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Development of a Zero Water Discharge (ZWD) – Recirculating Aquaculture

System (RAS) Hybrid System for Super Intensive White Shrimp ( Litopenaeus
vannamei ) Culture under Low Salin...

Article  in  Aquacultural Engineering · April 2018

DOI: 10.1016/j.aquaeng.2018.04.002

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 Aquacultural Engineering 82 (2018) 12–24

Contents lists available at ScienceDirect

Aquacultural Engineering
journal homepage: www.elsevier.com/locate/aque

Development of a zero water discharge (ZWD)—Recirculating aquaculture T

system (RAS) hybrid system for super intensive white shrimp (Litopenaeus
vannamei) culture under low salinity conditions and its industrial trial in
commercial shrimp urban farming in Gresik, East Java, Indonesia
⁎
Gede Suantikaa, , Magdalena Lenny Situmoranga, Jonathan Berlian Kurniawanb,
Sherly Arista Pratiwib, Pingkan Aditiawatia, Dea Indriani Astutia, Fahma Fiqhiyyah Nur Azizahc,
Yovita Astuti Djohana, Usman Zuhrid, Togar Mangihut Simatupange
a
Microbial Biotechnology Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung, 40132, Indonesia
b
Biology Study Program, School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung, 40132, Indonesia
c
Biomanagement Study Program, School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung, 40132, Indonesia
d
Commercial Shrimp Nursery and Grow-out “UD. Popular”, Desa Cerme Lor, Kec. Cerme, Kab, Gresik, East Java, Indonesia
e
Operation and Performance Management, School of Business and Management, Institut Teknologi Bandung, Jalan Ganesha No. 10 Bandung, 40132, Indonesia

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

Keywords: This study aims to develop a hybrid zero water discharge (ZWD) - recirculating aquaculture system (RAS) system
Litopenaeus vannamei to improve water quality, as well as the growth, survival, and productivity, of the super-intensive white shrimp
Hybrid system culture under low salinity conditions at semi-mass and the industrial level. The study consisted of two parts: (1) a
Recirculation aquaculture systems semi-mass trial for the optimization of shrimp production using a hybrid ZWD-RAS system with a total volume of
Zero water discharge system
2.7 m3 at the different shrimp stocking densities of 500 PL/m3, 750 PL/m3, and 1,000 PL/m3 and (2) an in-
Low salinity
dustrial trial at a commercial shrimp urban farming facility in Gresik, East Java, with total volume of 110 m3 at
Super intensive
Industrial the optimum shrimp stocking density from the semi-mass trial. Both the semi-mass and industrial trials were
performed in five steps: (1) preparation and installation of the RAS and ZWD system components; (2) pre-
paration of microbial components including nitrifying bacteria, the microalgae Chaetoceros muelleri, and the
probiotic heterotrophic bacteria Bacillus megaterium; (3) acclimatization of white shrimp post larvae from the
salinity level of 32 ppt to 5 ppt; (4) conditioning of the biofilter used in the RAS and shrimp tank (microbial loop
manipulation in ZWD); and (5) shrimp grow-out rearing for 84 days and 60 days for the semi-mass trial and the
industrial trial, respectively. The hybrid system combined a ZWD system and an RAS. Shrimp tanks were con-
ditioned with the addition of microbial components for ZWD at the beginning of the culture period. The RAS was
operated when NH4+ and NO2−-N levels in shrimp culture reached above 1 ppm until the levels decreased to
0–0.5 ppm. The culture performance in the semi-mass trial at 500 PL/m3, 750 PL/m3, and 1,000 PL/m3 stocking
densities was not significantly different for final mean body weight (12.06 ± 5.72, 11.84 ± 3.58,
12.04 ± 3.71 g/ind, respectively) and productivity (4.205 ± 0.071, 4.691 ± 0.025, 4.816 ± 0.129 kg/m3,
respectively). Significant differences in survival (70 ± 7%, 53 ± 3%, 40 ± 4%, respectively) and feed con-
version ratios (1.54 ± 0.01, 1.82 ± 0.00, 2.16 ± 0.03, respectively) were observed between the three dif-
ferent stocking densities. Water quality parameters and microbial loads during the semi-mass trial were similar
for all stocking densities and were within the tolerance levels for white shrimp grow-out production. The results
of the semi-mass trial showed that the hybrid ZWD-RAS system can maintain water quality and a microbial load
up to a 1,000 PL/m3 stocking density; however, the optimum performance based on survival, feed conversion
ratio, and productivity was reached at the 500 PL/m3 stocking density. The industrial trial of the application of
the hybrid ZWD-RAS system using the optimal stocking density of 500 PL/m3 resulted in a comparable shrimp
survival of 78% with a total production of 298 kg shrimp biomass (equal to a productivity level of 2.7 kg/m3).
The overall results of both the semi-mass and industrial trials showed that the application of a hybrid ZWD-RAS

⁎
Corresponding author.
E-mail address: gsuantika@sith.itb.ac.id (G. Suantika).

