Received: 31 January 2017 | Revised: 8 June 2017 | Accepted: 11 June 2017

DOI: 10.1111/are.13432

ORIGINAL ARTICLE

Performance of a photo-heterotrophic, hypersaline system for

intensive cultivation of white leg shrimp (Litopenaeus
vannamei) with minimal water replacement in lined ponds
using a stochastic approach

Luis Daniel Moreno-Figueroa
Naranjo-P

| Jose aramo | Alfredo Hern

andez-
Llamas
Andre
| Mayra Vargas-Mendieta | Jose
s Hern

andez-Gurrola |
Humberto Villarreal-Colmenares

BioHelis, Innovation and Technology Park,

Northwest Biological Research Center

cnico
(CIBNOR), Calle Instituto Polite
Nacional # 195, La Paz, Mexico
Correspondence
Humberto Villarreal-Colmenares, BioHelis,
Innovation and Technology Park,
Northwest Biological Research Center

cnico
(CIBNOR), Calle Instituto Polite
Nacional # 195, La Paz, Mexico.
Email: humberto04@cibnor.mx
Funding information
FINNOVA project, Grant/Award Number:
192666
Abstract
The aim of the study was to evaluate the performance of Litopenaeus vannamei in
an intensive photo-heterotrophic hypersaline system with minimal seawater replace-
ment, and establish relationships between parameters of a stochastic production
model and relevant water quality variables. Six experimental 1000 m2 lined ponds
were stocked at a density of 120 shrimp m
2 for a 105-day trial. Salinity increased
from 37 to 45  2 g/L, and the water level was maintained with the weekly
addition of filtered seawater, equivalent to 1.6% per day. The stochastic model
predicted that, at harvest, there is 95% confidence that the system produces
between 12.1 and 14.7 t/ha with a mean final individual weight of 13.1 g and a
mean survival of 84.2%. Sensitivity analyses showed that dissolved oxygen and
individual final weight of shrimp were the main variables influencing yield variance.
Nitrogenous compounds were maintained between optimal cultivation levels
(NH3–NH4+ = 0.73  0.43 mg/L, N–NO2
= 0.09  0.05 mg/L, N–NO3
= 3.22
 0.11 mg/L). Heterotrophic bacteria (6.6  3.4 9 105 CFU/ml) and chlorophyll-a
concentration (108.5  80.2 lg/L) showed a similar development pattern, indicating
a strong relationship between bacteria and microalgae during cultivation. Vibrio
spp. concentrations were low (1.24  1.42 9 103 CFU/ml). It was shown that
the photo-heterotrophic system could be used in hypersaline conditions, typical
of semi-arid regions, to consistently produce between 12.1 and 14.7 t/ha in
15 weeks.

KEYWORDS
hypersaline, intensive cultivation, Litopenaeus vannamei, minimal seawater replacement,
photo-heterotrophic, stochastic modelling

1 | INTRODUCTION technologies that generate more efficient production strategies

(Boyd & Clay, 2002; Chamberlain, 2012; FAO 2006). Most intensive
Biosecurity, environmental and economic concerns in the shrimp systems rely on supplemental feed quality (Tacon, 2002), hetero-
industry have resulted in a concerted effort to develop new trophic floc development (Avnimelech, 2007), and water exchange

Aquaculture Research. 2017;1–11. wileyonlinelibrary.com/journal/are © 2017 John Wiley & Sons Ltd | 1
2 |
reduction (Pruder, Mcalpine-Moss & Tacon, 2001; Sandifer & Hop-
kins, 1994; Shishehchian, 2012). However, in semiarid regions, such
as in Northwest Mexico, high evaporation rates represent a chal-
lenge for a significant reduction in water exchange as salinities
increase.
Several authors have reported no loss of production from
reduced water exchange in such environmental conditions. Martinez-
Cordova, Porchas-Cornejo, Villarreal-Colmenares, Calderon-Perez
and Naranjo-Paramo (1998), for example, reported no negative
effects from seawater exchange reduction in semi-intensive systems
in the semi-arid region of Northwest Mexico. The state of Baja Cali-
fornia Sur is located in the region, and intensive shrimp production
relies on night aeration and daily seawater exchanges of at least

