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Characterization of microbial communities in

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DOI: 10.1016/j.aquaculture.2010.10.019

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Contents lists available at ScienceDirect

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

Characterization of microbial communities in minimal-exchange, intensive

aquaculture systems and the effects of suspended solids management
Andrew J. Ray a,, Gloria Seaborn b, John W. Lefer c, Susan B. Wilde d, Alisha Lawson a, Craig L. Browdy e
a
Waddell Mariculture Center, South Carolina Department of Natural Resources, 211 Sawmill Creek Rd., Bluffton, SC 29910, USA
b
Center for Coastal Environmental Health and Biomolecular Research, NOAA, 219 Fort Johnson Rd., Charleston, SC 29412, USA
c
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

a r t i c l e i n f o a b s t r a c t

Article history: Minimal-exchange, intensive culture systems require little, if any, water exchange and have high animal stocking
Received 25 April 2010 densities. Intensive nutrient inputs lead to an abundant community of microorganisms. These microbes are
Received in revised form 8 October 2010 partially contained within suspended biooc particles and contribute to water quality maintenance and
Accepted 13 October 2010
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
Keywords:
Biooc
short review of microbial groups important for intensive marine aquaculture and descriptions of three methods
Shrimp for quantifying their abundance. The document also describes an experiment during which these methods were
Biomarkers used to monitor the effects of partial biooc removal on microbe abundance. The rst method uses light
Epiuorescence microscopy, with the option of epiuorescence, along with a ranking system to enumerate the abundance of
Intensive aquaculture microbial taxa. The second method exclusively uses epiuorescence to illuminate chlorophyll and cyanobacteria
Microbial ecology 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 tilapia, that can result in improved growth rate, feed conversion ratio
(FCR), and weight gain (Azim and Little, 2008; Burford et al., 2004;
1.1. Minimal-exchange, intensive systems Moss and Pruder, 1995; Wasielesky et al., 2006).

Minimal-exchange, intensive aquaculture systems offer an envi- 1.2. Microorganism groups

ronmentally 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).
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).