https://doi.org/10.1016/j.aquaeng.2018.04.002
Received 11 December 2017; Received in revised form 22 April 2018; Accepted 26 April 2018
Available online 27 April 2018
0144-8609/ © 2018 Elsevier B.V. All rights reserved.
G. Suantika et al.
system allows optimal shrimp survival and growth at the stocking density of 500 PL/m3 and has high potential
for application in commercial shrimp grow-out production at low salinity levels.
1. Introduction
Crustacean commodities are the second biggest aquaculture product
in the world after finfish commodities. Crustaceans significantly con-
tribute to the high growth of the food industry, with an annual growth
rate of 8.6%. In 2014, Indonesia’s crustacean production was greater
than 610.000 tonnes or approximately 10% of world’s crustacean pro-
duction (FAO, 2016). Indonesia produced more than 353 thousand
tonnes of shrimp in 2015, and the production of shrimp is predicted to
increase up to 506 thousand tonnes in 2020 (Ipsos, 2016), comprising
almost 15% of the total aquaculture value worldwide (FAO, 2016). The
production of white shrimp (Litopenaeus vannamei) now accounts for
approximately 75% of the total shrimp production in Indonesia (FAO,
2016).
The techniques used to produce white shrimp in Indonesia are still
dominated by conventional rearing strategies using flow through, out-
door, and earthen pond systems. Despite their relatively simple and
low-cost operations, several disadvantages are still attributed to these
conventional systems, such as a lack of water quality management,
biosecurity issues, and disease outbreak, which lead to unpredictable
culture performance (Otoshi et al., 2003). Environmental issues that
challenge the sustainability of shrimp aquaculture in Indonesia are
mainly due to wastes with high inorganic nitrogen contents, i.e., am-
monium (NH4+) and nitrite (NO2−), which are toxic to aquatic or-
ganisms and can lead to the eutrophication of the aquatic ecosystem
(Eng et al., 1989). To address this sustainability issue, a new strategy
for shrimp production needs to be developed and implemented im-
mediately in the aquaculture industry. This strategy should consider the
balance of three main components of sustainable aquaculture devel-
opment: economic, social, and environment components.
One potential alternative production strategy is the use of a closed
aquaculture system, which is a controlled and environmentally friendly
system that offers excellent water quality management and water effi-
ciency as well as site and culture dimension flexibility. Generally, there
are two types of closed systems in the aquaculture industry: (1) the
recirculation aquaculture system (RAS) and (2) the zero water dis-
charge (ZWD) or biofloc system (Funge-Smith and Phillips, 2001). The
basic principle of the RAS is the treatment of effluent culture water
through a series of consecutive processes: (1) filtration, including
physical and mechanical filtration (using protein skimmers for fine
particulate organic/protein matter removal and activated carbon filters
for the absorption of possible toxic substances) and biofiltration units
(using biofilters for toxic ammonium and nitrite removal); (2) disin-
fection; and (3) oxygenation before being reinjected into the rearing
tank. Even though the RAS can maintain stable water quality for long
periods, its operational cost, specifically its electricity costs (Suantika
et al., 2003), makes this system less attractive for application at the
farm level.
On the other hand, in ZWD or biofloc systems, the culture water is
maintained by means of microbial manipulation in the shrimp tank;
therefore, the microbial consortium can be functional in the nutrient
cycle, for water purification, for bacteria control, and as a feed sup-
plementation source (Suantika et al., 2015). In the ZWD system de-
scribed by Suantika et al. (2015), water quality is maintained by means
of microbial loop manipulation including (1) Bacillus megaterium to
enhance the rate of ammonification from accumulated organic matter;
(2) nitrification bacteria to convert toxic ammonium into less toxic
nitrate; and (3) the microalgae Chaetoceros muelleri to uptake nitrate,
improve the dissolved oxygen (DO) level, provide shading, and act as a
source of live feed for shrimp culture. Even though the operational cost
13
Aquacultural Engineering 82 (2018) 12–24
to maintain water quality in a ZWD system is relatively low, the car-
rying capacity of the system is not big enough to support a long period
of super intensive shrimp/fish culture (Suantika et al., 2015). There-
fore, the application of both systems using hybrid technology to com-
plement each system’s disadvantages needs to be studied and evaluated.
This study aims to evaluate the application of a hybrid ZWD-RAS
system to improve water quality and the growth, survival, and pro-
ductivity of super-intensive white shrimp culture under low salinity
conditions at the semi-mass and industrial levels.
2. Materials and methods
2.1. Semi-mass trial
2.1.1. Hybrid ZWD-RAS system preparation
A hybrid ZWD-RAS system was developed by combining two shrimp
rearing strategies, the RAS and the ZWD system. At the initial stage, the
hybrid system was run as a ZWD system in which no water discharge
was conducted during the operation as long as the system was capable
of supporting the water quality. The only water added during the ZWD
phase was to compensate for water losses due to evaporation. The RAS
was operated to maintain shrimp water quality, including the range of
NH4+ and NO2− levels in shrimp tanks of 0.5–1.0 ppm. In the RAS, a
series of effluent water treatment was conducted prior to reinjection
into the culture tank, including (1) physical filtration, (2) protein re-
moval in skimmers, (3) activated carbon filtration, and (4) biofiltration
(Suantika et al., 2003).
2.1.2. Compartment preparation for the hybrid ZWD-RAS system
The hybrid ZWD-RAS system consisted of several compartments: (1)
nine 500-L flat bottom cylindrical fibre shrimp tanks, each equipped
with two lines of aeration using air stones; (2) one 100-L rectangular
settling tank equipped with a filter carpet for the physical filtration of
effluent water; (3) one 100-L protein skimmer to remove fine organic
particulate matter; (4) one 50-L activated carbon tank to remove con-
taminants and impurities, and (5) one 1000-L flat bottom cylindrical
biofilter tank aerated and inoculated with a nitrification bacterial
consortium to degrade ammonium and nitrite in the effluent water.
2.1.3. Cultivation and activation of microbial components: nitrifying
bacteria, Bacillus megaterium, and Chaetoceros muelleri
Microbial components used in the ZWD system consisted of (1) a
chemoautotroph nitrifying bacterial consortium of ammonium oxi-
dizing bacteria (AOB) and nitrifying oxidizing bacteria (NOB), (2) the
heterotroph B. megaterium, and (3) the photoautotrophic marine diatom
C. muelleri. All microbial isolates used in this study were obtained from
the Laboratory of Aquatic Ecosystem Analysis, School of Life Sciences
and Technology, Institut Teknologi Bandung, Indonesia. The nitrifying
bacterial consortium was used to ensure the nitrification process oc-
curred in the ZWD system. Nitrifying bacteria stock cultures were
grown using Winogradsky medium and scaled to a total volume of
100 L using 5 ppt diluted sea water containing 10% (v/v) CaCO3 in a
fully aerated condition. As an inorganic nitrogen source, 2 ppm NH4Cl
was added to the bacterial stock culture (Fig. 1) (Suantika et al., 2015).
B. megaterium was cultured using 8 g/L nutrient broth medium at
room temperature for 24 h and then scaled using 12 g/L commercial
broth for another 24 h (Suantika et al., 2015). C. muelleri was cultured
using 0.1% Walne's medium (v/v) and 0.1% of 30 g/L sodium silicates
(Na2SiO3) at a 5 ppt salinity level (Suantika et al., 2015).
G. Suantika et al.
Fig. 1. Scheme of the nitrifying bacteria reactor to produce active nitrifying
bacteria used in the hybrid ZWD-RAS system.
2.1.4. Conditioning of the RAS biofilter and the microbial loop in the ZWD
system
Conditioning of the biofilter tank was conducted by inoculating a
biofilter tank containing 600 L of chlorinated diluted seawater at a 5 ppt
salinity level with 40 L of a nitrifying bacterial consortium at a density
of 106 CFU/mL. Seventy-five kilograms of limestone (calcium carbo-
nate) gravel 3–4 cm in diameter was used in the biofilter as the im-
mobilization medium for the nitrification bacteria to become estab-
lished as biofilm. Spherical black plastic 32 mm bioballs were added to
cover the biofilter surface area in order to improve the performance of
the nitrifying bacteria through the reduction of light in the biofilter and
to enlarge the surface area for the entrapment of fine suspended par-
ticulate entering the biofilter. Activation of nitrifying bacteria was in-
itiated by the addition of 3 g NH4Cl (equal to 5 ppm NH4+). Once the
NH4+ concentration in the biofilter had decreased to 0 ppm, another 6 g
NH4Cl was added to the biofilter (equal to 10 ppm NH4+). The biofilter
was considered ready for addition to the RAS when the NH4+ and
NO2− concentrations had both decreased to 0–0.5 ppm, indicating that
AOB and NOB were already active in oxidizing ammonium to nitrite
and nitrite to nitrate, respectively (Suantika et al., 2016).
During the initial stage, the shrimp tank was used as a ZWD system.
The ZWD system conditioning was initiated by the addition of 300 L
chlorinated diluted seawater at 5 ppt salinity into each 500-L shrimp
tank, followed by the addition of 12.5 kg limestone gravel 3–4 cm in
diameter to the tank bottom surface to retain the nitrifying bacteria
through biofilm establishment. Limestone gravel has an acid buffering
capacity due to the acidification that occurs from organic acid released
during decomposition (Suantika et al., 2016). Subsequently, the tank
was inoculated with 10 L of a nitrifying bacterial consortium consisting
of AOB and NOB at 106 CFU/mL, and 0.3 g NH4Cl (equal to 1 ppm
NH4+) was added to each tank as an ammonium source. After the ac-
tivation of AOB and NOB, indicated by the decrease of the NH4+ and
NO2− concentration to 0–0.5 ppm and increased NO3− level, 0.1% (v/
v) C. muelleri culture (at density of 106 cell/mL) and 150 mL B. mega-
terium (at a density of 107 CFU/mL) were added to the system. The
system was homogenized and oxygenated by means of two aeration
stones each with a 76 mL O2/sec aeration rate. The system was ready to
be used once the colour of culture water turned brownish and the in-
oculated diatom was found in the shrimp tank at the density of 104 cell/
mL. Before stocking, a circular feeding tray device with a 1.3-mm-
diameter mesh net was installed in the culture tank to control the
suitability of the daily shrimp feeding regime (Fig. 2) (Suantika et al.,
2015).
14
Aquacultural Engineering 82 (2018) 12–24
Fig. 2. Scheme of the ZWD system preparation and conditioning.
2.1.5. Acclimatization of ten-day-old post larvae (PL10) shrimp
The ten-day-old post larvae (PL10) shrimp used in this study were
obtained from the commercial hatchery of PT. Suri Tani Pemuka,
Indramayu, West Java, Indonesia. Shrimp acclimatization was achieved
by the gradual addition of chlorinated tap water to gradually lower the
water salinity from 32 ppt to 5 ppt over 13 days at the rate of ± 2 ppt/
day.
2.1.6. System set up and operation
In the hybrid RAS-ZWD system, the effluent water was transferred
from the shrimp tank into the settling tank by gravity before being
pumped into the protein skimmer and then the activated carbon filter
tank. Subsequently, the water was treated using a biofilter for ammo-
nium and nitrite removal before being transferred into the shrimp
culture tank (Suantika et al., 2003). The schematic overview of the
hybrid ZWD-RAS system at the semi-mass level is presented in Fig. 3.
During the initial stage, the hybrid system was run as a ZWD system
in which no water discharge occurred. To compensate for water losses
due to evaporation, chlorinated diluted seawater at 5 ppt salinity was
added weekly at a volume of 10% of the total culture volume. When the
ammonium and nitrite levels in the culture water had reached above
0.5 ppm, the water quality was improved by means of the RAS.
Intermittent use of the RAS was performed until the ammonium and
nitrite levels in the culture water had decreased to 0.0–0.5 ppm. The
recirculation rate was increased from 85% to 200% of the daily water
renewal rate during the course of the 84-day culture period (Table 3).
The culture tank was oxygenated by means of two aeration stones each
with a 76 mL O2/sec aeration rate. Water temperature was maintained
at 28 ± 2 °C by means of an electric submersible 110v/1000 w water
heater. The day time light intensity was maintained by means of LED
lighting in the range of 1700–1,900 lx at the water surface. Plastic
shading net was used to cover the surface of the culture tank to prevent
cyanobacteria blooms (Neilan et al., 2013).
2.1.7. Shrimp stocking and feeding management
For the semi-mass trial, PL10 shrimp were stocked at the three
different densities, 500 PL/m3, 750 PL/m3, and 1,000 PL/m3, and cul-
tured for 84 days. Each stocking density was tested in triplicate.
Feeding management was done by calculating the daily feed amount
(blind feeding) based on an estimation of mean body weight, survival
estimation and feeding rate using the following equation:
Daily Feed (gr) = SD × MBW × FR × SR
where SD is the initial stocking density, MBW is the average mean body
weight of shrimp (grams), SR is estimated survival (%), and FR is the
feeding rate. The estimated survival and the feed ration for each group
G. Suantika et al. Aquacultural Engineering 82 (2018) 12–24

