JOURNAL OF THEWORLD AQUACULTURE SOCIETY

Vol. 43, No. 6December, 2012

Effects of Biofloc Reduction on Microbial Dynamics inMinimal-exchange, Superintensive Shrimp, Litopenaeus vannamei ,

Culture Systems

Andrew J. Ray1,2

Waddell Mariculture Center, South Carolina Department of Natural Resources, 211 SawmillCreek Road, Bluffton, South Carolina 29910, USA

Gloria Seaborn

Center for Coastal Environmental Health and Biomolecular Research, NOAA, 219 FortJohnson Road, Charleston, South Carolina 29412, USA

Luis Vinatea

Laboratorio de Camaroes Marinhos, Departamento de Aquicultura, Universidade Federal deSanta Catarina, CEP 88040-900, Florianopolis, Santa Catarina, Brazil

Craig L. Browdy

Novus International, 5 Tomotley Ct., Charleston, South Carolina 29407, USA

John W. Leffler

Marine Resources Research Institute, South Carolina Department of Natural Resources, 217Fort Johnson Road, Charleston, South Carolina 29412, USA

AbstractThe microbial community in minimal-exchange, superintensive culture systems should be managed

to cycle nutrients and enhance production. This paper explores the effects of biofloc concentrationreduction and a fish-free diet on several microbial community characteristics. In 16, 3.5-m diameter,71-cm deep outdoor tanks, shrimp were stocked at 460/m3. Eight of the tanks received a fish-free,plant-based feed and eight received a conventional feed containing fishmeal and fish oil. Withineach diet type, biofloc concentration was reduced in four of the tanks and was not reduced in theother four tanks. Photosynthetically active radiation (PAR) extinction coefficients, photosyntheticoxygen production, chlorophyll-a (chl-a) concentrations, pheophytin-a (pheo-a) concentrations, andthe sum of odd and branched chain fatty acid concentrations as a bacterial abundance indicator (BAI)were measured. Biofloc reduction significantly (P ≤ 0.003) decreased PAR extinction coefficients,chl-a concentration, pheo-a concentration, and BAI concentration, while significantly increasingphotosynthetic oxygen production. Diet did not significantly affect (P > 0.05) any of these measuredparameters. The observed changes in microbial community characteristics corresponded with, andmay help to explain, significantly improved shrimp feed conversion ratios, growth rate, final weight,and biomass yield in the tanks with biofloc reduction.

1 Corresponding author.

2 Present address: The University of Southern Mississippi, Gulf Coast Research Laboratory, 703 East Beach Drive,Ocean Springs, Mississippi 39564, USA.

© Copyright by the World Aquaculture Society 2012

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BIOFLOC REDUCTION AND MICROBIAL DYNAMICS 791

In minimal-exchange, superintensive culturesystems, little or no water is exchanged with thesurrounding environment, substantially reduc-ing the risk of disease introduction, pollutantdischarge, and animal escapement. Reducedwater use may also permit inland culture ofmarine species, providing fresh seafood tometropolitan markets and utilizing less expen-sive land than that of coastal areas (Browdy andMoss 2005).

In minimal-exchange, superintensive culturesystems, a dense microbial community includ-ing algae, bacteria, zooplankton, and fungidevelops and is responsible for detoxifyingand cycling otherwise harmful nutrients. Themicroorganisms may also provide nutritionallybeneficial components to culture animals suchas amino acids and vitamins (Moss et al. 2006;Ju et al. 2008). A portion of the microbialcommunity is contained within, and adherentto, biofloc particles. Biofloc particles vary insize but are visible with the naked eye; theyare composed of microbes, uneaten feed parti-cles, feces, and detritus, all held together byphysiochemical forces and polymer matrices(Avnimelech 2012).

Microalgae assimilate ammonia and nitratein aquatic systems and utilize the nitrogenfrom these compounds to build cellular pro-teins. Some algae, most notably diatoms,may provide nutritional benefits for shrimp(Ju et al. 2009). Many heterotrophic bacte-ria can assimilate ammonia to form cellu-lar proteins and the resulting biomass maythen be available for shrimp consumption (DeSchryver et al. 2008). Another bacterial group,chemoautotrophic nitrifiers, oxidizes ammoniato ultimately form the much less toxic nitratecompound (Kuhn et al. 2010).

Water containing biofloc particles and theassociated microbiota can significantly improveshrimp growth compared to water lacking thesecomponents (Moss 1995; Wasielesky et al.2006). However, controlling the concentra-tion of biofloc particles by using simple side-stream settling chambers can improve shrimpproduction (Ray et al. 2010a). Although theunderlying reasons for this improvement areunclear, one hypothesis is that biofloc reduction

encourages beneficial changes in the microbialcommunity.