0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.10.019
 A.J. Ray et al. / Aquaculture 310 (2010) 130138
Zooplankton consume algae and bacteria and can play an important
role in the transfer of nutrients from primary producers to secondary
consumers. Zooplankton such as rotifers can contribute signicantly to
the protein and energy requirements of shrimp (Focken et al., 1998).
However, zooplankton also consume oxygen and can cause a reduction
in alkalinity through respiration.
Two functional categories of bacteria are primarily responsible for
water quality maintenance in minimal-exchange, intensive systems:
heterotrophic ammonia-assimilative and chemoautotrophic nitrifying
bacteria (Ebeling et al., 2006; Hargreaves, 2006). The heterotrophic
group removes TAN from the water column to build cellular proteins.
Nitrifying bacteria acquire energy through the oxidation reactions of
TAN to nitrite-N and nitrite-N to nitrate-N, the latter of which is much
less toxic than its predecessors in this sequence. Both the bacterial
assimilation and nitrication processes consume oxygen and reduce
alkalinity, thereby often requiring the supplementation of those two
components.
A portion of the microbial community in minimal-exchange,
intensive culture systems is contained on or within particles,
commonly called biooc (Fig. 1A). Biooc is composed of a variety
of microorganisms, uneaten feed, feces, and detritus, and the particles
are kept in suspension with water propulsion and aeration. Biooc
offers numerous ecological advantages for microbes, including
protection from predators, direct access to nutrients, and necessary
substrate area (De Schryver et al., 2008). Biooc is thought to provide
a packaging of microbial proteins and nutrients that is directly
accessible to culture animals (Avnimelech, 2009; Burford et al., 2004).
Biooc technology (BFT) can be considered a culture technique in
which water quality is maintained and in situ animal feed is
simultaneously produced in the form of biooc particles (Crab et al.,
2007). Although biooc can be benecial, some level of control over
the concentration of particles is likely required for optimal system
performance. Ray et al. (2010) demonstrated that shrimp biomass
production (kg m 3) was increased 41% when biooc concentration
was managed through the use of external settling chambers. They also
showed a 60% reduction in nitrate-nitrogen concentration and a 61%
reduction in phosphate concentration when biooc concentration
was managed.
To optimize system function, a benecial microbial community
should be developed and sustained. This requires knowledge of
microbial community composition and the ability to monitor changes.
By investigating microbial composition, inferences may be made of
the underlying reasons for differences in community structure and the
effects of management techniques, such as biooc removal, on that
structure. Monitoring of microorganisms may help characterize
opportunities for supplemental nutrition, and help managers recog-
nize the occurrence of problematic organisms, thereby guiding
informed management decisions.
The purpose of this document is to describe three techniques that
can be used to characterize microbial communities in minimal-
exchange, intensive aquaculture systems. To illustrate these techni-
ques in a practical application they were used in an experiment that
explored the effects of removing suspended solids (biooc) on
microorganism abundance.
1.3. Visual microscopy abundance quantication
In this case, abundance quantication describes a system of
ranking the relative abundance of organismal groups. Organisms are
categorized and ranked according to a predetermined scale, similar to
the previous work of researchers. Newall et al. (2006) used such a
scale to rank the abundance of diatoms in a river system. DeVantier
et al. (1998) described trained researchers who used a subjective scale
to rank macro-invertebrate abundance.
The current document explores the use of an abundance quanti-
cation system to describe the relative abundance of six microorganism