Fig. 3. Schematic overview of the hybrid ZWD-RAS system at the semi-mass level.

Total Plate Count (CFU / mL) = ∑ Colony × (1/ dilutionfactor )

Table 1
Feed management applied during the 84-day grow-out period of the semi-mass × (1/ Volume )
trial (Tacon et al., 2013).
Bacteria isolates, which were obtained from the enumeration pro-
Mean Body Feeding Rate Survival (%) Feeding Tray Monitoring cess, were purified and further identified using molecular approaches.
Weight (g) (%) Intervals (hours) Bacteria identification consisted of 4 steps: bacterial DNA extraction
<1 10.0 100 3.5
using the commercial Qiagen DNeasy blood and tissue kit; polymerase
1–3 8.0 98 3.5 chain reaction (PCR) amplification using the 27F/1492R primer; bac-
3–5 6.0 96 2.5 terial rRNA 16S gene sequencing using the 785F/907R primer; and
5–7 5.0 94 2 sequence homology testing using Nucleotide Blast versus GenBank and
7–9 4.0 92 2
Ribosomal Database Project (RDP) data (the first three steps were done
at Macrogen Inc., Korea). The results were validated with phylogenetic
analysis using the Winclada program and the Ratchet method (Island
size was based on Tacon et al. (2013). The daily feed amount was de-
Hopper) (Suantika et al., 2017).
livered at fixed feeding times four times per day at 09:00, 12:00, 16:00,
and 21:00. The feed was placed on the feeding tray, which was reg-
ularly checked every feeding time to evaluate the accuracy of the 2.1.10. Biological parameter measurement
shrimp daily feeding intake. The feeding strategy used in this trial is Biological culture parameters were assessed based on shrimp pro-
presented in Table 1. duction performance during the 84-day grow-out period. Production
performance parameters, including survival rate (SR), food conversion
ratio (FCR), and total biomass per tank, were calculated using the fol-
2.1.8. Measurement of physicochemical water parameters lowing equations (Suantika et al., 2015):
Water physicochemical parameters, including pH, temperature, DO, SR = Nt / No × 100%
and salinity, were directly measured in the water body twice per week
using a YSI 556 Multiparameter metre (YSI Incorporated, Yellow where SR = survival, No = initial shrimp number, Nt = final shrimp
Springs, OH). The measurement of NH4+, NO2− and NO3− con- number, and t = culture period.
centrations was performed using the Nessler, diazotization, and nitrate
HCl methods, respectively (Eaton and Franson, 2005). 2.1.11. Statistical analysis
All data obtained from this study were subjected to statistical ana-
lysis using one-way analysis of variance (ANOVA) with Tukey ad hoc
2.1.9. Microbiological parameter measurement analysis.
Measurement of microbiological parameters was done once per
week by using the total plate count (TPC) method on the water samples 2.2. Industrial trial
(Neilan et al., 2013). For each treatment group, 50 mL of culture water
from each shrimp replicate tank was sampled and pooled. Microbial Similar to the semi-mass trial, in the hybrid RAS-ZWD system at the
analysis of the pooled sample of culture water was performed in tri- industrial level, the culture effluent water was settled and physically
plicate. The heterotrophic bacteria load was enumerated by plating filtered in one 2,000-L rectangular settling tank before being pumped
samples of culture water following serial dilution on nutrient agar (NA) into two 100-L protein skimmers (Fig. 4a) and then one 250-L activated
medium, while the total number of Vibrio was measured by plating carbon filter tank (Fig. 4b). Subsequently, the water was treated in a
using thiosulphate citrate bile salt (TCBS) agar medium. The total 6000-L flat bottom concrete rectangular biofilter tank (Fig. 4c) before
number of bacteria was calculated by following the equation below being transferred into eight rectangular concrete shrimp tanks with a
(Cappucino and Sherman, 2011): total water volume of 112 T: seven tanks with the dimensions of
4 m × 4 m × 0.8 m and one tank with the dimensions of

