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transcript
Nutrient removal efficiency of Hydropuntia cornea in
an integrated closed recirculation system with pink
shrimp Farfantepenaeus brasiliensis
Daniel Robledo*, Leonardo Navarro-Angulo, David Valdes Lozano & Yolanda Freile-Pelegr�ın
Departamento de Recursos del Mar, Cinvestav, M�erida, Yucat�an, M�exico
Correspondence: D Robledo, Departamento de Recursos del Mar, Cinvestav, Km 6 carretera Antigua a Progreso C.P. 97133,
Cordemex, M�erida, A.P. 73, Yucat�an, M�exico. E-mail: robledo@mda.cinvestav.mx
Abstract
In this study, we have tested the effect of seaweed
stocking density in an experimental seaweed bio-
filter using the economically important red sea-
weed Hydropuntia cornea integrated with the
cultivation of the pink shrimp Farfantepenaeus bra-
siliensis. Nutrient removal efficiency was evaluated
in relation to seaweed stocking density (2.5, 4, 6
and 8 g fw L�1). Total ammonia nitrogen (TAN)
was the main nitrogen source excreted by F. bra-
siliensis, with concentrations ranging from 41.6
to 65 lM of NH4+-N. H. cornea specific growth
rates ranged from 0.8 � 0.2 to 1.4 � 0.5%
day�1 with lowest growth rates at higher sea-
weed stocking density (8 g fw L�1). Nutrient
removal was positively correlated with the culti-
vation densities in the system. TAN removal
efficiency increased from 61 to 88.5% with
increasing seaweed stocking density. Changes in
the chemical composition of the seaweed were
analysed and correlated with nutrient enrichment
from shrimp effluent. The red seaweed H. cornea
can be cultured and used to remove nutrients
from shrimp effluents in an integrated multi-tro-
phic aquaculture system applied to a closed recir-
culation system. Recirculation through seaweed
biofilters in land-based intensive aquaculture
farms can also be a tool to increase recirculation
practices and establish full recirculation aquacul-
ture systems (RAS) with all their known associ-
ated benefits.
Keywords: biofiltration, Farfantepenaeus brasili-
ensis, Hydropuntia cornea, IMTA, RAS, seaweed,
shrimp
Introduction
The shrimps of the Family Penaeidae are known
around the world as valuable resources for aqua-
culture, but the majority of research and develop-
ment efforts have been directed to few species of
shrimp that dominate world production (FAO
2007). In America, shrimp farming have devel-
oped based mainly on Litopenaeus vannamei
(Boone) and L. stylirostris (Stimpson), two species
that have been subjected to advances in domesti-
cation and to a selective brood stock programme.
Mexico is presently the second largest shrimp pro-
ducer in the western hemisphere with most of the
industries located in the north-west, Gulf of
California and Pacific coast (P�aez-Osuna, Gracia,
Flores-Verdugo, Lyle-Fritch, Alonso-Rodr�ıguez,
Roque & Ruiz-Fern�andez 2003). The shrimp aqua-
culture industry is less developed on the Caribbean
coast of the Gulf of Mexico, in spite of a multi-spe-
cific shrimp fishery decline in the Yucatan Penin-
sula that has historically been an important
source of employment for rural coastal communi-
ties (Gullian, Aramburu, Sanders & Lope 2010). In
this tropical region, five species of shrimp have
been described: Farfantepenaeus duorarum (Burken-
road), F. brasiliensis (Latreille), F. notialis (P�erez
Farfante), F. aztecus (Ives) and L. schmitti (Burken-
road). Interest in F. brasiliensis culture has been
recently examined motivated by the hardiness and
resistance in brood stock capture (Gaxiola, Gal-
lardo & Simoes 2010) and culture management,
as growth rates under culture conditions are
relatively good, and maximum adult size is large
comparable to other penaeid species (Braga, Lopes,
Krummenauer, Poersch & Wasielesky 2011).
