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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Waddell Mariculture Center, South Carolina Department of Natural Resources, 211 Sawmill Creek Rd., Bluffton, SC 29910, USA
Center for Coastal Environmental Health and Biomolecular Research, NOAA, 219 Fort Johnson Rd., Charleston, SC 29412, USA
Marine Resources Research Institute, South Carolina Department of Natural Resources, 217 Fort Johnson Rd., Charleston, SC 29412, USA
d
School of Forestry and Natural Resources, The University of Georgia, Athens, GA 30602, USA
e
Novus International, 5 Tomotley Ct., Charleston, SC 29407, USA
b
c
a r t i c l e
i n f o
Article history:
Received 25 April 2010
Received in revised form 8 October 2010
Accepted 13 October 2010
Keywords:
Biooc
Shrimp
Biomarkers
Epiuorescence
Intensive aquaculture
Microbial ecology
a b s t r a c t
Minimal-exchange, intensive culture systems require little, if any, water exchange and have high animal stocking
densities. Intensive nutrient inputs lead to an abundant community of microorganisms. These microbes are
partially contained within suspended biooc particles and contribute to water quality maintenance and
provision of supplemental nutrition to the culture species. Optimal function of minimal-exchange, intensive
systems is likely dependent on the structure of the microbial communities within them. This document offers a
short review of microbial groups important for intensive marine aquaculture and descriptions of three methods
for quantifying their abundance. The document also describes an experiment during which these methods were
used to monitor the effects of partial biooc removal on microbe abundance. The rst method uses light
microscopy, with the option of epiuorescence, along with a ranking system to enumerate the abundance of
microbial taxa. The second method exclusively uses epiuorescence to illuminate chlorophyll and cyanobacteria
pigments. Images are taken of each uorescing group of pigments and processed using image analysis software to
quantify the respective abundance of the two pigment types. Using the third method, changes in bacterial
abundance were determined by gas chromatographic measurement of bacteria-specic fatty acids in solvent
extracted water column lipids. Using these techniques, it was determined that removing solids from the culture
water signicantly (P 0.01) reduced the abundance of nematodes, rotifers, cyanobacteria, and bacteria.
Understanding microbial composition and the effects that management protocols have on that composition may
help system managers make better informed decisions.
2010 Elsevier B.V. All rights reserved.
1. Introduction
1.1. Minimal-exchange, intensive systems
Minimal-exchange, intensive aquaculture systems offer an environmentally attractive means of shrimp and sh production, allowing
for high density culture and little or no water exchange. With high
animal density comes intensive nutrient input in the form of feed.
When water is not exchanged with surrounding water bodies,
expensive nutrients from feed are retained within the system. In
minimal-exchange, intensive systems excess nutrients are assimilated
and mineralized by a dense microbial community in the water
column, thus alleviating potential toxicity (Avnimelech, 2006;
Bratvold and Browdy, 1998; Ebeling et al., 2006; Ray et al., 2009).
The microorganisms not only remove excess nutrients, but have been
implicated in nutritional provision for animals, including shrimp and
Corresponding author. Gulf Coast Research Laboratory, 703 East Beach Dr., Ocean
Springs, MS 39564, USA. Tel.: + 1 843 367 9407.
E-mail address: AndrewJRay@gmail.com (A.J. Ray).
0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.10.019
tilapia, that can result in improved growth rate, feed conversion ratio
(FCR), and weight gain (Azim and Little, 2008; Burford et al., 2004;
Moss and Pruder, 1995; Wasielesky et al., 2006).
1.2. Microorganism groups
Important microorganism groups in minimal-exchange, intensive
aquaculture systems include algae, zooplankton, and bacteria. Algae
utilize toxic total ammonia nitrogen (TAN), as well as less dangerous
nitrate-nitrogen and phosphate compounds to construct cellular
structures such as proteins and sugars. Various forms of algae, most
notably diatoms, are nutritious and can benet shrimp production by
contributing qualities such as essential amino acids and highly
unsaturated fatty acids (Ju et al., 2009; Moss et al., 2001). However,
potentially harmful algae can also be found in aquaculture systems.