15
G. Suantika et al.
Fig. 4. The hybrid ZWD-RAS system components: (a) protein skimmers (upper left), (b) activated carbon filter tank (upper right), (c) biofilter (bottom left), and (d)
shrimp culture tank (bottom right).
4 m × 3 m × 0.8 m (Fig. 4d). The schematic overview of the hybrid
ZWD-RAS system at the industrial level is presented in Fig. 5.
PL10 shrimp were collected from PT. Suri Tani Pemuka,
Banyuwangi, West Java, Indonesia and pooled in 100-L containers,
aerated for good mixing, sampled and counted. For the industrial trial,
the hybrid ZWD-RAS system was operated as in the semi-mass pro-
duction using a shrimp density of 500 PL/m3 with similar feeding
management, as shown in Table 1. Aeration and the water renewal rate
were set to values similar to those in the semi-mass trial. During the 60-
day grow-out culture period, the water quality parameters (tempera-
ture, pH, DO, and NH4+, NO2− and NO3− concentrations) were ob-
served weekly. The temperature, pH, and DO levels were directly
measured in the water body using a YSI 556 Multiparameter metre (YSI
Incorporated, Yellow Springs, OH). The measurement of NH4+, NO2−
and NO3− concentrations was done using the Nessler, diazotization,
and nitrate HCl methods, respectively (Eaton and Franson, 2005). At
the end of the culture period, the shrimp were harvested from all eight
culture tanks and pooled. Shrimp growth parameters including the final
total biomass and average final individual body weight were calculated.
16
Aquacultural Engineering 82 (2018) 12–24
3. Results and discussion
3.1. Semi-mass trial
3.1.1. Biofilter (RAS) and ZWD system conditioning
The RAS system was started with the conditioning of the biofilter, as
one of the most important compartments where the nitrification process
occurs. Ammonium (NH4+), nitrite (NO2−), and nitrate (NO3−) levels
during biofilter (RAS) conditioning are presented in Fig. 6. Based on the
nitrification performance during conditioning, the biofilter was able to
completely breakdown all of the 15 ppm ammonium added over 6 days
to nitrate ions at a rate of approximately 2.5 ppm/day. Nitrite levels as
a result of nitrification increased until day 5, and subsequently, they
gradually decreased to 0 ppm on day 7 of the conditioning period. In
total, it took a 7-day conditioning period to facilitate the nitrification
process in the biofilter. The NH4+ level of up to 15 ppm used during the
biofilter conditioning was based on the estimated total ammonium
accumulated from 9 shrimp tanks. Using the assumption that each
shrimp tank will contribute up to 1.5 ppm ammonium daily, the total
G. Suantika et al.
Fig. 5. Schematic overview of the hybrid ZWD-RAS system at the industrial level.
Fig. 6. Concentrations of NH4+, NO2−, and NO3− during the 7 days of biofilter
conditioning.
ammonium accumulated in the system was estimated to be up to
13.5 ppm daily. This calculation is based on the report by Muhammad
et al. (2016), in which the NH4+ level in each shrimp tank in a ZWD
system can reach up to 3 ppm following 70 days of grow-out culture,
while the use of a biofilter with limestone gravel and bioball substrates
may lower the NH4+ level by 50% (Suantika et al., 2016). The results of
AOB and NOB bacteria consortium activation in the biofilter was in-
dicated by the decrease of ammonium level in the biofilter. Subse-
quently, the gradual decrease of nitrite followed by the incremental
increase of nitrate level showed that the NOB was active and involved
in oxidizing nitrite to nitrate (Suantika et al., 2016).
The initial step for the ZWD system preparation was the inoculation
of the consortium of nitrifying bacteria in the culture tank. Then, 1 ppm
ammonium was added to consecutively activate the AOB and NOB in-
volved in the nitrification process. The ammonium (NH4+), nitrite
(NO2−), and nitrate (NO3−) levels during the ZWD system preparation
measured in the culture tanks are presented in Fig. 7.
Based on the result, the ZWD system could decrease ammonium
from 1 ppm to 0 ppm in the 5-day preparation period with a NH4+
breakdown capacity of ± 0.2 ppm/day. Nitrite was degraded into
17
Aquacultural Engineering 82 (2018) 12–24
Fig. 7. Concentrations of NH4+, NO2−, and NO3− during the ZWD system
conditioning through microbial manipulation.
nitrate within a 9-day period with a NO2− breakdown capacity of ±
0.1 ppm/day. The sixth day of ZWD preparation was when the nitrate
level reached the highest level, and 0.1% (v/v) C. muelleri culture at
density of 106 cell/mL was added and contributed to the lowering of the
nitrate level over the following days. As a major nutrient is needed for
microalgae assimilation, nitrate is the major nutrient needed to produce
biomass (Müller-Feuga et al., 2003).
Based on the result from the conditioning of the biofilter and the
ZWD system, it can be observed that the ammonium oxidation rate was
much faster than the nitrite oxidation rate, as AOB has a relatively
faster growth ( ± 8 h) than NOB ( ± 12 h) (Gerardi, 2002). The biofilter
also provides more favourable conditions, such as pH and salinity, for
the ammonium oxidation process than the nitrite oxidation process
(Jeon et al., 2010). In addition, the NOB activity depends on the
availability of the end product of ammonification, nitrate, by AOB. This
delay also contributes to the prolongation of the biofilter and ZWD tank
conditioning (Suantika et al., 2016). During the 84-day culture period,
all of the water physicochemical parameters observed in the three
treatment groups were in the range of tolerance for white shrimp
(Table 2).
G. Suantika et al.
Table 2
Water quality parameters, pH, temperature, DO, and salinity, during the 84-day
culture period.
Parameter 500 PL/m3 750 PL/m3 1,000 PL/m3
pH 6.5–8.7 6.4–8.6 6.4–8.6
Temperature (°C) 26.5–30.1 26.2–30.4 26.7–30.4
DO (ppm) 5.4–8.5 4.9–8.4 5.3–8.5
3.1.2. Ammonium, nitrite, and nitrate parameters
As one of the chemical water parameters in shrimp culture, dis-
solved inorganic nitrogen in the forms of ammonium, nitrite, and ni-
trate should be monitored tightly during the culture period. The con-
centrations of NH4+, NO2− and NO3− during the 84-day culture period
are shown in Fig. 8. The ammonium level could be stably maintained at
a level between 0.5–1.8 ppm during the culture period and still be
below its toxicity level at above 3.95 ppm at 30 ppt (Lin and Chen,
2001) or above 2 ppm at 10 ppt salinity (Schuler et al., 2010). For the
nitrite parameter, a stable level was able to be maintained during the
first 20 days of the culture period. Then, the differences of nitrite dy-
namics were obtained at three different stocking densities. The highest
nitrite level was obtained at the highest shrimp stocking density,
1,000 PL/m3 (∼3.8 ppm NO2− on day 47 of the culture period), and it
decreased in line with the decrease of the stocking density (3.5 ppm and
2.1 ppm at 750 PL/m3 and 500 PL/m3, respectively). As a final product
of the nitrification process, the nitrate level accumulated during the
culture period, and the highest level at the three different stocking
densities ranged between 100–150 ppm. The relatively high nitrate
level in this study may have been caused by a lower rate of nitrate
uptake by marine diatoms at low salinity levels (Gordillo et al., 2002).
The level of nitrite accumulation during the culture period was still
acceptable for shrimp culture since the toxicity level of this compound
Fig. 8. Concentrations of NH4+ (a), NO2− (b), and NO3− (c) in treatments with
different shrimp stocking densities during the 84-day culture period.
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Aquacultural Engineering 82 (2018) 12–24
is above 25.7 ppm at 30 ppt salinity (Lin and Chen, 2003) or 154 ppm,
which is the 48 h LC50 at the temperature of 29 °C, pH 7.9, and 10 ppt
salinity reported by Schuler et al. (2010). The combination of microbial
loop manipulation in the ZWD system and a series of water treatments
in the RAS resulted in the maintenance of relatively stable and low
ammonium and nitrite levels in the culture water. The limiting capacity
of the ZWD system in breaking down ammonium and nitrite in the high-
density shrimp culture can be compensated for by the periodic use of
the RAS. The use of the hybrid system for shrimp culturing allowed for
the synchronization of the maintenance of water quality and the effi-
ciency of energy costs for RAS operation. The concentrations of am-
monium and nitrite in all stocking density treatments were in the range
of tolerance of white shrimp (Lin and Chen, 2001, 2003; Schuler et al.,
2010), even though there were differences between ammonium and
nitrite levels for the stocking densities of 500 PL/m3 and 1,000 PL/m3
(p < 0.05). This system can maintain ammonium and nitrite below
their toxicity levels up to a stocking density of 1,000 PL/m3.
In general, this hybrid system could support shrimp culture and
maintain an acceptable water quality for a stocking density up to
1,000 PL/m3. However, to maintain water quality, a longer period of
RAS application was conducted at higher shrimp stocking densities.
This longer period of water treatment through the RAS was used be-
cause of the higher accumulation of organic matter during the culture
period. Higher organic accumulation in the water will directly affect the
accumulation of ammonium in the water because it is transformed by
decomposer bacteria into inorganic forms (ammonia or ammonium)
(Ghaly and Ramakrishnan, 2015). To maintain the ammonium level at
an acceptable level for shrimp culture, the RAS was run more frequently
in the hybrid system at higher stocking densities. For this trial, the total
RAS operational periods at 500 PL/m3, 750 PL/m3 and 1,000 PL/m3
were 484 h, 643 h, and 684 h, respectively (Table 3).
The higher stocking densities required higher circulation times be-
cause total organic matter in higher stocking densities is higher than in
lower stocking densities. Most organic matter comes from feed residue
and shrimp faeces. Based on the estimation calculation of TAN (total
ammonia nitrogen) only produced by feed during the 84-day culture
period (Somerville et al., 2014), the total amount of NH4+ and the
average amount per day are shown in Table 4. In Table 4, it is shown
that the ammonium input in higher stocking densities was higher than
in lower stocking densities. Since the ZWD system has a limitation in
oxidizing high ammonium input (Suantika et al., 2015), a more fre-
quent and longer water circulation using the RAS was needed at higher
stocking densities.
3.1.3. Microbiological parameters
The total plate count results of water samples for heterotrophic
bacteria and Vibrio sp. abundance in the hybrid system at three different
stocking densities, 500 PL/m3, 750 PL/m3 and 1,000 PL/m3, are shown
in Table 5.
The microbial abundance was relatively stable and ranged between
2 log units and 5 log units for heterotrophic bacteria and 1 log unit and
4 log units for Vibrio sp. In this system, the proportion of heterotrophic
bacteria was always higher than that of Vibrio sp., indicating that the
use of the hybrid system was able to maintain the stability and balance
among microbial components in the water. In terms of Vibrio load in the
system (both yellow and green isolates), the maximum abundance of
104 CFU/mL was counted during the 84-day culture period, and this
value was almost 2 log units lower than the pathogenicity level of Vibrio
on water of ∼106 CFU/mL (Pena et al., 2001). Therefore, the use of the
hybrid system could also keep the Vibrio sp. bacteria levels low and
stable in the hybrid system. The more frequent and longer water re-
circulation time in groups with higher densities (750 and 1,000 PL/m3)
could have contributed to the stability in the microbial load in the
culture, even though its total organic matter was higher than that at the
lower stocking density (500 PL/m3). This appropriate culture condition
is a very important consideration for the sustainability of shrimp
G. Suantika et al. Aquacultural Engineering 82 (2018) 12–24