1
20% day (Villarreal et al., 2015). This management is based on set
schedules, rather than on monitoring of BOD5, ammonia or nitrite
levels. Hopkins, Sandifer and Browdy (1995) stressed that rapid
water exchange flushes the phytoplankton population and disrupts
the system’s ability to reach equilibrium. On the other hand, high
solar radiation in the region is more conducive to phototrophic
organism development, making the development of full hetero-
trophic floc systems difficult, even when large amounts of carbon
are added (Burford, Thompson, McIntosh, Bauman & Pearson, 2004).
We have been testing a photo-heterotrophic system (also called
semi-biofloc or green-floc, Huda, Ispinanto, Bahri & Decamp, 2013;
McIntosh, 2001) in semi-arid conditions. Bioflocs can provide a wide
range of nutrients depending on environmental conditions, carbon
source used, total suspended solids level, salinity, stocking density,
phytoplankton and bacteria communities (Emerenciano, Gaxiola &
Cuzon, 2013). The aim is to take advantage of the phototrophic and
heterotrophic processes working in tandem by maintaining active
particle suspension in the water column through continuous aera-
tion. This allows for minimal or no water exchange, leading to the
stabilization of the system, and the effective recycling of nutrients
(Avnimelech, 2012; Huda et al., 2013; Ju, Forster & Dominy, 2009;
Ray, Dillon & Lotz, 2011; Wasielesky, Atwood, Stokes & Browdy,
2006).
Although previous studies report that high salinities negatively
affect growth of shrimp (Perez-Vel
azquez, Gonz
alez-Fe

lix, Jaimes-

rdova & Trujillo-Villalba, 2007; Villarreal,
Bustamante, Mart
ınez-Co
Hernandez-Llamas & Hewitt, 2003), it was expected that the physio-
logical resilience of L. vannamei (Bartlett, Bonilla, Quiros & Takano,
1990; D
ıaz, Re, Sierra & D
ıaz-Iglesias, 2004; Villarreal & Rivera,
1993), would contribute to obtaining adequate growth rates when
using a photo-heterotrophic system in high salinities.