One group that has been problematic for shrimp culture is
cyanobacteria, also known as blue-green algae. Some cyanobacteria
are capable of producing toxins that may diminish shrimp growth or
directly cause mortality (Alonso-Rodriguez and Paez-Osuna, 2003;
Zimba et al., 2006).
131
132
Fig. 1. Common components of minimal-exchange, intensive culture systems. (A) A biooc particle, which contains some of the microbial components in minimal-exchange,
intensive culture systems; some evidence suggests that adult shrimp may be able to consume these particles. (B) A high abundance of chlorophytes, also called green algae, (C) three
types of diatoms commonly observed at the Waddell Mariculture Center, (D) a heterotrophic dinoagellate has recently consumed green algae, (E) a nematode, (F) a rotifer. Images
B through F are organisms used for the visual microscopy quantication method. The cyanobacteria quantied for that method were best visualized using epiuorescence (Fig. 2).
include Alfaro et al. (2006), Budge et al. (2001), Kastovska et al. (2007),
Parrish et al. (2000), and Harvey and Macko (1997). Because these
specic fatty acids represent only a portion of the total FAs found in the
bacteria prole and the major FAs are those commonly found in other
organisms, these sums are not necessarily related to absolute bacteria
biomass. However, relative differences in the proportion of bacteria
from water samples can be readily assessed. Because the entire fatty acid
prole for the biooc is obtained with this method, the potential
availability of various fatty acids for animal nutrition may also be
assessed. For instance, the concentration of eicosapentaenoic acid (EPA),
an essential fatty acid, can be obtained simultaneously while evaluating
bacterial abundance.
133
Table 1
Scoring system used for visual microscopy quantication. Each of the six categories of
microorganisms was assigned a numerical score as a method of quantifying their
observed abundance.
Abundance
Numerical score
Description
Absent
Rare
0
1
Moderate
Common
2
3
Abundant
No organism observed
As few as one organism observed, as abundant as
being present in 4 elds of view
Present in between 5 and 9 elds of view
Present in every eld of view, occupies up to 50%
of each
Occupies over 50% of every eld of view
Fig. 2. Two images taken in the same location on a slide using different epiuorescence wavelengths. Image A was taken using the chlorophyll wavelength, which causes chlorophyll-a,
contained in both eukaryotic algae and cyanobacteria, to uoresce and image B was taken using the cyanobacteria wavelength, which causes only the cyanobacteria pigment phycocyanin
to uoresce. These images were assessed using image analysis software that allowed quantication of the amount of uorescing material in each image, reported in uorescing pixels
mL 1. In this particular example, there is a relatively high abundance of cyanobacteria, a sign of potentially poor culture conditions.
134
exposure time, and brightness so that the clearest possible image was
obtained.
Images were imported into ImageJ (Image Processing and Analysis in
JAVA, National Institutes of Health, Bethesda, Maryland, USA) as .TIFF
(Tagged Image File Format) les. Each picture was converted into an
eight bit, grayscale image, and then inverted. Contrast was enhanced by
normalizing the image to further improve image quality and maintain
continuity between images. The Entropy Threshold tool was applied to
help resolve the amount of material that was counted as uorescing.
Finally, the option analyze particles was turned on, which generated
the number of pixels in each image that were uorescing.
The mean number of uorescing pixels in the ten images of each
uorescence type on each slide was determined. Based on the number
of pixels uorescing, the number of pixels available in each image, and
the total volume of sample ltered, these numbers were converted to
uorescing pixels mL 1. To determine the abundance of photosynthetic pigments excluding cyanobacteria (eukaryotic algae only),
cyanobacteria uorescence was subtracted from the chlorophyll
uorescence value.
2.3. Bacterial fatty acid assessment by GC
Fifty mL of sample water was ltered through 47 mm diameter,
0.7 m pore-size, GF/F Whatman, borosilicate, glass microber lters
(Sigma-Aldrich Incorporated, St. Louis, Missouri, USA). Filters were
placed into a centrifuge tube containing 7 mL of chloroform and
immediately stored in a 20 C freezer for subsequent analysis. An
alternative to ltering that has proven successful is to centrifuge
samples and collect the pellet.