© 2012 Blackwell Publishing Ltd 1
Aquaculture Research, 2012, 1–11 doi:10.1111/are.12111
Shrimp farming, however, is generally perceived
as an activity that negatively impacts the environ-
ment. The direct discharge of waste nutrients from
shrimp farms into adjacent waters has raised glo-
bal concerns regarding adverse environmental
impacts from such practices (Naylor, Goldburg,
Primavera, Kautsky, Beveridge, Clay, Folke, Lubch-
enco, Mooney & Troell 2000). At a semi-intensive
shrimp farm in Mexico for each 1822 kg ha�1 of
shrimp harvested, only 35.5% of nitrogen and
6.1% of phosphorus inputs to the ponds are recov-
ered as shrimp biomass (P�aez-Osuna, Guerrero,
Ruiz-Fern�andez & Espinoza-Angulo 1997). In addi-
tion, for intensive shrimp farming, only 18–27%
of nitrogen and 6–11% of carbon applied to the
pond are assimilated (Funge-Smith & Briggs
1998). This causes an obvious wastage of nutri-
ents, of which 24% is retained in the sediments
and 27% is discharged in the water (Briggs & Fun-
ge-Smith 1994). Thus, there is an enormous risk
of eutrophication in water bodies receiving farm
effluents.
Adaptations of Chinese manure-based integrated
multiple species farming principle to the treatment
of intensive marine aquaculture effluents have
recently became one of the main goals in sustain-
able coastal aquaculture. Marine aquaculture sys-
tems integrating fed and extractive organism have
been defined as Integrated Multi-Trophic Aquacul-
ture (IMTA, Chopin, Buschmann, Halling, Troell,
Kautsky, Neori, Kraemer, Zertuche-Gonzalez,
Yarish & Neefus 2001). Most existing IMTA studies
have been developed for fish farms and tested in
temperate waters (see review by Troell, Halling,
Neori, Chopin, Buschmann, Kautsky & Yarish
2003). However, the rapid development of tropical
coastal aquaculture, particularly shrimp farming,
requires the urgent adaptation of such systems
using tropical species. Although previous studies
have shown enhanced growth of seaweeds and
shrimp in co-culture (Shang & Wang 1985; Wei
1990), integrated aquaculture of shrimp and sea-
weed requires relevant research as it is highly site
specific (i.e. latitude, climate) and species specific
(i.e. strains), and must evaluate factors that affect
seaweed growth and nutrient uptake capacity for
extrapolation to commercial scale. Most of the
studies using seaweeds to treat shrimp effluents
used seaweed stocked at discharge channels,
ditches or outflow ponds, with no recirculation
back to the shrimp cultivation system (Nelson,
Glenn, Conn, Moore, Walsh & Akutagawa 2001;
Jones, Preston & Dennison 2002; Marinho-Sori-
ano, Morales & Moreira 2002). Therefore, IMTA
principles applied to recirculation of shrimp efflu-
ent through seaweed biofilters in land-based inten-
sive aquaculture farms can also be a tool to
increase recirculation practices and establish full
recirculation aquaculture systems (RAS) with all
its known associated benefits (Cahill, Hurd & Lok-
man 2010; Robledo & Freile-Pelegr�ın 2011). As a
first step in this direction, we have tested the effect
of seaweed stocking density in an experimental
seaweed biofilter using the economically important
agar-producing red seaweed Hydropuntia cornea
integrated with the cultivation of the pink shrimp
F. brasiliensis. Nutrient removal efficiency in rela-
tion to seaweed stocking density in the recircula-
tion system and seaweed chemical composition
changes were analysed and correlated with nutri-
ent enrichment provided by the integration with
shrimp cultivation.
Material and methods
Experimental system
The experimental IMTA system applied to RAS
consisted of two identical and independent systems
from each other running simultaneously under
the experimental conditions tested in this study
(Fig. 1). One experimental unit consisted of 1-m2
rectangular tank (180 L) for shrimp, sedimenta-
tion tank (120 L) to remove particulate organic
matter, pumping reservoir (120 L) and the sea-
weed biofiltration unit (SBU). The SBU was con-
structed using six 90-L transparent fibreglass
cylinders (algae reactors) supplied with continuous
aeration and water drainage at the bottom. The
air-flow inlet and water drainage outlet of each
cylinder were controlled using a modified plastic-
valve. Three cylinders were used to maintain sea-
weeds, while the rest were used as a control to test
for background total ammonia nitrogen (TAN) vol-
atilization from aerated cylinders. Aeration mixed
the water, thus ensuring oxygenation to shrimp
tank and the representative collection of water
samples from each treatment. Water flow through
each cylinder could be stopped allowing individual
evaluation of water nutrient concentration over
time. Water from the pumping reservoir was
pumped through a PVC pipe (1/2 inch) to the SBU
using a submersible pump. Water flowed back to the
shrimp tank via a 1-inch stand pipe arrangement.
