Tratamiento de lodos con melaza en cultivo de camarón

1. Aquacultural Engineering 76 (2017) 1–8
Contents lists available at ScienceDirect
Aquacultural Engineering
journal homepage: www.elsevier.com/locate/aqua-online
Nutrient discharge, sludge quantity and characteristics in biofloc
shrimp culture using two methods of carbohydrate fertilization
Rafael Arantesa,∗
, Rodrigo Schveitzerb
, Walter Quadros Seifferta
, Katt Regina Lapaa
,
Luis Vinateaa
a
Department of Aquaculture, Federal University at Santa Catarina (UFSC), Laboratório de Camarões Marinhos, Florianópolis, SC CEP 88062-601, Brazil
b
Departmentof Marine Sciences, Federal University at São Paulo (UNIFESP), Avenida Saldanha da Gama, 89, Ponta da Praia, Santos, São Paulo CEP
11030-400, Brazil
a r t i c l e i n f o
Article history:
Received 3 February 2016
Received in revised form 4 August 2016
Accepted 15 November 2016
Available online 16 November 2016
Keywords:
BFT
Litopenaeus vannamei
Effluent
Sludge
Water use
a b s t r a c t
The aim of this study was to evaluate the effect of two methodologies of carbohydrate fertilization on the
volume and characteristics of effluent from intensive biofloc shrimp cultivation. Six fiberglass circular
tanks (50 m2
each) were divided into two treatments. In the treatment called continuous (CONT), the
tanks received daily molasses fertilization throughout the entire rearing period. In the treatment named
initial (INI), molasses was used only in the early weeks of cultivation. Juvenile Litopenaeus vannamei
(0.87 ± 0.10 g) were stocked at a density of 180 animals m−2
and cultured during 12 weeks until they
reached an average weight of 12 g. The tanks were operated with no water exchange and the total sus-
pended solids concentration were kept between 300 and 400 mg L−1
using settling chambers. The sludge
produced and the wastewater at harvest were quantified and their characteristics were determined. The
production of TSS in the CONT treatment was higher (0.25 kg of solids per kg of applied feed) than in
the INI treatment (0.16 kg kg−1
) (P < 0.05). The analysis of the sludge revealed a high amount of volatile
solids in both treatments, between 636 and 702 g kg−1
. However, due to the elevated sludge nitrogen
content, the carbon to nitrogen (C:N) ratio was low, with values of 6.4 ± 1.4 and 7.5 ± 1.6 for INI and
CONT respectively. The BOD:TSS ratio was also low in both treatments, but the INI showed lower values
(10.3 ± 0.6%) than the CONT (14.9 ± 0.0%) (P < 0.05). Both fertilization strategies were able to modify the
characteristics of sludge produced during cultivation. Moreover, the high nitrogen and sulfate content of
the sludge in both treatments indicated that it may be difficult to use an anaerobic digestion process to
treat sludge. In the INI treatment tanks, the sludge is partially stabilized, while in the CONT there was a
greater need for stabilization.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
Biofloc technology culture systems (BFT) allow intensive shrimp
production using low water quantities for cultivation (Boyd and
Clay, 2002; Browdy et al., 2001). Advantages of this technique
include reduced risk of pathogen introduction and low volume of
waste generated per kg of shrimp produced (Browdy et al., 2012;
Avnimelech, 2009).
The management practices used in BFT assure controlling tank
water quality without exchanging water (Browdy et al., 2001;
∗ Corresponding author at: Federal University at Santa Catarina (UFSC), Labora-
torio de Camarões Marinhos, Beco dos Coroas, CEP: 88061-600, Florianopolis, Santa
Catarina, Brazil.
E-mail address: arantesr75@gmail.com (R. Arantes).
Hopkins et al., 1993). In BFT systems, total ammonia nitrogen is
controlled by increasing the substrate’s carbon to nitrogen (C:N)
ratio (Avnimelech, 1999). The use of a carbon rich fertilizer in
association with the feed promotes ammonia nitrogen uptake by
the heterotrophic bacterial biomass (Crab et al., 2007). Sugarcane
molasses is a fertilizer that can be used for this purpose (Samocha
et al., 2007).
Among different water fertilization practices, it would be pos-
sible to use molasses only during the initial weeks of the rearing
period in order to reduce ammonia nitrogen concentrations until
the stablishment of a sufficiently large bacterial population that
realizes nitrification (Samocha et al., 2007; Ray and Lotz, 2014).
Thereafter, molasses input can be reduced and ammonia nitro-
gen can be converted into nitrate, which is a less toxic compound
(Cohen et al., 2005). Another fertilization practice is the contin-
uous use of molasses throughout the entire rearing period (Ray
http://dx.doi.org/10.1016/j.aquaeng.2016.11.002
0144-8609/© 2016 Elsevier B.V. All rights reserved.

2. 2 R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8
et al., 2011). In this case, an improved retention of nitrogen in the
heterotrophic bacterial biomass can be expected to keep nitrogen
available to shrimp as a natural food source in the form of bioflocs
(Gao et al., 2012). In addition, culture tanks that are continuously
enriched with an organic carbon source can have a lower rate of
nitrate formation, resulting in a less concentrated final effluent
(Ray and Lotz, 2014). On the other hand, using an additional car-
bon source a higher biofloc production rate is expected (Schveitzer
et al., 2013). One way to remove the excess biofloc concentration
produced is to use settling chambers (Ray et al., 2010). Although,
this practice produces sludge, an organic rich effluent (Fontenot
et al., 2007; Ray et al., 2011; Schveitzer et al., 2013).
Besides nitrate, other types of environmentally harmful com-
pounds such as total suspended solids (TSS) and biochemical
oxygen demand (BOD) can accumulate in the water during cultiva-
tion and reach concentrations above values permitted for effluent
discharge (Boyd and Clay, 2002). The use of different molasses
fertilization protocols could alter the concentration of these com-
pounds in tank culture water or in the sludge and there are few
studies that evaluate the amount and characteristics of these efflu-
ents produced in BFT culture under these different condittions. This
information can be important for the establishment of effluent con-
trol strategies, seeking to avoid excessive nutrient discharge in the
effluent (Boyd and Clay, 2002).
As observed for the final effluent discharged from harvest water,
the sludge produced must also be properly managed (Hopkins et al.,
1993; Sharrer et al., 2010). The sludge can be disposed of directly to
a drying bed or previously treated, if necessary (Chen et al., 1997;
Mirzoyan et al., 2012). In both cases, it is more economical if the
sludge stream is as concentrated as possible (Chen et al., 1997). In
case of direct disposal, the sludge should have a high concentration
of solids, between 50 and 100 g L−1, to have a reduced volume of
material to be handled (Sharrer et al., 2010; Couturier et al., 2008;
Chen et al., 1997). On the other hand, if the sludge must be treated
before disposal, the process used will depend on chemical char-
acteristics of the sludge (Mirzoyan et al., 2012). Among different
treatment methods, the anaerobic digestion process can be used
for the treatment of sludge with a high organic matter content, as
that found in aquaculture (Gebauer, 2004; Gebauer and Eikebrokk,
2006; Mirzoyan et al., 2008). The suitability of this sludge for anaer-
obic treatment option for example, depends on the sludge‘s carbon
to nitrogen ratio (C:N) and the chemical oxygen demand to sul-
fate ratio (COD:SO4−2) (Mirzoyan and Gross, 2013; Mirzoyan et al.,
2008).
