A and distributed by a microperforated hoses system allocated

A 42 day-trial was conducted at the Marine Station of
Aquaculture, University of Rio Grande, Rio Grande do Sul State, Brazil (32º 19′
S, 52º 15′ W). Twelve-800 L rectangular tanks were stocked with 320 juveniles
of Pacific white shrimp (1.17g ±0.55) at stocking density of 400/m³. The tanks
were filled with natural seawater (salinity = 33) filtered and treated with
chlorine solution and dechlorinated by aeration. The aeration was provided by
blower and distributed by a microperforated hoses system allocated in each
tank. Water exchange was made every time total ammonia levels exceed 7,0 mg/L
and nitrite exceed 20mg/L (approximately twice the safe level of each one
REFERENCIA) or whenever TSS exceeded 500 mg/L (REFERENCIA). Prior to the
experiment, the percentage of Carbon, Nitrogen and Hydrogen in feed and
molasses were determined using a CNHS Elemental Analyser (2400 CHNS/O Series II
PerkinElmer). T

 

Four treatments (three replicates) were tested using
four different C:N rates: 7,5:1, where no supplemental carbon was added, 10:1,
12,5:1 and 15:1, in accordance with nominal C:N rate proposed by Ebeling et al
(2006). Organic fertilization was made every time total ammonia nitrogen (TAN)
lecture exceed 1,0 mg/L with the addition of molasses powder.

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Water quality

 

Temperature, dissolved oxygen and pH were monitored
twice a day using a digital multiparameter. Water samples were collected daily
to determine total ammonia nitrogen (TAN = NH4+ + NH3) and nitrite (N-NO2-) according
to UNESCO (1983) and Strickland & Parsons (1972), respectively. Nitrate
(N-NO3-) concentrations were quantify every seven days (Aminot e Chaussepied, 1983).
Salinity was measured weekly using a portable refractometer. When necessary,
disinfected fresh water was added to adjust salinity or restore water losses by
evaporation. Alkalinity was measured each three days following the methodology
recommended by APHA (1998). Every time pH reached values below 7,3 and/or
alkalinity reached values below 100 mg/L, adjustments were made to correct them in accordance
with Furtado et al. (2014). Total settleable solids were measured twice a week (AOAC,
1999). Water turbidity was measured once a week using a digital turbidimeter
(Hach® 2100P). Measures of chlorophyll a was performed weekly. Water samples
(10 ml) was collected from each unit and filtered in TIPO DE FILTRO in a dark
room and then stored in 90% acetone in dark bottles at -12 ° C. After 24 hours
the concentration of chlorophyll a was determined with the aid of fluorimeter,
according to the methodology described in Welschmeyer (1994).

 

Feeding and monitoring

Shrimp were fed twice a day with a commercial diet
(Guabi Potimar/38 Active) spread in the tank and in feeding trays (one per
tank) to check consumption. Initial feed rate was adjusted by recommendations
of Jory et al. (2001) and then adjusted based on feed consumption and shrimp
growth. Weekly, 30 animals were randomly collected from each tank and
individually weighed. At the end of the study, 60 animals of each tank were
sampled and weighed for final weight. Survival was obtained by counting the
total survival individuals in each tank. Feed conversion rate was calculated by
dividing total feed offered by biomass increase. Yield was calculated by total
biomass/tank volume.

 

FISH

Each two days, 4,5 ml of BFT water samples were
collected from each tank and then fixed with 0,5ml of 20% paraformaldehyde
solution (final concentration: 2%) to detect the growth of the population of
nitrifying and heterotrophic bacteria by Fluorescent in situ Hybridization (FISH)
methodology (Cottrell & Kirchman 2003 adapted by Del Duca et. al., 2013) with
RNA probes selected to identify the groups of bacteria. In order to evaluate
the efficiency of the hybridization, a negative control was used with no
specificity for any bacterial group. All probes were marked with Cy3
fluorochrome. Abundance of bacteria was obtained by direct counting under
epifluorescence microscopy (Olympus® BX-60).

 

Statistical Analysis

The data was submitted to tests of normality
(Shapiro-Wilk’s test) and homoscedasticity (Levene’s test). After verifying
these premises, ANOVA One-way was performed. If significant differences were
detected (p

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