Lipids were extracted with a 2:1 ratio of chloroformmethanol
(Folch et al., 1957) as described by Budge and Parrish (2003) with
modication to accommodate sample size and the use of an internal
standard (IS). Briey, just prior to analysis, samples were allowed to
reach room temperature. After addition of 3.5 mL methanol and C23:0
methyl ester (ME) as an IS, the samples were vortexed until sample
material appeared to be fully dispersed, then sonicated for 10 min.
Aqueous sodium chloride (0.73%) was added with volume calculated
to achieve a chloroformmethanolwater ratio of 8:4:3 as specied by
Folch et al. (1957), and the contents were thoroughly mixed and
centrifuged. The lower chloroform layer was removed, the aqueous
layer washed with 3 2 mL of chloroform and chloroform extracts
combined. The chloroform was dried over anhydrous sodium sulfate,
ltered into a clean, dry tube and the chloroform removed under a
gentle stream of dry N2. The residue was dissolved in 1 mL hexane and
fatty acid methyl esters (FAMEs) prepared using BF3 (10% in
methanol, Supelco) as described by Metcalfe et al. (1966). The hexane
layer containing the FAMEs was concentrated for instrumental
analysis.
The FAMEs were analyzed by gas chromatography with splitless
injection. Separation was achieved with oven temperature programming as follows: Initial temperature 50 C with a 2 min hold, ramped at
20 min 1 to 150 C followed by a 1.25min 1 ramp to 220 C. With the
use of dual injection modules, samples were simultaneously analyzed on
dual DB225ms columns (50%-cyanopropylphenyl-methylpolysiloxane
30 m 0.25 mm, J&W Scientic, Folsom, California, USA) with detection
by ame ionization (FID) and mass spectrometry (MS). Scans from the
MS detector were used in conjunction with comparison of retention
times to those of known standards for peak identication. Correction
factors (determined by analysis of quantitative standards, GLC 85 and
GLC 411 (Nu-Chek Prep, Elysian, Minnesota, USA)), and verication
against theoretical correction factors were applied to fatty acid peak
areas from the FID. The amount of each fatty acid was calculated using IS
methodology and reported as g analyte L 1 of water. The sum of the
branched and odd chain fatty acids (iso 13:0, anteiso 13:0, C13:0, iso
14:0, anteiso 14:0, iso 15:0, anteiso 15:0, C15:0, C15:1, iso 16:0, iso 18:0,
iso 18:0, and C19:1) was used as a measure of the levels of bacteria in the
samples. Because the C17 fatty acids (C17:0 and C17:1n-8) are
commonly found in a variety of organisms they were omitted from the
sum.
2.4. Practical method application
2.4.1. Experimental setting and design
An experiment that assessed the effects of suspended solids
management on microbial communities in minimal-exchange, superintensive shrimp culture systems illustrates the employment of the
described microbial characterization techniques. The project was
carried out using 32 outdoor, polyethylene, 3.5 m diameter tanks,
lled with 6.3 m3 of ltered seawater at approximately 20 ppt salinity,
receiving blown air delivered through ceramic air stones. The
experimental system was the same as that described by Ray et al.
(2010) except that more tanks were used. The settling chambers
described by Ray et al. (2010) were used to remove suspended solids
from half of the experimental tanks, the other half received no solids
removal. Water was airlifted to the settling chambers, where
turbulence slowed and solids were allowed to settle, water near the
surface of the settling chambers owed back into shrimp culture
tanks. The suspended solids removed are synonymous with biooc
particles. Within each treatment (solids removal versus no solids
removal), half of the tanks were stocked at approximately
140 shrimp m 3 and the other half were stocked at approximately
420 shrimp m 3. The shrimp in this experiment were Litopenaeus
vannamei with a mean SE initial weight of 1.16 0.01 g. For the
purpose of this document, data from both shrimp stocking densities
were combined and only comparisons between treatments with and
without solids removal are made.