In biofloc technology, the use of a supplementary carbon source
in the culture tank water can result in differences of biofloc com-
position and energy content (Crab et al., 2009a). Consequently,
it is expected that by manipulating the C:N ratio of the organic
matter that enters the tanks, the sludge can be improved in qual-
ity, thus allowing a better performance for its use in an anaerobic
treatment processes. This study aims to identify whether the con-
tinuous application of molasses throughout the entire cultivation
period can improve biofloc shrimp culture performance and change
effluent characteristics and quantities of the sludge and final efflu-
ent when compared to a management practice that uses molasses
applications only at the beginning of the culture period.
2. Material and methods
2.1. Shrimp source and nursery
The study was initiated using ten-day-old post-larvae (PL10)
of Litopenaeus vannamei with initial weight of 0.024 g. The post-
larvae were obtained from a commercial hatchery (Aquatec LTDA,
in Brazil), and cultured in a round eight-meter diameter nursery
tank, filled with 43 m3 of filtered natural seawater at a density
of 2200 PL m−2. To stimulate heterotrophic bacterial production,
dried molasses with 69% of carbohydrate content was added daily
to maintain a carbon to nitrogen (C:N) ratio of 12.2 and control
ammonia-nitrogen build-up (Avnimelech, 1999). The C:N ratio was
calculated according to Avnimelech (2009), and using 43.5% of
carbon in the feed; 36.6% of carbon, and 3.0% of protein in the
molasses. The shrimp were cultured for 33 days, and then grad-
ually acclimated from 34 to 21 g L−1 salinity according to Roy et al.
(2010) using tap water filtered at 25 ␮m. After 3 days at a salinity
of 21 g L−1, the shrimp were stocked in experimental tanks.
2.2. Experimental units, experimental design and system
management
The units consisted of six 50 m2 circular fiberglass tanks (water
volume of 43.5 m3). A 7.4 HP regenerative blower was used to aerate
all six tanks, and a central aeration ring, 50 cm in diameter, made
of PVC pipe (32 mm) perforated with 1.0 mm holes was provided to
maintain the solids in suspension and to increase dissolved oxygen
levels. Another circular PVC pipe placed at the periphery of the
tank was equipped with 15 airlift pumps (100 mm in diameter) to
provide water aeration and movement in a circular pattern. Each of
the tanks had a transparent PVC liner cover and a 70% shade cloth
to reduce light intensity.
The six tanks were divided into two treatments, three repli-
cates each. Initially, each experimental unit was supplied with full
strength seawater mixed with fresh water to achieve a salinity con-
centration of 21 g L−1. The tanks were stocked with juvenile Pacific
white shrimp with an initial weight of 0.87 ± 0.10 g at a stocking
density of 180 m−2. Shrimp were fed three times per day with a
35% crude protein (CP) commercial diet (Guabi, Brazil). Two feed-
ing trays received a total of 50% of the feed applied to each tank,
and were used to check the amount of feed consumed after 1.5 h
to avoid overfeeding (Casillas-Hernandez et al., 2006). The shrimp
were cultured for 12 weeks until those in the fast growth treatment
reached an average size of 12 g.
The two treatments were used to evaluate the effect of different
molasses fertilization methods on the volume of effluents produced
and their characteristics. The treatment named continuous (CONT)
received daily sugarcane molasses additions throughout the entire
rearing period. Molasses was used to stimulate the growth of het-
erotrophic bacteria and was calculated to immobilize 50% of the
nitrogen added from feed, using the ratio of 20 g of carbohydrates
per gram of TAN (Avnimelech, 1999). This treatment was designed
to keep TAN levels close to zero, and whenever TAN concentrations
rose above this, an additional input of dried molasses was added
to reduce ammonia nitrogen. In the other treatment, called initial
(INI), molasses was used only during the initial weeks to avoid TAN
build up until the nitrification process was complete (i.e., the nitrite
peak began to drop). In order to allow an increase of nitrifying
bacterial biomass the percentage of molasses input was reduced
gradually, until it was terminated at the 6th week. This reduction
was made taking into consideration that TAN concentrations could
not exceed 0.5 mg L−1, and whenever TAN concentrations increased
above this value, an additional input of dried molasses was used to
momentarily increase the C:N ratio and avoid higher TAN concen-
trations. In both treatments, the organic fertilization was divided
and applied to the tanks three times a day, 1.5 h after feeding, to
prevent a drop in dissolved oxygen concentration.
The concentration of total suspended solids (TSS) of each tank
was controlled through the use of one radial-flow settling tank,
0.61 m in diameter or 0.10 m3. These settling tanks were adapted
from Davidson and Summerfelt (2005) and Johnson and Chen
(2006), and were operated continuously to maintain the TSS con-

3. R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8 3
centration between 300 and 400 mg L−1. The flow rate of the
settling chambers was adjusted according to the feeding input and
monitored twice daily to prevent abrupt changes in TSS concen-
tration (Arantes et al., 2016). Tank water volume and the amount
of sludge removed were monitored daily as described in item 2.4.
Brackish water additions were used to replace the sludge removed,
and freshwater was added to replace evaporative loss, its volume
was monitored and used to calculate the evaporation rate.
2.3. Water quality monitoring
Dissolved oxygen and water temperature (YSI 550A, USA) were
measured twice a day, salinity (YSI 30 salinity meter) was measured
daily, and pH (YSI model100 pHmeter) twice a week. The alkalinity
of the water based on CaCO3 (APHA, 1998 – 2320B), total suspended
solids (TSS) (APHA, 1998 – 2540D) and volatile suspended solids
(VSS) (APHA, 1998 – 2540E) were analyzed weekly using 0.6 ␮m
fiberglass microfilters (GF-6 Macherey-Nagel). The settleable solids
volume (SS) was monitored daily with an Imhoff cone (APHA, 1998)
using a 15-min settling time as described by Avnimelech, (2007).
Total ammonia nitrogen (TAN), nitrite (NO2-N), nitrate (NO3-
N) and sulfate (SO4
2−) were analyzed weekly according to
Strickland and Parsons (1972) and APHA (1998). Total nitrogen
(TN) was determined weekly using the methodology proposed
by Valderrama (1981) with recommendations from Burford et al.
(2003). These recommendations consisted of two 35-min oxida-
tion steps, with samples being removed and stirred to dissolve the
hydroxide precipitates.
2.4. Effluent production and characteristics
The amount of water used per tank was calculated by adding the
volume of sludge produced during cultivation, the amount of water
used to replace evaporative losses and the total water discharged
from the tanks at harvest (the final effluent). To determine the vol-
ume of sludge produced, each day the supernatant water of each
settling tank was pumped back to the culture tank and the retained
solids were removed from the base of the cone. The sludge volume
produced daily was determined using a graduated 40-l bucket.
