Nutrient removal efficiency of Hydropuntia cornea in an integrated closed recirculation system with...

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: [email protected]

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.

© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–112

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



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.



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).



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).



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



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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