then decreased sharply, while the nitrite concentra-
tions increased in both systems (Fig. 3B). The maximum nitrite
concentration in the treatment system was approximately one-
fourth of that in the control system. The nitrate concentration in
the control system then gradually increased from day 16 until the
end of the experiment with a final value of 19.05 mg/L, whereas the
nitrate concentration in the treatment system increased from day
16 to day 36 (8.95 mg/L) and was then maintained at the same level
until the end of the experiment (Fig. 3C). Thus, the final nitrate
concentration in the treatment system was only 50% of the control
system.
To evaluate whether Ettlia sp. consumed the nitrogenous com-
pounds in the treatment system, we calculated both differences in
the corresponding time points of the total dissolved nitrogen (TDN)
and DCW between the control and the treatment and designated
these differences as DTDN (TDNtreatment  TDNcontrol) and DDCW
(DCWtreatment MT  DCWcontrol MT), respectively. These two values
significantly correlated with each other (r ¼ 0.70, P < 0.05) (Fig. 4).
At the end of the experiment (day 73), PN concentrations were
0.51 ± 0.04 mg-N/L and 6.69 ± 0.41 mg-N/L in the control and
treatment MT, respectively. DPN (difference in PN between the
control and treatment, i.e., PNtreatment MT  PNcontrol MT) accounted
for 65% of DTDN in RAS.
The phosphate concentration increased steadily from phase 1
and peaked at an early stage of phase 2, with maximum values of
116 S.-J. Chun et al. / Water Research 135 (2018) 112e121

Fig. 3. Mean concentrations of dissolved nitrogenous compounds and phosphate in systems. (A) TAN, (B) Nitrite, (C) Nitrate, and (D) Phosphate. Water samples were collected from
the RT of each RAS. Vertical arrows indicate sampling points for microbial community analysis. Asterisks indicate significant differences in the control and the treatment.

1.22 mg/L and 0.86 mg/L in the control and treatment systems,
respectively (Fig. 3D). Similar to the nitrate concentrations, the
phosphate concentrations in the treatment system were lower
(approximately 30%) than those in the control system. The phos-
phate concentrations in both systems then declined gradually
during phase 2, reaching final concentrations below 0.2 mg/L after
60 days.
3.3. Dynamics of bacterial community compositions
The shifts of the bacterial community structure in the three
different components of the RAS were determined using the Illu-
mina MiSeq sequencing approach. A total of 891,264 sequence
reads of the 16S rRNA gene were obtained after trimming low-
quality sequences, and removing chimeras and single/doubletons.
These sequences were further divided into 4,801 OTUs (99% simi-
larity cut-off).
The bacterial community compositions showed a significant
difference among the MT, BSFs, and RT and among the experi-
mental phases (Fig. 5). Proteobacteria, mainly Xanthomonadales
(19.3%), Caulobacterales (15.7%), Burkholderiales (12.0%), Rhizobiales
(11.5%), and Oligoflexales (7.5%), dominated the bacterial
 S.-J. Chun et al. / Water Research 135 (2018) 112e121 117

in the treatment MT at day 60 as minor groups (<0.1%), but their

Fig. 4. Correlation between DDCW of MT and DTDN. DDCW ¼ DCWtreatment
MT  DCWcontrol MT,DTDN ¼ TDNtreatment  TDNcontrol.
community in the Ettlia sp. seed. Among these 6 dominant groups,
Xanthomonadales, Caulobacterales, and Burkholderiales decreased to
1.9 ± 0.8%, 1.6 ± 0.4%, and 3.2 ± 0.1%, respectively, at day 60 in the
treatment MT, while the relative abundance of some minor groups
increased during the experiment. For instance, Rhodospirillales,
Phycisphaerae, and Cytophagales represented 12.8 ± 1.5%,
14.3 ± 4.8%, and 12.5 ± 7.6%, respectively, of the bacterial commu-
nity in the treatment MT at day 60. In addition to these major
bacterial groups, Nitrosomonadales and Nitrospirales were observed
proportions were approximately 6 times higher than those in the
control system. To evaluate whether the bacteria, which were
initially associated with Ettlia sp. seed, could also contribute to the
improvement of water quality, a Venn diagram was constructed to
explore the shifts of the shared bacteria in the seed and those in the
later phase of the experimental treatment (Fig. S3). A total of 404
different OTUs were observed in the Ettlia sp. seed; thus, theoret-
ically, at least 404 different bacteria carried out the treatment in the
RAS. The overlapping bacterial OTUs in the initial seed and in the
later MT samples decreased during the experiment (from 189 OTUs
at day 29e133 OTUs at day 60).
The bacterial community compositions in the control MTs
showed a high variation between the identical RASs at day 60,
where one control MT was dominated by Aeromonadales (49.1%)
and Alteromonadales (24.2%), while the bacterial community
composition in the other control MT closely resembled that in the
treatment MTs. In addition, the inoculation of the Ettlia sp. in the
treatment system tended to increase the bacterial species richness
(Chao1 and ACE) and diversity (Simpson's index and Shannon's
index) in the MT compared to that in the control MT (Table S1).
The bacterial community compositions did not vary greatly
between the control and treatment BSFs after 29 days. However,
clear differences appeared at day 60. Chlorobiales (15.4 ± 2.9%),
Rhodospirillales (13.8 ± 3.6%), and Rhodocyclales (9.9 ± 4.0%) were
the major groups in the treatment BSFs, while Burkholderiales
(16.3 ± 0.4%), Planctomycetales (16.1 ± 12.6%), and Sphingobacter-
iales (12.0 ± 9.9%) were the major groups in the control BSFs at day