Sludge samples were taken daily to analyze pH and DO, TSS and
VSS; and the total solids (TS) and volatile solids (VS) were analyzed
twice a week (APHA, 1998). To analyze the alkalinity, TAN, NO2-
N, NO3-N and SO4
2− of the sludge, aliquots were centrifuged at
7000 rpm for 20 min and the supernatant was filtered with a 0.6
micrometer glass-fiber filter. Raw samples (in duplicate) were used
to analyze the total nitrogen (Valderrama, 1981). The sludge’s five-
day biochemical oxygen demand (BOD5) was analyzed in triplicate
samples and determined weekly according to APHA (1998) using
the WTW OxiTop system.
The total amount of TSS produced per treatment was calculated
using the increase in the mass of TSS in the tank water, added to
the mass of TSS removed from the settling tank. The amount of TSS
produced per kg of feed applied to each tank was calculated by plot-
ting the cumulative amount of TSS produced and the cumulative
amount of feed supplied to the tanks.
The organic content of the sludge was determined by the
amount of volatile solids, and total organic carbon (TOC) was cal-
culated according to Boyd (1995) and Mirzoyan et al. (2008). Based
on the understanding that all the organic carbon is oxidized to
CO2 in the furnace, the chemical oxygen demand (COD) was calcu-
lated by the following equation: COD (g kg−1) = [molecular weight
of oxygen (32 g)/molecular weight carbon (12 g)] × TOC (g kg−1).
This estimate was used because of the low accuracy associated with
the COD analysis in brackish and saline solutions (Mirzoyan et al.,
2008). To determine final effluent characteristics, water samples
(in triplicate) were taken during harvest and presented as mean
values.
2.5. Shrimp monitoring
Weight gain was monitored weekly by weighing three groups
of at least 80 shrimp per tank using a digital scale. The final weight
(g), survival (%), growth rate (g week−1) and final biomass (kg m−3)
were recorded at the end of the experiment and used to assess the
tank performance. The feed conversion ratio (FCR) was estimated as
the total dry weight of the feed supplied/shrimp wet weight gain.
Due to shrimp mortality observed in the CONT treatment tanks,
bacteriological analyses were performed on shrimp from one of the
tanks of this treatment. The hemolymph was collected by insert-
ing a 21G needle (precooled to 4 ◦C to prevent clotting) with a 1 ml
syringe into shrimp ventral sinus. Samples of hemolymph (10 ␮l)
were spread on TCBS agar (Thiosulfate Bile Salt Sucrose, Oxoid)
under sterile conditions to check for the presence of Vibrio spp.
The total number of colony forming units (CFU) was analyzed after
24 h of incubation at 30 ◦C.
2.6. Statistical analysis
Culture performance data, effluent volume and effluent char-
acteristics were analyzed using one-way ANOVA. Mean weight
(g), feed intake (kg week−1), tank water quality,and sludge mass
data were compared using one-way ANOVA with repeated mea-
sures. The treatments were considered the main factor and the
weeks of cultivation the additional factor (Gomez and Gomez,
1984). Significant differences were analyzed using Tukey’s test at a
5% significance level. Normality and homoscedasticity were tested
using Shapiro-Wilk (Zar, 1999) and Bartlett (Gomez and Gomez,
1984) tests, respectively. Percentage data were analyzed using data
transformed into arc-sine (y0.5); variables without homogeneous
variances were transformed to log (x + 1). Statistical analyses were
conducted using the STATISTICA Version 8, (StatSoft South America
– Brazil) and the results were presented as means ± SD (standard
deviation). Data for suspended solids production during cultiva-
tion were obtained and compared by linear regression analysis of
covariance, using ANCOVA (Zar, 1999).
3. Results
3.1. Water quality in the culture tanks
The chemical and physical variables of the water were not
statistically different between the treatments (P > 0.05). The aver-
age temperature in all tanks was 28.9 ± 0.3 ◦C ranging from 25.8
to 30.5 ◦C. The dissolved oxygen concentration was 5.4 ± 0.5 and
5.2 ± 0.6 mg L−1, pH 7.5 ± 0.1 and 7.6 ± 0.0, and salinity 21.7 ± 0.3
and 21.6 ± 0.2 respectively for the INI and CONT treatments.
The carbon to nitrogen ratio (C:N) of the organic matter added
to the tanks (feed + molasses) had average values of 8.7 ± 0.1 in
the INI treatment and 11.7 ± 0.1 in the CONT. Starting from the
3rd week of culture, the C:N ratio was significantly lower in the
INI treatment, and after the 6th week of culture, the organic car-
bon input was interrupted in tanks of this treatment (Fig. 1). The
concentrations of TAN, NO2-N and NO3-N had similar variations
in both treatments and did not demonstrate significant differences
between them (P > 0.05).
TSS increased until the 4th week of culture. After this, the set-
tling tanks were operated continuously and the TSS concentration
was maintained from 300 to 400 mg L−1 until the end of the rear-
ing period. The average values were 322.3 ± 27.4 mg L−1 for the INI
treatment and 325.9 ± 13.9 mg L−1 for the CONT treatment.

4. 4 R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8
Fig. 1. Carbon to nitrogen ratio C:N (a), total ammonia nitrogen TAN (b), nitrite (c),
nitrate (d), during 12 weeks of culture using two methods of sugarcane molasses
additions: initial (INI) and continuous (CONT).
3.2. Shrimp production
Shrimp from the INI treatment had a higher survival rate and
weight gain than those in the CONT treatment (Table 1). The feed
conversion ratio was lower and the final biomass harvested was
higher in the INI treatment.
3.3. Volume of effluent produced
The amount of water used during cultivation was similar in
both treatments (Table 2). Most of the effluent produced was from
the water discharged from the tanks during harvest (final efflu-
ent) (>80%), and no significant differences were observed between
the treatments. Sludge drained from the settling tanks represented
2.2% of the total water consumed in the INI treatment, which was
statistically lower than the 3.4% produced in the CONT (P < 0.05).
Table 1
Shrimp production in tanks with L. vannamei cultured using two molasses fertiliza-
tion strategies: Initial (INI) and continuous (CONT). Initial shrimp stocking density
180 m−2
, 78 days of culture with an initial weight of 0.84 g.
Parameter Treatment1,2
INI CONT
Final average weight (g) 12.8 ± 1.3a
8.0 ± 1.2b
Survival (%) 76.9 ± 6.7a
57.0 ± 8.6b
FCR 1.5 ± 0.1a
2.4 ± 0.0b
Average weekly growth (g) 1.0 ± 0.1a
0.7 ± 0.0b
Final biomass (kg m−3
) 1.8 ± 0.3a
0.8 ± 0.0b
1
Values are mean ± standard deviation.
2
Different letters indicate significant difference by Tukey´ıs test (P < 0.05).
Table 2
Water consumption and effluent produced (liters per tank) in intensive L. van-
namei biofloc culture using two molasses fertilization strategies: Initial (INI) and
continuous (CONT).
