Aquaculture research, tập 41, số 10, 2010

Aquaculture Research, 2011, 42, 1415^1423

doi:10.1111/j.1365-2109.2010.02730.x

Bioremediation and reuse of shrimp aquaculture
effluents to farm whiteleg shrimp, Litopenaeus

vannamei : a first approach
Luis R Mart|¤ nez-Co¤rdova1, Jose¤ A Lo¤pez-El|¤ as1, Guadalupe Leyva-Miranda2, Luis Armenta-Ayo¤n2 &
Marcel Martinez-Porchas3
1

Departamento de Investigaciones Cient|¤ ¢cas y Tecnolo¤gicas de la Universidad de Sonora, Sonora, Me¤xico
Maestr|¤ a en Biociencias, Universidad de Sonora, Sonora, Me¤xico

2
3

Centro de Investigacio¤n en Alimentacio¤n y Desarrollo, Sonora, Me¤xico

Correspondence: M Mart|¤ nez-Porchas, Centro de Investigacio¤n en Alimentacio¤n y Desarrollo, Km. 0.6 Carretera a La Victoria, Hermosillo, Sonora, Me¤xico. E-mail:

Abstract
Shrimp aquaculture e¥uents were bioremediated in
a two-phase system (System A) using the black clam
Chione £uctifraga and the benthic microalgae Navicula sp., and then reused to farm whiteleg shrimp Litopenaeus vannamei. In the experimental design,
Systems B and C had an identical structure as System
A, but no clams or microalgae were added. System B
received the same shrimp e¥uents while System C
received only estuarine water. Shrimp raw e¥uents

had a poor water quality. System A improved
the water quality by decreasing the concentrations
of total nitrogen, total ammonia nitrogen (TAN), nitrites, nitrates, phosphates, total suspended solids
(TSS) and organic suspended solids (OSS). System B
also decreased the concentration of TAN, TSS
and OSS via sedimentation, but the e¡ect was less
pronounced than that observed in System A. Shrimp
reared in the bioremediated e¥uents (System A) had
better production (3166 kg ha À 1) and higher survival
(89.2%) than those reared in e¥uents from Systems B
(2610 kg ha À 1,75.1%) and C (2874 kg ha À 1, 82.1%). It
is concluded that the bioremediation system was
moderately e⁄cient and the bioremediated e¥uents
were suitable to farm L. vannamei.

Keywords: bioremediation, shrimp e¥uent, Chione
£uctifraga, Navicula sp.
Introduction
Aquaculture has experienced vigorous growth
worldwide in the last two decades. Its contribution

r 2010 Blackwell Publishing Ltd

to the global production of aquatic organisms grew
from 3.9% in 1970 to more than 36% in 2006 (FAO
2009). The Crustaceans are the group with the highest growth rate (almost 17% per year from 2000 to
2006) and penaeid shrimp are by far the most important in terms of volume and value of production (FAO
2009).
However, the explosive development of shrimp
aquaculture has caused some serious problems, such

as competition for water and land (PaŁez-Osuna, Gracia, Flores-Verdugo, Lyle-Fritch, Alonso-Rodr|¤ guez.,
Roque & Ruiz-FernaŁndez 2003), environmental impacts, including deforestation, eutrophication of receiving ecosystems, modi¢cation of habitat for
terrestrial and aquatic animals, modi¢cation of landscape and hydrological patterns (GonzaŁlez-Ocampo,
Morales, CaŁceres-Mart|¤ nez, Aguirre, HernaŁndez-VaŁzquez, Troyo-Dieguez & Ortega-Rubio 2006; Thomas,
Courties, El Helwe, Herbland & Lemonnier 2010), the
dependence of formulated shrimp feed from ¢sh meal
as the main protein ingredient (Tacon 2002) and the
continuous presence of epizooties (SaŁnchez-Mart|¤ nez, Aguirre-GuzmaŁn & Mej|¤ a-Ruiz 2007).
As an example of the potential impact of aquaculture e¥uents, Tacon (2002) shows data regarding
how much organic matter (OM), nitrogen (N) and
phosphorous (P) is discharged into the environment
for each tonne of shrimp harvested, depending on
the feed conversion ratio (FCR). In Mexico, shrimp
aquaculture operates with a mean FCR of about 1.8;
considering the annual production and the data provided by Tacon (2002), it is calculated that 112 million kg of OM,7.8 million kg of N and 2.5 million kg of

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Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

P are discharged into the receiving ecosystems each
year (Mart|¤ nez-Co¤rdova, Mart|¤ nez-Porchas & Corte¤sJacinto 2009). These are huge amounts and it is absolutely necessary to stop and if possible to reverse this
process if we wish to have a sustainable aquaculture.
Some strategies have been used or suggested to
minimize these impacts, including settling lagoons
(Mart|¤ nez-Co¤rdova & Enriquez-Ocanìa 2007), septic
tank treatments (Summerfelt & Penne 2007), low or
zero water exchange (Balasubramanian, Pillai & Ravichandran 2005), recirculation systems (Timmons,
Ebeling, Wheaton, Summerfelt & Vinci 2002; Lezama-Cervantes, Paniagua-Michel & Zamora-Castro

2010), the use of mangrove forests as nutrient sinks
(Rivera-Monroy, Torres, Bahamon, Newmark & Twilley 1999), polyculture practices (Martinez-Cordova &
Martinez-Porchas 2006; Mart|¤ nez-Porchas, Mart|¤ nezCo¤rdova, Porchas-Cornejo & Lo¤pez-El|¤ as 2010) and
bioremediation (Paniagua-Michel & Garcia 2003).
Bioremediation is the use of individual or
combined organisms (animal, vegetal, bacteria, etc.)
to minimize the polluting charge of e¥uents from
aquaculture or any other activity, taking advantage
of the natural or modi¢ed abilities of those organisms
to reduce and/or transform waste products (ChavezCrooker & Oberque-Contreras 2010). Bioremediation
can be conducted in di¡erent forms: in situ, ex situ,
biostimulation, bioagmentation and others. Some
examples of successful bioremediation practices are
the use of plants (phytoremediation), macroalgae,
microalgae, ¢lter feeders, bio¢lters (polymer spheres
with immobilized microorganisms), bio¢lms^bio£ocs
(De Schryver, Crab, Deforidt, Boon & Verstraete 2008;
Kuhn, Boardman, Craig, Flick Jr & Mclean 2009) or
combined systems including two or more of these
practices. Although it has been demonstrated that
some bivalves and micro- or macroalgae are capable
of bioremediating e¥uents, many of these studies
have been focused on the bioremediation of ¢sh e¥uents (Hussenot 2003; Zhou,Yang, Hu, Liu, Mao, Zhou,
Xu & Zhang 2006; Liu,Wang & Lin 2010).
The use of endemic species of bivalve and microalgae to bioremediate e¥uents would prevent the introduction of exotic species, which may cause other
problems. It is important to study di¡erent combinations of these species in order to achieve the greatest
e⁄ciency.
The black clam (Chione £uctifraga) inhabits estuaries and shallow coastal waters in the Gulf of California. It tolerates high concentrations of OM in the
water column and can withstand a wide range of
temperatures and salinities (Mart|¤ nez-Co¤rdova

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Aquaculture Research, 2011, 42, 1415–1423

1988), conditions that are similar to those prevailing
in shrimp farm e¥uent; also, the species has commercial importance in north-western Mexico,
mainly as an artisanal ¢shery, but it is beginning to
be farmed in the region (Tinoco-Orta & CaŁceres-Mart|¤ nez 2003). In the case of microalgae, Navicula sp. is a
diatom that has been found in shrimp ponds and can
be a food source for shrimp; also, it is reported to have
the ability to act as a bioremediator of water (Paniagua-Michel & Garcia 2003).
Based on the above information, the study was
focused on evaluating an integrated system using
benthic microalgae (Navicula sp.) and clam (C. £uctifraga) to bioremediate shrimp aquaculture e¥uents and
reuse the bioremediated e¥uents to farm whiteleg
shrimp Litopenaeus vannamei at a microcosm level.

Material and methods
Organisms
The shrimp were obtained from a commercial farm
(Maricultura del Pac|¤ ¢co S.A., Mazatlan, Me¤xico)
and maintained under the laboratory conditions of
dissolved oxygen (DO) 6.05 mg L À 1, temperature
28 1C, total ammonia nitrogen (TAN) 0.01mg L À 1,
s
fed ad libitum (35% crude protein; Purina Me¤xico ,
Hermosillo, Me¤xico) and a daily water exchange of
100%. These conditions were maintained until the
shrimp achieved an individual biomass of 5 g.

Adult black clams (C. £uctifraga) with an average
size of 3.0 Æ 0.4 cm were hand collected from the La
Cruz estuary (28148 05700 N, 111155 03000 O); organisms
of a lower size were discarded, because we aimed to
evaluate only the capacity of adult clam, which have
a greater ¢ltration capacity. The clam was maintained under the above laboratory conditions for 2
weeks. During this period, the organisms were fed
with the diatom Chaetoceros muelleri.
The microalga Navicula sp. was obtained from an
aquaculture laboratory at Centro de Estudios Superiores del Estado de Sonora (CESUES, Navojoa, Sonora,
Me¤xico). The microalgae were scaled up from 10 mL
to 200 L using an F/2 medium with a double concentration of silicates. Thereafter, the experimental units
were inoculated with the microalgae 1 week before
beginning the trial.