Table 3
Water recirculation frequency and average recirculating time, daily water renewal rate and total water volume treated weekly of the hybrid system at three different
stocking densities.
Week 500 PL/m3 750 PL/m3 1,000 PL/m3

Recirculating Daily Total water Recirculating Daily Total water Recirculating Daily Total water
Frequency (day/ renewal rate volume treated Frequency (day/ renewal rate volume treated Frequency (day/ renewal rate volume treated
week){h/day} (%) weekly (L) week) {h/day} (%) weekly (L) week) {h/day} (%) weekly (L)

0–1 0 0 0 0 0 0 0 0 0
1–2 (1){10} 85 1,275 (1){10} 85 1,275 (1){10} 85 1,275
2–3 (1){10} 85 1,275 (1){10} 85 1,275 (1){10} 85 1,275
3–4 (1){10} 85 1,275 (1){10} 85 1,275 (1){10} 85 1,275
4–5 (1){10} 85 1,275 (1){10} 85 1,275 (1){12} 85 1,275
5–6 (1){10} 85 1,275 (2){12} 85 2,550 (2){12} 85 2,550
6–7 (1){12} 100 1,500 (3){12} 100 4,500 (3){12} 100 4,500
7–8 (1){12} 100 1,500 (4){12} 100 6000 (5){12} 100 7,500
8–9 (2){15} 125 3,750 (5){15} 125 9,375 (6){15} 125 11,250
9–10 (4){18} 150 9,000 (6){18} 150 13,500 (7){18} 150 15,750
10–11 (7){21} 175 18,375 (7){21} 175 18,375 (7){21} 175 18,375
11–12 (7){24} 200 21,000 (7){24} 200 21,000 (7){24} 200 21,000

Table 4 Table 6
Estimation of the total ammonium production based on applied feeding Diversity of culturable bacterioplankton.
(Somerville et al., 2014).
Isolates Species Proportion (%)
Stocking density Estimation of total Estimation of ammonium
(PL/m3) ammonium (ppm) production (ppm/d) Isolate 1 Shewanella seohaensis 25.60
Isolate 2 Brevibacterium sanguinis 40.89
500 295 3.47 Isolate 3 Acinetobacter radioresistens 15.92
750 387 4.55 Isolate 4 Staphylococcus saprophyticus 16.67
1,000 466 5.48 Isolate 5 Shewanella amazonensis 0.07
Isolate 6 Methylobacterium aquaticum 0.15
Isolate 7 Brevibacterium casei 0.66

culture. The use of the hybrid ZWD-RAS system in this study allowed
for the maintenance of a low abundance of Vibrio, the bacteria that
causes serious damage in the shrimp industry through vibriosis syn-
drome (Lavilla-Pitogo et al., 1998).
3.1.4. Diversity of culturable bacterioplankton
The diversity and proportion of culturable bacteria in the culture
water are presented in Table 6. From the culture isolation, 7 isolates
were identified. There were four predominant species (abundance >
10%), including Brevibacterium sanguinis (40.89%), Shewanella seo-
haensis (25.6%), Staphylococcus saprophyticus (16.67%), and Acineto-
bacter radioresistens (15.92%), isolated from the hybrid system. Brevi-
bacterium sp. common bacteria that is found in white shrimp guts
(Goodwin, 2005) and can produce poly-hydroxyl butyrate biopolymer
(PHB) that can act against Vibrio pathogenicity (Kiran et al., 2014).
Shewanella sp. are also indigenous probiotic bacteria that can inhibit
Vibrio pathogenicity (Suantika et al., 2013). Staphylococcus sp. are
common bacteria that are found in white shrimp intestines (Goodwin,
2005). These bacteria, in certain circumstances like high culture den-
sity, could promote the production of enterotoxin in the intestine that
might harm the shrimp culture (Beckers et al., 1985). Acinetobacter sp.
are denitrifying bacteria with nitrite removal capacity, and their use
was reported as an alternative method for the prevention and control of
nitrite accumulation in intensified shrimp aquaculture (Cao et al.,
2012). Based on the observed bacterial diversity and proportion, it is
suggested that most of the bacterial species found in the hybrid system
were potential probiotic candidates.
3.1.5. Diversity index
Stability and predictable bacterial diversity are very important in
intensive shrimp culture. In this study, a low and stable level of mi-
crobial diversity between 0–1 was able to be maintained during the
culture period (Table 3). The low and predictable bacteria diversity of
the hybrid ZWD-RAS system shows that this system was able to mini-
mize the unexpected culture failure due to the unpredictability of mi-
crobial community structure contributed by the unpredictability of
water quality. In this system, the microbial community structure was
dominantly composed of functional bacteria, such as Brevibacterium
sanguinis and Shewanella seohaensis, that are known as probiotic bac-
teria for shrimp culture. The very controllable bacteria structure and
diversity dominantly composed of probiotic bacteria allows an appro-
priate culture condition for shrimp (Suantika et al., 2015). The concept
of diversity is particularly important because it is commonly considered
an attribute of natural or organized communities (Hairston, 1959).
Diversity has been said to increase in a successional sequence to a
maximum climax, to enhance community stability and to relate to
community productivity, integration, evolution, niche structure and
competition (Fisher et al., 1943). The diversity index in the hybrid
ZWD-RAS system is shown in Table 7.
Based on the diversity index, the bacterial community of the hybrid
ZWD-RAS system could be categorized as low because the diversity
index value (H’) was below 1 (Sudarma and Suprapta, 2011). During
the culture period, the bacterial succession would still occur, but the
continuous removal of organic matter and continuous water make up

Table 5
Dynamics of culturable bacterioplankton population.
Medium 500 PL/m3 (CFU/mL) 750 PL/m3 (CFU/mL) 1,000 PL/m3 (CFU/mL)

NA 3.95 × 102–3.5 × 104 4.5 × 102–2.51 × 105 6.6 × 102–1.2 × 105

TCBS 0.5 × 101–1.23 × 104 4.2 × 102–4.55 × 104 1.9 × 102–6.35 × 104

*NA = Nutrient Agar (NA); and TCBS = thiosulphate citrate bile salt.

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G. Suantika et al. Aquacultural Engineering 82 (2018) 12–24