Variable Treatments1,2,3
INI CONT
Total water use 47,904.3 ± 903.1 47,866.7 ± 604.1
Final effluent 40,800.0 ± 818.5 (85.2%) 40,150.0 ± 1202.1 (83.9%)
Evaporation 6060.2 ± 617.4 (12.6%) 6095.8 ± 453.7 (12.7%)
Sludge volume 1044.1 ± 153.8a
(2.2%) 1620.8 ± 144.2b
(3.4%)
1
Values are mean ± standard deviation.
2
Different letters indicate significant difference by Tukey´ıs test (P < 0.05).
3
Values in parentheses indicate the percentage of water used.
Fig. 2. Relationship between cumulative TSS produced and the cumulative amount
of feed applied to the culture tanks in intensive L. vannamei BFT systems using two
different molasses fertilization strategies: Initial (INI) and continuous (CONT).
The amount of nitrogen supplied by the feed was higher
in the INI (6.9 ± 0.9 kg) than the CONT treatment (4.5 ± 0.1 kg).
Meanwhile, the total amount of solids produced (TSS from har-
vested water + TSS from sludge) was similar in both treatments:
23.9 ± 6.7 kg of TSS in the INI and 25.9 ± 0.5 of TSS in the CONT
treatment (P > 0.05). The TSS production per kilogram of feed added
to the tanks, calculated from the regression plot, was higher in
the CONT treatment, which produced 0.25 kg of TSS per kg of feed
(Fig. 2), compared to the INI treatmentin which each kg of feed
produced 0.16 kg of TSS.
3.4. Nutrient discharge
The net nutrient discharge produced is shown in Table 3. The
concentration of total nitrogen (TN) and BOD discharged per shrimp
biomass by the CONT treatment was respectively 2.2 and 3.5 times
higher than that observed in the INI (P < 0.05). Of the TN exported
by the INI treatment, 27% was associated with sludge, compared to
46% in the CONT.

5. R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8 5
Table 3
Nitrogen and five-day biochemical oxygen demand (BOD5) exported per kilogram
of shrimp produced with two fertilization strategies using molasses: Initial (INI) and
continuous (CONT).
Variables1,2
Final effluent Sludge Total
(Nitrogen g kg−1
)
INI 28.2 ± 8.9 10.4 ± 1.4a
38.7 ± 8.3a
CONT 47.4 ± 10.5 40.8 ± 6.1b
88.1 ± 16.6b
(BOD5 g kg−1
)
INI 18.5 ± 3.3a
17.9 ± 2.8a
36.5 ± 0.6a
CONT 39.7 ± 5.4b
87.9 ± 8.07b
127.6 ± 2.6b
1
Values are mean ± standard deviation.
2
Different letters indicate significant difference by Tukey´ıs test (P < 0.05).
3.5. Characteristics of the final effluent and sludge
The final effluent water presented similar characteristics for
both treatments except for the TSS concentrations that were higher
in the INI treatment; 405.0 ± 30.0 mg L−1, compared to the CONT
303.0 ± 13.0 mg L−1 (Table 4). Values of BOD5 were similar, and the
concentration of 36.0 ± 3.6 mg L−1 observed inthe INI was statisti-
cally the same as the CONT (32.5 ± 3.5 mg L−1). The nitrate nitrogen
concentration in the INI treatment, 32.7 ± 5.0 mg L−1, was also not
statistically different than that in the CONT (28.0 ± 1.4 mg L−1).
Considering the sludge removed from the settling tanks, in both
treatments the concentrations of TN, TSS and BOD were respec-
tively 18, 36 and 45 times higher than that observed in the final
effluent (water discharged at harvest) (Table 4). On the other hand,
the sulfate concentrations (1.6 g L−1) were the same in both the tank
water and in the sludge (P ᮀ 0.05).
Considering the sludge produced, the amount of BOD5 was
higher in the CONT treatment (149.8 ± 0.7 g kg−1) than in the INI
(103.2 ± 13.5 g kg−1) (P < 0.05). The values for the BOD:TSS percent
ratio were higher in the CONT treatment, 14.9 ± 0.0%, when com-
pared to the 10.3 ± 0.6% observed in the INI (P < 0.05; Table 4). The
sludge removed from the CONT also had higher VSS and TOC con-
centrations (P < 0.05) than the INI (Table 4). Meanwhile, the higher
input of molasses did not increase the C:N ratio, which was statis-
tically similar in each treatment. The same pattern was observed
for the chemical oxygen demand to sulfate ratio (COD:SO4
−2)
for which no statistically significant differences were observed
between treatments (Table 4).
4. Discussion
4.1. Culture water quality
The water temperature, dissolved oxygen, pH, and TSS were
within the ranges considered suitable for intensive biofloc shrimp
culture (Burford et al., 2003; Schveitzer et al., 2013; Correia et al.,
2014). Salinity was constant in both treatments due to freshwater
additions made to compensate for evaporative losses. The highest
observed values for total ammonia nitrogen in the third week of
culture did not exceed critical concentrations for L. vannamei BFT
cultivation (Burford et al., 2003; Cohen et al., 2005). The same was
observed for nitrite nitrogen, which had concentrations lower than
the 6.1 and 15.8 mg L−1, which was described as safe for L. vannamei
culture at salinities between 15 and 25 gL−1(Lin and Chen, 2003).
The increase of nitrite and nitrate in both treatments indicates
that the nitrification process occurred regardless of the molasses
application practiced. It is evident that the C:N ratio used in the
CONT tanks was not high enough to reduce the development of
nitrifying bacteria. Gao et al. (2012) showed that it is possible to pre-
vent nitrate production with the use of a carbon to nitrogen ratio of
20. However, the maintenance of such a high C:N ratio throughout
the entire culture cycle (C:N > 15) is difficult, due to the high input
of carbohydrates, which can increase the risk of dissolved oxygen
reduction (Ray and Lotz, 2014), especially when using gross bubble
diffusion, as was used in this trial (Arantes et al., 2010).
4.2. Shrimp development
The shrimp growth rate and survival in the INI treatment are
close to the range reported by other biofloc shrimp cultures (Ray
et al., 2010; Samocha et al., 2007). Although water quality variables
were maintained at values suitable for L. vannamei culture (VanWyk
and Scarpa, 1999), shrimp development in the INI was higher than
that in the CONT treatment.
Considering that the initial culture conditions were the same for
both treatments, and that the major difference between them was
the amount and frequency of molasses application, it is possible
that the poorer shrimp performance recorded in the CONT treat-
ment was associated with the use of this fertilizer in the water.
Shrimp in the CONT tanks exhibited a lack of appetite, lethargic
behavior and consistently soft exoskeletons, recognized as symp-
toms of vibrio infection (Barraco et al., 2008). The microbiological
Table 4
Qualitative characteristics of the final effluent and sludge produced from L. vannamei intensive biofloc culture using two fertilization strategies with molasses: Initial (INI)
and continuous (CONT).