131
categories important and common to intensive marine aquaculture
systems: chlorophytes (green algae), diatoms, dinoagellates, nema-
todes, rotifers, and cyanobacteria. The abundance of organisms
belonging to these categories was assessed using light and uorescence
microscopy with live samples. The categories used were selected based
on the authors' experience with shrimp culture systems at the Waddell
Mariculture Center (WMC) in Bluffton, South Carolina, USA. Other
categories may be needed, depending on location and endemic biota.
Broad classications such as these save time, minimize the amount of
taxonomical training required by the analyst, and can be less susceptible
to biases (DeVantier et al., 1998).
This technique can be performed with an ordinary compound light
microscope, although epiuorescence technology is advantageous.
Many of the cyanobacteria and algal cells in intensive culture systems
are small and become concealed by particles in the water, making
visualizing and discerning between these two groups difcult.
Epiuorescence can illuminate the chlorophyll-a pigment, contained
by both eukaryotic algae and cyanobacteria, and can uoresce the
cyanobacteria pigment phycocyanin exclusively. This facilitates ease
of observation and differentiation between eukaryotic algae and
cyanobacteria. Kastovska et al. (2007) described using epiuores-
cence in this manner to discern between the two groups. Epiuor-
escence functions by powering a broad-spectra mercury light bulb;
lters are then used to select the specic wavelengths of light that
cause chlorophyll pigments, cyanobacteria pigments, or stains such as
46-diamidino-2-phenylindole (DAPI), a DNA stain, to uoresce. An
advantage that epiuorescence has over other pigment analyses is
that many types of lters are available to discern a variety of biological
components, not limited to pigments.
An inverted compound microscope allows the observer to look up,
through a larger sample than would be possible with an ordinary
compound microscope. This technology allows the microorganisms in
a relatively large sample (3 mL in this case) to settle down onto one
plane. The three-dimensional biooc particles that are common in
intensive culture systems are better visualized using an inverted
scope because the sample is not compressed onto a at slide.
1.4. Epiuorescence with image analysis quantication
This procedure utilizes the epiuorescence technology described
above, except that rather than using live samples, images of uorescing
chlorophyll (chlorophyll-a) and cyanobacteria pigment (phycocyanin)
are captured using preserved samples on at slides. An image of
uorescing chlorophyll is taken, and an image of cyanobacteria pigment
is then taken in the same location on the slide. This enables a direct
comparison between chlorophyll (including cyanobacteria) and cyano-
bacteria pigment exclusively. The details of wavelengths used for
pigment excitation and uorescence emission are given in Section 2.1.
To determine the relative abundance of uorescing pigments in each
image, the pictures are processed using the computer program ImageJ.
Researchers have used this technique in other applications.
Selinummi et al. (2005) processed DAPI-stained images with image
analysis software to quantify bacterial cells. Verity and Sieracki (1993)
outlined the general procedures for measuring plankton biomass
using this methodology.
1.5. Bacterial fatty acid assessment by gas chromatography
In this method, gas chromatography with mass spectrometry
(GCMS) is used to separate, identify, and quantify fatty acids (FAs)
derived from the lipid component obtained by solvent extraction of
biooc. The sum of odd and branched chain fatty acids thus obtained
serves as a measure of the relative abundance of bacteria. Odd and
branched chain fatty acids, produced primarily by both aerobic and
anaerobic bacteria, are widely accepted as biomarkers for bacteria and
sums are frequently used to estimate bacterial contributions; examples
132 A.J. Ray et al. / Aquaculture 310 (2010) 130138