Table 7 shrimp culture, such as S. seohaensis and B. sanguinis, are beneficial

Diversity index. bacteria for shrimp culture. It has been reported that S. seohaensis can
Shrimp Density (PL/m3) Hʹ improve shrimp survival (Suantika et al., 2013), while B. sanguinis was
reported to be able to produce PHB that could inhibit Vibrio
500 0.12–0.89 pathogenicity (Kiran et al., 2014).
750 0–1
Predominant species at this density were B. sanguinis (52.42%), S.
1,000 0.07–1
seohaensis (22.89%), and A. radioresistens (14.92%). All three bacteria
genera have a significant role in shrimp culture because they have the
were able to maintain and control the bacteria involved in this process ability to control pathogenic bacteria in water. Brevibacterium has a
(Suantika et al., 2003). significant role in controlling vibriosis in shrimp culture through an
antibiofilm and/or anti adhesive process to Vibrio sp. by the PHB
compound that can be produced by Brevibacterium sp. bacteria (Kiran
3.1.6. Dynamics of culturable bacterioplankton composition
et al., 2014). Based on the similarity index clustering analysis, pre-
3.1.6.1. 500 PL/m3 stocking density. The dynamics of the bacterial
sented in Fig. 12, all culturable bacteria in the community obtained
community in the hybrid ZWD-RAS system at a 500 PL/m3 stocking
during the 84 days of the culture period were in the same group. The
density are presented in Fig. 9. During a 4-week culture period, it was
dynamics recorded during the culture period have no significant effect
observed that the microbial community still fluctuated, in which 3–4
on the change in bacterial community since the similarity index of the
species were observed in the culture, including S. seohaensis, S.
communities was higher than 50% on a weekly basis. The balance en-
saprophyticus, B. casei, and M. aquaticum. A more stable bacterial
vironment in the hybrid ZWD-RAS system due to continuous water
community was observed from week 5 until the end of the culture
treatment and renewal contributed to the stability of the water quality
period (week 12), indicated by low fluctuation, and bacterial succession
in this system. The stability of nitrogen compounds, pH, DO, etc. could
was found when only two predominant bacteria species, S. seohaensis
maintain favourable culture conditions and balance interactions among
and S. saprophyticus, were frequently observed during the culture
bacterial components in the culture (Suantika et al., 2015).
period. Both of these bacteria were predominant because these
bacteria are indigenous bacteria that live in shrimp intestines
3.1.6.3. 1,000 PL/m3 stocking density. The dynamics of the culturable
(Goodwin, 2005).
bacteria community obtained in the hybrid ZWD-RAS system at a
A high proportion of S. seohaensis and S. saprophyticus of 20.24 (up
1000 PL/m3 stocking density is shown in Fig. 13. In general, during 8
to 70.2% and 20.24% respectively) in the bacterial community struc-
weeks of the culture period, the bacterial community was relatively
ture enables the system to improve shrimp culture performance because
stable, in which S. seohaensis was the predominant species and always
one beneficial function of these two bacteria, specifically the Shewanella
present during this period. Starting from week 10, a new bacterial
group, is the ability to improve the survival rate for shrimp culture.
species, B. sanguinis, was observed in the culture and became the
Shewanella is well known as indigenous bacteria and was present every
dominant species by week 12 of the culture period. In this study, the
week during the culture period. This presence gives a positive effect for
presence of S. seohaensis during the culture period may have supported
shrimp due its ability to control pathogenic bacteria in water through
the shrimp growth as this genus was reported to be able to control
biofilm, bacteriocin production, and the pyomelanin (pigment com-
pathogenic bacteria, as well as facilitate the synergistic interaction with
pound) production mechanism (Suantika et al., 2013). Based on the
other bacterial components in the culture (Suantika et al., 2013).
community similarity index of culturable bacteria obtained, all of the
Based on the similarity-based community clustering analysis, it
bacteria community was in the same cluster during the 84-day culture
could be stated that all bacteria communities recorded during culture
period, even though the similarity decreased due to the presence of B.
period were not different, with a similarity index above 50%, even
sanguinis at week 10 (Fig. 10).
though the presence of B. sanguinis from weeks 10–12 lowered the si-
milarity of the bacterial community compared to the previous weeks
3.1.6.2. 750 PL/m3 stocking density. The bacteria community dynamics
(Fig. 14).
obtained from the hybrid ZWD-RAS system at 750 PL/m3 is shown in
Fig. 11. In general, during the 7-week culture period, the bacterial
3.1.7. Culturable bacteria proportion of the hybrid ZWD-RAS system at all
communities were predominantly composed of two species, S.
densities
seohaensis and S. saprophyticus. This structure changed when the
The presence and proportion of culturable bacteria obtained in the
culture entered weeks 8 and 9, where a new species B. casei was
hybrid ZWD-RAS system cultivation at all densities for 84 days of cul-
involved in bacterial succession. From weeks 10–12, B. sanguinis
tivation are summarized in Table 8. In total, seven culturable bacteria
appeared and almost replaced S. seohaensis in the culture. Even
were isolated from the culture water at all different stocking densities.
though the change in bacterial community was documented during
The bacteria S. seohaensis and S. saprophyticus were always predominant
this period, the bacteria species involved in microbial interactions in
(with abundances above 10%) in all treatment groups with different
densities. In this study, it is suggested that S. seohaensis may have in-
creased the shrimp survival in the 500 PL/m3 treatment group due to its
role in the inhibition of pathogenic bacteria, including Vibrio sp.
(Suantika et al., 2013). The abundance of S. seohaensis decreased in line
with the increasing of stocking density. On the other hand, Brevi-
bacterium may have also supported shrimp survival due to its ability to
produce PHB with antibiofilm and/or antiadhesive activity against
pathogenic Vibrio sp. (Kiran et al., 2014), even though its presence was
only observed at the end of the culture period.

3.1.8. Biological parameters

Biological parameters, including mean individual shrimp body
weight (MBW) and length, final biomass, survival (SR) and feed con-
Fig. 9. Proportion of culturable bacterioplankton obtained by the hybrid ZWD- version ratio (FCR), can be seen in Table 9. There were no significant
RAS system for every week of cultivation at 500 PL/m3. differences in shrimp growth (final mean body weight and length)

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G. Suantika et al. Aquacultural Engineering 82 (2018) 12–24

Fig. 10. Clustering similarity of culturable bacterioplankton obtained by the hybrid ZWD-RAS system for every week of cultivation at 500 PL/m3.

Fig. 13. Proportion of culturable bacterioplankton obtained by the hybrid

Fig. 11. Proportion of culturable bacterioplankton obtained by the hybrid
ZWD-RAS system for every week of cultivation at 750 PL/m3.
among all treatments. It is shown that the stocking densities sig-
nificantly affected shrimp survival, where the highest survival of 70%
was obtained at the 500 PL/m3 stocking density, followed by 750 PL/
m3 (SR∼52.9%) and 1,000 PL/m3 (SR∼40%). The significantly lower
survival in the higher stocking densities may be explained by the en-
vironmental stress due to the space competition and crowed effect
caused by the high shrimp population (Wickin and Lee, 2002). This
environmental stress could increase the cannibalistic behaviour of
white shrimp during the moulting period (Muhammad et al., 2016).
ZWD-RAS system for every week of cultivation at stocking density of 1,000 PL/
m3.
The significant differences in survival significantly contributed to
the shrimp productivity. The shrimp productivity among the different
stocking densities was not significantly different, even though the
productivity tended to increase at higher stocking densities (4.2 kg/m3,
4.7 kg/m3, and 4.8 kg/m3 at 500, 750, and 1,000 PL/m3 stocking den-
sities, respectively). Survival has a very important role in achieving
economical effectiveness in shrimp culture, as high survival will in-
crease the total biomass produced and thus lower the feed conversion
ratio. In this study, even though there were no differences in the shrimp
final MBW, the significant difference in survival may have led to the

Fig. 12. Clustering similarity of culturable bacterioplankton obtained by the hybrid ZWD-RAS system for every week of cultivation at 750 PL/m3.

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G. Suantika et al. Aquacultural Engineering 82 (2018) 12–24

Fig. 14. Clustering similarity of culturable bacterioplankton obtained by the hybrid ZWD-RAS system for every week of cultivation at 1,000 PL/m3.