Variable Final Effluent1
Sludge1
INI CONT INI CONT
DO (mg L−1
) 4.8 ± 0.2 5.7 ± 0.0 0.1 ± 0.0 0.1 ± 0.0
TAN (mg L−1
) 0.2 ± 0.0 0.3 ± 0.0 4.5 ± 0.7 4.4 ± 0.1
NO2-N (mg L−1
) 0.1 ± 0.0 0.2 ± 0.0 0.75 ± 0.0 0.76 ± 0.0
NO3-N (mg L−1
) 32.7 ± 5.0 28.0 ± 1.4 20.8 ± 4.2 26.9 ± 1.2
Sulfate (mg L−1
) 1699.3 ± 44.1 1641.0 ± 11.0 1622.2 ± 21.9 1670 ± 31.4
TN (mg L−1
) 53.7 ± 4.7 38.9 ± 9.7 813.5 ± 184.3 826.0 ± 49.9
BOD5 (mg L−1
) 36.0 ± 3.6 32.5 ± 3.5 1370.0 ± 246.4a
1750 ± 28.3b
TSS (mg L−1
) 405.0 ± 30.0a
303.0 ± 13.0b
13687 ± 3452 12195 ± 546
VSS (%TSS) 44.9 ± 0.6 44.9 ± 1.4 64.4 ± 0.43a
70.5 ± 0.58b
Total solids(g L−1
) – – 16.2 ± 0.9 14.1 ± 1.2
VS (g kg−1
) – – 636 ± 9.8a
702 ± 12.0b
TOC (g kg−1
) – – 318.0 ± 4.9a
351.0 ± 6.0b
C:N – – 6.38 ± 1,4 7.52 ± 1.6
COD:SO4
2−
– – 8.5 ± 0.4 7.9 ± 0.6
BOD5:TSS (%) 9.0 ± 0.0 10.7 ± 0.0 10.3 ± 0.6a
14.9 ± 0.0b
pH 7.5 ± 0.0 7.7 ± 0.0 7.3 ± 0.0 7.3 ± 0.0
BOD5 Five-day Biochemical Oxygen Demand; DO: Dissolved oxygen; TAN: Total ammonia nitrogen; TSS: Total suspended solids; NO2-N: Nitrite nitrogen; NO3-N: Nitrate
nitrogen; TN: Total nitrogen; VSS: Volatile suspended solids; VS: Volatile solids. TOC: Total organic carbon; COD: Chemical oxygen demand; SO4
2−
: Sulfate.
1
Different letters in the same row indicate significant difference by Tukey´ıs test (P < 0.05).

6. 6 R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8
analysis of the shrimp revealed the presence of Vibrio spp. on the
hemolymth and on the hepatopancreas, suggesting infection by
this bacteria genus. An adverse effect from the continuous use
of molasses was also reported by Ray and Lotz (2014), who par-
tially attributed lower shrimp survival to the high concentration
of unusable components of the molasses, which fouled the water.
The hypothesis about the negative effects of molasses is also sup-
ported by the fact that in the INI tanks, after stopping molasses use
in week 6, food consumption by the shrimp increased and conse-
quently shrimp growth rate. For this reason it is a better option to
use molasses only at the beginning of the cycle when nitrification
may be slow and later on, only in cases when TAN are not con-
trolled by the nitrification process or algal absorption. In this case,
it should be properly adjusted according to the TAN concentration
observed in the water.
4.3. Volume of effluent produced
In the CONT treatment, the amount of sludge produced was
higher because of the higher values of TSS produced per kg of feed,
which may be associated with the higher heterotrophic bacterial
biomass production and the higher FCR observed in this treatment.
A fraction of nitrogen not incorporated by shrimp may have been
transformed into microbial biomass due to the higher C:N ratio
used in these tanks. This biomass increased the TSS concentration,
which in turn was removed as sludge in the settling tanks (Gaona
et al., 2016; Ebeling et al., 2006).
Despite the differences in sludge volume between the treat-
ments, sludge only accounted for a small portion, from 2.2 to 3.4%,
of the total water used during cultivation. The small volume can
be attributed to the low amount of solids generated in relation to
the amount of feed applied to the tanks and can indicate that a sig-
nificant fraction of organic matter added to the tanks, as feed and
molasses, may have degraded inside the tank (Hopkins et al., 1993).
The observed values of solids production, between 160 and 250 g
per kilogram of the applied feed, are close to the observed range for
other completely mixed and aerated systems, where the produc-
tion of microbial biomass was 11–38% of the applied feed (Hopkins,
1994; Avnimelech, 2009). These results corroborate with Brune and
Drapcho (1991), who noted that stimulating an in-pond digestion
process of organic matter can prevent the transfer of assimilative
capacity to the receiving stream. This reinforces the importance of
BFT as an alternative technology to reduce possible environmen-
tal impacts that may be caused by intensive shrimp production
(Jackson et al., 2003; Browdy et al., 2012).
4.4. Nutrient discharge
The total amount of nitrogen exported by the CONT treatment
(88 g per kg of shrimp produced) reveals that nutrient use within
this treatment was less effective, probably related to the higher
shrimp mortality and to poorer FCR. In the INI, better FCR resulted
in a lower amount of nitrogen discharged (38.7 ± 8.3 g kg−1). These
values are also lower than those found in other intensive sys-
tems that use continuous water exchange to control water quality
(Funge-Smith and Briggs, 1998), and even in semi intensive sys-
tems (Paez-Osuna et al., 1997), and show that the management
employed in the INI is an appropriate action to avoid nitrogen
waste from intensive shrimp culture. Although nitrogen retention
by bioflocs is reported to be improved when a carbon source is used
to fertilize water (Crab et al., 2009b; Avnimelech, 2009; Silva et al.,
2013), the relationship between a carbon source addition and the
amount of time that nitrogen remains inside the biofloc biomass
should be better elucidated (Avnimelech and Kochba, 2009). BOD5
analysis is an indicator of organic material in water and reflects the
discharge of substances that can be biologically degraded and con-
sume oxygen, to potentially reduce DO levels in the receiving water
(Samocha et al., 2004). Because of the higher input of feed per kg of
shrimp produced and the continuous input of carbohydrates, the
amount of BOD5 exported by CONT was higher.
The use of settling tanks for sludge removal proved to be an
important tool as a first step in final effluent water treatment. In
the INI, 27% of the nitrogen input from feed was retained as sludge,
preventing its discharge at the time of shrimp harvest. Moreover,
the amount of BOD removed was 49% in the INI treatment and 69%
in the CONT, which indicates that sludge removal during cultivation
can reduce the impact of the higher volume of final effluent on
the receiving body of water (Hopkins et al., 1993). On the other
hand, this solids removal did not eliminate the polluting potential
of the culture, it just re-located nutrients to improve their further
processing (Piedrahita, 2003).
4.5. Characteristics of final effluent and sludge
The attempt to reduce nitrate discharge from the final effluent
by using molasses was not successful in this trial, since nitrate nitro-
gen concentrations were the same in both treatments (Table 4).
This may be related to an elevated leaching of nitrogen that proba-
bly occurs because of the higher shrimp mortality in this treatment.