Shrimp culture system
The e¥uents used for the study were obtained from a
semiintensive culture of white shrimp L. vannamei

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Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

Aquaculture Research, 2011, 42, 1415^1423

(25 shrimp m À 2) reared in tanks with estuarine
water.
The shrimp were farmed in six rectangular, plastic
tanks with an area of 6 m2 and ¢lled with water

pumped from the estuary; each tank was provided
with 0.9 m3 of sediment to achieve a height of 5^
6 cm. The culture conditions were as follows: time of
culture 50 days, initial size 5.0 Æ 0.3 g, stocking density 25 org m À 2, feeding supply and frequency twice
a day to satiation (in feeding trays), the feed used was
Camaronina with 35% of crude protein (Purina Me¤xs
ico ) and the daily water exchange was 20%.

plastic tanks (1000 L) with black clams (C. £uctifraga)
distributed on the bottom (35 org m À 2). In Phase II,
the water treated in each tank with clams £owed into
subsequent tanks (1000 L) containing the benthic
microalgae Navicula sp. at an initial concentration of
50 000 cells mL À 1 (Fig. 1). The microalgae were attached to the walls and to arti¢cial substrates (plastic
nets) introduced into the tanks, with a surface area of
1.65 m2.
The untreated e¥uents £owed through the System
B, which had a structure identical to the bioremediation system (System A), but with no clams or microalgae in the 1000 L tanks (Fig. 1).
A third system (System C) was constructed to determine the quality of the inlet estuarine water and
was used as a control of the shrimp rearing in the ef£uents. The structure of the System C was identical to
that of Systems A and B, but shrimp were not reared
in the rectangular tanks that received the estuarine
water; also, no clams or microalgae were introduced
into the 1000 L tanks.
Every system was constructed with three replicates with a water £ow of 0.56 L min À 1 for every ex-

Bioremediation system
One half of the e¥uent was sent to a bioremediation
system and the other half was sent to an identical
physical system but was not treated. Both types of

water were then used to cultivate white shrimp.
The bioremediation system (System A) consisted of
two phases. In Phase I, the e¥uents were equally distributed and made to £ow through three di¡erent

ESTUARY

Semiintensive
culture of
white
shrimp
Effluents
Phase I:
Black clams
Phase II:
Microalgae
Reservoir

SYSTEM A

SYSTEM B

Treatment 1

Treatment 2

Figure 1 Scheme of the system used for the bioremediation of shrimp e¥uents (System A) with bivalves (Phase I) and
microalga (Phase II). System B only tested the e¡ect of the pools on the decrease in the water quality parameters.

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Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

perimental unit. The £ow rates of the experimental
systems were controlled by water valves. The hydraulic retention time (HRT) (yh) was 29.7 h for each circular tank containing clams or microalgae and 184.5 h
for each complete system.

Reuse of treated e¥uents to cultivate white
shrimp
After being treated in Systems A^C, the water was
then reused for rearing shrimp (L. vannamei) in plastic tanks (6 m2) identical to those mentioned above.
Nine pools were used to farm the shrimp in the treated water. The treated water from System A was distributed into three pools with a shrimp stocking
density of 25 shrimp m À 2 (Fig.1); the water from System B also £owed into the other three culture pools,
and the same was done for System C. Particularly for
System C, the water used was pumped directly from
the estuary, but previously £owed through all the
pools without animals or microalgae. The culture
conditions were the same as mentioned above for
the shrimp culture system.

Aquaculture Research, 2011, 42, 1415–1423

duction method (Wood, Armstrong & Richards 1967),
and the orthophosphates were measured using the
PhosVer 3 method (HACH, Loveland, CO, USA).
To measure the TSS and OSS, 1L of the sampled
water was ¢ltered through GFC 47 mm Whatman ¢lters, which were then washed and dried at 90 1C for
4 h. The di¡erence in weight between the dried ¢lters

before and after ¢ltration was estimated as the TSS.
The OSS were determined by incineration of the dried
¢lters in a mu¥e furnace at 450 1C for 8 h, and then
cooled and weighed. The same process was carried
out with clean seawater (previously ¢ltered and sterilized) from the estuary, which was a basepoint subtracted for the results of TSS and OSS.
The concentration of microalgae cells (Navicula sp.)
was determined by treating samples of water with a
Branson 2210 ultrasonic bath and the concentration
of cells per millilitre was determined in a hematocytometer.
The shrimp production variables, growth, survival, ¢nal biomass and FCR were evaluated in each of
the pools. The FCR was estimated as the weight of
feed administered/weight gain of the shrimps.

Statistical analysis
Analysis of water quality and production
variables
The environmental and water quality variables were
measured in the estuarine inlet water and e¥uents
(before and after bioremediation). The estuarine inlet
water was sampled in the rectangular tanks of System C, which did not have shrimp and received water
directly from the estuary. The raw e¥uents were
measured at the exit of each shrimp culture tank before the water entered into System A or B. The e¥uents from each system were then analysed as they
exited the tanks of Phase II.
The variables were monitored periodically for temperature, salinity, DO, pH and chlorophyll a twice a
day, using a multisensor YSI 6600 series (Yellow
Springs, OH, USA). Total nitrogen (TN), N-NO2, NNO3, TAN and P-PO4, total suspended solids (TSS)
and organic suspended solids (OSS) were all determined once a week.
The TN was measured using the micro-Kjeldahl
method. The concentration of TAN was evaluated
using the ammonia-salicylate method (Bower &

Holm-Hansen 1980). The nitrite concentration was
determined using the colorimetric method described
by Strickland and Parsons (1972, NitriVer Method). Nitrate was determined using the cadmium^copper re-

1418

A one-way analysis of variance was performed to evaluate the production variables of the shrimp cultivated
in the e¥uents from Systems A, B and C, and a post hoc
Tukey test was used to detect signi¢cant di¡erences. A
con¢dence level of 95% was established. The results
are presented as means (standard deviation). To evaluate the water quality variables, a repeated-measures
analysis of variance was performed.

Results
Bioremediation
Signi¢cant di¡erences in some of the water quality
parameters were observed among the raw e¥uents,
the di¡erent Systems of e¥uent processing (A, B or
C) and the inlet estuarine water (Figs 2 and 3).
Most of the parameters monitored for water quality were signi¢cantly higher in the raw e¥uents and
the e¥uents from System B that were not bioremediated, while lower concentrations of those parameters were observed in System A (bioremediated
e¥uents), System C (control) and the Estuary.
The TN increased with time in all the systems,
but signi¢cantly higher values were found in the
raw e¥uents and System B, while no di¡erences were

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Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

Aquaculture Research, 2011, 42, 1415^1423

TAN

Concentration (mg·L–1)

Total Nitrogen

NO2

2.4

0.04

1.2

1.8

0.03

0.9

1.2

0.02

0.6

0.6

0.01

0.3

0

0

1 2 3 4 5 6 7

0

1 2 3 4 5 6 7

NO3

PO4

0.5

0.05

0.4

0.04

0.3

0.03

0.2

0.02

0.1

0.01

0

1 2 3 4 5 6 7

0

1 2 3 4 5 6 7

1 2 3 4 5 6 7

Time (Weeks)

Figure 2 Concentrations of water quality parameters found throughout time in the raw e¥uents, the bioremediated
e¥uents with bivalve and microalga (System A), the nonbioremediated e¥uents (System B), the estuarine that £owed into
a similar system of tanks but without animals (System C) and the estuarine water (Estuary). Di¡erent letters on the left of
each marker in each graphic indicate signi¢cant di¡erences.
Total Suspended Solids

300
250
200

150
100
Concentration (mg·L–1)

observed among the rest of the Systems (A and C) and
the estuarine water (Fig. 2).
The concentration of TAN recorded a similar
dynamic in all the treatments through the
experimental period; however, the raw e¥uents
and the e¥uents from System B had higher
concentrations of TAN that those found in System C
and the Estuary. In the case of the bioremediated ef£uents (System A), the concentrations of TAN were
signi¢cantly lower than the raw e¥uents but no differences were found with regard to the rest of the
treatments.
For nitrite levels, the raw e¥uents and System B
showed the highest concentrations during the experiment. Although System A showed results similar
to those of System B (P 5 0.069), they were also similar to those observed in System C and Estuary
(P40.7). A similar tendency was observed for nitrates as that for TAN and nitrite concentrations
(Fig. 2).
The phosphates were also higher in the nonbioremediated e¥uents (raw e¥uents and System B) as
compared with the rest of the treatments. System A
had lower values than the nonbioremediated e¥uents, but higher than those of System C and the Estuary (Fig. 2).
The TSS were the highest in the raw e¥uents, followed by those found in System B. Both nonbioremediated e¥uents had higher values of TSS than
Systems A and C and the estuarine water. Systems A
and C and the estuarine water showed statistically similar values (Fig. 3). The OSS were also the highest in

50
0

1

2

3

4

5

6

c
a
b
a
a

Raw Effluents
System A
System B
System C
Estuary

c
ab
b
a
a

Raw Effluents

System A
System B
System C
Estuary

7

Organic Suspended Solids
50
40
30
20
10
0

1

2

3 4 5 6
Time (Weeks)

7

Figure 3 Concentrations of suspended solids found throughout time in the raw e¥uents, the bioremediated e¥uents with
bivalve and microalga (System A), the nonbioremediated e¥uents (System B), the estuarine that £owed into a similar system
of tanks but without animals (System C) and the estuarine
water (Estuary). Di¡erent letters on the left of each marker in
each graphic indicate signi¢cant di¡erences.

the raw e¥uents and those from System B. No di¡erences were found among the OSS levels of System A
and the rest of the treatments (Fig. 3).

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Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

Navicula sp.

100000

Table 1 Environmental variables in the microcosm culture
of shrimp in the bioremediated e¥uents (System A), the nonbioremediated e¥uents (System B) and the estuarine water
(System C)

Cells·mL–1

80000
60000

System A

40000
20000
0

1

2

3

4

5

6

Temperature ( 1C)
Salinity (%)
DO (mg L À 1)
pH
NO3 (mg L À 1)
NO2 (mg L À 1)
TAN (mg L À 1)
PO4 (mg L À 1)

7

Time (Weeks)
Chione fluctifraga

Survival (%)

100

26.1

41.2
5.6
8.2
0.30
1.04
0.68
0.16

Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ

1.1a
2.1a
0.5a
0.3a
0.06a
0.55a
0.35ab
.04a

System B
26.2
41.5
5.7

8.2
0.41
1.51
1.05
0.21

Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ

1.2a
2.0a
0.6a
0.2a
0.05b
0.76b
0.6b
.09a

System C
25.9
41.0
5.9
8.3
0.28

1.01
0.60
0.15

Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ

0.9a
1.9a
0.6a
0.2a
0.05a
0.62a
0.32a
0.06a

80
Di¡erent letters in the same row indicate signi¢cant di¡erences
(Po0.05).
DO, distilled water; TAN, total ammonia nitrogen.

60
40
20

0

1

2

3

4

5

6

Table 2 Production parameters of white shrimp farmed at
microcosms in the bioremediated e¥uents (System A), the
nonbioremediated e¥uents (System B) and the estuarine
water (System C)

7

Time (Weeks)

Figure 4 Concentration of the benthic microalga (Navicula sp.) and survival of clams (Chione £uctifraga) during
the experiment.