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Seaweed integrated to shrimp closed recirculation D Robledo et al. Aquaculture Research, 2012, 1–11
The recirculation system was operated 24 h at a
water exchange rate of 1 volume day�1 (total flow
through the treatment Q = 510 L day�1). After a
predetermined period (7 days), sedimentation tank
was siphoned out to eliminate particulate matter.
The experimental system was in a temperature
controlled room, which maintained water temper-
ature at 30°C � 0.5; during the entire experimen-
tal period, water pH was maintained at 8.0–8.4
and salinity at 32.5 psu. Light was controlled to
provide a 12-h light and 12-h dark photoperiod at
a light intensity measured outside the SBU of
100 lM photons m�2s�1 that is the saturation
irradiance for photosynthesis reported for H. cornea
(Ordu~na-Rojas, Robledo & Dawes 2002) giving an
available daily light dosage of 43 mol
photons m�2 day�1.
Experimental organisms
Pink shrimp F. brasiliensis juveniles were obtained
from R�ıa Lagartos, Yucat�an M�exico, and stocked
in the experimental tanks for acclimation during
3 weeks, before the experimental trials com-
menced. The average shrimp weight at the begin-
ning of the experiment was 7.0 � 0.67 g
(mean � SD; N = 150). Organisms were stocked
at an initial density of 60 organism m�2, total
biomass per tank averaged 436 � 32 g m�2
simulating intensive shrimp farming density (>50organism m�2). Shrimp were feed daily using com-
mercial pellets (40% protein, Camaronina 40,
Purina®, Ciudad Obregon, Sonora, Mexico.) at a
feeding rate between 5 and 10% of total shrimp
biomass daily in four rations at times 8:00, 12:00,
(a)
(b)
Figure 1 Top (a) and side view (b) of the integrated closed recirculation system. Each experimental unit consisted
of shrimp tank (1 m2), sedimentation tank and a pumping reservoir attached to six 90-L algae reactors: seaweed
biofilter unit (SBU). Units were kept indoors under controlled conditions and were run simultaneously at each one
of the seaweed stocking densities tested. The ‘shrimp effluent’ flowed through the SBU at a seawater exchange rate
of 1 volume day�1.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–11 3
Aquaculture Research, 2012, 1–11 Seaweed integrated to shrimp closed recirculation D Robledo et al.
16:00, 20:00 hours. To calculate the amount
of food, shrimps were weekly measured and
weighted, using an electronic balance OHAUS
(precision � 0.01 g).
The red seaweed H. cornea (homotypic synonym
Gracilaria cornea J. Agardh) was obtained from nat-
ural beds at Dzilam de Bravo, Yucat�an M�exico,
and acclimated for 2 weeks in the SBU. Clean and
healthy vegetative material was used to seed four
stocking densities (2.5, 4, 6 and 8 g fw L�1) in
the integrated closed recirculation system. Each
week, seaweed was weighed with an electronic
balance OHAUS® (precision � 0.01 g) and
adjusted to experimental stocking density. Hydro-
puntia cornea growth was evaluated by calculating
the specific growth rate based on seaweed fresh
weight (SGR% day�1) as: SGR = 100 9 [ln(Wt/
W0)]/t where Wt is the fresh weight biomass after
t day in culture and W0 is the initial fresh weight
biomass.
Water quality measurements
Daily measurements of temperature (mercury ther-
mometer, �0.5°C), salinity (AtagoTM optical
refractometer, Tokyo, Japan), pH and dissolved
oxygen (YSI, Model 556, YSI Incorporated, Yellow
Spring, OH, USA) were taken at the integrated
closed recirculation system. Light intensity was
measured inside the SBU for each stocking density
with an underwater PAR Sensor Spherical Quan-
tum sensor (LI-193SA LI-COR, Lincoln). Water
samples were collected weekly over a month for
each of the SBU at the corresponding stocking
density tested. Nutrient-rich water from the shrimp
tanks entering the SBU, hereafter referred as
‘shrimp effluent’, and biofiltered water from the
outflow of the SBU, hereafter referred as ‘seaweed
effluent’, were collected from each cylinder to ana-
lyse dissolved inorganic nitrogen (DIN) in the form
of nitrite nitrogen (NO2�-N), nitrate nitrogen
(NO3�-N) and total ammonia nitrogen
(TAN = NH3 + NH4+) according to methods
described by Strickland and Parsons (1972). The
ionized (NH4+) and non-ionized (NH3) forms of
ammonia are interrelated through the chemical
reaction equilibrium (NH4+ = NH3 + H+), thus the
relative concentration of both forms of ammonia
relies on pH and water temperature, and was cal-
culated according to Millero (2006).