rez, Ruiz-Velazco, Hern
andez-
On the other hand, Estrada-Pe
Llamas, Zavala-Leal and Mart
ınez-C
ardenas (2015); Hernandez-
~ oz (2013); Hern
andez-Llamas
Llamas, Ruiz-Velazco and Gomez-Mun
and Zara
ın-Herzberg (2011) and Ruiz-Velazco, Hern
andez-Llamas,
~oz and Magallon (2010), among others, have used stock
Gomez-Mun
models, to describe growth and survival of cultivated shrimp and its
correlations with water quality variables. Using such stock models
from a stochastic approach allows analysing the information more
completely, compared with conventional statistical tools.
MORENO-FIGUEROA ET AL.
The aim of the study was to evaluate the potential for intensive
shrimp production in hypersaline conditions on a photo-hetero-
trophic system with limited seawater replacement in lined ponds.
We did not find previous reports in the literature of such a study.
Also, we used stochastic and stock models to study the influence of
water quality parameters on growth and survival of shrimp and to
predict variability in yield.
2 | MATERIALS AND METHODS
2.1 | Experimental setting
The trial was conducted at BioHelisâ, the Innovation and Technology
Park, located at the Northwest Biological Research Center in La Paz,
Baja California Sur, Mexico (24°080 32 “N, 110°180 39″ W). Six
1,000 m2 plastic lined ponds (50 9 20 m) and 1.35 m deep were
filled with seawater (37 g/L salinity). Fourteen days before stocking,
ponds were fertilized daily with a commercial mix (Nutrilake-STDâ,
SQM Company, Chile) of nitrate (15%), silicon oxide (3.5%) and
sodium (25%), at a rate of 5 Kg/ha. In addition, a commercial probi-
otic mix (Alibio 2135â and Alibio ACâ Mexico City, Mexico) was
added to each pond (70 L/ha), to promote a heterotrophic microbial
complex, daily the first week and twice per week until the end of
the trial. The probiotic mix contained Lactobacillus sporogenes,
Saccharomyces cerevisiae, Saccharomyces fragilis, Bacillus brevis
(1 9 108 CFU/g), Bacillus subtilis, Bacillus polymyxa, Bacillus
megaterium, Nitrobacter vulgaris (1 9 107 CFU/g). A target of
400,000 total heterotrophic bacteria (THB) (CFU/ml) was expected
(Hern
andez-Gurrola, 2016).
No water exchange was done during the trial. After the first
month, a weekly seawater addition of 1.6% per day per pond was
implemented for evaporation recovery and salinity level mainte-
nance. Aeration was provided by two 2 HP aspirator aerators per
pond (Aire-O2â, Chaska, MN, USA) 24 h per day. A rate equivalent
to 20 HP/ha was used during the first 30 days, increasing the rate
to 40 HP/ha until the end of the trial.
2.2 | Water quality parameters
Temperature (T), dissolved oxygen (DO), pH and salinity (S) were
measured at approximately 70 cm of pond depth using a multipa-
rameter YSI Professional Plus probe (YSI Incorporated, Yellow
Springs, OH, USA) at 8, 12, and 18 h. The dissolved oxygen sensor
was calibrated preparing 8 g of Na2SO3 diluted in 500 ml of distilled
water. After 60 min the sensor was immersed in the solution for
0 mg/L calibration. After rinsing, the second point was completed in
water saturated air. The salinity-conductivity sensor was calibrated
using 2 YSI standards (1,000 lS/cm and 50,000 lS/cm conductivity
solutions), and calibration for the pH sensor was accomplished with
pH7 and pH10 YSI provided buffers. Once per week, dissolved
ammonia, nitrate, nitrite, phosphate and alkalinity were measured
following methodologies described by the manufacturer using an
optical photometer YSI 9500 (YSI Incorporated, Yellow Springs, OH,
MORENO-FIGUEROA ET AL.
USA) (1% precision). Ammonia determination is based on the
indophenol method, where ammonia reacts with alkaline salicylate in
the presence of chlorine to form a green-blue indophenol complex.
Equipment calibration for ammonia used a stock solution with
3.82 g of NHCl4, dried at 105°C, in ammonia-free reagent water,
and diluted to 1 L, where 1 ml = 1 mg NH3–N. Standard solutions
1/10, 1/100 and 1/1000 are prepared by dilution in water and read
at 690 nm. Nitrate is first reduced to nitrite, using a zinc-based solu-
tion. Nitrite is then determined by reaction with sulphanilic acid in
the presence of N-(1-naphthyl)-ethylene diamine to form a reddish
dye. A standard curve was done using dilutions of a solution of
200 lM NaNO2 and read at 540 nm absorbance. Phosphate analysis
is based on the vanadomolybdate method where phosphate reacts
with ammonium molybdate in the presence of ammonium vanadate,
to form the yellow phosphovanadomolybdate. Calibration for a range
between 0 and 35 mg P/L is linear at 470 nm absorbance. For all
nutrient tests, resulting colour intensity is directly proportional to
concentration. The YSIâ photometer is pre-programmed with calibra-
tions for each test parameter. This enables the instrument to select
the appropriate wavelength filter automatically and allows the pho-
todiode response to be converted to a concentration reading. Bio-
logical oxygen demand (BOD5) and total suspended solids (TSS)
analysis were performed weekly following APHA (2005). Chloro-
phyll-a (CHL-a) was measured once per week at approximately
70 cm of pond depth using a Hydrolab data probe DS5X (Hachâ
Company, Loveland, Colorado, USA) (0.1 lg/L precision). The
chlorophyll-a sensor is an in-situ optical fluorometer, where the sam-
ple is irradiated using blue (460 nm) light. Chlorophyll-a absorbs the
blue light energy and fluoresces red (620–715 nm) light. The sensor
directly measures the amount of red light emitted by the chloro-
phyll-a in the water sample and displays as lg/L. Calibration was
done for two points, 0 and 500 lg/L, using a stock solution of
chlorophyll-a (SIGMA C6144) in ethanol, diluted in water. Turbidity
(TB) and sedimentary solids (SS) were measured using a Secchi disk
and an Imhoff cone once per week. Heterotrophic marine and Vibrio
spp. bacteria were determined weekly following APHA (2005). All
water samples were collected approximately at 8 a.m. Water samples
for analysis of bacteria were stored in sterile-plastic bags and kept at
4 °C until analysis. Afterwards, a 1:10 dilution of each sample was
used for inoculation with 100 ll in Petri dishes. The agar media used
was TCBS for Vibrio and 2216 for marine heterotrophic bacteria.
The incubation period was 48 h at 35 °C. Finally, the colonies were
counted and the results were expressed in CFU/ml.
Water samples for DBO5 were obtained from the surface of each
pond and transported to the laboratory for analysis. After 5 days of
incubation at 20 °C, the difference between the initial and final dis-
solved oxygen concentration was calculated. Water samples (50 ml)
for TSS were obtained from the central pond area at 70 cm of depth,
and were filtered using constant weight Whatman 1.2 lm pore diam-
eter filters and placed at 105 °C in an oven for 2 hr. Water samples
for measurement of sedimentary solids (SS) (1 L) were obtained from
the central pond area at 70 cm of depth, placed in an Imhoff cone
and settled for 45 min. Results were expressed in ml/L.
| 3
2.3 | Shrimp source, nursery, and feeds
Litopenaeus vannamei postlarvae (PL 14) were obtained from the
“Gran Mar Larvas” hatchery (La Paz, Baja California Sur, Mexico),
and stocked at 120 PL/m2 in the ponds. Shrimp were fed a
ZieglerTM intensive-35 pellet v-pak (35% crude protein) by
evenly distributing the ration four times per day at 8, 12, 16 and
20 h.
The feed rate was based on shrimp size, estimated weekly from
a 100 shrimp sample, and expected survival. The initial feed rate was
10% of total biomass and progressively decreased to 2% by the end
of the trial (Molina-Poveda & Villarreal-Colmenares, 2008). Final sur-