The differences in final effluent only appeared in the TSS concentra-
tions, which were higher in the INI treatment, reflecting the sudden
increase of the feed input in this treatment in the final weeks of
cultivation. Despite the differences in TSS, values of BOD5 con-
centrations were similar in both treatments, suggesting a greater
bacterial respiration rate in the CONT treatment, related with the
daily carbohydrate additions (Schveitzer et al., 2013). Concentra-
tions presented for BOD5 in both treatments were similar to those
observed in other intensive shrimp farming systems (Cohen et al.,
2005) and, despite the values around 30 mg L−1, are within the suit-
able range for the discharge of aquaculture effluents (Boyd, 2003).
TSS and nitrate nitrogen values are well above the permitted values
(Boyd, 2003). This could reinforce the need for treatment applica-
tions to reduce the effluent discharge load at the time of shrimp
harvest, as described elsewhere (Jackson et al., 2003; Boyd and Clay,
2002; Krummenauer et al., 2014; Ray and Lotz, 2014).
In relation to the sludge removed from the settling chambers,
the frequency of removal (∼24 h) sought to minimize reactions of
solids degradation, and thus avoid nutrient dissolution and their
return to the tank water (Van Rijn and Nussinovitch, 1997; Sharrer
et al., 2010). However, the high concentrations of total ammo-
nia nitrogen observed indicate fast mineralization of nutrients, as
reported by Conroy and Couturier (2010). Furthermore, the sludge
showed oxygen concentrations below the detection limit, indi-
cating an anaerobic sludge at the time of drainage. Given this
condition, it was expected that the sulfate concentration was lower
than that observed in the culture water tank. However, this differ-
ence was not detected, indicating that there was no conversion of
these compounds to other reduced forms, such as sulfide, an unde-
sirable odorous compound (Turovskiy and Mathai, 2006). This was
supported by the fact that using a drainage frequency of 24 h, no
offensive odors were observed in the sludge drained.
Sludge disposal in intensive aquaculture systems can be per-
formed directly or require a previous treatment process to reduce
costs of disposal and prevent offensive odors (Chen et al., 1997).
In this study, the total solids concentration, 13 to 16 g L−1, can be
considered low for the sludge to be directly disposed (Sharrer et al.,
2010). Although this value is close to that obtained in other BFT sys-
tems, between 9 and 33 g L−1 (Schveitzer et al., 2013; Lyles et al.,
2008; Boopathy et al., 2007; Ray et al., 2011), if settling tanks are
used for TSS control, a thickening step would be necessary before
final disposal of this sludge (Bergheim et al., 1998; Sharrer et al.,
2010).

7. R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8 7
On the other hand, the sludge had high organic matter content
in both treatments (>60%), suggesting that a previous treatment
method is necessary before it could be disposed of (Turovskiy
and Mathai, 2006; Mirzoyan et al., 2008). In the CONT treatment,
the higher amount of carbohydrate added as molasses resulted in
changes to the sludge composition, which had a higher VSS con-
tent, showing greater availability of organic matter to be used in
anaerobic digestion processes (Gavala et al., 2003; Gebauer, 2004;
Gebauer and Eikebrokk, 2006). However, the C:N ratio did not sig-
nificantly increase (C:N = 7.5) due to the high total nitrogen content
observed (Gebauer and Eikebrokk, 2006). It is important to note that
a C:N ratio, lower than 10, could be considered to be inappropri-
ate for the use of anaerobic processes (Mirzoyan and Gross, 2013;
Turovskiy and Mathai, 2006; Durmaz and Sanin, 2003). A high nitro-
gen content can harm anaerobic digestion due to the elevated rates
of ammonia production (Couturier et al., 2008), which can lead to
failures in reactors designed for sludge digestion due to its toxicity
(Gebauer and Eikebrokk, 2006). Likewise, the COD:SO4
−2 ratio does
not significantly increase with the continuous use of molasses, and
was kept below 10 in both treatments. A COD:SO4
−2 ratio lower
than 10 is considered unsuitable for anaerobic sludge digestion,
mainly due to the potential for sulfide production and its toxicity,
as well as its inhibitory effect on methane production (Mirzoyan
et al., 2008; Hulshoff Pol et al., 1998).
The BOD5 determination is used to indicate the amount of
biodegradable carbon (Tchobanoglous and Burton, 1991) and the
BOD5:TSS ratio serves as a measure of the degree of sludge stabi-
lization (Chen et al., 1997). In this study, BOD5:TSS values of the
sludge in the CONT treatment were significantly higher, exhibit-
ing a greater need for stabilization. On the other hand, for both
treatments, the BOD5:TSS ratio was at the lower limit of the range
reported by Chen et al. (1997), suggesting that a fraction of the
organic matter removed as sludge had been previously stabilized
within the culture tank (Hopkins et al., 1993). Thus, it is suggested
that thickening the sludge with a subsequent alkaline stabilization
process using lime could be sufficient for the sludge disposal from
both treatments (Bergheim et al., 1998; Sharrer et al., 2010).
5. Conclusions
A fertilization strategy using carbohydrates only at the begin-
ning of the rearing period allows high shrimp biomass production,
with a relatively low net loss of nitrogen to the effluent. The unnec-
essary use of molasses to improve nitrogen retention can threaten
production results, indicating that molasses should only be used
to control ammonia concentration levels and not in an attempt
to improve nitrogen retention in the biofloc biomass. Consider-
ing the C:N ratio used (12:1), the frequency of carbon application
does not affect the nitrate concentration in the final effluent water
discharged after harvest. Furthermore, the continuous application
of molasses can increase the amount of exported nutrients and
the sludge volume due to the negative effects of this carbohydrate
source on shrimp survival and growth. The continuous fertilization
with molasses can change the characteristics of the sludge, making
it less stable, although, these changes are not sufficient to facilitate
the use of anaerobic stabilization processes.
Acknowledgments
The authors would like to thank the Coordenac¸ ão de
Aperfeic¸ oamento de Pessoal de Nível Superior (CAPES) for the doc-
toral fellowship and financial support (Ciências do Mar – CAPES – no
09/2009). The authors would also like to thank the Laboratório de
Camarões Marinhos (LCM-UFSC) and Rafael Pacheco Derner, Fran-
cisco Pchara, Hadja Hadtke Nunes, Jesus Malpartida Pasco, Gabriel
Bandeira and Frank Belletini, for their support in conducting the
experiment. Luis Vinatea and Walter Quadros Seiffert are research
fellows of the National Council for Scientific and Technological
Development CNPq.
References
APHA, 1998. Standard Methods For The Examination Of Water And Wastewater,
20th ed. Byrd Prepress, Springfield.
Arantes, R., Schveitzer, R., Scopel, B., Vinatea, L., Lapa, K.R., Seiffert, W.Q., 2010.
Preliminary results of a pump driven water circulation system for shrimp
production on a super-intensive recirculating biofloc culture. In: Rakestraw,
T.T., Douglas, L.S. (Eds.), Proceedings of the 8th International Conference on
Recirculating Aquaculture. Virginia Tech University, Blacksburg, V.A, pp.
156–158.
Arantes, R., Schveitzer, R., Magnotti, C., Lapa, K.R., Vinatea, L., 2016. A comparison
between water exchange and settling tank as a method for suspended solids
management in intensive biofloc technology systems: effects on shrimp
(Litopenaeus vannamei) performance, water quality and water use. Aquac.
