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International Journal of Aquaculture 2012, Vol.2, No.6, 29-39
http://ija.sophiapublisher.com
36
(160 mg/L as CaCO
3
) was used to control alkalinity
and pH. A YSI 5 200 multi-parameter sonde (YSI
Inc., Yellow Springs, OH, USA) was placed in each
raceway and data uploaded remotely. Daily and
weekly water quality data from each treatment was
analyzed using a repeated measures ANOVA. Mean
survival, FCR, total yields, weekly growth and final
shrimp weights were analyzed using one-way
ANOVAs and an
α
value of 0.05. All statistical
analyses were conducted using SPSS statistical
software (V. 15 for Windows, SPSS Inc., Chicago, IL,
USA). Mixing of the ambient air with bottled oxygen
did not begin until Day 68 of the study. For a period of
40 days (Days 68 through 108) air was enriched with
oxygen only intermittently, specifically for 30~60 minutes
following the day-time feeding and only if DO levels
dropped below 3 mg/L, at a flow rate of l.0 L/min.
During the final week (Day 102 through termination
at Day 108), air was constantly mixed with oxygen at
a flow rate of 0.3~0.5 L/min.
4.3 Analysis of microbial community
The algal and bacterial communities associated with
each raceway were monitored weekly in order to
correlate particulate control methods (e.g., FF to ST)
to the changes in the populations of algae and
bacteria present within the system. Algal monitoring
began on July 24 and ended October 2, 2009 while
bacterial communities, specifically gram-negative
and gram-positive, were monitored only during the
final five weeks of the study. Water samples were
collected from each raceway in sterile 50 mL conical
FalconTM tubes in the morning at ~9:00 AM and
transported to Texas A&M University-Corpus Christi
to be analyzed within 2 h of collection. A fluorescence
activated cell sorter (FACS) flow cytometer VantageSE
(Becton, Dickinson and Company, San Jose, CA, USA)
in combination with a COHERENT Innova Enterprise
II Laser (488 nm) (COHERENT, Inc., Auburn, CA,
USA) was used to analyze the water samples. Due to
the interspersed use of the FACS, quality control
occurred between sampling weeks with BioSure®
chicken red blood cells (CRBC) in phosphate buffered
saline (PBS) and standard instrument settings to insure
that instrument parameters remained within acceptable
limits between sampling dates.
Algal communities were distinguished using fluorescence
characteristics inherent to the individual cells within
(534
±
34) nm (FL1) and (630
±
22) nm (FL3) fluorescence
ranges as well as the forward scatter (FSC) physical
parameter, which is indicative of cell size. Prior to
analysis, cells were passed through a 100 μm nylon
mesh filter to decrease the prevalence of cell
aggregates and remove debris. Ten thousand events
(one event=one cell passing through the laser path)
were acquired for each sample. The greatest
community resolution was acquired with the FL1 and
FL3 parameters when used on a 4 log-scale plot as
well as the FL1 and FSC parameters (Trask et al.,
1982; Olson et al., 1989).
A procedure analogous to gram-staining was used to
determine the percentage of gram-positive vs.
gram-negative cells following methods adapted
from Holm and Jespersen (2003). Briefly, a 1.5 mL
aliquot of filtered raceway water sample was
transferred to a micro-centrifuge tube (~10
6
cells/mL)
and centrifugated at 700×g for 10 min. The
supernatant was decanted and re-suspended in 1.5 mL
of a 3 M KCl solution (3 M KCl, 0.035 M EDTA
[pH 7.0], Sigma Aldrich, St. Louis, MO, USA). This
solution served to permeabalize the cell wall of
gram-negative bacteria. Two hundred and fifty
microliters of the suspension was stained with 50 μL
of hexididum iodide (HI; 200
μ
g/mL in 0.1 M
NaHCO
3
, Invitrogen, Grand Island, NY, USA) and
incubated at 50
℃
for 15 min. Since HI binds to all
bacterial DNA (Holm and Jespersen, 2003) gram-
positive bacteria resist HI staining due to their
thicker cell wall. Twenty-five microliters of the
secondary stain, Oregon Green 488 nm-conjugated
wheat germ agglutinin (WGA; 200 μg/mL in 0.1 M
NaHCO3, Invitrogen, Grand Island, NY, USA), was
then added to the suspension and incubated at room
temperature for 4 min. WGA binds selectively to
gram-positive cells (Holm and Jespersen, 2003).
Ten thousand events were analyzed in the FL1 and
FL3 parameters for each sample.
4.4 Statistical analysis
FACS data was analyzed with FlowJo v. 8.8.6 (Tree
Star, Inc., Ashland, OR, USA). In order to determine
the effects of this system on the biofloc community
experiments were carried out in fixed factor three-way
ANOVA designs. Solid control method and sampling
date were used with microorganism (gram-stain),
particle (size) or region (autofluorescence and size) as