vival was determined from harvested biomass and final average
weight.
2.4 | Stochastic model
Production data from the grow-out trials was used to calibrate a
stock model for shrimp biomass as a function of time, where bio-
mass was the product of the individual weight of shrimp and the
number of survivors. The equation developed by Ruiz-Velazco et al.
(2010) was used to predict growth of shrimp:
wt ¼ wi þ ðwf
wi Þ½ð1
kt Þ=ð1
kh Þ3 (1)
where, wt is mean individual weight predicted after t time units, wi is
the initial weight, wf is the final weight, k is a growth coefficient, and
h is the number of time units at harvest time.
Survival was calculated as a function of time, using the exponen-
tial equation:
nt ¼ n0 e
zt (2)
where nt is the number of survivors, n0 is the initial population, t is
time and z is the instantaneous mortality rate, which was estimated
as:
z ¼
Lnðnf=n0 Þ=tf (3)
where nf is the final surviving population at harvest time (tf). Nonlin-
ear regression procedures available in Statistica 6.0 (StatSoft, Tulsa,
OK) were used to fit the growth equation; significance was set at
p < .05 for regression ANOVA. Outlier values in the growth datasets
were detected with box plot graphs available in Stata 13.0 software
(StataCorp, College Station, TX, USA) and discarded from the
datasets.
Analyses were performed to determine possible correlations
between production parameters, water quality and nutrients. Rela-
tionships between parameters and variables that resulted signifi-
cantly correlated were established using simple linear regression. The
mean values of the predictor variables recorded during the experi-
ment were used for regression analyses. Statistica 6.0 (StatSoft,
Tulsa, OK, USA) was used for analyses, setting significance at
p < .05.
Two sources of stochastic variability were considered and incor-
porated into the stock model. One corresponds to the parameters of
the biomass model (wf, k, z) that resulted significantly related to
4 |
water quality and nutrients. In these cases, the stochastic parameter
value (QS) was calculated as follows:
QS ¼ a þ b  v þ e
where a and b are regression coefficients, v is any of the water qual-
ity or nutrient variables, and e is the residual error resulting from the
corresponding regression analysis. In cases where a significant rela-
tionship was not found, a probability distribution was directly fitted
to the parameter values.
The second source of stochastic variability corresponded to the
dependent variable wt. In this case, the deterministic value of wt cal-
culated with Equation (1) was modified by adding the residual error
resulting from fitting the equation to the growth data. The “en-
velope” method described in Vose (2001) was used to incorporate
residual error as a stochastic element.
The stock model was programmed in Excel 10.0. Output proba-
bility distribution of shrimp biomass was inferred, using Monte Carlo
simulation. The coefficient of variation (CV = standard deviation/
mean) was used as an index of uncertainty regarding production
(Ayyub, 2014).
Sensitivity analysis was used to analyse the importance of pro-
ductions parameters, water quality variables and nutrient concentra-
tion in determining the variability in total biomass. The coefficient of
multiple linear regressions was used as an indicator of sensitivity,
where a high absolute value of the coefficient indicates high impor-
tance of the variability in biomass production. Procedures in @Risk
6.0 (Palisade, Ithaca, NY, USA) were used for simulation and
sensitivity analysis.
3 | RESULTS
3.1 | Water quality
Weekly variability in temperature, dissolved oxygen, pH, salinity,
ammonia, nitrate, nitrite and chlophyll-a in the lined pond waters
are shown in Figure 1, while averages for, phosphate, total alkalin-
ity, BOD5, TSS, turbidity, heterotrophic bacteria and Vibrio spp.
are presented in Table 1. Table 2 shows final weight, survival,
feed conversion ratio, growth rate and yield for shrimp during the
trial.
3.2 | Stochastic model
Equation (1) fitted adequately to shrimp growth data, and yielded
significant results regarding regression ANOVA (Figure 2). With 95%
confidence, final individual weight was estimated to vary from 12.36
to 14.49 g (mean = 13.10 g) and survival ranged from 78.7% to
89.8% (mean = 84.2%).
Results showed that the growth coefficient (k) and the mortality
rate (z) were negatively correlated with water temperature (r =
.93)
and dissolved oxygen (r =
.85) respectively. The relationships
established for k and z with temperature (T) and dissolved oxygen
(DO) resulted in regression equations are:
MORENO-FIGUEROA ET AL.
k ¼ 1:067
0:0031ðTÞ (5)
z ¼ 0:017
0:0030ðDOÞ (6)
(4)
Results from the Monte Carlo simulation for calculation of bio-
mass yields showed that the coefficient of variation (CV) diminished
as time advanced (Figure 3). The output distribution of biomass after
105 days of cultivation is shown in Figure 4. There is 95% confi-
dence that the cultivation system could produce between 12.1 and
14.7 t/ha (mean = 13.2 t/ha).
Results from sensitivity analyses of shrimp biomass after 45, 75
and 105 days are shown in Table 3. According to the stochastic
model, temperature, final weight of shrimp and dissolved oxygen
were the most important factors influencing the variability in shrimp
production. Final weight tended to gain importance as time
advanced, while mortality rate had medium or low importance
throughout the period of cultivation.
4 | DISCUSSION
Biosecurity and costs are two elements frequently associated with
the need to reduce water exchange. Semi-intensive shrimp cultiva-
tion systems use 5% to 20% water exchange per day (FAO 2006) to
maintain acceptable water quality, mainly because they lack artificial
aeration to support carrying capacity. Despite the use of artificial
aeration, some intensive systems still rely on water exchange to
maintain water quality, needing to remove excess organic matter
developed by the addition of carbon sources in an attempt to
develop heterotrophic systems. While this may improve carrying
capacity (Avnimelech, 2012), Hopkins et al. (1995) indicated that sig-
nificant water exchanges flush phytoplankton and destabilize the
production system. Burford et al. (2004) mention that there could be
a loss of nitrification capacity. Reduced water exchange is thus,
desirable.
Water requirement to produce a kilogram of shrimp is a good
indicator of the efficiency of the cultivation system (Boyd & Clay,
2002). Casillas-Hern
andez, Nolasco-Soria, Garc
ıa-Galano, Carrillo-
Farnes and P
aez-Osuna (2007) reported that 71.5 m3 of water were
needed to produce one kg of shrimp in a 203-day cycle, with a den-
sity of 15 PL m2 in semi-intensive farms in Sonora, Mexico. On the
other hand, Boyd and Clay (2002) reported that an intensive farm in
Belize only required 1 m3 per kg of shrimp in a 139-day cycle, with a
density of 120 org m2 and a mean salinity of 28 g/L. In our work, we
used an average of 2.3 m3 of sea water per kg of shrimp, which is still
significantly low, considering that the higher temperatures, dry cli-
mate and high solar irradiance (791
799 cal cm
2 day
1; INIFAP,
2016), result in an evaporation rate averaging 10 mm of the water
column per day (Villarreal et al., 2016; this study).
Venero et al. (2009) reported that FCR values ranging from 1.2
to 2.5 in intensive shrimp production are considered successful. In
hypersaline conditions, energy expenditure for routine metabolism in
shrimp is expected to increase (Villarreal et al., 2003), so the average
MORENO-FIGUEROA ET AL. | 5