In terms of e⁄ciency, System A with clams and microalgae removed 17.3% of TN, 24.5% of TAN, 19.2% of
nitrites, 13.5% of nitrates, 21.6% of phosphates, 22.3%
of TSS and 23.2% of OSS from the raw e¥uents. System
B showed a removal e⁄ciency of o4% of TN, nitrites,

nitrates and phosphates, while the removal of TAN,TSS
and OSS was 10.2%,10.1% and 8.6% respectively.
The concentration of the benthonic microalgae remained at levels above the 50 000 cells mL À 1, during
the ¢rst 5 weeks of the experiment, but declined during the last week (Fig.4). By the last week, the presence
of Navicula sp. was replaced in part by an unidenti¢ed
diatom. The chlorophyll levels were signi¢cantly
higher in Phase II of System A (23.5 Æ 3.8 mg m À 3),
which had the benthonic microalgae, followed by
Phase II from Systems B (7.0 Æ 2.6 mg m À 3) and C
(4.0 Æ 2.7 mg m À 3) respectively. The survival of the
clams was constant during the entire experiment,
with an average survival above 90% in every week
(Fig. 4); as adult clams were used for the experiment,
the growth during the experimental period was not
signi¢cant (0.5^1.0 mm).
Shrimp rearing in e¥uents
No signi¢cant di¡erences were detected among the
treatments with respect to the following environ-

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Aquaculture Research, 2011, 42, 1415–1423

Final weight (g)
Growth rate
(g week À 1)
Survival (%)
Final biomass
(kg ha À 1)
FCR

System A

System B

System C

14.2 Æ 1.3a
1.15 Æ 0.04a

13.8 Æ 1.2a
1.10 Æ 0.05a

14.0 Æ 1.6a
1.12 Æ 0.06a

89.2 Æ 2.1a
3166 Æ 206a

75.1 Æ 3.6c
2610 Æ 280b

82.1 Æ 3.8b
2874 Æ 205ab

1.47 Æ 0.06a

1.73 Æ 0.05b

1.51 Æ 0.05a

Di¡erent letters in the same row indicate signi¢cant di¡erences
(Po0.05).
FCR, feed conversion ratio.

mental variables: temperature, salinity, DO, pH and
PO4 (Table 1). However, higher values of TAN, NO2
and NO3 were observed in the tanks where shrimp
from System B were reared.
Regarding the production parameters of the
shrimps farmed in di¡erent types of e¥uents, the ¢nal weight and the growth rate were statistically similar among all the treatments (Systems A, B and C).
However, the highest survival was found in shrimps
reared in the bioremediated e¥uents (System A), followed by those reared in estuarine water (System C),
while the lowest survival was observed in the
shrimps cultivated in nonbioremediated e¥uents
(System B) (Table 2).
The highest biomass was also achieved in System
A and the lowest in System B, while no di¡erences
were observed among System C and the rest of the
treatments. The biomass of shrimp from System A
was 21% higher than that of System B (Table 2).

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Aquaculture Research, 2011, 42, 1415^1423

Finally, FCRs were lower in Systems A and C compared with those obtained for the shrimps reared in
System B (Table 2).
Discussion

Shrimp rearing at a semiintensive scale is activity
practice capable of exerting a signi¢cant impact on
the environment. Some of the parameters of water
quality increased by 100% or more from the estuarine water to the raw e¥uents. Although this was demonstrated at an experimental scale, similar results
have been observed in commercial farms (Jackson,
Preston & Thompson 2004). Jackson et al. (2004) studied the discharge nutrient loads at di¡erent shrimp
farms, ¢nding concentrations of TN and TSS as high
as 3 and 200 mg L À 1, respectively; similar results
were observed in this experiment.
The bioremediation system appeared to have a
moderate e⁄ciency, as nitrogenous compounds and
the phosphates were lower after the e¥uent £owed
through the pools with clams and the benthonic microalgae. Some authors have documented that the
presence of bivalves and microalgae can decrease
the concentration of di¡erent compounds that contaminate receiving ecosystems. For instance, Hernandez, Bashan and Bashan (2006) found a P
removal e⁄ciency of around 25% for Chlorella spp.
alone, and up to 72% for Chlorella spp. co-immobilized with Azospirillum braziliense. In addition, Jones,
Dennison and Preston (2001) evaluated a multiphase system (sedimentation, ¢lter feeders and microalgae) to treat shrimp e¥uents and achieved an
overall improvement in water quality as follows: TSS
À
(12%), TN (28%), P (14%), NH1
4 (76%), NO3 (30%),
À
PO4 (35%), bacteria (30%) and chlorophyll a (0.7%);
however, the HRT of this system was lower than that
observed in our experiment, which suggests that the
e⁄ciency of our system was lower that of Jones et al.
(2001). Also, the density of bivalves they used was
much higher, although the retention time used in
our system corresponded to a daily water exchange

similar to that used in commercial farms (10^20%).
The bioremediation e⁄ciency in the above-mentioned experiments as well as in our experiment was
estimated by measuring the number of nutrients
removed from the raw e¥uents. The results suggest
that the quality of water was signi¢cantly improved
after being treated by the bioremediation system (A);
however, System B, which did not have clams or microalgae, also showed some e⁄ciency in removing
TAN,TSS and OSS. These results may be attributed to

Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

the sedimentation in the pools. Hence, it can be hypothesized that the e⁄ciency of System A in removing nitrogenous metabolites and phosphates can be
attributed to the presence of Navicula sp., while the
removal of suspended solids may be caused in part
by the presence of clams and by sedimentation in
the tanks (sediments were observed in the treatment
pools of Systems A and B, although they were not
measured). Vymazaj (1988) found that microalgae
species such as Navicula sp. were capable of removing
nutrients from polluted streams with a maximum ef¢ciency of 80% and 70% for ammonium and orthophosphates respectively. Navicula sp. has been used as
part of bio¢lms to improve the water quality, due to its
ability to remove nitrogenous compounds and phosphates (Thompson, Abreu & Wasielesky 2002). Regarding suspended solids, the presence of bivalves
and the use of tanks as sedimentation units have
been shown to decrease the concentration of suspended solids and TN from aquaculture e¥uents
(Jones et al. 2001; Bernal-Jaspeado 2006; Li, Veilleux
& Wikfors 2009).
The greater e⁄ciency of System A in removing OSS
than TSS could be explained by the ¢ltration activity
of the clams; in this regard, it has been demonstrated
that bivalves preferably ingest organic and reject inorganic materials (Newell & Jordan 1983).

Although the bioremediation system with C. £uctifraga and Navicula sp. had acceptable e⁄ciency, the
concentrations of the microalgae decreased during
the last week. This decrease may be attributed to the
high turbidity observed in System A during the last 2
weeks, caused by the increase in TSS and OSS. It has
been reported that the abundance of some microalgae species such as Navicula sp. depends on the turbidity and the concentration of suspended solids
(Unrein & Vincour 1999). Some alternatives to solve
this problem might be to increase bivalves in Phase I
or include a sedimentation tank, as suggested by
Jones et al. (2001) to decrease the concentration of
suspended solids. Although Navicula sp. levels decreased by the last week, the nitrogenous metabolites
continued decreasing in System A, which may be attributed to the remaining concentration of Navicula
sp. and the unidenti¢ed diatom that was predominant during the last few days.
As a ¢rst approach, it was observed that the
bioremediation system had lower e⁄ciency than
those using macroalgae, in terms of nutrient removal
and biomass production (Xu, Fang & Wei 2008;
Marinho-Soriano, Nunes, Carneiro & Pereira 2009;
Mart|¤ nez-Porchas et al. 2010). However, it is important

r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1415^1423

1421


Bioremediation and reuse of e¥uents L R Mart|¤ nez-Co¤rdova et al.

to continue studying the ability of C. £uctifraga and
Navicula sp. as potential bioremediators of shrimp
(or ¢sh) e¥uents, at di¡erent densities and using different system designs, to achieve a better e⁄ciency.

Regarding the production parameters of the
shrimp reared on the three systems, it was observed
that the bioremediated e¥uents (System A) were
very suitable for the culture of white shrimp.
The growth rates were similar to or higher than the
0.9^1.0 g week À 1, reported as commercially feasible
(Mart|¤ nez-Co¤rdova 1999). The productive response of
the shrimp reared in the bioremediated e¥uents was
better than that observed in those cultivated in e¥uents from Systems B and C. The di¡erence in survival
and biomass among the shrimps reared in bioremediated e¥uents (from System A) and those reared
in the nonbiormediated e¥uents (System B) may be
attributed to the improvement in the water quality.
Although none of the water quality parameters
reached the lethal concentration (LC50) in the nonbioremediated e¥uents, they were almost twofold
higher than those from the bioremediated e¥uents.
Di¡erent authors have documented that the chronic
exposure of penaeid shrimps to high concentrations
of nitrogenous compounds and suspended solids can
diminish their growth and food intake (Frias-Espericueta, Harfush-Melendez & Paez-Osuna 2000; Ray,
Lewis, Browdy & Le¥er 2009). Moreover, the slight
increase in the productive response of shrimps from
System A as compared with those reared in System C
could be attributed to the higher survival and to the
presence of OM in System A, such as bio£ocs and microalgae (Navicula sp. and other diatoms), which
could be an alternative source of food.
The biomass obtained in Systems A and C was
higher than the mean reported in most semiintensive
farms of the region. The FCRs in the same treatments
are considered to a pro¢table value for commercial
purposes (Juarez 2008). The results suggest that the

shrimp can thrive in bioremediated e¥uents, which
indicates that the treated water (by clams and microalgae) can be reused by recirculation. This practice
would reduce the environmental impact caused by
the massive discharges of shrimp aquaculture.
Furthermore, the black clam have commercial value
and may represent and extra economical income for
farmers (Mart|¤ nez-Porchas et al. 2010).
It can be concluded that the bioremediation system
was moderately e⁄cient in removing nutrients and
solids (TSS and OSS) from shrimp aquaculture e¥uents. Also, the tanks themselves are useful due to
the sedimentation activity.

1422

Aquaculture Research, 2011, 42, 1415–1423

It is necessary, however, to improve System A with
some modi¢cations in the design. The production
parameters obtained in the present study strongly
suggest that the bioremediated e¥uents can be used
for farming white shrimp without a negative e¡ect on
its survival and growth.