Fig. 5. Bacterial communities from various system components. (A) Relative abundance of OTUs at class level across samples, (B) Heatmap displaying relative abundances of specific
OTUs across samples. Only OTUs with average abundance of >1% assigned sequences across all samples were used. Color scale represents log-transformed relative abundance of
number of sequences. Classes with <1% abundances were pooled together and are shown as 'Others'. 'C0 is control and 'T0 is treatment. 'DXX0 is sampling date. '10 and '20 represent
replicate of each sample.
118 S.-J. Chun et al. / Water Research 135 (2018) 112e121

An NMDS 2-dimensional plot of the bacterial communities,

based on the metric of dissimilarity among the MT, BSFs, and RT in
the control and treatment systems, is shown in Fig. 6. Along the first
axis, the samples were distinguished according to the sampling
day: samples from days 29 and 60 were separated from the samples
from day 0 (tap water and Ettlia sp. seed). Along the second axis, the
samples from the control system were separated from the samples
from the treatment system. In addition, the treatment MT samples
were separated from the RT and BSFs samples during the experi-
ment. The statistical significance of the above clustering was
further confirmed with ANOSIM tests (P < 0.001).

3.4. Network analysis based on Ettlia sp., water quality, and

bacterial community
Fig. 6. Non-metric multidimensional scaling (NMDS) ordination plot of BrayeCurtis
community dissimilarities based on top 100 OTUs from 16S rRNA gene sequences.
To reveal the interactions between the bacteria and the Ettlia sp.
Small gray circles represent OTUs (stress value ¼ 0.104).
in the RAS, an associated network analysis was performed based on
Spearman's rank correlation coefficient (r). Several thousand sig-
nificant correlations (P < 0.05) were found within the bacterial
60. Fusobacteriales accounted for 28.7 ± 9.8% of the bacterial com-
groups and among the bacteria, Ettlia sp. and water quality pa-
munity in both systems at day 29, but this decreased to 4.4 ± 2.0% in
rameters. However, this study focused on the correlations between
the control, which was approximately 3 times higher than that in
the Ettlia sp. and directly connected bacterial groups, and correla-
the treatment system at day 60.
tions between the water quality parameters and directly connected
The bacterial community in the tap water was dominated by
bacterial groups. A total of 60 nodes and 87 edges were obtained,
Proteobacteria: mainly Pseudomonadales (26.5%), Enterobacteriales
based on the criteria described above (Fig. 7). A total of 35 direct
(23.1%), and Rhizobiales (13.7%). These groups gradually shifted to
correlations (24 positive correlations and 11 negative correlation)
Betaproteobacteria (mainly Burkholderiales), which dominated at
were established between the Ettlia sp. and 27 bacterial orders,
day 60 (64.8 ± 6.9%) in the treatment RT. Fusobacteriales repre-
most of which belonged to Gammaproteobacteria (25.7%) and
sented 21.9 ± 10.1% of the bacteria in the control RT at day 60, which
Alphaproteobacteria (20.0%). Among these bacterial groups,
was approximately 3 times higher than that in the treatment RT.
approximately half of the directly linked bacterial groups were
The effect of the inoculated Ettlia sp. on the species richness and
from the MT samples, and approximately one-third were from the
diversity in the treatment RT varied during the experiment: all
BSFs. For instance, in the MT, the Ettlia sp. was positively correlated
indices were higher in the treatment RT at day 29, but were lower at
with Nitrospirales, Rhodospirillales, and Acidobacteria subgroup 6
day 60 compared with the control RT (Table S1).
but was negatively correlated with Xanthomonadales, Oligoflexales,