Res., http://dx.doi.org/10.1111/are.12984 (in press).
Avnimelech, Y., Kochba, M., 2009. Evaluation of nitrogen uptake and excretion by
tilapia in bio floc tanks, using 15
N tracing. Aquaculture 287, 163–168.
Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture
systems. Aquaculture 176, 227–235.
Avnimelech, Y., 2007. Feeding with microbial flocs by tilapia in minimal discharge
bio-flocs technology ponds. Aquaculture 264, 140–147.
Avnimelech, Y., 2009. Biofloc Technology – A Practical Guide Book. The World
Aquaculture Society, Baton Rouge, Louisiana, USA.
Barraco, M.A., Perazzolo, L.M., Rosas, R.D., 2008. In: Morales, V., CuéllarAnjel, J.
(Eds.), Guía Técnica – Patología e Inmunología de Camarones Penaeidos.
Programa CYTED Red II-D Vannamei, Panamá, Rep. de Panamá. 270 pp.
Bergheim, A., Cripps, S.J., Liltved, H., 1998. A system for the treatment of sludge
from land-based fish-farms. Aquat. Liv. Res. 11, 279–287.
Boopathy, R., Bonvilain, C., Fontenot, Q., Kilgen, M., 2007. Biological treatment of
low-salinity shrimp aquaculture wastewater using sequencing batch reactor
internationa. Biodeterior. Biodegrad. 59, 16–19.
Boyd, C.E., Clay, J.W., 2002. Evaluation of Belize Aquaculture, Ltd: A Superintensive
Shrimp Aquaculture System. Report prepared under the World Bank, NACA,
WWF and FAO Consortium Program on Shrimp Farming and the Environment.
Work in Progress for Public Discussion. The consortium, p. 17.
Boyd, C.E., 1995. Bottom Soils Sediment and Pond Aquaculture. Chapman & Hall,
New York.
Boyd, C.E., 2003. Guidelines for aquaculture effluent management at the
farm-level. Aquaculture 226, 101–112.
Browdy, C.L., Bratvold, D., Stokes, A.D., McIntosh, R.P., 2001. Perspectives on the
application of closed shrimp culture systems. In: Browdy, C.L., Jory, D.E. (Eds.),
The New Wave, Proceedings of the Special Session on Sustainable Shrimp
Culture. The World Aquaculture Society, Baton Rouge, L.A, USA, pp. 20–34.
Browdy, C.L., Ray, A.J., Leffler, J.W., Avnimelech, Y., 2012. Biofloc-based aquaculture
systems. In: Tidwell, J.H. (Ed.), Aquaculture Production Technologies.
Wiley-Blackwell, Oxford, UK, pp. 278–307.
Brune, D.E., Drapcho, C.M., 1991. Fed pond aquaculture. In Aquaculture systems
engineering. In: Procedings of the World Aquaculture Society and the
American Society of Agricultural Engineers Jointly Sponsored Session, ASAE,
American Society of Agricultural Engineers, St. Joseph, Michigan USA.
Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., 2003.
Nutrient and microbial dynamics in high-intensity, zero-exchange shrimp
ponds in Belize. Aquaculture 219, 393–411.
Casillas-Hernandez, R., Magallõn-Barajas, F., Portillo-Clarck, G., Paez-Osuna, F.,
2006. Nutrient mass balances in semi-intensive shrimp ponds from Sonora,
Mexico using two feeding strategies: trays and mechanical dispersal.
Aquaculture 258, 289–298.
Chen, S., Coffin, D.E., Malone, R.F., 1997. Sludge production and management for
recirculating aquacultural systems. J. World Aquacult. Soc. 28, 303–315.
Cohen, J.M., Samocha, T.M., Fox, J.M., Gandy, R.L., Lawrence, A.L., 2005.
Characterization of water quality factors during intensive raceway production
of juvenile Litopenaeus vannamei using limited discharge and biosecure
management tools. Aquacult. Eng. 32, 425–442.
Conroy, J., Couturier, M., 2010. Dissolution of minerals during hydrolysis of fish
waste solids. Aquaculture 298, 220–225.
Correia, E.S., Wilkenfeld, J.S., Morris, T.C., Wei, L.W., Prangnell, D.I., Samocha, T.M.,
2014. Intensive nursery production of the Pacific white shrimp Litopenaeus
vannamei using two commercial feeds with high and low protein content in a
biofloc-dominated system. Aquacult. Eng. 59, 48–54.
Couturier, M.F., Fraser, S., Conroy, J., Singh, K.S., Desbarats, A., 2008. Anaerobic
digestion of aquaculture waste. In: Rakestraw, T.T., Douglas, L.S., Marsh, L.,
Granata, L., Flick, G.J. (Eds.), Proceedings of the 7th International Conference on
Recirculating Aquaculture. Virginia tech University, Blacksburg, V.A, pp.
366–373.
Crab, R., Avnimelech, Y., Defoirdt, T., Bossier, P., Verstraete, W., 2007. Nitrogen
removal techniques in aquaculture for a sustainable production. Aquaculture
270, 1–14.

8. 8 R. Arantes et al. / Aquacultural Engineering 76 (2017) 1–8
Crab, R., Chielens, B., Wille, M., Bossier, P., Verstraete, W., 2009a. The effect of
different carbon sources in the nutritional value of bioflocs, a feed for
Macrobrachium rosembergii postlarvae. Aquacult. Res. 41, 559–567.
Crab, R., Kochva, M., Verstraete, W., Avnimelech, Y., 2009b. Bio-flocs technology
application in over-wintering of tilapia. Aquacult. Eng. 40, 105–112.
Davidson, J., Summerfelt, S.T., 2005. Solids removal from a coldwater recirculating
system-comparison of a swirl separator and a radial-flow settler. Aquacult.
Eng. 33, 47–61.
Durmaz, B., Sanin, F.D., 2003. Effect of carbon to nitrogen ratio on the physical and
chemical properties of activated sludge. Environ. Technol. 24, 1331–1340.
Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the
stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of
ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346–358.
Fontenot, Q., Bonvillain, C., Kilgen, M., Boopathy, R., 2007. Effects of temperature,
salinity, and carbon: nitrogen ratio on sequencing batch reactor treating
shrimp aquaculture wastewater. Biores. Technol. 98, 1700–1703.
Funge-Smith, S.J., Briggs, M.R.P., 1998. Nutrient budgets in intensive shrimp ponds:
implications for sustainability. Aquaculture 164, 117–133.
Gao, L., Shan, H.-W., Zhang, T.-W., Bao, W.-Y., Ma, S., 2012. Effects of carbohydrate
addition on Litopenaeus vannamei intensive culture in a zero-water exchange
system. Aquaculture 342–343, 89–96.
Gaona, C.A.P., Serra, F.P., Furtado, P.S., Poerch, L.H., Wasielesky, W., 2016. Biofloc
management with different flow rates for solids removal in the Litopenaeus
vannamei BFT culture system. Aquacult. Int., http://dx.doi.org/10.1007/
s10499-016-9983-2 (in press).