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Aquaculture Research, 2011, 42, 1424^1430

doi:10.1111/j.1365-2109.2010.02734.x

Evaluation of corn distillers dried grains with solubles
and brewers yeast in diets for channel catfish

Ictalurus punctatus (Rafinesque)
Menghe H Li, Daniel F Oberle & Penelope M Lucas
Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS, USA
Correspondence: M Li,Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, PO Box 197, Stoneville, MS
38776, USA. E-mail:

Abstract
A study was conducted to examine the use of distillers grains with solubles (DDGS), ethanol extracted
DDGS (EDDGS), and brewers yeast in channel cat¢sh,
Ictalurus punctatus, diets. Diets containing these ingredients were compared with all-plant and ¢sh meal
control diets. Juvenile channel cat¢sh (initial weight:
9.1 Æ 0.2 g ¢sh À 1) were stocked in £ow-through
aquaria and fed one of six practical diets for 8 weeks.
Diets containing1% brewers yeast or 30% DDGS supported the same level of growth and feed e⁄ciency
ratio (FER) as the diet containing 5% ¢sh meal. Ethanol extraction e¡ectively removed most of the fat and
yellow pigments in DDGS. The diet containing 30%
EDDGS resulted in signi¢cantly lower growth and
FER compared with the diet containing DDGS. However, the weight gain of ¢sh fed the EDDGS diet was
intermediate compared with ¢sh fed the all-plant
control, ¢sh meal control, and 1% and 2% brewers
yeast diets. The EDDGS could potentially be used at

high levels as a substitution for soybean meal without causing yellow pigment deposition in cat¢sh
£esh, provided that the ethanol extraction process is
proven economical. Brewers yeast, used at 1^2% of
the diet, appears to be e¡ective at improving weight
gain and FER of channel cat¢sh over the all-plant diet.

Keywords: channel cat¢sh, distillers dried grains,
brewers yeast, growth, feed e⁄ciency
Introduction
Distillers dried grains with solubles (DDGS) from
corn is a by-product of ethanol production. It is relatively high in protein (27%) and highly palatable to

1424

channel cat¢sh, Ictalurus punctatus. With the rapid
expansion of ethanol production in the United States,
the prices for DDGS have become more competitive
compared with soybean meal and other plant protein
sources. Use of this by-product in cat¢sh feeds would
reduce feed cost.
Several studies have been reported on the use of
DDGS in channel cat¢sh diets. Early studies demonstrated that up to 35% DDGS without lysine supplementation (Webster, Tidwell & Yancey 1991; Webster,
Tidwell, Goodgame, Yancey & Mackey 1992; Webster,
Tidwell, Goodgame & Johnsen 1993) and up to 70%
with lysine supplementation (Webster et al. 1991)
could be used to partially replace soybean meal
and ¢sh meal in channel cat¢sh diets without a¡ecting ¢sh growth. Recently, Lim, Yildirim-Aksoy and
Klesius (2009) also reported no di¡erences in the
growth of juvenile channel cat¢sh fed diets containing up to 40% DDGS with supplemental lysine. In a
pond study with channel cat¢sh, Robinson and Li

(2008) found that up to 30^40% DDGS with supplemental lysine could be used in food ¢sh diets. They
noted that feed e⁄ciency ratio (FER) was improved
in ¢sh fed diets containing 30^40% DDGS. However,
it was not clear whether the improved FER of ¢sh fed
diets containing DDGS was caused by the increased
dietary fat level, because of high levels of fat (about
9%) contained in the DDGS, or by other compounds
present in the product.
Li, Robinson, Oberle and Lucas (2010) examined
the use of several corn distillers by-products including DDGS, distillers solubles and high-protein DDGS
in diets and the e¡ects of additional dietary fat on
juvenile channel cat¢sh performance. They found
that elevated fat levels in diets containing distillers

r 2011 Blackwell Publishing Ltd


Aquaculture Research, 2011, 42, 1424^1430

Distillers grains, brewers yeast in cat¢sh diets M H Li et al.

by-products were only partially responsible for the
improvement in FER of ¢sh fed the distillers byproducts. The presence of the distillers solubles in
the diet, possibly due to the brewers yeast, Saccharomyces cerevisiae, further improved FER, and also
improved weight gain over the control diets with or
without additional fat.
Distillers grains with solubles contain up to three
times of the amount yellow pigments lutein and zeaxanthin found in yellow corn. The high level of yellow
pigments may limit its use in cat¢sh diets because
high dietary yellow pigment levels can result in

pigment deposition in the £esh, rendering it less
appealing to the general consumer in the United
States (Lee1987; Li, Robinson & Oberle 2009). If levels
of pigmented compounds in DDGS can be reduced,
more DDGS could be used without adversely a¡ecting marketability of the cat¢sh product. Therefore,
the present study was conducted to examine the
e¡ect of DDGS, de-pigmented DDGS and brewers
yeast in the diet on the growth, FER and body proximate composition of juvenile channel cat¢sh.

cial Analytical Chemists International (AOAC)
(2000) using the Soxtec System (Foss North America,
Eden Prairie, MN, USA). The resulting material was
dried at 60 1C for 60 min to evaporate the solvent
residue. The hexane extracted DDGS was not used in
the feeding study because only a small amount of
yellow pigment was removed during the process.
The experimental diets were prepared as sinking
pellets according to procedures described previously
(Li, Johnson & Robinson 1993).
Juvenile channel cat¢sh were obtained from the
USDA Agriculture Research Service’s Cat¢sh Genetics Research Unit (Stoneville, MS, USA). Thirty ¢sh
were stocked into each of thirty 110 L £ow-through
aquaria at the Thad Cochran National Warmwater
Aquaculture Center (NWAC), Mississippi State
University (Stoneville, MS, USA). The aquaria were
supplied with well water (£ow rate: approximately
1L min À 1) and continuous aeration.Water temperature and dissolved oxygen were monitored in the
system once daily using a YSI oxygen meter (Yellow
Springs Instruments, Yellow Springs, OH, USA)
and averaged at 29.8 Æ 0.2 1C and 6.8 Æ 0.2 mg L À 1

respectively. A diurnal light:dark cycle was regulated
at 14:10 h.
Before initiation of the experiment, the ¢sh were
acclimated for 2 weeks and fed an all-plant conditioning diet once daily to apparent satiation at
08:00 hours. After acclimation, all ¢sh were pooled
and graded to a uniform size, and 15 ¢sh were collectively weighed and restocked in each aquarium.
Initial ¢sh weight was determined and averaged
9.1 Æ 0.2 g ¢sh À 1 (mean Æ SD). Fish were fed to
apparent satiation (in about 40 min) once daily for
8 weeks. Satiation was achieved by ¢rst feeding an
amount of diet based on the percentage of ¢sh body
weight (less than satiation), followed by feeding
several times from a pre-weighed diet container.
Diet consumption was monitored and recorded at
each feeding. Dead ¢sh, if any, were removed daily
from the aquarium and weighed. Aquaria were
cleaned weekly.
At the end of the feeding period, feed consumption
and weight gain per ¢sh, FER and survival were calculated. Feed e⁄ciency ratio was determined as follows:

Materials and methods
Six practical diets containing 28% crude protein and
5% crude fat (Table 1) were formulated to meet or
exceed all known nutrient requirements of channel
cat¢sh (National Research Council 1993). Diet descriptions follow:
Diet 1 ^ all-plant control diet.
Diet 2 ^ ¢sh meal control diet (similar to Diet 1 except with 5% menhaden ¢sh meal in replacement
of part of soybean meal).
Diet 3 ^ 30% ethanol extracted DDGS (EDDGS).
Diet 4 ^ 30% DDGS.

Diet 5 ^ 1% brewers yeast (similar to Diet 1 except
with brewers yeast in replacement of part of soybean meal).
Diet 6 ^ 2% brewers yeast (similar to Diet 1 except
with brewers yeast in replacement of part of soybean meal).
The DDGS was provided by Poet, LLC (Sioux Falls,
s
SD, USA) and Brewtech dried brewers yeast was
provided by International Ingredient Corporation
(St Louis, MO, USA). Remaining dietary ingredients
were obtained from the Delta Western Feed Mill
(Indianola, MS, USA) and were from commercial
sources. The DDGS was extracted with hexane and
ethanol, respectively, in a manner similar to the ether
extraction method described by Association of O⁄-

r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1424^1430

FER ¼ ð½final fish weight; g tankÀ1 Š
À ½initial fish weight; g tankÀ1 Š
þ ½weight of dead fish; g tankÀ1 ŠÞ=
ðtotal feed fed; g tankÀ1 Þ

1425


Distillers grains, brewers yeast in cat¢sh diets M H Li et al.

Aquaculture Research, 2011, 42, 1424^1430

Table 1 Ingredient and proximate compositions of experimental diets (expressed percentage on an as-fed basis)

Ingredient
Soybean meal (dehulled)
Menhaden fish meal
EDDGSÃ
DDGSw
Brewers yeast
Corn meal (cooked)
Wheat middlings
Lysine HCl
Dicalcium phosphate
Corn oil
Other ingredientsz
Proximate analysis (%)‰
Dry matter
Crude proteinz
Crude fatz
Lutein1zeaxanthinz (mg kg À 1)
DE:P ratio (kcal g À 1 protein) k

All-plant
control

Fish meal
control

30%
EDDGSÃ

30%

DDGSw

1% brewer
yeast

2% brewer
yeast

44.60
0.00
0.00
0.00
0.00
30.71
10.00
0.00
1.50
2.49
10.70

37.85
5.00
0.00
0.00
0.00
33.50
10.00
0.00
1.00
1.95

10.70

26.60
0.00
30.00
0.00
0.00
22.40
6.31
0.40
1.30
2.29
10.70

29.75
0.00
0.00
30.00
0.00
23.00
5.00
0.30
1.25
0.00
10.70

42.90
0.00
0.00
0.00

1.00
31.52
10.00
0.00
1.50
2.38
10.70

42.05
0.00
0.00
0.00
2.00
31.37
10.00
0.00
1.50
2.38
10.70

88.67 Æ 0.01
27.59 Æ 0.11
4.64 Æ 0.04
4.16 Æ 0.11
10.2

88.30 Æ 0.02
28.22 Æ 0.10
4.50 Æ 0.00
3.54 Æ 0.10

9.2

87.80 Æ 0.02
28.25 Æ 0.18
5.01 Æ 0.01
9.78 Æ 0.48
9.5

88.37 Æ 0.03
27.38 Æ 0.09
4.49 Æ 0.00
3.90 Æ 0.06
10.2

87.64 Æ 0.06
27.35 Æ 0.18
4.49 Æ 0.03
4.00 Æ 0.06
10.2

87.9
27.7
4.68
3.91
10.2

Æ
Æ
Æ
Æ

0.02
0.10
0.02
0.00

ÃEthanol extracted distillers dried grains with solubles.
wDistillers dried grains with solubles.
zIncludes 7.5% cottonseed meal, 1% menhaden ¢sh oil, 2% carboxymethyl cellulose (pellet binder), 0.05% vitamin premix, 0.05% L-ascorbyl
monophosphate and 0.1% trace mineral premix.Vitamin and trace mineral premixes were the same as described by Robinson and Li (2007).
‰Values represent mean Æ SD (n 5 2, two batches per diet).
zExpressed as 900 g kg À 1 dry matter basis.
kEstimated digestibility to protein ratio.