2.4.2. Water quality and microbial analyses
Temperature, dissolved oxygen, pH, and salinity were each
measured twice per day at approximately 0830 and 1600 h in each
tank using a YSI Model 556 (YSI Incorporated, Yellow Springs, Ohio,
USA). Total ammonia nitrogen (TAN), nitrite nitrogen (NO2-N),
nitrate nitrogen (NO3-N), orthophosphate (PO4), alkalinity (as
CaCO3), total suspended solids (TSS), volatile suspended solids
(VSS), and turbidity were each measured once per week in each
tank. Aliquots for these eight water quality parameters and for the
microbial analyses were taken from 500 mL samples collected just
below the water surface in each tank.
The three nitrogen species were measured using Hach Methods
8155, 8507, and 8039, respectively (Hach Company, 2003). Dilutions
were made as necessary due to some high nitrogen concentrations
and samples were read in a Hach DR/4000 V Spectrophotometer
(Hach Company, Loveland, Colorado, USA). Absorbance was measured
at 655 nm, 507 nm, and 500 nm for TAN, NO2-N, and NO3-N,
respectively, and concentrations were determined using standard
curves derived from the analysis of known standards. Because of the
variability that can be associated with these tests, two replicate
samples were assessed for TAN and NO2-N, and three replicates were
used for NO3-N. Orthophosphate concentration was measured using
the PhosVer 3 (ascorbic acid) method outlined in Hach Method 8048
(Hach Company, 2003). Absorbance was measured at 890 nm using
the Hach DR/4000 V Spectrophotometer. Dilutions were made for
orthophosphate analysis as necessary and two replicates were used
for each test. Known standards were analyzed with each sample set
during nitrogen and orthophosphate analyses to monitor accuracy.
Alkalinity was measured following the Potentiometric Titration to
Preselected pH procedure, outlined in section 2320 B by the APHA
(1989). Sodium bicarbonate was added as needed to maintain alkalinity
above 100 mg CaCO3 L 1 throughout the study. TSS and VSS were
measured following ESS Method 340.2 (ESS, 1993). Turbidity was
measured using a LaMotte model 2020e Turbidity Meter (LaMotte
Company, Chestertown, Maryland, USA).
135
Table 2
Overall water quality throughout the shrimp culture period. Data are presented as
mean standard error (range). Nitrate, orthophosphate, total suspended solids (TSS),
volatile suspended solids (VSS), and turbidity were all generally lower, and alkalinity
was generally higher in the solids removal treatment; Ray et al. (2010) showed that
settling chambers can signicantly affect these parameters.
Treatment
Solids removal
Temperature (C)
AM
27.0 0.4 (22.230.5)
PM
28.2 0.4 (23.531.7)
Dissolved oxygen (mg L 1)
AM
6.0 0.2 (3.89.2)
PM
6.8 0.2 (4.611.6)
pH
AM
7.5 0.1 (6.38.2)
PM
7.9 0.1 (6.48.6)
Salinity (g L 1)
AM
20.3 0.7 (13.925.4)
PM
20.4 0.7 (13.925.8)
1
Ammonia (mg TAN L )
0.4 0.2 (0.04.7)
Nitrite (mg NO2-N L 1)
0.5 0.2 (0.04.8)
1
Nitrate (mg NO3-N L )
38.6 9.4 (0.4266.9)
Orthophosphate
23.5 2.4 (5.950.4)
(mg PO4 L 1)
135.3 11.6 (40.0360.0)
Alkalinity
(mg CaCO3 L 1)
1
397.0 42.5 (60.01065.0)
TSS (mg L )
VSS (mg L 1)
270.1 24.5 (43.3573.3)
Turbidity (NTU)
39.9 4.7 (5.2104.1)
No solids removal
26.8 0.4 (21.330.3)
28.1 0.4 (22.631.7)
6.1 0.2 (3.49.3)
6.6 0.2 (4.710.5)
7.4 0.1 (6.28.2)
7.7 0.1 (6.48.6)
19.7 0.7 (13.724.9)
19.7 0.7 (13.724.8)
0.3 0.1 (0.04.5)
0.3 0.2 (0.06.2)
53.0 12.6 (0.7293.3)
40.7 6.4 (10.2115.8)
127.5 11.1 (40.0250.0)
616.0 57.9 (51.71215.0)
427.7 44.9 (26.7880.0)
69.8 5.1 (6.7111.6)
80
60
40
20
136
A.J. Ray et al. / Aquaculture 310 (2010) 130138
b
4