F I G U R E 1 Weekly morning (8 AM) average (standard deviation) of temperature, dissolved oxygen, pH, salinity, ammonia, nitrate, nitrite
and chlophyll-a for lined ponds used for intensive photo-heterotrophic cultivation in hypersaline conditions of Litopenaeus vannamei in Baja
California Sur, Mexico
6 | MORENO-FIGUEROA ET AL.

T A B L E 1 Average (standard deviation) phosphate, total

alkalinity, BOD5, TSS, SS, turbidity, heterotrophic and Vibrio spp.
bacteria for intensive photo-heterotrophic cultivation of shrimp
Litopenaeus vannamei in experimental 1,000 m2 lined ponds
Parameter Value
PO43
(mg/L) 2.9  1.5
Alkalinity (mg/L CaCO3) 193  45
BOD5 (mg/L) 15.8  8.9
TSS (mg/L) 81.1  59.7
SS (ml/L) 5.1  3.5
Turbidity (cm) 30  15
Heterotrophic bacteria (105 CFU/ml) 6.60  3.40
Vibrio spp. (103 CFU/ml) 1.24  1.42
F I G U R E 3 Biomass production of Litopenaeus vannamei
throughout the photo-heterotrophic cultivation period. The dashed
lines indicate 95% confidence intervals. The coefficient of variation
is represented by the line with triangles
T A B L E 2 Average (standard deviation) for final weight, survival,
food conversion ratio, growth rate and yield for shrimp Litopenaeus
vannamei in intensive photo-heterotrophic cultivation in
experimental 1,000 m2 lined ponds for 105 days of cultivation
Parameter Value
Final weight (g) 13.33  0.35
Survival (%) 84.3  1.2
FCR 1.52  0.01
Growth rate (g/week) 0.95  0.05
Yield (t/ha) 13.44  0.23