Gavala, H.N., Yenal, U., Skiadas, I.V., Westermann, P., Ahring, B.K., 2003. Mesophilic
and thermophilic anaerobic digestion of primary and secondary sludge: effect
of pre-treatment at elevated temperature. Water Res. 37, 4561–4572.
Gebauer, R., Eikebrokk, B., 2006. Mesophilic anaerobic treatment of sludge from
salmon smolt hatching. Biores. Technol. 97, 2389–2401.
Gebauer, R., 2004. Mesophilic anaerobic treatment of sludge from saline fish farm
effluents with biogas production. Biores. Technol. 93, 155–167.
Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research,
second ed. John Wiley, New York.
Hopkins, J.S., Richard II, D.H., Paul, A.S., Craig, L.B., Alvin, D.S., 1993. Effect of water
exchange rate on production, water quality, effluent characteristics and
nitrogen budgets of intensive shrimp ponds. J. World Aquacult. Soc. 24,
304–320.
Hopkins, J.S., 1994. An Apparatus for continuous removal of sludge and foam
fractions in intensive shrimp culture ponds. Progressive Fish Cult. 56, 135–139.
Hulshoff Pol, L., Lens, P.L., Stams, A.M., Lettinga, G., 1998. Anaerobic treatment of
sulphate-rich wastewaters. Biodegradation 9, 213–224.
Jackson, C., Preston, N., Thompson, P.J., Burford, M., 2003. Nitrogen budget and
effluent nitrogen components at an intensive shrimp farm. Aquaculture 218,
397–411.
Johnson, W., Chen, S., 2006. Performance evaluation of radial/vertical flow
clarification applied to recirculating aquaculture systems. Aquacult. Eng. 34,
47–55.
Krummenauer, D., Samocha, T.M., Poersch, L., Lara, G., Wasielesky, W., 2014. The
reuse of water on the culture of pacific white shrimp, Litopenaeus vannamei, in
BFT system. J. World Aquacult. Soc. 45, 3–14.
Lin, Y.-C., Chen, J.-C., 2003. Acute toxicity of nitrite on Litopenaeus vannamei
(Boone) juveniles at different salinity levels. Aquaculture 224, 193–201.
Lyles, C., Boopathy, R., Fontenot, Q., Kilgen, M., 2008. Biological treatment of
shrimp aquaculture wastewater using a sequencing batch reactor. Appl.
Biochem. Biotechnol. 151, 474–479.
Mirzoyan, N., Gross, A., 2013. Use of UASB reactors for brackish aquaculture sludge
digestion under different conditions. Water Res. 47, 2843–2850.
Mirzoyan, N., Parnes, S., Singer, A., Tal, Y., Sowers, K., Gross, A., 2008. Quality of
brackish aquaculture sludge and its suitability for anaerobic digestion and
methane production in an upflow anaerobic sludge blanket (UASB) reactor.
Aquaculture 279, 35–41.
Mirzoyan, N., McDonald, R.C., Gross, A., 2012. Anaerobic treatment of
brackishwater aquaculture sludge: an alternative to waste stabilization ponds.
J. World Aquacult. Soc. 43, 238–248.
Paez-Osuna, F., Guerrero-Galvan, S.R., Ruiz-Fernandez, A.C., Espinoza-Angulo, R.,
1997. Fluxes and mass balances of nutrients in a semi-intensive shrimp farm in
north-western Mexico. Mar. Pollut. Bull. 34, 290–297.
Piedrahita, R.H., 2003. Reducing the potential environmental impact of tank
aquaculture effluents through intensification and recirculation. Aquaculture
226, 35–44.
Ray, A.J., Lotz, J.M., 2014. Comparing a chemoautotrophic-based biofloc system and
three heterotrophic-based systems receiving different carbohydrate sources.
Aquacult. Eng. 63, 54–61.
Ray, A.J., Lewis, B.L., Browdy, C.L., Leffler, J.W., 2010. Suspended solids removal to
improve shrimp (Litopenaeus vannamei) production and an evaluation of a
plant-based feed in minimal-exchange, superintensive culture systems.
Aquaculture 299, 89–98.
Ray, A.J., Dillon, K.S., Lotz, J.M., 2011. Water quality dynamics and shrimp
(Litopenaeus vannamei) production in intensive: mesohaline culture systems
with two levels of biofloc management. Aquacult. Eng. 45, 127–136.
Roy, L.A., Davis, D.A., Saoud, I.P., Boyd, C.A., Pine, H.J., Boyd, C.E., 2010. Shrimp
culture in inland low salinity waters. Rev. Aquacult. 2, 191–208.
Samocha, T.M., Lopez, I., Jones, E., Jackson, S., Lawrence, A.L., 2004. Characterization
of intake and effluent waters from intensive and semi-intensive shrimp farms
in texas. Aquacult. Res. 35, 321–339.
Samocha, T.M., Patnaik, S., Speed, M., Ali, A.-M., Burger, J.M., Almeida, R.V., Ayub, Z.,
Harisanto, M., Horowitz, A., Brock, D.L., 2007. Use of molasses as carbon source
in limited discharge nursery and grow-out systems for Litopenaeus vannamei.
Aquacult. Eng. 36, 184–191.
Schveitzer, R., Arantes, R., Costódio, P.F.S., do Espírito Santo, C.M., Arana, L.V.,
Seiffert, W.Q., Andreatta, E.R., 2013. Effect of different biofloc levels on
microbial activity, water quality and performance of Litopenaeus vannamei in a
tank system operated with no water exchange. Aquacult. Eng. 56, 59–70.
Sharrer, M., Rishel, K., Taylor, A., Vinci, B.J., Summerfelt, S.T., 2010. The cost and
effectiveness of solids thickening technologies for treating backwash and
recovering nutrients from intensive aquaculture systems. Biores. Tech. 101,
6630–6641.
Silva, K.R., Wasielesky, W., Abreu, P.C., 2013. Nitrogen and phosphorus dynamics in
the biofloc production of the Pacific white shrimp, Litopenaeus vannamei. J.
World Aquacult. Soc. 44, 30–41.
Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis,
vol. 167. Bulletin of Fisheries Research Board, Canada.
Tchobanoglous, G., Burton, F., 1991. Wastewater Engineering,
Treatment/Disposal/Reuse, 3rd ed. McGraw Hill, New York.
Turovskiy, I.S., Mathai, P.K., 2006. Wastewater Sludge Processing. John Wiley &
Sons, Inc., New Jersey.
Valderrama, J.C., 1981. The simultaneous analysis of total nitrogen and total
phosphorous in natural waters. Mar. Chem. 10, 109–122.
Van Rijn, J., Nussinovitch, A., 1997. An empirical model for predicting degradation
of organic solids in fish culture systems based on short-term observations.
Aquaculture 154, 173–179.
VanWyk, P., Scarpa, J., 1999. Water quality and management. In: VanWyk, P. (Ed.),
Farming Marine Shrimp in Recirculating Freshwater Systems. Florida
Department of Agriculture and Consumer Services, Tallahassee, pp. 128–138.
Zar, J., 1999. Bioestatistical Analysis, 3rd ed. Pentice Hall, New Jersey.