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.
 A.J. Ray et al. / Aquaculture 310 (2010) 130138
2. Materials and methods
2.1. Visual microscopy abundance quantication
All microscopy was performed with an Olympus IX-51 Inverted
Microscope (Olympus America Incorporated, Center Valley, Pennsylvania,
USA) outtted with epiuorescence technology. It was equipped with a
lter which enabled chlorophyll-a excitation at 480 nm and uorescence
emission at 660 nm. The microscope also had a lter enabling excitation
of the cyanobacterial pigment phycocyanin at 570 nm and uorescence
emission at 620 nm. The chlorophyll and cyanobacteria pigment lters
were used extensively to distinguish between eukaryotic algae and
cyanobacteria, to illuminate minute cells, and to visualize cells that
otherwise would be hidden by particles in the water, they were also used
for the method described in Section 2.2 of this document.
Three mL of thoroughly mixed sample water was placed in one
5 mL well of a two square well Chamber Slide (Lab TekNalge Nunc
International, Naperville, Illinois, USA). Each sample was carefully
examined by moving the stage of the microscope from the bottom of
the well to the top along the y-axis, then horizontally across the x-axis
and back down from the top of the well to the bottom along the y-axis.
The well was then examined from side to side across the x-axis and
one corner was inspected. The microscope stage was moved along the
described course and randomly stopped at ten unique elds of view to
screen for organisms in each sample. The observer helped to ensure
randomness in choice of elds of view by not looking through the
microscope oculars when moving the slide.
During visual microscopy abundance quantication, six groups of
organisms were recorded: chlorophytes, diatoms, dinoagellates,
nematodes, rotifers, and cyanobacteria (Figs. 1B through E and 2B).
The presence or absence of each group of organisms was noted and all
groups were ranked numerically, relative to their abundance
following the quantication system presented in Table 1.
2.2. Epiuorescence with image analysis quantication
Epiuorescence and image analysis software were used to quantify
the abundance of chlorophyll pigment and cyanobacteria pigment.
Samples were preserved in 2% gluteraldehyde solution (nal
concentration). The gluteraldehyde xed samples were stored in a
10 C refrigerator to be examined at a later date.
A dilution was created by combining 2.0 mL preserved sample water
with 3.0 mL of sterile seawater. The solution was then mixed using a
vortex mixer (Fisher Scientic, Pittsburg, Pennsylvania, USA) for 30 s to
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.
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 0 No organism observed
Rare 1 As few as one organism observed, as abundant as
being present in 4 elds of view
Moderate 2 Present in between 5 and 9 elds of view
Common 3 Present in every eld of view, occupies up to 50%
of each
Abundant 4 Occupies over 50% of every eld of view
break up biooc particles. The diluted sample was ltered using a
25 mm diameter, black 0.22 m pore-size, Poretics polycarbonate lter
(GE Osmonics, Minnetonka, Minnesota, USA) placed atop a 0.7 m pore-
size GF/F glass microber lter (Whatman, Maidstone, Kent,
United Kingdom). The GF/F lter acted as a cushion to protect the
polycarbonate lter, upon which ltered material collected. The
polycarbonate lter was transferred to a pre-cleaned 1.0 mm thick
glass microscope slide (VWR International, West Chester, Pennsylvania,
USA). Several drops of Type FF immersion oil (Cargille Laboratories,
Cedar Grove, New Jersey, USA) were applied between the slide, lter,
and a cover slip to prevent desiccation.
Photo-micrographs were taken of the prepared slides using the
Olympus IX51 inverted microscope with an attached four megapixel
digital camera (Qimaging, Surrey, British Columbia, Canada)
connected to a desktop computer (Dell Corporation, Round Rock,
Texas, USA). A slide was placed on the microscope stage and the
chlorophyll (chlorophyll-a) uorescence lter was engaged. Without
looking at the image created, the stage was moved so that light
penetrated the lter at approximately the bottom center point of
ltered material and a photograph was taken (Fig. 2A). After taking an
image with the chlorophyll lter engaged, the cyanobacteria
(phycocyanin) lter was engaged without moving the microscope
stage and another image was taken (Fig. 2B) so that two images, one
of each uorescence type was taken in a single location. Both
epiuorescence lters are described in more detail in Section 2.1 of
this document. The stage was then moved without looking at the
image so that the slide moved upward, along the y-axis, into a unique
eld of view, and the same set of two different uorescence images
was captured. This was performed ten times so that a total of ten
chlorophyll and ten cyanopigment images were created per slide.
Picture quality adjustments were made as needed such as focus,
134 A.J. Ray et al. / Aquaculture 310 (2010) 130138
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 photosyn-
thetic 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 program-
ming 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, super-
intensive 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).
 A.J. Ray et al. / Aquaculture 310 (2010) 130138 135