After the ¢nal ¢sh number and weight were determined, ¢ve ¢sh from each aquarium were euthanized
by an overdose (500 mg L À 1) of tricaine methanesulphonate (MS-222TM, Argent Chemical Laboratories,
Redmond,WA, USA), and ¢llet samples were removed.
Digital pictures were taken of one ¢llet from each ¢sh
using an EOS 1D Mark II digital SLR camera (Cannon
USA, Lake Success, NY, USA). The yellow intensity values [Commission Internationale de I’Eclairage (CIE)
bà (negative: blueness; positive: yellowness)] were determined from the digital picture of the ¢llet at three
locations along the dorsal line of the ¢llet using
Adobe Photoshop CS3 image editing software (Adobe
Systems, San Jose, CA, USA). The ¢llets were then
pooled by aquarium, and stored at À 80 1C for subsequent proximate and pigment analyses.
The ¢llet samples were homogenized into a paste
using a Grindomix GM-200 Knife Mill (Retsch
GmbH, Haan, Germany) and part of the sample was
lyophilized with a Freezone Freeze Dry System (Labconco, Kansas City, MO, USA) for 16^18 h for protein
and fat analyses.

Proximate analyses were performed in duplicate
on diet and pooled ¢llet samples from each aquarium
with methods described by AOAC (2000). Crude pro-

1426

tein of diet and ¢llet samples was analysed by the
combustion method with the FP-2000 protein determinator (Leco, St Joseph, MI, USA), crude fat by ether
extraction with the Soxtec System (Foss North America, Eden Prairie, MN, USA) and moisture by oven drying with a mechanical convection oven (Precision,
Winchester, VA, USA). Diet and ¢llet samples were
analysed for lutein and zeaxanthin concentrations
using high-performance liquid chromatography
(Moros, Darnoko, Cheryan, Perkins & Jerrell 2002).
Data were subjected to one-way analysis of variance (ANOVA) and the Fisher’s protected least signi¢cant di¡erence procedure (Steel, Torrie & Dickey
1997) with the STATISTICAL ANALYSIS SYSTEM version 9.1
software (SAS Institute 2004). Aquaria were the experimental units and variation among aquaria within a treatment was used as the experimental error in
tests of signi¢cance. An a level of 0.05 was used.

Results
Hexane extraction method used in the present study
removed almost all fat (from 9.49% to 0.06%) in
DDGS, but was not e¡ective in removing the yellow

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Aquaculture Research, 2011, 42, 1424^1430

Distillers grains, brewers yeast in cat¢sh diets M H Li et al.

pigments lutein and zeaxanthin (from 30.9 to
26.7 mg kg À 1). Ethanol extraction of DDGS removed
most of the fat (from 9.49% to 1.89%) and almost all
yellow pigments (from 30.9 to 0.6 mg kg À 1).
The feeding study showed no signi¢cant di¡erences among dietary treatments for feed consumption and survival (Table 2). The overall mortality was
2.5% and the cause was not known. Weight gain of
¢sh fed diets containing 30% DDGS, and 1% and 2%
brewers yeast was signi¢cantly higher than that of
¢sh fed the all-plant control diet, but not signi¢cantly
di¡erent from that of ¢sh fed the ¢sh meal control diet
(5% menhaden meal).Weight gain of ¢sh fed the diet
containing 30% EDDGS was signi¢cantly lower than
that of ¢sh fed the diet containing 30% DDGS, but
was intermediate with that of ¢sh fed the all-plant
and ¢sh meal control diets. Feed e⁄ciency ratio of ¢sh
fed diets containing 30% DDGS and1% brewers yeast
was signi¢cantly higher than that of ¢sh fed the allplant control diet and the diet containing 30%
EDDGS, but not signi¢cantly di¡erent from that of
¢sh fed the ¢sh meal control diet. Feed e⁄ciency ratio
of ¢sh fed the diet containing the 2% brewers yeast
diet was intermediate, not signi¢cantly di¡erent from
that of ¢sh fed other diets.
Fish fed the ¢sh meal control diet had a signi¢cantly higher protein, but had similar levels of fat
and moisture compared with ¢sh fed the all-plant
control diet (Table 3). Fish fed diets containing 30%
EDDGS and 30% DDGS had similar levels of ¢llet protein, fat and moisture levels. Fillet protein levels of
¢sh fed the EDDGS and DDGS diets were similar to
that of ¢sh fed the all-plant control diet, but were low-

er than that of ¢sh fed the ¢sh meal control diet and

brewers yeast diets. Fillet fat levels of ¢sh fed the
EDDGS diet were similar to that of ¢sh fed the allplant control and ¢sh meal control diets, but were
lower than that of ¢sh fed the brewers yeast diets. Fillet moisture levels of ¢sh fed the EDDGS diet were
higher than that of ¢sh fed the all-plant control, ¢sh
meal control and brewers yeast diets. Fillet protein,
fat and moisture levels of ¢sh fed the 1% and 2%
brewers yeast diets were similar.
Fish fed the diet containing 30% EDDGS had a signi¢cantly lower CIE bà value than ¢sh fed the allplant control diet and the diet containing 30% DDGS
(Table 4). The CIE bà value of ¢sh fed the all-plant control diet was signi¢cantly lower than that of ¢sh fed
the 30% DDGS diet. Lutein1zeaxanthin levels in the
£esh of ¢sh fed the all-plant control and the diet containing 30% EDDGS were signi¢cantly lower than
that of ¢sh fed the 30% DDGS diet.

Discussion
Results from the present study support the observation by Li et al. (2010) that the use of 30% DDGS in
the diet improved weight gain and FER over an
all-plant control diet. In addition, the present study
demonstrated that the 30% DDGS diet provided the
same level of growth and FER as the ¢sh meal control
diet. Li et al. (2010) suggests that the improvement of
weight gain and FER by feeding 30% DDGS is likely
caused by the presence of distillers solubles, possibly
due to the brewers yeast. In the present study, ¢sh

Table 2 Mean feed consumption, weight gain, feed e⁄ciency ratio and survival of juvenile channel cat¢sh fed various experimental diets for 8 weeksÃ

Diet description

Feed consumption
(g fish À 1)w

Weight gain
(g fish À 1)z

Feed efficiency
ratiow

Survival (%)

All-plant control
Fish meal control
30% EDDGS‰
30% DDGSz
1% brewers yeast
2% brewers yeast
Pooled SEM

102.1
103.3
106.6
108.7
104.9
110.1
2.2

61.3
68.7
64.8
71.3
68.2

68.7
1.9

0.600
0.665
0.607
0.656
0.651
0.624
0.013

96.0
98.3
98.7
98.7
100.0
93.3
1.6

c
ab
bc
a
ab
ab

b
a
b
a

a
ab

ÃMeans represent average values of ¢ve tanks per diet. Means within each column followed by di¡erent letters were di¡erent (P
the Fisher’s protected least signi¢cant di¡erence procedure).
wBased on 900 g kg À 1 dry matter of the diet.
zInitial weight was 9.1 Æ 0.2 g ¢sh À 1.
‰Ethanol extracted distillers dried grains with solubles.
zDistillers dried grains with solubles.

0.05,

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1427


Distillers grains, brewers yeast in cat¢sh diets M H Li et al.

Aquaculture Research, 2011, 42, 1424^1430

Table 3 Mean ¢llet protein, fat and moisture concentrations of juvenile channel cat¢sh fed various experimental
diets for 8 weeksÃ

are derived (Tacon & Jackson1985). The brewers yeast
used in the present study contained 3.0% nucleotides
(analysed by Euro¢ns Scienti¢c, Des Moines, IA, USA).
The nucleotides included adenosine-, cytidine-,
guanosine-, uridine-5 0 -monophosphate and a trace
amount of inosine-5 0 -monophosphate. Dietary supplementation of a mixture of nucleotides has been

shown to increase the height of villa in the intestine
of rat (Uauy, Stringel, Thomas & Quan 1990) and
Atlantic salmon, Salmo salar (Burrells, Williams,
Southgate & Wadsworth 2001). As a result, the mucosal surface area of the intestine is increased and
therefore nutrients are more e⁄ciently absorbed and
utilized. Li, Gatlin III and Neil (2007) reported that
dietary supplementation of a mixture of nucleotides
enhanced the growth and FER of red drum, Sciaenops
ocellatus during the ¢rst week of feeding, but the improvement diminished during the following 3 weeks
of feeding. Lin,Wang and Shiau (2009) found that the
addition of individual and a mixture of nucleotides in
the diet improved the growth and FER in grouper, Epinephelus malabaricus after 8 weeks of feeding.
Nucleotides such as adenosine-, inosine- and uridine-5 0 -monophosphate have been shown to stimulate gustatory sensory cells in several ¢sh species
(Ishida & Hidaka 1987; Ikeda, Hosokawa, Shimeno &
Takeda 1991; Kubitza, Lovshin & Lovell 1997). Li et al.
(2010) reported that the use of DDGS or distillers
solubles in the diet increased feed intake in channel
cat¢sh, which they attributed to the possible chemoattractive e¡ects of nucleotides present in the yeast
cells. However, no signi¢cant di¡erences in feed
consumption were observed among ¢sh fed various
diets in the present study. The discrepancy between
responses of the present study and Li et al. (2010) cannot be easily explained, but could be due to the larger
variation in feed consumption of ¢sh in various replicated tanks in the present study.
In the present study, ¢sh did not perform well on
the all-plant control diet as compared with ¢sh fed
diets containing ¢sh meal, DDGS and 1% brewers
yeast. Although results from previous studies are inconclusive, more evidence appears to indicate that
the inclusion of ¢sh meal in the diet improves the
growth and FER of juvenile channel cat¢sh (Mohsen
& Lovell 1990; Li, Peterson, Janes & Robinson 2006;

Li, Robinson, Peterson & Bates 2008). Fish muscle is
rich in nucleotides (Ikeda, Hosokawa, Shimeno &
Takeda 1988), which may contribute to the growthenhancing e¡ect of ¢sh meal on channel cat¢sh.
Several studies have demonstrated that relatively
high levels of DDGS can be used in channel cat¢sh

Diet description

Fillet
protein (%)w

Fillet
fat (%)w

Fillet
moisture (%)

All-plant control
Fish meal control
30% EDDGSz
30% DDGS‰
1% brewers yeast
2% brewers yeast
Pooled SEM

16.6
17.3
16.3
16.3
17.0

17.1
0.2

5.88
5.23
5.08
5.55
6.32
6.33
0.31

76.5
76.3
77.7
77.1
75.4
75.4
0.3

bc
a
c
c
ab
ab

ab
b
b
ab

a
a

b
b
a
ab
c
c

ÃMeans represent average values of ¢ve tanks with ¢ve ¢sh per
tank. Means within each column followed by di¡erent letters
were di¡erent (P
0.05, the Fisher’s protected least signi¢cant
di¡erence procedure).
wOn wet-tissue basis.
zEthanol-extracted distillers dried grains with solubles.
‰Distillers dried grains with solubles.

