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International Journal of Aquaculture 2012, Vol.2, No.6, 29-39
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34
Isolating and culturing a majority of the associated
flora of shrimp has proved difficult and therefore
research has focused on growth independent
identification techniques (Lau et al., 2002) or those
techniques designed to monitor and identify particular
microorganisms (Tendencia and de la Peña, 2001;
Nakayama et al., 2006). We investigated the
application of a rapid technique, flow cytometry, as a
means to assess changes in bacterial as well as
auto-fluorescent pelagic communities of an RAS
system. We found that using the gram-stain like
procedure developed by Holm and Jespersen (2003),
and initially utilized for milk associated bacteria, the
relative densities of gram-negative to gram-positive
bacteria could be estimated. We were able to detect
weekly fluctuations in this community of bacteria.
Specifically, we were able to determine that the two
solids control methods employed, foam fractionation
and conic cylindrical settling tanks, and did not result
in a significant change in the ratio of gram-positive to
gram-negative bacteria when using pre-conditioned
culture water. The fluctuations we observed within
each treatment indicate a level of system homeostasis
with respect to gram-negative and gram-positive
bacteria. Fluctuations within a system were observed
when the ratio of gram-positive:gram-negative
bacteria reached 2:8. Gram-positive bacteria cultured
from wild habitats (Rengpipat et al., 2003) as well as
produced commercially (Wang et al., 2005), have
received attention regarding their ability to kill or
inhibit the growth of gram-negative organisms within
marine aquaculture systems. It is therefore critical
that a population of these organisms remain within the
system to balance potential overgrowth of pathogenic
gram-negative organisms. Gram-positive organisms
also play an important role in minimizing the
accumulation of phosphorous and nitrogen species.
Decreased reactive phosphorous as well as total
nitrogen and chemical oxygen demand have been
reported in systems with gram positive probiotic use
(Wang et al., 2005).
Additionally to bacterial staining, we were able to
distinguish three major classes of particles in the
biofloc using flow cytometry; pico particles (<2 m),
nano particles (2~8 m), and macro particles (7~20 m).
Pico particles displayed a range of red fluorescence
spanning three log scale regions from 10
0
~10
3
relative fluorescence units. Nano and macro particles
were found within a more narrow range: 10
1
~10
2
and
10
2
~10
3
, respectively. Although we found that macro
particles were in the highest concentration, FACS does
not differentiate between single and clustered cells or
cells attached to debris. Flow cytometry methods will
only detect these conglomerates as a single large
“particle”. Macro particles should therefore be
considered a diverse group that may require greater
resolution. Systems such as closed zero exchange
super-intensive culture systems tend to produce
suspended aggregates or biofloc. The larger the
flocculated material the greater its porosity tends to be
as well as it’s potential to interact with the external
environment producing a more efficient system.
Uptake of nutrients by microorganisms has been
found to be more efficient when flocculated than as a
single cell (Logan and Hunt, 1987). It would not be
surprising if these particles supported a diverse group
of organisms.
The settling tank (ST) particulate control resulted in a
slightly decreased but insignificant concentration of
nitrate in the respective raceways. This was most
likely a product of denitrification by bacteria
occurring in the sludge within the STs. In a similar
study raising
L. vannamei
, Holl et al (2011) reported
that super-intensive systems relying on FF for
particulate control resulted in greater accumulation of
nitrogen in the system. Further, their study utilized a
bead filter method of solids removal, which would
produce a denitrifying community of bacteria similar
to our STs. Particulate control methods such as
settling tank clarifiers have also been demonstrated to
promote increased shrimp growth (Lewis et al., 2009;
Ray et al., 2010). These two groups also suggest that
STs and bead filters are more efficient than FF for
maintaining adequate water quality with respect to
solids and potentially harmful NO
2
-N and NH
3
. In our
study there was a negligible difference between the
capacity of the FF and the ST to change the microbial
community within the raceway water column when
general bacterial community characteristics were used
as a proxy. This may have been an effect of using
pre-conditioned water.
3 Conclusion
Heterotrophically dominated super-intensive zero
exchange greenhouse covered RAS represent an