Water nutrient content was also measured in
the system during evaluation of the daily rhythm
of nutrient excretion by shrimp without and with
seaweeds in the SBU. A diel (24-h cycle) set of
samples were collected at each one of the SBU to
examine day/night nutrient concentrations every
2 h for 24 h. Dissolved inorganic nitrogen was
measured over the period from 8:00 to 20:00
(light period) and from 20:00 to 8:00 (dark per-
iod). Nutrient measurements from ‘shrimp effluent’
at the integrated closed recirculation system with-
out seaweed evaluated the concentration of each
nitrogen compound. Excretion rates of dissolved
inorganic nitrogen in ‘shrimp effluent’ were also
measured under the same conditions with seaweed
stocked in the integrated closed recirculation sys-
tem at the highest stocking density (8 g fw L�1)
tested.
Experimental design
Nitrogen removal efficiency of H. cornea at the SBU
was evaluated at four stocking densities (2.5, 4, 6
and 8 g fw L�1), corresponding to 0.7, 1.1, 1.6
and 2.2 kg seaweed per square meter of shrimp
cultivation area. Each stocking density was evalu-
ated over 4-week period in the experimental sys-
tem. The efficiency of the H. cornea biofilter at
each stocking density was evaluated by measuring
removal efficiency (%) as the missing ratio
between ‘seaweed effluent’ and the ‘shrimp
effluent’ TAN flux and calculated as N-
removal = (N0�Nf/N0) 9 100, where N0 is the
‘shrimp effluent’ TAN concentration and Nf is the
‘seaweed effluent’ TAN concentration measured at
the SBU; removal rate (lM L�1 h�1) was calcu-
lated as the difference between ‘shrimp effluent’
and ‘seaweed effluent’ TAN concentration in rela-
tion to water exchange rate in the system as V = f
(N0�Nf) where f is water flow rate, N0 is the
‘shrimp effluent’ TAN and Nf is the ‘seaweed efflu-
ent’ TAN concentration measured at the SBU; and
biomass uptake rate (lM TAN g fw�1 h�1) was
calculated as TAN uptake in relation to seaweed
stocking density.
Hydropuntia cornea samples collected at the end
of each seaweed stocking density trial were used
to analyse chlorophyll a and phycobiliproteins con-
tent in fresh samples according to Jeffrey and
Humphrey (1975) and Beer and Eshel (1985)
respectively. Another set of samples were oven-
dried at 60°C for 24 h to analyse tissue total
nitrogen (Strickland & Parsons 1972), protein
(Lowry, Rosebrough, Farr & Randall 1951) and
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Seaweed integrated to shrimp closed recirculation D Robledo et al. Aquaculture Research, 2012, 1–11
carbohydrate content (Dubois, Gilles, Hamilton,
Rebers & Smith 1956).
Statistics
Data were tested for normality (Kolmogorov-Smir-
nov, P < 0.05) and homocedasticy (Cochran,
P < 0.05) tests. Experimental stocking densities
(2.5, 4, 6, 8 g fw L�1) were compared using a test
of independent samples (t-student) to evaluate dif-
ferences between densities and time (4 weeks).
The effect of the stocking density with four levels
was analysed using ANOVA followed by a post-hoc
comparison test (Tukey-HSD, P < 0.05). Parson’s
correlation coefficient was used to determine corre-
lation between environmental and biological
parameters of the integrated closed recirculation
system.
Results
Water quality of ‘shrimp effluent’
Daily changes in the concentration of nitrite nitro-
gen (NO2�-N), nitrate nitrogen (NO3
�-N) and
ammonia nitrogen (NH4+-N) from ‘shrimp effluent’
are shown in Fig. 2. Nitrogen concentration (lM)
excreted by F. brasiliensis over the 24-h cycle fluc-
tuated as follows: TAN was the main nitrogen
source excreted in the system accounting for
43.4 � 3.26% of the total DIN. Concentrations
ranged from 41.6 to 65 lM of NH4+-N (mean
53 � 6.0 lM). Nitrite nitrogen corresponded to
35.1 � 3.60% of the total DIN with concentra-
tions ranging from 39.1 to 47.4 lM of NO2�-N
(mean 41.8 � 3.45 lM). Nitrate nitrogen was
found in lowest concentration with mean values of
25.8 � 5.92 lM of NO3�-N accounting for nearly
21% of the DIN in the system. Feeding resulted in
increased total ammonia nitrogen (TAN) concen-
tration during morning feeding (8:00), whereas
nitrate decreased and nitrite increased at the end
of the diel cycle.