Table 4 Mean CIE bà value and lutein plus zeaxanthin
concentrations of juvenile channel cat¢sh fed experimental
diets containing distillers dried grains with solubles (DDGS)
and ethanol extracted distillers dried grains with soluble
(EDDGS) for 8 weeksÃ

Diet description

CIE bÃ

Lutein1zeaxanthin

(lg g À 1)w

All-plant control
30% EDDGS
30% DDGS
Pooled SEM

15.1 b
13.7 c
18.2 a
0.4

0.83 b
0.83 b
1.03 a
0.01

ÃMeans represent average values of ¢ve tanks with ¢ve ¢sh per
tank. Means within each column followed by di¡erent letters
were di¡erent (P
0.05, the Fisher’s protected least signi¢cant
di¡erence procedure).
wOn wet-tissue basis.

fed 1% and 2% brewers yeast had similar weight
gain and FER as ¢sh fed the 30% DDGS diet. This
indirectly con¢rms that brewers yeast in the DDGS
may play a role in the improvement of ¢sh growth
performance.
During ethanol production, brewers yeast is typically used to ferment corn to produce ethanol. Distillers dried grains with solubles have been estimated to

contain about 3.9% yeast cells (Ingledew 1999). Previous studies with sea bass, Dicentrarchus labrax
(Oliva-Teles & Goncalves 2001) and hybrid striped
bass, Morone chrysops  Morone saxatilis (Li & Gatlin
III 2005), showed that the use of brewers yeast in the
diet improved ¢sh growth and FER. Yeast cells contain 5^12% nucleic acids from which nucleotides

1428

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Aquaculture Research, 2011, 42, 1424^1430

Distillers grains, brewers yeast in cat¢sh diets M H Li et al.

diets without adversely a¡ecting ¢sh performance
(Webster et al. 1991; Robinson & Li 2008; Lim et al.
2009), but there is a concern about high yellow
pigment levels in DDGS. Studies have shown that
feeding diets containing lutein1zeaxanthin levels
higher than 7^10 mg kg À 1 can deposit enough
pigment in cat¢sh £esh to be visible (Lee 1987; Li
et al. 2009). Fish fed the DDGS diet in the present
study only showed slightly yellow colouration by
visual examination. Also, the CIE bà values and
lutein1zeaxanthin concentration in ¢sh £esh were
at low levels. This is mostly due to the short feeding
period and small ¢sh size used in the present study.
However, both the CIE bà value and lutein1zeaxanthin concentration in ¢sh fed EDDGS were signi¢cantly lower than in ¢sh fed DDGS. This is anticipated
because yellow pigments lutein and zeaxanthin in

the EDDGS were e¡ectively removed by ethanol
extraction. Analyses of yellow pigments conducted
at our laboratory on various batches of DDGS showed
that the pigment varied from 22 to 40 mg kg À 1.
Longer periods of feeding of DDGS containing a high
level of the pigment at 30% would result in yellow
pigment deposition that may reduce marketability of
the cat¢sh product.
Ethanol extraction does not appear to extract the
nucleotides from the DDGS. Total nucleotide (the same
four main nucleotides found in brewers yeast) concentrations were 0.17% for DDGS and 0.25% for EDDGS.
However, ¢sh fed the 30% EDDGS diet had lower
weight gain and FER than ¢sh fed the 30% DDGS diet.
This response cannot be easily explained with regard
to the nucleotide concentrations of the diets.
Signi¢cant di¡erences were observed in ¢llet proximate composition of ¢sh fed various experimental
diets. The di¡erences cannot be easily explained.
However, these di¡erences were relatively small and
¢llet protein, fat and moisture levels were within the
normal range for this size of ¢sh. Although all diets
were formulated to be isonitrogenous and isolipidic,
di¡erences in dietary ingredient composition, digestible energy and other nutrients may have a¡ected
the nutrient retention of these ¢sh.
With the method used in the present study, ethanol
extraction e¡ectively removed most of yellow pigments in DDGS, while hexane did not. Further investigations are needed to optimize extraction conditions
using hexane as a solvent to remove oil and pigments
because hexane is commonly used as the solvent in
extracting oil from oilseeds and other feedstu¡s.
In summary, the present study demonstrates that
diets containing1% brewers yeast or 30% DDGS sup-

port the same level of growth and FER as a diet containing 5% ¢sh meal for juvenile channel cat¢sh. The
diet containing EDDGS resulted in signi¢cantly lower
growth and FER compared with the diet containing
DDGS. However, the weight gain of ¢sh fed the
EDDGS diet was intermediate compared with ¢sh
fed the all-plant control, ¢sh meal control and 1^2%
brewers yeast diets. Ethanol extracted DDGS could
potentially be used at high levels as a substitution of
soybean meal without causing yellow pigment deposition in cat¢sh £esh, provided that the ethanol extraction process is proven economical. Brewers yeast,
used at 1^2% of the diet, appears to improve weight
gain and FER over the all-plant control diet used in
the present study.

Acknowledgments
We thank Sandra Philips and Cli¡ Smith for daily
management of the experiment. Special thanks to
Poet, LLC of Sioux Falls (South Dakota, USA), which
provided the distillers dried grains with solubles and
International Ingredient Corporation (St Louis, MO,
USA) for providing the brewers yeast. This manuscript is approved for publication as Journal Article
No. J-11855 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. This project is supported under Project Number
MIS-371390 through a grant from USDA National Institute of Food and Agriculture.

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Aquaculture Research, 2011, 42, 1431^1439

doi:10.1111/j.1365-2109.2010.02735.x

A new system for the culture and stock enhancement
of sea cucumber, Apostichopus japonicus (Selenka),
in cofferdams
Libin Zhang1, Hongsheng Yang1, Qiang Xu1, Kun Xing1,3, Peng Zhao1,2 & Chenggang Lin1,4
1

Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,

Qingdao, China
2

Chinese Academy of Sciences, Graduate University, Beijing, China

3

Dalian Fisheries University, Dalian, China

4

College of Marine Life Sciences, Ocean University of China, Qingdao, China

Correspondence: H Yang, Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of
Sciences, Nanhai Road 7, Qingdao 266071, Shandong Province, China. E-mail:

Abstract

Introduction

A new multilayer, plate-type system for the culture
and stock enhancement of sea cucumbers in co¡erdam was developed. To optimize and evaluate the system, four experimental designs were implemented
using polyethylene (PE)-corrugated sheets of various
colours, interval spacing and shapes/styles. Results
showed that a system equipped with black PE-corrugated sheets attracted more animals than either blue,
green, transparent or a selection of mixed sheets (six
transparent sheets in the upper layer and ¢ve black
sheets in the lower layer) (Po0.05). Also, more animals gathered in the system with oblique-angled
sheets (301 to the base plate) than either a wavy (the
bottom and every second sheet was at an angle of 101
to the base plate) or parallel arrangement (Po0.05),
and more animals assembled in the system with
2 cm between sheets than spacings of 3, 4 or 5 cm
(Po0.05). As expected, the upper layers of the systems attracted more animals than lower layers in
most cases except for those with transparent and

mixed oblique-angled sheets with a 3 cm spacing
(Po0.05). Thus, a system with black, oblique-angledcorrugated sheets and 2 cm spacing is recommended
forApostichopus japonicus (Selenka) culture and stock
enhancement in co¡erdams or ponds.

Since the early1980s, due to its high nutritional value
and the development of successful hatchery techniques (Liao 1997), farming of the sea cucumber, Apostichopus japonicus Selenka (Liao 1980), has become a
signi¢cant mariculture sector in North China (Chen
2004; Yuan, Yang, Zhou, Mao, Zhang & Liu 2006).
This species of sea cucumber inhabits reefs or gritty
substrates with a gentle current of high-quality seawater, an abundance of natural food and no freshwater input (Chang, Ding & Song 2004). Thus, the
addition of structures, such as arti¢cial substrates or
reefs, is a simple method of habitat improvement and
stock enhancement. The functions of such systems
are (1) to protect broodstock and larvae from predators, (2) to increase the availability of benthic algae
and organic debris and (3) to improve the habitat for
aestivation and hibernation (Chen 2003).
In China, many materials have been utilized as arti¢cial substrates or reefs for sea cucumber culture,
such as stones (Chen 2004, 2007; Li, Hao, Gao & Yin
2004; Sun & Chen 2006), tiles (Chen 2003; Wang,
Zhang, Zhang, Qu & Song 2004; Qin, Dong, Niu,Tian,
Wang, Gao & Dong 2009), concrete structures (Zhao
1995; Sun 2004; Qin et al. 2009), scallop lantern nets
(Li & Huo 2007), woven fabrics (Lin 2007), plastic
components (Li & Huo 2007) and even Chinese oak
branches (Yang & Shan 2007). Although these
substrates were mainly developed through trial and

Keywords: sea cucumber, Apostichopus japonicus,
culture system, stock enhancement

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1431


New system for the culture of sea cucumber L Zhang et al.

error, they had no regular structure and could not be
easily removed from ponds, for example, for cleaning
and harvesting.
Many factors in£uence habitat selection in animals, such as habitat features, characteristics of the
animal and the presence/absence of food, predation,
competition, etc. (Riechert 1976; Abramsky, Al¢a,
Schachak & Brand 1990; Ward & Porter 1993; Yan &
Chen 1998). Apostichopus japonicus, as a sedimentary
feeder, ingests organic matter, including bacteria,
protozoa, diatoms and detritus from plants and animals (Zhang, Sun & Wu 1995; Yang, Yuan, Zhou,
Mao, Zhang & Liu 2005) and re-utilizes residual food
and faeces (Yang, Wang, Zhou, Zhang, Wang, He
& Zhang 2000; Yang, Zhou, Wang, Zhang, Wang,
He & Zhang 2000; Yang et al. 2005). Thus, arti¢cial
reefs are commonly used for sea cucumber ranching
in China (Chen 2003), as they provide protection
against predators (Ambrose & Anderson 1990), supply shelter and food, in addition to sites for aestivation
(Chen 2004, 2007; Qin et al. 2009).
An increasing demand for beche-de-mer, together
with evidence of a world-wide decline in natural
stocks (Conand 2004), suggests that the development
of e⁄cient enhancement methods, especially new

types of structures, are urgently required. This study
describes the development and trials of a new culture
system, a multilayer, plate-type reef, which can be
easily varied in colour, shape/style or spacing, for
pond culture of sea cucumbers.