Fig. 7. Network analysis showing bacterial orders, Ettlia sp., and water quality parameters. The red and gray lines represent negative and positive correlation, respectively. Node size
represents relative abundance.
 S.-J. Chun et al. / Water Research 135 (2018) 112e121
and Caulobacterales. Meanwhile, in the RT, the Ettlia sp. was nega-
tively correlated with Pseudomonadales and Enterobacteriales.
Among the bacterial groups that directly correlated with Ettlia sp.,
approximately 40% of them were also observed in initial Ettlia sp.
seed. For phosphate, six direct correlations with bacterial groups
were found: phosphate showed a strong negative correlation with
Obscuribacterales in the BSFs.
4. Discussion
Greenwater technology is referred to one of the photosynthetic
suspended-growth systems (PSGS), in which animals are cultured
in water containing high concentrations of microalgae. This system
has several advantages: for instance, it entails lower capital costs
relative to other RAS and offers increased control over stock man-
agement relative to conventional static ponds (Hargreaves, 2006).
However, the main disadvantages of this system compared to other
RAS are the difficulties associated with controlling water quality
and microalgae density, metabolism, and community composition
(Hargreaves, 2006; Tseng et al., 1991). In addition, algae-induced
turbidity decreases water transparency and adversely affects the
ability to capture free-swimming prey under low light conditions
(Carton, 2005; Naas et al., 1996). To overcome these disadvantages,
we applied a highly settleable microalga, Ettlia sp., to the RAS to
control and maintain the water quality without increasing algae-
induced turbidity. The concept of integrating a microalga in an
RAS differs from other studies that have applied a microalga to the
rearing tank. The microalgal and rearing tanks were separated in
this study due to the autoflocculating characteristic of Ettlia sp. The
present study demonstrated that the sedimentation of the inocu-
lated Ettlia sp. was not disturbed and that the microalgae remained
in the MT. The turbidity was under 0.1 NTU in the treatment, a
comparable grade to the control system. Further, water in the
treatment RAS never turned green throughout the experiment,
even though DCW of the treatment MT was as high as ~1.2 g/L at the
end of experiment.
The accumulation of high concentrations of nitrate is another
serious problem in an RAS. Recently, even a low nitrate concen-
tration was reported to be problematic (Camargo et al., 2005). In
addition, the TAN and nitrite concentrations in an RAS are consid-
ered indispensable criteria for evaluating the success of an RAS
Fig. 8. Schematic representation of microalga Ettlia sp.-based RAS. Black arrows refer to nitrogen flow. Red arrows refer to interaction. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)
119
(Rojas-Tirado et al., 2016). Microalgae can utilize nitrogenous
compounds, which are byproducts of bacterial degradation and the
ammonification of organic compounds (Li et al., 2008). Previous
studies have already demonstrated that nitrogenous compounds
could be reduced by up to 50% compared to the control when
microalgae (e.g. Spirulina sp., Nostoc sp.) were applied to RAS
(Kamilya et al., 2006; Sombatjinda et al., 2014). Our results showed
that after an initial stabilization period (phase 1), an average of
34 ± 4% of nitrogen remained in the control system which was
consistent with the previous findings that approximately half of the
input nitrogen remained in RAS without water exchange
(Hargreaves, 1998; Hu et al., 2013). Importantly, the remaining ni-
trogen concentration was reduced by over 50% when Ettlia sp. was
inoculated in the treatment system. PN in the treatment MT, mainly
from Ettlia sp. and its associated bacteria, accounted for 65% of
DTDN in the RAS. In addition, DDCW and DTDN were significantly
correlated with each other. These results imply that Ettlia sp. and its
associated bacteria could be responsible, through assimilation, for
the removal of nitrogenous compounds in RAS, with at least 65% of
nitrogen assimilated to its biomass in this study. In summary, Ettlia
sp. and its associated bacteria utilized the problematic nitrogenous
compounds efficiently, thereby improving the water quality in an
aquaculture system. Ettlia sp. flocs were also easily harvestable to
prevent any nutrient release by microalgal death or degradation.
Previous research and development in RASs has been mostly
focused on bacteria-based systems (Qiu et al., 2016; Schreier et al.,
2010). It is already known that microalgae and their associated
bacterial community develop a complex interaction network that
forms a highly dynamic ecosystem (Ramanan et al., 2016). How-
ever, little information is available about the shift of the bacterial
community during microalgal cultivation in an RAS. Phycisphaerales