F I G U R E 4 Output probabilty distribution of biomass production

of Litopenaeus vannamei after 105 days of photoheterotrophic
cultivation in hypersaline conditions. The dashed lines indicate 95%
confidence intervals

result is a self-cleaning system, which limits waste releases to the

F I G U R E 2 Fit of Equation (1) to growth data of Litopenaeus
vannamei shrimp in intensive photo-heterotrophic cultivation ponds.
Curves were fitted separately for data from each replicate
FCR in this trial (1.52) was not unexpected. Shrimp farmers are look-
ing for management methods that allow reducing feed conversion
ratios to limit wastes and costs, lessening the impact on the environ-
ment (Villarreal, 2005). In this regard, the minimal water replacement
and constant mechanical aeration used in the photo-heterotrophic
system, promotes the development of microbial communities (Ray
et al., 2011) that contribute to degrading organic matter and other
wastes (Kumaraguru-Vasagam, Suresh & Chamberlain, 2009).
Together with shrimp consumption of the flocculated mix, the net
environment. Our system does not remove solids from the pond
during cultivation, yet it discharged approximately 13,500 m3 ha
1 cy-
cle
1 of sewage, which is considerably lower than the 38,000 m3
ha
1 cycle
1 reported by Thi, Kroeze, Bush and Mol (2010) for an
intensive shrimp farm in Vietnam, and 208,000 m3 ha
1 cycle
1
reported by Casillas-Hern
andez et al. (2007) for semi-intensive farms
in Northwest Mexico. In our trial, an average of 5.1 ml/L of sedimen-
tary solids (SS) was produced in the ponds; which would only repre-
sent 69 m3 ha
1 of solid wastes per cultivation cycle.
Decamp, Cody, Conquest, Delanoy and Tacon (2003) state that
in culture systems without water exchange, the dynamics of nitro-
gen does not appear to be significantly impacted by the salinity.
Average concentrations of total ammonia nitrogen (0.73 
0.43 mg/L) and nitrite nitrogen (0.09  0.05 mg/L) recorded during
MORENO-FIGUEROA ET AL. | 7

T A B L E 3 Sensitivity analysis of shrimp biomass after 45, 75 and The weekly values for Chlorophyll-a (Figure 1) and heterotrophic
105 days of cultivation of Litopenaeus vannamei, in intensive photo- bacteria show a strong relationship, where microalgae produce organic
heterotrophic lined ponds exudates that heterotrophic bacteria use as substrate (Ohara, Fukam
Time (days) Parameter Regression coefficient & Ishida, 1993), contributing to the optimization of the productive sys-
45 T 0.80 ~ o-Herrera, 2003). Concentration of hetero-
tems (Riquelme & Avendan
k
0.46 trophic bacteria in our study was 6.6  3.4 9 105 CFU/ml, which is
wf 0.38 two orders of magnitude lower than the 4 9 107 CFU/ml reported for
DO 0.13 some biofloc systems (e.g. Burford et al., 2004) that use addition of

z
0.08
75 wf 0.65
T 0.55
DO 0.36
k
0.32
z
0.23
105 wf 0.74
DO 0.57
z
0.36
T, temperature; k, growth coefficient; wf, final weight; DO, dissolved oxy-
gen; z, mortality rate.
the experiment were within the general acceptable levels for grow-
out of L. vannamei when reared in ponds (0.2–2 mg/L and
<0.23 mg/L respectively) (Boyd, 2001, 2002). Low concentration of
ammonia and nitrite have been reported in studies with zero water
exchange systems, as a result of the removal of these compounds
by the microbial complex (Burford et al., 2004; Rajkumar et al.,
2015; Vinatea et al., 2010; Wasielesky et al., 2006). The higher
average concentration of nitrate (3.22  0.11 mg/L), compared to
nitrite (0.09  0.05 mg/L) in this trial suggests efficient nitrification
processes in the cultivation system due to the oxygenation of the
water column (Avnimelech, 2012; Rajkumar et al., 2015). In the
photo-heterotrophic system, lack of addition of a supplemental car-
bon source (e.g. molasses) improves the nitrification process (Zhu &
Chen, 2001), and reduces operational costs by avoiding the
expense of its supplementation (e.g. 1.6 kg of molasses per kg of

nez & Durruti-Lagunes, 2013))
food added per day (Valenzuela-Jime
and later removal of excess solids. Samocha et al. (2007) recom-
mend TSS values of less than 500 mg/L for intensive penaeid
shrimp cultivation. Values for this trial were well below this limit.
Mean Chlorophyll-a concentration (108.5  80.27 lg/L) was
similar to that reported by Burford, Thompson, McIntosh, Bauman
and Pearson (2003) (105–170 lg/L) for an intensive system
(120 org/m2) with salinity ranging from 38 to 42 g/L, but lower than
2
that reported for super-intensive biofloc systems (130 org/m ) with
concentrations ranging from 400 to 550 lg/L for a salinity of 25 g/L
(Rajkumar et al. 2015).
BOD5, increased the first 50 days reaching 30 mg/L. After that,
BOD5 decreased showing the system is consuming excess organic
matter, which is assimilated by the shrimp (Huda et al., 2013), thus
reducing the nutrient release into the environment (McIntosh et al.,
2001).
molasses to effect the C:N ratio that favours bacteria development
(Avnimelech, 2007).
The lack of elevated carbon sources limited the development of
Vibrio spp. in our trial (1.24  1.42 9 103 CFU/ml) (see Eiler,
Gonz
alez-Rey, Allen & Bertilsson, 2007), to levels below those
reported for intensive shrimp farming in Brazil (2.20 9 104 CFU/ml,
Paiva-Maia, Alves-Modesto, Octavio-Brito, Olivera & Vascolcelos-
Gesteira, 2013), and well below reported levels of vibriosis outbreaks
(10 9 103 CFU/ml, Ching, Portal & Salinas, 2014).