Aquaculture Research, 2011, 42, 1431^1439

Materials and methods
Multilayer, plate-type sea cucumber reef
system
The new multilayer, plate-type sea cucumber reef
(Fig. 1) consists of a base plate (supporting the system), polyethylene (PE)-corrugated sheet (stacked on
the base plate), polyvinyl chloride (PVC) cannulae
(acting as spacers for the PE-corrugated sheet) and
tie wires (holding the system in place).
The base plate is a cast-reinforced concrete slab of
75 Â 70 Â 8 cm containing nine holes of10 cm in diameter arranged in a regular pattern. Three strut bars,
made from galvanized tubes, 70 cm in length and
2.5 cm external diameter, were cast vertically near
the centre of the base plate. The shade netting system
consisted of 2.0 mm high-density polyethylene netting
supported (sown) on a polypropylene U-shaped frame,
70 cm in height,60 cm in width and 2.5 cm in external
diameter, cast on one side of the base plate.
The PE-corrugated sheet was 70 cm2 and 2 cm in
wave height. Circular holes of 4.0 cm in diameter
were punched symmetrically in the troughs. Three
other circular holes, 3.0 cm in diameter, were
punched near the centre of the corrugated sheet to

accommodate the strut bars and load the sheet e⁄ciently. The colour of the corrugated sheet was
decided by experimental design.
The cannulae or spacers were made from PVC
tubing, 4 cm in external diameter and 0.2 cm in wall

Figure 1 Multilayer, plate-type sea cucumber reef: (a) graphic, (b) lateral view. 1, Base plate; 2, polypropylene (PPR) piping; 3, high-density polyethylene (HDPE) netting; 4, shade netting; 5, strut bar; 6, polyethylene (PE)-corrugated sheet; 7,
polyvinyl chloride (PVC) cannula; 8, tie wire.

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Aquaculture Research, 2011, 42, 1431^1439

New system for the culture of sea cucumber L Zhang et al.

thickness. The length of the cannulae depended on
the spacing required between the corrugated sheets.
The corrugated sheets were loaded on the base
plate via the three holes onto the three strut bars
and spaced with the PVC cannulae. The shape and
style of the corrugated sheets can also be varied by
using di¡erent-length cannulae. Finally, a tie wire
was fastened around the strut bars to secure the multilayer, plate-type sea cucumber reef system.

Deployment of the new system
Forty new structures were grouped into10 categories
by sheet colours, spacing between corrugated sheets
and shape/styles of the sheets (Table 1). The number

and location of each structure are also listed in
Table1. Depending on the arrangements of the corrugated sheets, each shape/style was classi¢ed as ‘oblique’, ‘parallel’ and ‘wavy’. ‘Oblique’ means that all
sheets were parallel and at an angle of 301 to the base
plate.‘Parallel’ means that all corrugated sheets were
parallel to the base plate. ‘Wavy’ means that every
second sheet was at an angle of 101 and the bottom
sheet was also at an angle of 101 to the base plate.
All 40 structures were placed at depths 41.8 m in
a 467 000 m2 co¡erdam in Haiyang (Yantai Province,

China) between 3 July 2008 and 4 July 2008 (Table1).
All assigned sites had near-identical physical habitat
characteristics. During deployment, the shade nettings in all systems faced south, so that structures
with the ‘oblique’arrangement had more shading.

Experimental design
Experiments were performed more than 8 months
after deployment of the new structures to allow acclimation in the co¡erdam. The ¢rst experiment was
carried out between 18 March 2009 and 4 May
2009 to access which colour was the most attractive
to sea cucumbers among systems BO3, TO3, BLO3,
GO3 and MO3. From the result of ¢rst experiment,
another experiment was carried out between 20 October 2009 and 29 November 2009 to ascertain the
optimum spacing among the BO2, BO3, BO4 and
BO5 categories and shape/style among BO3, BP3
and BW3 (Table 1). All experiments were conducted
during the high mobility phases of sea cucumbers.

Experimental conditions
During deployment of the systems in the co¡erdam,

seawater temperature ranged 24.5^26.0 1C, dissolved

Table 1 Number and location of each structure
Features of polyethylene-corrugated sheets
Colour

Spacing (cm)Ã

Style

Quantity

Site

Number of structures

System abbreviation

Black

2

Oblique

11

4

BO2

Black

3

Oblique

11

4

BO3

Black

4

Oblique

11

4

BO4

Black

5

Oblique

11

4

BO5

Black

3

Parallel

11

4

BP3

Black

3

Wavy

11

4

BW3

Transparent

3

Oblique

11

4

TO3

Blue

3

Oblique

11

4

BLO3

Green

3

Oblique

11

4

GO3

Mixedw

3

Oblique

11

36135 0 1300 N
120157 0 4900 E
36135 0 2000 N
120157 0 5200 E
36135 0 1400 N
120157 0 5000 E
36135 0 1200 N
120157 0 4900 E
36135 0 1400 N
120157 0 5500 E
36135 0 1600 N
120157 0 5500 E
36135 0 2100 N
120157 0 5000 E
36135 0 2000 N
120157 0 5000 E

36135 0 2000 N
120157 0 5300 E
36135 0 1500 N
120157 0 5400 E

4

MO3

ÑSpacing’ indicates the distance between neighbouring sheets.
w‘Mixed’ indicates six transparent sheets in the upper layers and ¢ve black sheets in bottom layers.

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New system for the culture of sea cucumber L Zhang et al.

oxygen ranged 5.00^5.34 mg L À 1, pH ranged 8.09^8.18
and salinity ranged 28.0^28.4%. During the ¢rst and
the second experiments, respectively, seawater temperatures ranged 6.6^18.0 and 3.8^18.8 1C, dissolved
oxygen ranged 4.39^5.89 and 4.29^5.69 mg L À 1, pH
ranged 7.72^8.13 and 7.81^8.05, and salinity ranged
29.3^30.0 and 28.5^30.0%. Over the experimental period, dissolved oxygen of surface seawater was 0.20^
0.60 mg L À 1 higher than that of the bottom.

Sample collection
All sea cucumbers in each experimental structure
were separately collected by a SCUBA diver on 18

March, 9 April, 4 May and 20 October, 10 November
and 29 November 2009. Individuals were weighed
and the numbers on each layer were noted. Although
numbers on every layer were included in the ¢eld
study, for analysis, the upper six sheets and lower ¢ve
sheets plus the base plate were combined to form an
‘upper’and ‘lower layer’category respectively.
As a control, ¢ve samples were collected randomly
on every observation day at a distance from the experimental systems using a 70 Â 70 cm quadrat
(the same area as the new structure) to assess density
of sea cucumbers. All collected animals were replaced near the sampling zones.

Statistical analysis
Di¡erences in numbers of sea cucumbers between
the ‘upper’ and ‘lower layer’ categories were ana-

Aquaculture Research, 2011, 42, 1431^1439

lysed by a paired t-test (SPSS V 13.0). Numbers of
sea cucumbers were compared across each observation day. The three ¢xed factors were colour
of corrugated sheets (C, within subjects repeated
measure factor with ¢ve levels), spacing of corrugated sheets (I, within subjects repeated measure
factor with four levels) and shape/style of corrugated sheets (S, within subjects repeated measure
factor with three levels). ANOVA and pair-wise post
hoc LSD multiple range tests were performed using
SPSS V 13.0 software. Di¡erences were considered to
be signi¢cant at Po0.05.

Results
E¡ect of colour

Numbers of A. japonicus in systems with di¡erent colour corrugated sheets (BO3, TO3, BLO3, GO3 and
MO3) are shown in Fig. 2. Statistical analysis showed
the same pattern on di¡erent observation dates. The
number of animals in BO3,TO3, BLO3, GO3 and MO3
was signi¢cantly higher than the control group
(number of sea cucumber sampled by quadrat outside
the systems; Po0.05). The highest number was found
in BO3 and was signi¢cantly higher than the other
structures or the control group (Po0.05). The lowest
number occurred in the TO3 and was signi¢cantly
lower than the other systems (Po0.05). No signi¢cant di¡erences were found between numbers of animals in TO3, BLO3 or MO3 (P40.05).

Figure 2 Numbers of Apostichopus japonicus versus colour of polyethylene-corrugated sheets. Di¡erent letters indicate
signi¢cant di¡erences (Po0.05) and bars represent standard errors of means.

1434

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Aquaculture Research, 2011, 42, 1431^1439

New system for the culture of sea cucumber L Zhang et al.

ences among sea cucumber numbers in BO3, BW3
and BP3 (Po0.05).

E¡ect of spacing
Numbers of A. japonicus versus spacing of PE-corrugated sheets (BO2, BO3, BO4 and BO5) are presented
in Fig. 3. Statistical analysis showed the same trend

on di¡erent observation dates. Numbers of animals
in BO2, BO3, BO4 and BO5 were signi¢cantly higher
than in the control group. The highest number was
found in system BO2 and was signi¢cantly higher
than in the other structures or control group
(Po0.05). No signi¢cant di¡erences were found between numbers of animals in BO3, BO4 or BO5
(P40.05).

‘Upper’ versus ‘lower layer’ density

Numbers of A. japonicus versus shape/style of PE-corrugated sheets (BO3, BP3 and BW3) are illustrated in
Fig.4. Statistical analysis showed the same pattern on
di¡erent observation dates. Numbers of animals in
BO3, BP3 and BW3 were signi¢cantly higher than
in the control group (Po0.05), showing the relationship BO34BW34BP3. There were signi¢cant di¡er-

Table 2 showed the numbers of A. japonicus in the
upper and lower layers with di¡erent coloured sheets
(BO3,TO3, BLO3, GO3 and MO3). Numbers of sea cucumbers in the upper layers were signi¢cantly higher
than in lower layers for BO3, BLO3 and GO3 on all 3
observation days (Po0.05).
However, in MO3, the number of sea cucumbers
in the upper layers was signi¢cantly lower than in
the lower layers on all 3 observation days (Po0.05).
Furthermore, in TO3, there was no signi¢cant di¡erence in the number of sea cucumbers in upper layers
versus the lower layers on either 18 March or 9 April
(P40.05). In addition, the number of sea cucumbers
in the upper layers was signi¢cantly lower than in
the lower layers of TO3 on 4 May (Po0.05).
Table 3 showed the numbers of A. japonicus in the

upper and lower layers with di¡erent sheet spacing

Figure 3 Numbers of Apostichopus japonicus versus spacing of polyethylene-corrugated sheets. Di¡erent letters
indicate signi¢cant di¡erences (Po0.05) and bars represent standard errors of means.