and Rhizobiales in the MT and Burkholderiales (e.g. Rhizobacter) in
the RT, which are alga-associated and plant growth-promoting
bacteria (Fukunaga et al., 2009; Santoyo et al., 2016), increased
during the experiment in the treatment system (Fig. 5B). Pseudox-
anthomonas, a well-known bacterium involved in granule stability
enhancement (Wan et al., 2013), also appeared in the treatment MT.
These bacterial groups might have helped the inoculated Ettlia sp.
to adapt to the new environment and maintain its flocculation
structure during the experiment (De-Bashan et al., 2004).
Furthermore, the inoculated Ettlia sp. might also induce the
120 S.-J. Chun et al. / Water Research 135 (2018) 112e121
establishment of bacterial groups that are favorable for its survival.
Fusobacteriia, a fish feces-related bacterial group, decreased in the
treatment RT compared to control RT, indicating that undigested
fish feces remained in the control water, a sign of degenerating
water quality (Roeselers et al., 2011). Nitrifying bacteria and
microalgae have been applied to remove nitrogenous compounds
in aquaculture systems, where nitrifying bacteria convert TAN to
nitrate while microalgae convert nitrate to its biomass (Suantika
et al., 2015). We also found that Nitrospirales and Nitro-
somonadales, two well-known nitrifying bacteria, were more
enriched in the treatment MT and tightly coupled with Ettlia sp.,
suggesting that these nitrifying bacteria act as efficient converters
of ammonia to nitrate, the more favorable form of nitrogen for Ettlia
sp. (Isleten-Hosoglu et al., 2013), which indicates a tight mutualistic
interaction. Approximately 30% of the native bacterial OTUs in the
Ettlia sp. seed, which accounted for approximately 10% of total
bacterial OTUs in the MT, were still present at day 60, suggesting
that Ettlia sp. could function as a vessel for supporting bacteria.
However, the major bacterial groups in the seed became the minor
groups at the end, suggesting that the unique environment of the
RAS could reshape the bacterial community composition of the
associated groups in a way that could help Ettlia sp. better adapt to
the RAS.
The phosphate concentrations decreased during phase 2, then
maintained low concentrations in both the control and treatment
systems, being utilized by microalgae and other microbes. In
addition, various polyphosphate-accumulating organisms, such as
Obscuribacterales, can accumulate phosphorus in their cell, which
may also decrease the phosphate concentration (Oyserman et al.,
2017). In this study, Obscuribacterales was negatively correlated
with the phosphate concentrations (Fig. 7), further supporting this
hypothesis.
In summary, the newly designed microalgae-based RAS could
remove at least 65% of nitrogen without increasing algae-induced
turbidity (Fig. 8.) The inoculated Ettlia sp. and its associated bac-
teria were tightly coupled and showed mutualistic interactions
during the experiment. The autoflocculation characteristic of Ettlia
sp. made it easy to harvest the biomass, which could be further
used as high-value products and sustainable feed source for
aquaculture systems, especially for freshwater shrimp and fish (La
et al., 2016).
This study used Danio rerio, a model vertebrate organism, as an
aquaculture organism. No negative effects on the Danio rerio were
noted when the Ettlia sp. was inoculated in the MT, indicating that
the two organisms were able to co-exist in the RAS. However, Danio
rerio is not a commercial aquaculture species. Therefore, other
aquaculture organisms should be co-cultured with the Ettlia sp. to
further evaluate whether this microalga has any positive or nega-
tive effects on the growth, health conditions, and productivity of
the target aquaculture organisms.
5. Conclusions
This study produced four key results. First, a highly settleable
microalga, Ettlia sp., was applied to a newly designed RAS and grew
well without increasing the algae-induced turbidity. Second, the
Ettlia sp. and its associated bacteria improved the water quality,
especially by reducing the concentrations of nitrogenous com-
pounds, which can be toxic to aquatic organisms in an RAS. Third,
the Ettlia sp. induced a stable and favorable bacterial community
structure for its survival and growth. Fourth, more detailed re-
lationships in the aquatic ecosystem structure were revealed using
a network analysis method. In summary, the water quality was
improved by the Ettlia sp. and its associated bacteria. Furthermore,
the Ettlia sp. could be applied to a conventional RAS for additional
beneficial effects and used as a new effective technique to control
and maintain an RAS.
Acknowledgments
This research was supported by the Advanced Biomass R&D
Center, a Global Frontier Program, funded by the Korean Ministry of
Science and ICT (2010-0029723).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.watres.2018.02.007.
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