Integrated closed recirculation system performance
Table 1 summarizes the variation found in culture
parameters during operation of the integrated
closed recirculation system for each stocking den-
sity. No significant differences in these parameters
were found (P < 0.05). Mean water temperature
was 29.7°C � 0.6, and light intensity inside the
SBU varied according to the seaweed density
between 49.2 � 8.8 and 61.4 � 12.4 lM pho-
tons m�2 s�1. Dissolved oxygen was maintained
at 4.6 � 0.35 mg L�1, water pH at 8.16 � 0.11
and salinity at 32 psu. Specific growth rates of H.
cornea ranged from 0.8 � 0.2 to 1.4 � 0.5%
day�1 with lowest growth rates at higher seaweed
stocking density (8 g fw L�1). The similar stocking
rates and size distribution of F. brasiliensis used in
each experiment throughout the experiment
resulted in comparable excretion of total ammonia
nitrogen across all seaweed stocking densities
tested (Table 1). Final weight of F. brasiliensis was
around 9.77 � 0.92 g corresponding to a specific
growth rate of 0.97 � 0.25% day�1 with a sur-
vival rate of 68%. Nutrient concentration from
shrimp effluent was equivalent to 103.5 � 35 lMDIN comprising of 38.9 � 4.0 lM NH4
+-N,
36.4 � 11.9 lM NO2�-N and 29.0 � 11.6 lM
NO3�-N. Seaweed stocking density had a signifi-
cant negative correlation with ammonia
(r = �0.83; P > 0.05) and DIN (r = �0.93;
P > 0.05) availability, thus higher nitrogen in
‘shrimp effluent’ was available for seaweed growth
under low seaweed stocking density.
Comparison of ‘shrimp effluent’ versus ‘seaweed
effluent’ dissolved inorganic nitrogen concentra-
tions at the integrated closed recirculation system
in relation to seaweed stocking density are shown
in Fig. 3a–D. Dissolved inorganic nitrogen availability
Figure 2 Concentration of major forms of dissolved
inorganic nitrogen (lM) generated from ‘shrimp efflu-
ents’ over 24-h period at the integrated closed recircu-
lation system without seaweed. NH4+-N ammonia
nitrogen (○), NO2�-N nitrite nitrogen (�), and NO3
�-Nnitrate nitrogen (M) concentrations.
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–11 5
Aquaculture Research, 2012, 1–11 Seaweed integrated to shrimp closed recirculation D Robledo et al.
at the recirculation system was influenced by sea-
weed density. In particular, TAN removal effi-
ciency increased from 61 to 88.5% at increasing
seaweed stocking density. At seaweed stocking
densities of 2.5 and 4 g fw L�1 non-ionized form
of ammonia (NH3) represented between 11.6 and
8.5% of TAN, respectively; whereas, at 6 and
8 g fw L�1, NH3 did not exceed 7% of TAN. Con-
centration of nitrite decreased in the ‘seaweed
effluent’ when compared with ‘shrimp effluent’,
although its removal efficiency did not exceed 23%
(Fig. 3). On the contrary, nitrate content increased
at increasing seaweed stocking densities from 38.7
to 1.28% and was not entirely removed from the
system. The only significant differences between N
concentration between ‘shrimp effluent’ and ‘sea-
weed effluent’ were found for TAN (P < 0.05);
therefore, TAN was the preferred source of inorganic
Table 1 Mean � sd of parameters measured over four weeks at the integrated closed recirculation system. Hydropuntia
cornea growth rates, TAN concentration and availability were calculated in relation to seaweed stocking density tested
Seaweed stocking density (g fw L�1)
Parameter 2.5 4 6 8
Temperature (°C) 29.3 � 0.47a 29.4 � 0.50a 30.0 � .49a 30.1 � 0.53a
Light intensity (lM photons m�2 s�1) 49.2 � 6.64a 41.4 � 7.0a 61 � 14.9a 46.3 � 10.8a
pH 8.26 � 0.13a 8.20 � 0.10a 8.11 � 0.01a 8.07 � 0.02a
DO (mg L�1) 4.53 � 0.41a 4.51 � 0.50a 4.59 � 0.27a 4.78 � 0.22a
Specific growth rate (% day�1) 1.36 � 0.46a 1.09 � 0.59a 1.36 � 0.49a 0.76 � 0.19a
TAN0 (lM) 44.3 � 6.7a 36.6 � 16.3a 38.5 � 8.4a 43 � 16a
N-availability (lM gfw�1 day�1) 15.53 � 3.64b 8.67 � 3.70ab 5.88 � 1.31a 5.15 � 1.95a
Biomass production (g m�2) 257 327 570 480
Different letters indicate significant differences between densities (Tukey P < 0.05).