Table 8 Table 11
Proportion of culturable bacterioplankton obtained by the hybrid ZWD-RAS Biological culture performance of shrimp culture during the 60-
system at different densities following an 84-day grow-out period. day grow-out period at an industrial level.
Species Proportion (%) Parameter Unit

500 PL/m3 750 PL/m3 1,000 PL/m3 Total culture water volume (m3) 110
Stocking density (PL/m3) 500
Shewanella seohaensis 70.20 27.48 20.77 Culture period (days) 60
Staphylococcus saprophyticus 20.25 11.07 33.82 Survival (%) 78
Brevibacterium sanguinis 7.54 58.07 41.85 Shrimp final MBW(g) 7
Acinetobacter radioresistens 0.05 2.75 2.14 Total biomass (kg) 298
Brevibacterium casei 0.76 0.5 1.27 Feed Conversion Ratio 1.1
Methylobacterium aquaticum 0.94 0.06 0.09
Shewanella amazonensis 0.26 0.08 0.05
suggested in several studies (Perez-Velazquez et al., 2007; Gao et al.,
2016). This suggests that the applied feeding management in this study
Table 9
needs to be improved through a better estimation of the actual shrimp
Biological culture performance of shrimp culture at three different stocking
biomass during the grow-out period, as well as dietary modification
densities during 84 days of culture.
approaches (Roy et al., 2010). The FCR values in all treatments were
Parameter 500 PL/m3 750 PL PL/m3 1,000 PL/m3 still in the tolerance range of shrimp culture (below 2.6) (Woynarovich
et al., 2010). However, the trend of increasing FCR values in the cul-
Length (cm) 10.98 ± 0.49a 10.96 ± 0.54a 10.68 ± 0.73a
MBW (g) 12.06 ± 5.72a 11.84 ± 3.58a 12.04 ± 3.71a tures at high densities indicated a lower economic viability with higher
Survival (%) 70.00 ± 7.02a 52.89 ± 2.77b 40.00 ± 4.00c feed costs (Quintero and Roy, 2010). In farmer level applications, these
Final biomass (kg) 1.261 ± 0.006a 1.407 ± 0.002a 1.445 ± 0.012a results provide a clear view that the application of the hybrid ZWD-RAS
Productivity (kg/m3) 4.205 ± 0.071a 4.691 ± 0.025a 4.816 ± 0.129a
system will be most efficient at the stocking density of 500 PL/m3, as it
FCR 1.54 ± 0.01a 1.82 ± 0.004a 2.16 ± 0.03b
requires much less seed and feed (thus lower operational costs) to
*Different superscripts in the same row show significant differences produce shrimp productivity similar to that at higher densities up to
(p < 0.05). 1,000 PL/m3.

Table 10
3.2. Industrial trial
Water quality parameters during the 60-day grow-out period at an industrial
level.
3.2.1. Water quality parameters
Parameter 500 PL/m3 Tolerance Level Based on water quality measurements, it was shown that all water
pH 7.2–8.6 7-8.5 (Lin and Chen, 2001) quality parameters were in the tolerance level for white shrimp culture.
Temperature (°C) 28–30 28-32 (Lin and Chen, 2001) The only exception was the salinity level, because in this study, the
DO (ppm) 3.0–4.8a ≥4 (Lin and Chen, 2001) shrimp culture was performed at a low salinity of 5–6 ppt. Water quality
Salinity (ppt) 5–6 5-30 (Jayasankar et al., 2009) at this industrial level was comparable with that in the semi-mass trial,
Ammonium (ppm) 0–1.35 < 2 (Schuler et al. 2010)
and it can be stated that the hybrid ZWD-RAS system can maintain
Nitrite (ppm) 0.05–15 ≤25.7 (Lin and Chen, 2003)
Nitrate (ppm) 0–50 ≤232 (Lin and Chen, 2001) water quality similar to that in the semi-mass trial. The nitrite level was
increased in the last week of the culture period and reached up to
15 ppm NO2−. Unlike Nitrosomonas-like bacteria, Nitrobacter-like bac-
a
When DO reached < 4.0 ppm, aeration was adjusted and maintained at
4–5 ppm. teria are slower to develop and are much less hardy. They are not as
robust and are easily compromised by chemical treatments and changes
decrease of feed efficiency (increased FCR) in the treatments with in water quality, including temperature; thus, it is common to have high
higher densities. This may indicate that the treatments with high den- nitrite readings after a filter has fully cycled. This nitrite spike com-
sities might have been overfed. The overfeeding could have been re- monly occurs in aquaculture systems that rely on nitrification to cycle
lated to a lower feed utilization capacity by the shrimp at low salinity as nitrogen (Wang et al., 2004; Ray, 2012). This might also be due to the