Figure 4 Numbers of Apostichopus japonicus versus
shape/style of corrugated sheets. Di¡erent letters indicate
signi¢cant di¡erences (Po0.05) and bars represent standard errors of means.

E¡ect of shape/style

Table 2 Numbers of Apostichopus japonicus in di¡erent coloured upper and lower layers
18 March
SystemsÃ

Upper layer

BO3
TO3
BLO3
GO3
MO3

12.8
4.0
9.0
8.5
7.3

Æ

Æ
Æ
Æ
Æ

0.9a
0.4a
0.9a
0.6a
0.9b

9 April
Lower layer
9.8
5.8
6.3
6.5
9.8

Æ
Æ
Æ
Æ
Æ

0.9b
0.6a
0.6b
0.5b
0.5a

4 May

Upper layer
12.8
6.3
9.3
10.0
6.8

Æ
Æ
Æ
Æ
Æ

0.6a
5.9a
0.8a
0.8a
0.9b

Lower layer
10.3
5.0
7.3
8.3
10.5

Æ

Æ
Æ
Æ
Æ

0.8b
0.8a
0.6b
0.9b
0.3a

Upper layer
11.8
4.5
9.8
9.8
7.5

Æ
Æ
Æ
Æ
Æ

1.0a
0.3b
0.9a
0.9a
0.6b

Lower layer
9.8
6.5
7.5
7.0
10.0

Æ
Æ
Æ
Æ
Æ

0.9b
0.3a
0.5b
0.4b
0.7a

ÃValues are mean Æ SEM. Data with di¡erent letters in the same system and observation date indicate signi¢cant di¡erence between

di¡erent coloured upper and lower layers (Po0.05).

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New system for the culture of sea cucumber L Zhang et al.

Aquaculture Research, 2011, 42, 1431^1439

Table 3 Numbers of Apostichopus japonicus in upper and lower layers at di¡erent spacings and shape/style
20 October
SystemsÃ

Upper layer

BO2
BO3
BO4
BO5
BP3
BW3

13.0
11.3
10.8
9.8
5.8
9.5

Æ
Æ
Æ
Æ
Æ
Æ

0.4a

0.5a
0.6a
0.8a
0.9a
0.6a

10 November
Lower layer
9.5
7.8
7.3
7.5
3.8
6.3

Æ
Æ
Æ
Æ
Æ
Æ

0.6b
0.6b
0.5b
0.6b
0.5b
0.6b

Upper layer

13.5
11.5
10.8
10.3
5.8
8.5

Æ
Æ
Æ
Æ
Æ
Æ

0.9a
1.2a
0.6a
0.9a
0.9a
0.5a

29 November
Lower layer
10.0
7.3
8.0
7.8
4.0
5.5

Æ
Æ
Æ
Æ
Æ
Æ

0.4b
0.6b
0.4b
1.0b
0.7b
0.5b

Upper layer
12.5
10.3
10.3
9.5
6.8
8.3

Æ
Æ
Æ
Æ
Æ
Æ

0.6a

0.5a
0.9a
0.3a
0.5a
0.5a

Lower layer
9.3
8.3
7.5
7.8
3.8
5.5

Æ
Æ
Æ
Æ
Æ
Æ

0.6b
0.9b
0.9b
0.5b
0.3b
0.6b

ÃValues are mean Æ SEM. Data with di¡erent letters in the same system and at the observation date indicate signi¢cant di¡erence

among the di¡erent treatments (Po0.05).

(BO2, BO3, BO4 and BO5) and with di¡erent sheet
shape/style (BO3, BP3 and BW3). In all systems and
on all observation days, statistical analysis showed
that the number of sea cucumbers in the upper layers
was signi¢cantly higher than in the lower layers
(Po0.05).

Discussion
Attraction of sea cucumbers to di¡erent
colours
Animals select suitable habitats as protection against
predators and/or competition, better food availability,
etc. (Yan & Chen 1998). This includes matching body
colour with the background colour as a protective device. Zhang, Wang, Rong, Cao and Chen (2009) have
demonstrated that black and grey settlement substrates are more desirable to A. japonicus than red,
white, green or yellow substrates in laboratory experiments. In this study, it showed the similar colour
desirability to A. japonicus in the ¢eld, and A. japonicus tended to settled on the black material, similar in
body colour, of the ¢ve experimental coloured materials (Table 1). Thus, the number of animals in BO3 was
signi¢cantly higher than in TO3, BLO3, GO3 or MO3.
The number of animals in TO3 was lowest, as the
transparent corrugated sheets provided little shade
and hence were less attractive to sea cucumbers.

Attraction of sea cucumbers to di¡erent
spacing
A characteristic of living on rocky reefs (Zhang et al.
1995) is that A. japonicus tend to inhabit crevices and
holes, giving shade and protection against predation.

Apostichopus japonicus cannot tolerate high tempera-

1436

tures. It is a temperate water sea cucumber that is
known to aestivate when water temperatures rises
above a threshold level (Yang, Zhou, Zhang,Yuan, Li,
Liu & Zhang 2006; An, Dong & Dong 2007; Yuan,
Yang, Wang, Zhou, Zhang & Liu 2007; Dong, Dong &
Ji 2008; Ji, Dong & Dong 2008; Wang, Yang, Gabr &
Gao 2008; Yuan,Yang,Wang, Zhou & Gabr 2009) and
the animals conceal themselves in crevices during
aestivation. In this study, the number of sea cucumbers in BO2, with the smallest spacing, was signi¢cantly higher than the other BO3, BO4 and BO5
systems.
Attraction of sea cucumbers to di¡erent
shape/style
Light has a signi¢cant e¡ect on the diurnal rhythm,
migration, grouping behaviour, feeding, etc. of aquatic animals (Zhou, Niu & Li 1999; Chen, Gao, Liu,
Shao & Shi 2007). Migration of newly settled juveniles from sheltered nursery areas to exposed adult
habitats has been demonstrated in the northern sea
cucumber Cucumaria frondosa (Hamel & Mercier
1996). On the other hand, the larvae of Psolus chitonoides were found to settle initially near adults and
then relocate in nearby shaded habitats (Young &
Chia 1982). Young and Chia (1982) and Hamel and
Mercier (1996) found that the main factors regulating
the post-settlement migration of juvenile holothurians was the distribution of shaded substrates and
vulnerability to predatory pressure. Mercier, Battaglene and Hamel (2000) found that early-stage
pentactula larvae of Holothuria scabra showed a
negative phototaxic response and migrated to the
shaded side of the substrate. Zhang, Chen and Sun

(2006) found that when exposure to high light intensity, A. japonicus migrated to the shade of arti¢cial
reefs or onto the arti¢cial reefs.

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Aquaculture Research, 2011, 42, 1431^1439

In this study, varying the angle of the corrugated
sheets gave di¡erent degree of shading. Obviously,
more shade is provided by the oblique than either
the parallel or wavy design. It might be the reason
why the numbers of A. japonicus in the BO3 design
were signi¢cantly higher than in either BW3 or BP3.

Attraction of sea cucumbers to ‘upper’ and
‘lower’ layers
Distribution of aquatic animals has a spatial heterogeneity (Persson & Svensson 2006). In its natural environment, A. japonicus live in crevices in rocky reefs
close to sea grass (Zostera marina) and low light intensity, or a silty bottom with luxuriant meadows of Z.
marina (Zhang et al. 1995). However, when bottom
water is low of dissolved oxygen, sea cucumbers
climb up the rocky reefs. In this co¡erdam utilized in
this study, the level of dissolved oxygen of bottom
water was lower than that of surface water. In addition, as the study was carried out during the period of
high activity for sea cucumbers, the animals could
climb to the ‘upper layers’ within the experimental
structures. Clearly, there was more shade in the ‘lower’ than in the ‘upper’ layer of MO3; thus, sea cucumbers prefer the ‘lower’ layer to settle. In TO3, with
transparent corrugated sheets, all animals were exposed to the sun equally. Consequently, there was no
signi¢cantly di¡erent attraction of sea cucumbers to
the ‘upper’and ‘lower’ layer in 18 March and 19 April.

However, from 18 March, 9 April to 4 May, the sunlight was becoming higher day after day. Although
the system TO3 was assembled with transparency,
the illumination of the ‘lower’ layer was a little lower
than that of the‘upper’ layer. This might be the reason
why the number of sea cucumbers in the‘upper’ layer
was lower than that in the ‘lower’ layer.
The study indicated that the new system provided
sea cucumbers with a safer environment when seawater was low in dissolved oxygen in farming ponds,
especially during the summer or in periods of low
seawater quality.

Conclusion
The study demonstrated that the numbers of sea cucumbers, A. japonicus, in all of the new structural designs investigated was signi¢cantly higher than in
the natural environment. The results indicated that
a design of black, corrugated sheets with a 2 cm spacing and oblique orientation was the most attractive

New system for the culture of sea cucumber L Zhang et al.

for A. japonicus, and most suitable for culture and
stock enhancement in co¡erdams or ponds. The new
multilayer, plate-type sea cucumber reef can increase
the farming/habitat space. Besides, it is not only easily installed and removed, but can also be supplied
with di¡erent coloured sheets, at di¡erent spacings
and angles to match the pond environment and animal behaviour. The new system is less labour intensive and more cost e¡ective, and because it reduces
the frequency of feeding, it will improve water quality
in farming areas and protect benthic communities
and ecosystems.

Acknowledgments
The authors thank Shandong Oriental Ocean Sci-tech

Haiyang Branch for providing facilities for the present
study, and Edanz Writing for help in editing this manuscript. This work was supported by the National Marine
Public Welfare Research Project (No. 200805069), the
National High Technology Research and Development
Program of China (863 Program) (No. 2006AA100304)
and the National Key Project of Scienti¢c and Technical
of China (No. 2006BAD09A02).

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