DO, dissolved oxygen; TAN0, initial total ammonia nitrogen.
Figure 3 Efficiency of seaweed biofilter integrated to the closed recirculation system. Changes in the major forms of
dissolved inorganic nitrogen (lM): ammonia nitrogen (NH4+-N), nitrite nitrogen (NO2
�-N) and nitrate nitrogen
(NO3�N) in relation to seaweed stocking density (a) 2.5, (b) 4, (c) 6 and (d) 8 g fw L�1. Dark bars represent ‘shrimp
effluent’ and white bars ‘seaweed effluent’ nitrogen concentrations. Error bars indicate the standard deviation (*sig-nificant difference at P < 0.05).
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–116
Seaweed integrated to shrimp closed recirculation D Robledo et al. Aquaculture Research, 2012, 1–11
nitrogen used in the SBU by H. cornea under experi-
mental conditions. The seaweed biofiltering unit
removed between 27 and 38 lM N L�1 day�1
equivalent to nitrogen removal rates by seaweed bio-
mass between 4.55 and 9.58 lM N g fw�1 day�1.
Total ammonia nitrogen concentration in
‘shrimp effluent’ and removal efficiencies at the
higher seaweed stocking density (8 g fw L�1) over
a 24-h period are shown in Fig. 4. Average TAN
concentration varied between 27.7 and 51.9 lMincreasing during the night period. SBU removal
efficiencies did not exceed 82% at 8 g fw L�1 with
lower efficiencies and higher variability observed
during lower TAN availability (Fig. 4).
Hydropuntia cornea biochemical composition is
shown in Table 2. No significant differences in the
main nitrogen storage compounds were observed
at the different stocking densities (P > 0.05). Only,
protein and carbohydrate had significant changes
in relation to seaweed stocking densities at 4 and
6 g fw L�1 respectively. Chlorophyll a is also an
important reservoir of nitrogen in H. cornea tissue
that increased its content significantly at 4 and
8 g fw L�1 most probably because of reduced light
availability under these stocking densities.
Discussion
The aim of this study was to evaluate removal effi-
ciency of the red seaweed H. cornea in an inte-
grated closed recirculation system for shrimp
cultivation. It was observed that the daily pattern
of nutrient release by F. brasiliensis cultivated at
the integrated closed recirculation system fluctu-
ates in relation to shrimp metabolic activity. Most
of the nitrogen excreted by the shrimp was in the
form of NH4+ (43%) with their highest content
coinciding with feeding at early morning. On this
regard, it is well known that aquatic crustaceans
are ammonotelic, with ammonia making up to 60
–100% of the total excreted nitrogen, and exhibit-
ing higher nocturnal TAN excretion rates (Crear &
Forteath 2002). In the integrated closed recircula-
tion system, major sources of nutrients are shrimp
excretion and shrimp feed; when organic wastes
(unconsummated feed pellets, faeces, etc.) accumu-
lated in the sediment are degraded, ammonia,
nitrate and nitrite are formed (Martin, Veran, Gue-
lorget & Pham 1998). In particular, in penaeid
shrimp, ammonia excretion and oxygen consump-
tion tend to increase with increasing proteins in
the diet (Rosas, Sanchez, D�ıaz, Soto, Gaxiola, Brito,
Baes & Pedroza 1995). The observed fluctuation in
nutrient excreted by F. brasiliensis can be related
to endogenous cycle interactions, physical activity,
digestive processes or their combination.
In intensive aquaculture systems, where water
is re-utilized, biological filters perform nitrification,
oxidizing ammonia to form nitrate nitrogen (Troell
Figure 4 Concentration of TAN (lM) generated from
‘shrimp effluent’ over 24-h period at the integrated
closed recirculation system with seaweed at stocking
density of 8 g fw L�1. TAN availability (�) over 24 h,
and removal efficiency (○) of the SBU. Error bars indi-
cate the standard deviation.