22
G. Suantika et al.
dimensions of the biofilter used in this industrial trial, which might not
have the correct carrying capacity for the shrimp culture system.
Moreover, the conditioning of the biofilter in this study could have been
improved by adding different immobilization media, including more
porous media to increase the surface area for nitrifying bacteria im-
mobilization instead of increasing the biofilter dimension. A summary
of the water quality parameters at the industrial level is provided in
Table 10.
3.2.2. Biological performance
The stocking density applied in the industrial trial was based on the
results of the semi-mass trial with the optimum density of 500 PL/m3.
Based on the overall culture performance, shrimp survival at this in-
dustrial level was slightly higher (78%) than that in the semi-mass trial
(70%). However, with the shorter grow-out culture period in this in-
dustrial trial compared to the semi-mass trial, the survival in the in-
dustrial trial can be said to be similar. The shrimp productivity in the
industrial trial within the 60-day culture period was 2.71 kg/m3, with
an average final individual body weight of 7 g. The summary of bio-
logical parameters during the 60-day grow-out period at an industrial
level is provided in Table 11.
4. Conclusion
Based on the results of the semi-mass trial, it can be stated that the
hybrid ZWD-RAS system can maintain water quality and a microbial
load up to a 1,000 PL/m3 stocking density; however, the optimal cul-
ture performance based on shrimp survival, FCR and productivity was
reached at the 500 PL/m3 stocking density, with a productivity of
4.2 kg/m3 within the 84-day grow-out period. The industrial trial of the
application of the hybrid ZWD-RAS system in commercial shrimp urban
farming using the optimum stocking density of 500 PL/m3 resulted in a
shrimp productivity of 2.71 kg/m3, with an average final individual
body weight of 7 g within the 60-day grow-out period. Further opti-
mization of shrimp stocking density, as well as feeding management, is
needed to improve the growth and survival of super-intensive shrimp
culture using this hybrid ZWD-RAS system in low salinity conditions.
Declaration of interest
None.
Acknowledgements
The authors thank the Indonesia Endowment Fund for Education
(LPDP), the Ministry of Finance and the Ministry of Education and
Culture, Indonesia, for the financial support of the project under the
contract number of PRJ-637/LPDP/2016. The authors also thank PT.
Suri Tani Pemuka, Indramayu, West Java, Indonesia, for providing the
post larvae shrimp for this project.
References
Beckers, H.J., Van Leusden, F.M., Tips, P.D., 1985. Growth and enterotoxin production of
Staphylococcus aureus in shrimp. Epidemiol. Infect. 95, 685–693.
Cao, H., Wang, H., He, S., Ou, R., Hou, S., Yang, X., 2012. Isolation and characterization
of a denitrifying Acinetobacter baumannii H1 using NO2−-N as nitrogen source from
shrimp farming ponds. Afr. J. Microbiol. Res. 6, 2258–2264.
Cappucino, J., Sherman, N., 2011. Microbiology: A Labolatory Manual, 6th ed. Benjamin
Cummings Publishing Company, USA.
Eaton, A.D., Franson, M.A.H., 2005. Standard Methods for the Examination of Water and
Wastewater. American Public Health Association, USA.
Eng, C.T., Paw, J.N., Guarin, F.Y., 1989. The environmental impact of aquaculture and
the effects of pollution on coastal aquaculture development in Southeast Asia. Mar.
Pollut. Bull. 20, 335–343.
23
Aquacultural Engineering 82 (2018) 12–24
FAO, 2016. The State of world fisheries and aquaculture 2016. Contributing to Food
Security and Nutrition for All. FAO, Rome.
Fisher, R.A., Corbett, A.S., Williams, C.B., 1943. The relation between number of species
and the number of individuals in a random sample from an animal population. J.
Anim. Ecol. 12, 42–58.
Funge-Smith, S., Phillips, M.J., 2001. Aquaculture systems and species. In: Subasinghe,
R.P., Bueno, P., Phillips, M.J., Hough, C., McGladdery, S.E., Arthur, J.R. (Eds.),
Aquaculture in the Third Millennium. Technical Proceedings of the Conference on
Aquaculture in the Third Millennium. Bangkok, Thailand, 20–25 February 2000.
Gao, W., Tian, L., Huang, T., Yao, M., Hu, W., Xu, Q., 2016. Effect of salinity on the
growth performance, osmolarity and metabolism-related gene expression in white
shrimp Litopenaeus vannamei. Aquacult. Rep. 4, 125–129.
Gerardi, M.H., 2002. Nitrification and Denitrfication in the Active Sludge Process. Wiley-
Interscience, pp. 193.
Ghaly, A.E., Ramakrishnan, V.V., 2015. Nitrogen sources and cycling in the ecosystem
and its role in air, Water and soil pollution: a critical review. J. Pollut. Effect Control
3, 136.
Goodwin, S., 2005. Polyphasic characterization of bacteria isolated from shrimp larva. J.
Young Investig. 12, 1–4.
Gordillo, F.J.L., Dring, M.J., Savidge, G., 2002. Nitrate and phosphate uptake char-
acteristics of three species of brown algae cultured at low salinity. Mar. Ecol. Prog.
Ser. 234, 111–118.
Hairston, N.G., 1959. Species abundance and community organization. Ecology 40,
404–416.
Ipsos, 2016. Indonesia’S Aquaculture Industry. Ipsos Business Consulting. https://www.
ipsos.com/sites/default/files/2016-08/indonesia-aquaculture-industry.pdf.
Jayasankar, V., Jasmani, S., Nomura, T., Nohara, S., Do, H.T.T., Wilder, M.N., 2009. Low
salinity rearing of the pacific white shrimp Litopenaeus vannamei: acclimation, sur-
vival and growth of postlarvae and juveniles. Japan Agric. Res. Q. 4 (43), 345–350.
Jeon, B.Y., Seo, H.Y., Kang, S.W., Park, D.H., 2010. Effect of electrochemical redox re-
action on biochemical ammonium oxidation and chemical nitrite oxidation. J.
Microbiol. Biotechnol. 20, 483–493.
Kiran, G.S., Lipton, A.N., Priyadharshini, S., Anitha, K., Suárez, L.E.C., Arasu, M.V., Al-
Dhabi, N.A., 2014. Antiadhesive activity of poly-hydroxy butyrate biopolymer from a
marine Brevibacterium casei MSI04 against shrimp pathogenic vibrios. Microb. Cell
Fact. 13, 114.
Lavilla-Pitogo, C.R., Leano, E.M., Paner, M.G., 1998. Mortalities of pond-cultured juvenile
shrimp Penaeus monodon associated with dominance of luminescent vibrios in the
rearing environment. Aquaculture 164, 337–349.
Lin, Y.C., Chen, J.C., 2001. Acute toxicity of ammonia on Litopenaeus vannamei Boone
juveniles at different salinity levels. J. Exp. Mar. Biol. Ecol. 259, 109–119.
Lin, Y.C., Chen, J.C., 2003. Acute toxicity of nitrite on litopenaeus vannamei boone juve-
niles at different salinity levels. Aquaculture 224, 193–201.
Müller-Feuga, A., Robert, R., Cahu, C., Robin, J., Divanach, P., 2003. Uses of microalgae
in aquaculture. In: Støttrup, J.G., McEvoy, L.A. (Eds.), Live Feed in Marine
Aquaculture. Blackwell Science, pp. 263–269.
Muhammad, H., Situmorang, M.L., Djohan, Y.A., Aditiawati, P., Suantika, G., 2016.
Biological, technical, and financial feasibilities study of zero Water discharge (ZWD)
system application in Low salinity White shrimp (Litopenaeus vannamei Boone) Urban
aquaculture, study case: gresik District, East java, Indonesia. J. Fish. Livest. Prod. 4,
197.
Neilan, B.A., Pearson, L.A., Muenchhoff, J., Moffitt, M.C., Dittmann, E., 2013.
Environmental conditions that influence toxin biosynthesis in cyanobacteria.
Environ. Microbiol. 15, 1239–1253.
Otoshi, C.A., Arce, S.M., Moss, S.M., 2003. Growth and reproductive performance of
broodstock shrimp reared in a biosecure recirculating aquaculture system versus a
flow-through pond. Aquacult. Eng. 29, 93–107.
Pena, L.D., Celia, R.L., Mlagros, G.P., 2001. Luminescent vibriosis associated with mor-
tality in pond-cultured shrimp Penaeus monodon in the Philippines: species compo-
sition. Fish. Pathol. 36 (3), 133–138.
Perez-Velazquez, M., Gonzáléz-Félix, M.L., Jaimes-Bustamente, F., Martínez-Córdova,
L.R., Trujillo-Villalba, D.A., Davis, D.A., 2007. Investigation of the effects of salinity
and dietary protein level on growth and survival of pacific white shrimp Litopenaeus
vannamei. J. World Aquacult. Soc. 38, 475–485.
Quintero, H.E., Roy, L.A., 2010. Practical feed management in semi-intensive systems for
shrimp culture. In: Alday-Sanz, V. (Ed.), The Shrimp Book. Nottingham University
Press, Nottingham, UK.
Ray, A.J., 2012. Management of Biological and Chemical Constituents for the
Advancement of Intensive, Minimal-Exchange, Biofloc-based Shrimp (Litopenaeus
vannamei) Aquaculture. Dissertations. The University of Southern Mississippi, USA,
pp. 472.
Roy, L.A., Davis, D.A., Saoud, I.P., Boyd, C.A., Pine, H.J., Boyd, C.E., 2010. Shrimp culture
in inland low salinity waters. Rev. Aquacult. 2, 191–208.
Schuler, D.J., Boardman, G.D., Kuhn, D.D., Flick, G.J., 2010. Acute toxicity of ammonia
and nitrite to pacific white shrimp, Litopenaeus vannamei at low salinities. J. World.
Aquacult. Soc. 41, 438–446.
Somerville, C., Cohen, M., Pantanella, E., Stankus, A., Lovatelli, A., 2014. Small-scale
Aquaponic Food Production. Integrated Fish and Plant Farming. FAO.
Suantika, G., Dhert, P., Sweetman, E., O’Brien, E., Sorgeloos, P., 2003. Technical and
economical feasibility of a rotifer recirculation system. Aquaculture 227, 173–189.
Suantika, G., Lumbantoruan, G., Muhammad, H., Azizah, F.F.N., Aditiawati, P., 2015.
Performance of zero Water discharge (ZWD) system with nitrifying bacteria and
G. Suantika et al.
microalgae Chaetoceros calcitrans components in super intensive White shrimp
(Litopenaeus vannamei) culture. J. Aquacult. Res. Dev. 6, 359.
Suantika, G., Pratiwi, M.I., Situmorang, M.L., Djohan, Y.A., Muhammad, H., Astuti, D.I.,
2016. Ammonium removal by nitrifying bacteria biofilm on limestone and bioball
substrate established in Freshwater trickling biofilter. Poult. Fish. Wildl. Sci. 4, 157.
Suantika, G., Aditiawati, P., Astuti, D.I., Khotimah, Z.F., 2013. The use of indigenous
probiotic Halomonas aquamarina and Shewanella algae for white shrimp (Litopenaeus
vannamei Boone) hatchery productivity in zero water discharge system. J. Aquacult.
Res. Dev. 4, 1–8.
Suantika, G., Situmorang, M.L., Aditiawati, P., Khakim, A., Suryanarayan, S., Nori, S.S.,
Kumar, S., Putri, F., 2017. Effect of Red seaweed Kappaphycus alvarezii on growth,
salinity stress tolerance and vibriosis resistance in shrimp Litopenaeus vannamei
hatchery. J. Fish. Aquat. Sci. 12, 127–133.
Sudarma, I.M., Suprapta, D.N., 2011. Diversity of soil microorganism in banana habitats
24
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with and without fusarium wilt symptom. J. ISSAAS 17, 147–159.
Tacon, A.G.J., Jory, D.E., Nunes, A.J.P., 2013. Shrimp feed management: issues and
perspectives. In: Hasan, M.R., New, M.B. (Eds.), On-farm Feeding and Feed
Management in Aquaculture. FAO Fisheries and Aquaculture Technical Paper No.
583. FAO, Rome, pp. 481–488.
Wang, W.N., Wang, A.L., Zhang, Y.J., Li, Z.H., Wang, J.X., Sun, R.Y., 2004. Effects of
nitrite on lethal and immune response of macrobrachium nipponense. Aquaculture
232, 679–686.
Wickin, J.F., Lee, D.O., 2002. Crustacean Farming: Ranching and Culture, 2nd ed.
Blackwell Science, Oxford, London, Edinburgh, Malden, Carlton, Paris.
Woynarovich, A., Moth-Poulsen, T., Péteri, A., 2010. Carp Polyculture in Central and
Eastern Europe, the Caucasus and Central Asia: A Manual. FAO Fisheries and
Aquaculture Technical, FAO, Rome.