n and Hern
andez (2006) reported that L. vannamei
Buckle, Baro
hyperregulates between salinities of 10 and 20 g/L and hyporegu-
lates between 20 and 40 g/L. The isosmotic point when exposed to
constant salinities changed in relation to temperature (20–32 °C)
from 717 to 823 mmol/kg, and the combination of 32 °C and 28 g/
L produced the best growth.
At lower salinities, Bray, Lawrence and Leung-Trujillo (1994)
found that L. vannamei (1.6 g) reared for 35 days exhibited higher
mean final weights (12.36  0.32 g and 12.22  0.18 g) and growth
rates (2.14 and 2.12 g/week) at 5 and 15 g/L, respectively, than at
35 g/L (10.95  0.27 g, 1.86 g/week) and 49 g/L (9.79  0.43 g,
1.63 g/week). Although it has been reported that survival of marine
shrimp reared at low salinities varies and may be influenced by fac-
tors such as the ionic composition of the water (Fraga-Maica, De
Borba, Germano-Martins & Wasielesky, 2014), survival was not sig-
nificantly different among treatments in this trial.
At higher salinities, Perez-Vel
azquez et al. (2007) reported that
final weight (2.84  0.47 g) of L. vannamei was lower at 50 g/L
salinity in comparison to those reared at 35 g/L (3.40  0.33 g).
Survival was the same for both treatments (87.5  27.9%). Similarly
Sui, Ma and Deng (2015) found that shrimp reared at 30 g/L salinity
grew remarkably faster that those reared at 60 g/L, with mean
specific growth rates (SGR) of 5.8  0.4% and 4  0.4% respec-
tively. A higher mean survival rate was observed at 60 g/L
(73  2.8%) than at 30 g/L (61  11.3%). The growth reduction
was associated to the higher total ammonium concentration in the
culture medium and the extra energy consumption for osmoregula-
tion at hypersaline conditions. It has been indicated that at high
salinities, shrimp spends more energy to maintain haemolymph
osmolality and growth rate could be compromised (Buckle et al.,
2006; D
ıaz et al., 2004; Villarreal & Rivera, 1993).
For systems with salinities slightly above the optimum, L. van-
namei shows significant adaptation capabilities. Ayaz, Sumara, Vadher
and Ishakani (2015) reported good survival rates (100%–94.99%) and
relatively similar SGR values ranging from 1.54 to 1.99 in systems
with hypersaline water (35–45 g/L) in L. vannamei postlarvae reared
8 | MORENO-FIGUEROA ET AL.

62 days. Robertson, Lawrence and Castille (1993) reported that L.
vannamei juvenile (5.3 g) reared 42 days achieved 17.5  0.3 g at
46 g/L with a survival of 87%. Bartlett et al. (1990) and Mart
ınez-
Cordova, Vilarreal-Colmenares, Porchas-Cornejo, Naranjo-P
aramo