Week
10
h
1200
1000
800
600
400
200
12
Solids Removal
No Solids Removal
Week
10
12
No solids removal
Chlorophytes (visual)
3.98 0.02 (3.004.00)
3.97 0.02 (3.004.00)
Diatoms (visual)
0.14 0.04 (0.002.00)
0.21 0.04 (0.002.00)
Dinoagellates (visual)
0.96 0.11 (0.004.00)
0.74 0.09 (0.004.00)
Nematodes (visual)
0.24 0.04 (0.002.00)a
0.57 0.05 (0.002.00)b
Rotifers (visual)
1.66 0.07 (0.004.00)
2.16 0.08 (0.004.00)b
Cyanobacteria (visual)
2.58 0.87 (1.004.00)a
3.12 0.05 (1.004.00)b
Cyanobacteria
50.612.57 (32.8698.60)b
39.101.73 (20.4165.83)a
(pixels mL 1 106)
Bacteria fatty acid
828.13 53.02
395.00 23.08
indicators (g L1)
(240.001700.00)b
(140.001000.00)a
Fatty acid bacterial indicator concentrations were analyzed based on the values during
the nal week of the experiment using a MannWhitney Rank Sum test. All other
abundance data were assessed using a repeated measures ANOVA which evaluated
differences in abundance throughout the experiment.
137
Fig. 3. Graphs a through f depict the relative abundance of chlorophytes, diatoms, dinoagellates, nematodes, rotifers, and cyanobacteria, respectively, based on light microscopy
abundance quantication; the scoring system for this method is described in Table 1. Graph g depicts the results of cyanobacteria quantication using epiuorescence and image
analysis, shown as uorescing pixels per milliliter. Graph h represents the concentration of branched and odd chain fatty acids, used as bacterial indicators to assess the relative
abundance of bacteria. Week four is the rst week that settling chambers were used to remove suspended solids and this began the period that changes in microbial abundance were
explored.
138
4. Conclusion
Assessing the structure of the microbial communities upon which
minimal-exchange, intensive culture systems are heavily reliant is an
important step in understanding and predicting system function. This
document offers strategies of evaluation useful for the microbial
communities and conditions that occur in minimal-exchange, intensive
culture systems. These techniques were used to successfully demonstrate the effects that suspended solids removal can have on the
abundance of certain microorganisms. By removing suspended solids,
the abundance of nematodes, rotifers, cyanobacteria, and bacteria were
all signicantly reduced while chlorophyte, diatom, and dinoagellate
abundances were not signicantly affected. These results indicate the
effects that relatively simple management strategies can have on the
microbial communities in intensive aquaculture systems.
Acknowledgements
The techniques described in this document were adapted and
applied at the Waddell Mariculture Center in Bluffton, South Carolina,
USA as part of a Master of Science Thesis (Ray, 2008) through the College
of Charleston, Charleston, South Carolina, USA. Thank you Heidi Atwood,
Kirsten Ayers, Maggie Broadwater, Amy Dickson, Sylvia Galloway, Traci
Holstein, Martin Jones, Peter Kingsley-Smith, Beth Lewis, Brad McAbee,
Mark McConnel, Stacy Ray, Andrew Shuler, Jesus Venero, Joe Wade,
David White, and the staff of the Waddell Mariculture Center. Any
mention of a trademark or product manufacturer is in no way an
endorsement of that business or a suggestion that one product is
superior to another. This research was supported by grants from the
USDA Integrated Organic Program and the USDA US Marine Shrimp
Farming Program. This is contribution number 657 from the South
Carolina Department of Natural Resources Marine Resources Research
Institute.
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