At week four of this 13 week study, settling chamber operation Table 2

and sampling for microbial abundance data was initiated. Samples Overall water quality throughout the shrimp culture period. Data are presented as
mean standard error (range). Nitrate, orthophosphate, total suspended solids (TSS),
were collected and analyzed weekly, except the bacterial fatty acid volatile suspended solids (VSS), and turbidity were all generally lower, and alkalinity
assessment, which was conducted at 3-week intervals. was generally higher in the solids removal treatment; Ray et al. (2010) showed that
settling chambers can signicantly affect these parameters.
2.4.3. Statistical analyses
Treatment
The data collected were analyzed using SigmaPlot Version 11 (Systat
Solids removal No solids removal
Software, Incorporated, San Jose, California, USA). One-way Repeated
Measures (RM) ANOVAs were used to analyze the microbial abundance Temperature (C)
data. However, because there were only four samples of bacterial AM 27.0 0.4 (22.230.5) 26.8 0.4 (21.330.3)
PM 28.2 0.4 (23.531.7) 28.1 0.4 (22.631.7)
indicators, the power of the RM ANOVA for this data set was weak. Dissolved oxygen (mg L 1)
As a result, and because the data did not have equal variance, a AM 6.0 0.2 (3.89.2) 6.1 0.2 (3.49.3)
MannWhitney Rank Sum Test was used to compare the bacterial data PM 6.8 0.2 (4.611.6) 6.6 0.2 (4.710.5)
from only the nal sample date between tanks with and without solids pH
AM 7.5 0.1 (6.38.2) 7.4 0.1 (6.28.2)
removal. A Pearson Product Moment Correlation Test was used to
PM 7.9 0.1 (6.48.6) 7.7 0.1 (6.48.6)
test whether cyanobacteria abundance data collected using visual Salinity (g L 1)
microscopy and those collected using epiuorescence were correlated. AM 20.3 0.7 (13.925.4) 19.7 0.7 (13.724.9)
PM 20.4 0.7 (13.925.8) 19.7 0.7 (13.724.8)
1
3. Results and discussion Ammonia (mg TAN L ) 0.4 0.2 (0.04.7) 0.3 0.1 (0.04.5)
Nitrite (mg NO2-N L 1) 0.5 0.2 (0.04.8) 0.3 0.2 (0.06.2)
1
Nitrate (mg NO3-N L ) 38.6 9.4 (0.4266.9) 53.0 12.6 (0.7293.3)
By the end of this experiment, tanks with suspended solids Orthophosphate 23.5 2.4 (5.950.4) 40.7 6.4 (10.2115.8)
removal had a mean total suspended solids (TSS) concentration 28% (mg PO4 L 1)
lower than tanks without suspended solids removal (567 mg L 1 Alkalinity 135.3 11.6 (40.0360.0) 127.5 11.1 (40.0250.0)
(mg CaCO3 L 1)
versus 785 mg L 1, respectively). Overall water quality conditions 1
TSS (mg L ) 397.0 42.5 (60.01065.0) 616.0 57.9 (51.71215.0)
during the experiment are presented in Table 2. VSS (mg L 1) 270.1 24.5 (43.3573.3) 427.7 44.9 (26.7880.0)
Turbidity (NTU) 39.9 4.7 (5.2104.1) 69.8 5.1 (6.7111.6)
3.1. Visual microscopy abundance quantication