n-Noriega (1997) concluded that growth was not reduced
and Arago
within the range of 30–45 g/L salinity. It is clear that, within the
optimum temperature range, the tolerance to salinity is wide, and
good growth can be obtained between 25 and 45 g/L (Ponce-
Palafox, Mart
ınez-Palacios & Ross, 1997). The ability of L. vannamei
to regulate changes in osmolality according to cultivation conditions
appears to be significant, as evidenced by a number of reports show-
ing acceptable growth rates at different salinity and densities (see
Table 4).
In our study, the proposed stock model effectively predicted the
dynamics of production of L. vannamei. This was a consequence of
the adequacy of the growth and survival equations, as well as the
statistical models resulting from the regression analysis. According to
the model, with a 95% confidence, total biomass ranges from
12.1 t/ha to 14.7 t/ha, with a mean of 13.2 t/ha, approximating the
average yield value in the database of the experimental trial
(13.4 t/ha).
The relationship between parameters of the stock model and
water quality variables was consistent with reports by other authors
(Bett & Vinatea, 2009; Boyd & Gautier, 2000; Ponce-Palafox et al.,
1997; Zhang, Zhang, Li & Huang, 2006) regarding the influence of
temperature and dissolved oxygen on growth and survival of shrimp.
We found that the growth coefficient (k) was inversely related to
water temperature, while the mortality rate (z) diminished when dis-
solved oxygen was high. In Equation (1), a low value of k indicates
early fast growth and our results showed a positive influence of
higher temperatures for early growth of shrimp. As the cultivation
period advanced, however, this early positive impact of temperature
tended to diminish and the relevance of the variability in weight gain
and dissolved oxygen increased. By the end of the trial, variation in
yields was mainly explained by the random variability in the final
weight of shrimp observed among the ponds, and by the differences
in surviving populations resulting from the influence of dissolved
oxygen on the mortality rate.
We also found that the mortality rate (z) was negatively related
to dissolved oxygen, indicating a negative effect of low dissolved
oxygen on shrimp survival as reported by Allan and Maguire (1991),
Jiang, Pan and Bo (2005) and Hu, Luqing and Futao (2008).
Results from the model in our case, showed that, as time
advances, the coefficient of variation diminished, indicating that
there is more certainty in predicting higher yields. Sensitivity analy-
sis indicated the mortality rate (z) had low influence on shrimp pro-
duction, as a consequence of the relatively stable high survival in
our study, which was better than the reported maximum survival
levels (70%–80%) for semi-intensive farms in Mexico (SAGARPA,
2009).
We conclude that, the use a hypersaline system with 37–45 g/L,
with minimal seawater replacement, produces final weight, survival,
yield and FCR, comparable to those obtained in marine or brackish
systems for cultivation of shrimp. Maintaining a good water quality
by recycling nutrients from the photo-heterotrophic complex con-
tributed to obtaining promissory results.
Growing shrimp to a marketable size in fifteen weeks, as was the
case in this study, suggests that two crops of shrimp could be pro-
duced annually which is an economically attractive option for shrimp
producers in Northwestern Mexico. The results of our study are

T A B L E 4 Stocking density (SD), cultivation period (CP), salinity, initial weight (wi), growth rate (GR), yield and averages (standard deviation)
of survival, final weight (wf), and specific growth rate (SGR), for selected reports of Litopenaeus vannamei grow-out
a
CP Salinity GR Yield Survival
SD (PL/m) (days) g/L) wi (g) (g/week) (t/ha) (%) wf (g) b
SGR (%) References
30 42 46 5.3 2.03 4.57 87  5 17.5  0.9 29.5  2.4 Robertson et al. (1993)
14 35 35 1.6 1.86 1.17 77  6 10.9  0.3 26.8  0.7 Bray, Lawrence and Leung-Trujillo (1994)
49 1.64 1.07 78  4 9.8  0.4 23.3  0.5
20 98 43 0.01 1.05 1.61 55  6 14.7  0.8 14.9  0.3 Mart
ınez-Cordova et al. (1997)
15 203 35 0.001 1.06 4.03 87  2 30.9  2.0 15.2  0.7 Casillas-Hern
andez et al. (2007)
56 32 35 0.36 0.66 1.67 88  26 3.40  0.3 9.5  0.3 Perez-Vel
azquez et al. (2007)
50 0.54 1.39 88  30 2.84  0.5 7.7  0.4
95 63 35 0.03 0.12 1.09 100  0 1.15  0.0 1.8  0.1 Ayaz et al. (2015)
40 0.02 0.14 1.17 96  1 1.29  0.1 2.0  0.1
45 0.02 0.11 0.91 95  2 1.01  0.0 1.5  0.1
50 0.03 0.11 0.69 74  8 0.98  0.0 1.5  0.2
150 40 30 0.09 0.38 2.08 61  8 2.28 5.8  0.3 Sui et al. (2015)
60 0.26 1.70 73  2 1.56 4.0  0.3
120 105 45 0.01 0.89 13.44 84  1 13.3  0.4 12.7  0.2 This study
a
Yield was calculated from stocking density, survival, and final weight data.
SGR (specific growth rate) = (final weight
initial weight/cultivation period) 9 100.
b
MORENO-FIGUEROA ET AL.
representative of the conditions in La Paz, BCS, Mexico. In general,
our stochastic modelling approach could also be used on shrimp
farms located in other semi-arid regions, once it is adjusted and cali-
brated for the specific conditions of the farms.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of Jesus Aguilar of CIB-
NOR and the critical comments of an anonymous reviewer. This
work was funded by the FINNOVA project #192666 awarded to Dr.
Humberto Villarreal. Luis D. Moreno-Figueroa is recipient of a stu-
dent fellowship from Consejo Nacional de Ciencia y Tecnolog
ıa of
Mexico (CONACYT).
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How to cite this article: Moreno-Figueroa LD, Naranjo-
P
aramo J, Hern
andez-Llamas A, Vargas-Mendieta M,
Hern
andez-Gurrola JA, Villarreal-Colmenares H. Performance
of a photo-heterotrophic, hypersaline system for intensive
cultivation of white leg shrimp (Litopenaeus vannamei) with
minimal water replacement in lined ponds using a stochastic
approach. Aquac Res. 2017;1–11. https://doi.org/10.1111/
are.13432