Table 2 Mean � SD of the biochemical composition analyzed in Hydropuntia cornea cultured at each stocking density
at the integrated closed recirculation system
Seaweed stocking density (g fw L�1)
Parameter 2.5 4 6 8
Phycoerythrin (lg g dw�1) 3115 � 513a 2019 � 566a 2899 � 416a 2783 � 586a
Chlorophyll a (lg g dw�1) 17 � 9.5a 92 � 41.1ab 17 � 4.9a 143 � 75b
Total nitrogen (% N dw�1) 1.2 � 0.33a 0.9 � 0.23a 1.0 � 0.167a 0.9 � 0.166a
Protein (%) 7.0 � 0.68a 4.8 � 0.95b 7.1 � 0.53a 6.7 � 0.98a
Carbohydrates (%) 38.4 � 3.74a 44.8 � 4.95b 29.7 � 2.32c 37.8 � 1.76ab
Different letters indicate significant differences between densities (Tukey P < 0.05).
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–11 7
Aquaculture Research, 2012, 1–11 Seaweed integrated to shrimp closed recirculation D Robledo et al.
et al. 2003). In these systems, N becomes also
available in the form of NO3�; however, in recircu-
lation aquaculture systems long-term impacts of
nitrate on penaeid shrimp have been documented
(Kuhn, Drahos, Marsh & Flick 2010). During the
24-h cycle at the integrated closed recirculation
system without seaweeds, only 21% of total inor-
ganic nitrogen was observed in the form of nitrate
(Fig. 2). However, during the evaluation of biofil-
tration system, nitrate concentrations increased
particularly at higher seaweed stocking densities
(Fig. 3), without any adverse effects for cultured
shrimp. On this regard, nitrogen species can be
maintained at low levels if systems are designed
and managed properly.
In organic, semi-intensive and intensive shrimp
ponds, NH3 represents between 6 and 7% of TAN
(Ramos e Silva, Bezerra D�avalos, da Silveira, Stern-
berg, Soares de Souza, Constantino Spyrides & Lu-
cio 2010). In our study, the relative concentration
of NH3 was always low when compared with that
of NH4+. On this regard, volatilization of ammonia
from aerated cylinders in our system was negligi-
ble as in marine systems nitrogen lost through vol-
atilization and/or denitrification is much lower
than in freshwater systems (Hopkins, Hamilton,
Sandifer, Browdy & Stokes 1993).
Several studies have already demonstrated that
it is possible to cultivate economically valuable
seaweeds using wastewaters from intensive and
semi-intensive aquaculture, improving its water
quality and allowing re-circulation or the dis-
charge into the sea (Troell et al. 2003). In the
integrated closed recirculation system tested, it
was observed that at increasing H. cornea stocking
densities, TAN removal rates increased from 61 to
88.5%. There are relatively few studies investigat-
ing the feasibility or application of integrated cul-
tures of seaweed and shrimp (Jones et al. 2002;
Marinho-Soriano et al. 2002), although this
approach has been regarded as promising in recir-
culation aquaculture (Cahill et al. 2010). In our
experiment, direct uptake of nutrients is achieved
through the seaweed biofiltering unit, besides
maintaining proper conditions of dissolved oxygen
and pH by the seaweed photosynthetic process. At
the integrated closed recirculation system, water
quality parameters remained within ranges suit-
able for the growth of penaeid shrimp: dissolved
oxygen was greater than 4 mg L�1 and tempera-
ture was within the optimum for promoting rapid
growth. Best aquaculture practice standards (BAP)
for shrimp farming effluent management indicate
pH range between 6.0 and 9.5, dissolved oxygen
>4 (minimum requirement is 3.0) and TAN
<5 mg L�1 (equivalent to ca. 350 lM) (www.gaal-
liance.org).
The nutritional state of the seaweed influences
the kinetic absorption of nutrients, so initial low
nitrogen tissue content rapidly absorb available
nutrients, and consequently exhibited high growth
during the first few days (Table 1). For assessment
of water quality, the effects of nutrients on sea-
weeds may be more relevant than the instanta-
neous physical concentrations. Particularly,
relevant are the different sources of nitrogen
(NH4+, NO2
�/NO3� or organic forms such as urea)
in the water column, as these forms of N are the
preferred sources for many seaweed species (Neori,
Chopin, Troell, Buschmann, Kraemer, Halling,
Shpigel & Yarish 2004). On this regard, the culti-
vation of H. cornea is promising as these species
are capable of rapid uptake and storage of nitro-
gen (Navarro-Angulo & Robledo 1999; Ordu~na-
Rojas et al. 2002).