The results of this technique indicate that systems with solids

management had signicantly lower abundances of nematodes
(P=0.010), rotifers (P=0.004), and cyanobacteria (P=0.004) compared
to those without solids removal. By the last sample period of the study,
nematode abundance had been reduced by 60.0%, rotifer abundance by
18.6%, and cyanobacteria abundance had been reduced by 22.7% through
the use of settling chambers. The abundance of the other groups assessed
with visual microscopy: chlorophytes, diatoms, and dinoagellates, were
not signicantly affected by solids removal. The abundance of organisms
over time is presented in Fig. 3. Table 3 gives the overall mean, standard
error, and range of the abundance of each group throughout the study,
with the exception of bacterial abundance for which only data from the
last sample date is reported.
Chlorophytes were highly abundant throughout the study (Fig. 3a),
regardless of whether solids were managed, and most belonged to the
genus Oocystis sp. Based on visual observations, these organisms
appeared to be buoyant, perhaps preventing the cells from settling in
the settling chambers. Moreno-Ostos et al. (2008) observed that
Oocystis sp. were neutrally buoyant, which prevented settling. Diatoms
were present in low abundance near the beginning of the study and
became less common as the experiment progressed, regardless of
whether solids were removed (Fig. 3b). Shrimp may have consumed
a portion of the diatom community; previous authors have shown
that these nutritious algae are an important food item for shrimp (Moss
et al., 2001; Sullivan and Moncreiff, 1990). Some competition
for resources may have existed between chlorophytes and diatoms,
with chlorophytes being favored in these experimental systems.
Dinoagellates had a generally low relative abundance throughout the
study, regardless of whether solids were removed (Fig. 3c). These
organisms are free-swimming which may have prevented them from
settling in the settling chambers, potentially helping to explain why
their abundance was not different between the treatments.
The organisms that were reduced in abundance by removing solids
appeared to be closely associated with the biooc particles. Nematodes
(Fig. 3d) and rotifers (Fig. 3e) were seen almost exclusively grazing on
and within the particles and cyanobacteria were also principally located
within biooc particles. Both nematodes and rotifers consume oxygen
and reducing their abundance may consequentially reduce biochemical
oxygen demand, leaving greater oxygen resources available for the
target crop. They also ingest algae and bacteria, suggesting that a change
in the abundance of these primary consumers could have a top-down
trophic effect. However, the two zooplankton groups are thought to be
nutritious prey for shrimp. In terms of potential nutrition, a decrease in
the abundance of these zooplankton may not be desirable. The fact that
cyanobacteria (Fig. 3f) were removed with solids management is an
encouraging reason to manage solids concentration. Cyanobacteria can
hinder aquaculture efforts by producing lethal toxins and contributing
to off-avors of animal esh (Alonso-Rodriguez and Paez-Osuna, 2003;
Zimba et al., 2006).
The visual microscopy abundance quantication method is a
relatively simple technique. An analyst can be taught to distinguish
between broad groupings of organisms, such as the ones described
here, with minimal training.
Visual microscopy may also be used to perform algal and
zooplankton counts. This is achieved by counting the number of
organisms in a given area of a specially designed microscope slide or
hemacytometer, then extrapolating to determine the number of
organisms per volumetric unit. However, in limited exchange intensive
culture systems, microorganisms are more abundant than in typical,
natural aquatic systems as a result of hypereutrophic conditions. If
assessments of multiple culture units at a shrimp production facility or
research center are needed, routinely counting microorganisms in these
systems would likely be unreasonably time consuming.
It is best to use live samples for visual microscopy quantication as
most preservatives will damage organisms such as dinoagellates,
potentially rendering them unrecognizable. Using live samples allows
real-time assessment of the microbial community. Routinely
performing this procedure can alert the analyst to shifts in community
structure such as an algae crash or dinoagellate bloom, either of
which may have negative impacts on shrimp production. Recognizing
that such situations are the cause of diminished shrimp growth can
save system managers from making the mistake of over-feeding
shrimp to compensate for slow growth (Ray et al., 2009). Advanta-
geous events can also be detected, such as a bloom of diatoms, during
which time enhanced shrimp growth may be expected. Knowing the
 136

e
c

g
Cyanobacteria (fluorescence) pixels mL - 1 x 10 6 Rotifer Relative Abundance Dinoflagellate Relative Abundance Chlorophyte Relative Abundance
a

0
20
40
60
80
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4

4
6
8
Week
10
12
f

h
d
b

g L - 1 Bacterial Indicator Fatty Acids Cyanobacteria (visual) Relative Abundance Nematode Relative Abundance Diatom Relative Abundance

0
200
400
600
800
1000
1200

Solids Removal
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4

No Solids Removal
4
A.J. Ray et al. / Aquaculture 310 (2010) 130138

6
8
Week
10
12
 A.J. Ray et al. / Aquaculture 310 (2010) 130138
Table 3
The overall relative abundance of each microbial group throughout the study. Data are
presented as mean standard error (range). Different letters within a row indicate
signicant differences (P 0.01) between the two treatments.
Treatment
Solids removal 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 39.101.73 (20.4165.83)a 50.612.57 (32.8698.60)b
(pixels mL 1 106)
Bacteria fatty acid 395.00 23.08 828.13 53.02
indicators (g L1) (140.001000.00)a (240.001700.00)b
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.
abundance of nutritionally benecial or potentially toxic organisms
that can reduce growth may help system managers better calculate
feed rations based on expected shrimp growth. A drawback to this
technique is that it can be somewhat subjective; care must be taken to
eliminate bias and monitor consistency among analysts.
3.2. Epiuorescence with image analysis quantication
Cyanobacteria pigment abundance, as determined by epiuores-
cence with image analysis quantication, was signicantly reduced
during the study (P b 0.001) through the practice of solids removal
(Fig. 3g). By the nal week of the study, cyanobacteria pigments were
reduced by 17.1% with solids removal. The abundance of eukaryotic
algae pigments (chlorophyll uorescence minus cyanobacteria uo-
rescence) was not signicantly affected by solids removal.
The results of the epiuorescence with image analysis quantica-
tion method substantiated those of the visual microscopy assessment.
The relative abundance of eukaryotic algae (in this case chlorophytes,
diatoms, and photoautotrophic dinoagellates) did not appear to be
affected by solids removal. Eukaryotic algal groups other than
chlorophytes, diatoms, and photoautotrophic dinoagellates were
rarely seen during the current experiment. The abundance of the
eukaryotic algal groups that were monitored with visual microscopy
seemed unaffected by solids removal, the uorescence of eukaryotic
chlorophyll-a likewise appeared unchanged.
A signicant reduction in cyanobacteria abundance with solids
removal was indicated by both visual microscopy and by epiuores-
cence with image analysis. This correspondence helps to validate the
methods; however, cyanobacteria abundance data collected with the
two methods were not statistically correlated. Samples for the two
methods were collected on different days of the week, perhaps
hindering correlation. Also, the two methods measure different
things. Visual microscopy measures the abundance of cells, while
epiuorescence measures the abundance of uorescing pigments
within those cells. Therefore, epiuorescence may comparatively
underestimate cyanobacteria abundance, but still elucidate changes in
abundance.
This technique allows for direct comparison between eukaryotic
algae and cyanobacteria. Unlike visual microscopy, samples can be
preserved for later analysis, potentially helping to overcome time
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.
137
constraints. Images of live or immediately preserved samples can be
used for real-time, or near real-time assessments with this technique.
Preserved samples likely reduce errors associated with measuring
mobile organisms more than once.
Spectrophotometric analysis of pigments is another common means
of assessing pigment concentrations in water samples. An advantage
that epiuorescence has is that only active pigments are detected,
whereas spectrophotometry can include the inactive pigments of dead
cells. An alternative to using image analysis software with this technique
would be to count the uorescing microorganisms and arrive at a
concentration per volumetric unit. However, as with light microscopy
counts, this becomes exceedingly labor intensive. Other authors have
used epiuorescence to perform such cell counts (Kawahara et al., 2009)
or simply to visualize microorganisms (Azim and Little, 2008). However,
the technique presented here offers a means of quantication that is
more resistant to human bias and is relatively expedient.
3.3. Bacterial fatty acid assessment by GC
By the end of the experiment, fatty acid bacterial biomarkers were
signicantly reduced (P b 0.001) by solids removal (Fig. 3h). In systems
with solids management, the nal mean concentration of bacterial fatty
acids was 60.3% lower than in those systems without solids manage-
ment (411.3 44.1 g L 1 versus 1036.9 119.0 g L 1, respectively).
Table 3 shows the overall mean concentrations of bacterial fatty acids
throughout the study.
By reducing the abundance of bacteria in shrimp culture systems
respiration may be reduced, in turn reducing the total oxygen demand
of the system and the need for expensive supplemental oxygen
injection. Although these systems rely, in large part, on bacteria for
water quality maintenance, excess bacteria may reduce the amount of
oxygen available to shrimp. There was no indication of water quality
deterioration during this experiment (Table 2), suggesting that an
abundance of microbes sufcient to cycle harmful nutrients remained
in the systems. Bacteria associated with biooc particles may provide
a packaging of valuable nutrients (De Schryver et al., 2008). However,
some groups of bacteria are pathogenic to shrimp. Therefore, further
explorations of the effects of partial bacterial exclusion on shrimp
production are likely needed.
The patterns by which bacterial abundance changes over time and
with respect to management protocol have important implications for
system function; the described technique of bacterial fatty acid
assessment is effective at documenting those changes. Ju et al. (2008)
described using HPLC to assess muramic acid levels which served as a
biomarker to infer bacterial abundance. The same method to assess
muramic acid abundance can be used to determine amino acid
abundances. Assessing either amino acid or fatty acid proles of biooc
can also be useful for evaluating nutritional qualities of the material. In
this respect, both methods offer dual purposes. In terms of cost, both
amino acid and fatty acid characterizations require comparably
expensive equipment. This likely makes both methods better suited to
research interests; however, infrequent sampling by an outsourced
laboratory is an option for commercial aquaculture operations as well.
Both methods are likely equal in their effectiveness, but it is important to
develop a variety of tools for microbial assessment. The ability to use
multiple techniques allows more specic questions to be addressed, in
this case nutritional as well as biological. In the future it may be desirable
to describe entire fatty acid proles of microbial communities in
aquaculture systems, allowing for a more complete characterization of
those communities.
138 A.J. Ray et al. / Aquaculture 310 (2010) 130138
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 demon-
strate 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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