In the seaweed biofilter unit, the preference for
NH4+ was evident under all stocking densities, and
thus being reflected in the removal efficiency
(Fig. 3). The choice of cultured animals and biofil-
ters in integrated systems has to be made on the
basis of their nutrient release rates and the clear-
ance rate of each component of the system. In
recent studies, the use of seaweed for the design of
recirculation biofilter systems has been shown to
outperform traditional bacterial biofilms (Cahill
et al. 2010). Our study showed that H. cornea
independently of the growth rates attained (1.3%
day�1) had lower nitrogen fixation rates
(1.54 mg N g dw�1 day�1) in comparison with
those reported for green seaweed (4.2-
13.9 mg N g dw�1 day�1) by Yokoyama and Ish-
ihi (2010). Similar growth rates were also found
for Gracilaria parvispora fertilized by shrimp farm
effluent (Nelson et al. 2001), whereas other Graci-
laria species, including G. lemaneiformis, exhibited
growth below 4% at lower nitrogen availability
(Xu, Fang, Tang, Lin, Le & Liao 2008). Higher
seaweed biomass has been also obtained for G.
caudata (1418 � 708 g m2), which exhibited
higher growth rates at lower shrimp stocking den-
sities under higher nitrogen loads (Marinho-Sori-
ano et al. 2002).
In most of the abovementioned studies, low
water movement and large amount of suspended
© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–118
Seaweed integrated to shrimp closed recirculation D Robledo et al. Aquaculture Research, 2012, 1–11
particles in shrimp effluents contributed to sea-
weed biomass reduction due to deposition of silt
over thalli, which eventually block incident light
thus affecting growth performance in the long
term (Nelson et al. 2001; Jones et al. 2002; Marin-
ho-Soriano et al. 2002). This has been also
observed for G. vermiculophylla used to remove
nutrients from a land-based pilot scale turbot culti-
vation system (Abreu, Pereira, Yarish, Buschmann
& Sousa-Pinto 2011). On this regard, low growth
rates in H. cornea may be attributed most probably
to low light availability under high stocking densi-
ties and nitrogen loads, but not to sediment depo-
sition over thalli. In accordance with the
functional-form model, the higher growth rates
reported for other Gracilaria species studied so far
in these systems could be related to its thin, slen-
der and highly ramified morphologies, whereas H.
cornea is a coarse, cartilaginous and little branched
species, with lower growth rates, but higher agar
yield (Freile-Pelegr�ın 2000). It should be noted
that the suitability of seaweed species for maricul-
ture is determined not only by its production
capacity, but by the yield and the quality of their
agar and/or other interesting compounds.
The metabolic profile of H. cornea grown in this
experiment indicates storage of nutrients in the
form of pigment and protein that can be used dur-
ing nutrient limitation. So, if nitrogen is available
at concentrations such as that other factors
become limiting to growth, they will start to store
these reserves in the form of free amino acids and
pigments. In the seaweed biofilter unit, protein
and carbohydrate contents increased to 7.1% and
44.8% at densities of 6 and 4 g fw L�1 respec-
tively (Table 2). Chlorophyll a content also
increased, particularly at higher stocking densities,
but in this case, most probably influenced by low
light availability (Table 2).
Conclusions
The above result exemplifies how integrated multi-
trophic aquaculture of economically important red
seaweeds could be used as an efficient biological
nutrient removal in closed recirculation systems of
shrimp as an IMTA system applied to RAS. The
red seaweed H. cornea can be cultured and used to
remove nutrient-rich effluents from cultivation of
pink shrimp F. brasiliensis. The integrated growth
of H. cornea can add significant revenue to native
shrimp farming, such as F. brasiliensis, which can
then be harvested and used for agar or/and any
other useful product extraction. The feasibility of
seaweed cultivation at the commercial level in
shrimp farm effluents requires further investiga-
tion. Not only daily fluctuations of nutrient loads
are important but also seasonality will affect signif-
icantly nutrient removal efficiencies by seaweeds.
Acknowledgments
This study was supported by CONACYT
CB-83386. E. Real del Le�on skillfull technical
assistance during nutrient analysis is greatfully
acknowledged. M.L. Zaldivar Romero carried out
seaweed tissue chemical analysis in the Applied
Phycology Laboratory.
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