Using a fish-free diet rather than a con-ventional diet may be a more environmen-tally sustainable practice, with the opportunityfor reduced feed costs and niche marketing(Amaya et al. 2007). Ray et al. (2010a) demon-strated that a plant-based fishmeal and fishoil-free diet could generate equivalent shrimpproduction to that of a conventional diet inminimal-exchange, superintensive culture sys-tems. However, the impact of these two diettypes on the function and composition of themicrobial community is unclear.

Ray et al. (2010a) present general waterquality and shrimp production results fromthe above-referenced experiment. The currentpublication gives a brief overview of thesetopics while primarily focusing on a moredetailed study of the effects that suspendedsolids reduction (SR) and diet had on microbialdynamics during the same experiment.

This paper explores the availability of lightused for photosynthesis in the water column,algal productivity, chlorophyll-a (chl-a) andpheophytin-a (pheo-a) concentrations, and bac-terial abundance indicator (BAI) concentration.The availability of light in the water column canhave implications for algal dynamics such asoxygen production and pigment concentration(Agustí et al. 1994). Oxygen fluctuations are acritical consideration in superintensive culturesystems where the microbial community canconsume as much dissolved oxygen (DO) as theintended culture species (Browdy et al. 2012).Because algae have been implicated in provid-ing nutritional benefits to shrimp (Ju et al. 2009;Kent et al. 2011), understanding how systemmanagement can affect algal concentration andquality may be useful. The concentration ofchl-a is commonly used to assess algal abun-dance (Dolan et al. 1978) and the concentrationof pheo-a, a degradation product of chl-a, iscommonly used to infer the relative amount ofdead or dying algal cells (SooHoo and Kiefer1982). Finally, the concentration of bacterialfatty acids (FAs) can be used to infer the rela-tive abundance of bacteria in the water column(Budge et al. 2001; Alfaro et al. 2006).

792 RAY ET AL.

Table 1. Protein, lipid, ash, and moisture content of thetwo diets used for this study.1

Conventional feed Fish-free feed

Crude protein 35.7 36.4Total lipid 11.0 10.8Ash 6.6 6.9Moisture 9.7 8.2

1Values are given as percent total composition.

Materials and Methods

Experiment

The experimental design, diet characteris-tics, routine husbandry, water quality, andshrimp production during this experiment weredescribed in detail by Ray et al. (2010a). Theexperiment examines the effects of suspendedSR versus no SR. The experiment also evalu-ates two diets: a conventional feed (CF) diet,containing fishmeal and fish oil, and a fish-free feed (FF) diet, containing soybean meal asthe primary source of protein and algal extractsas the principal source of polyunsaturated FAs.Both diets were formulated to contain 35%crude protein and 11% total lipid (Table 1).

The formulation of the FF diet is presentedin Table 2 and the precise composition of theCF diet (Ziegler™ Hyperintensive 35; ZieglerBrothers Inc., Gardners, PA, USA) is propri-etary; however, the ingredients listed on thepackaging label are presented in Table 2.

The experiment was conducted using 16 out-door, 71-cm deep, 6.3-m3 plastic tanks, oper-ated at approximately 20 g/L salinity. Shrimp,with mean ± SEM weight of 1.31 ± 0.06 g,were stocked at 460/m3 and cultured for 12wk. Eight of the experimental tanks receivedthe commercially available CF and eight tanksreceived the experimental FF. Within each feedtype, four of the tanks had SR and four didnot. The combination of these factors led tofour treatments, each containing four randomlyassigned replicate tanks. The treatments aredenoted as CF (CF, no SR), CF-SR (CF, withSR), FF (FF, no SR), and FF-SR (FF, withSR). There was no water exchanged during thisexperiment, except in the tanks with solids man-agement a small amount of water was added toreplace the volume removed during SR. Con-stant aeration was provided using blown air

Table 2. Ingredients listed on the feed packaging label for the commercially produced conventional feed (CF) diet andthe specific formulation of the experimental fish-free feed (FF) diet.

CF FF

Ingredient Rank1 Ingredient Rank1 Ingredient Percentage2

Grain products 1 Di-α tocopheryl acetate 20 Soybean meal (expelled) 55Marine protein products 2 Vitamin supplement 21 Corn gluten meal 12Plant protein products 3 Riboflavin 22 Whole wheat 11.1Processed grain products 4 Niacin 23 Pea meal 10Poultry bi-product meal 5 Calcium pantothenate 24 Squid meal 2Brewer’s dried yeast 6 Menadione sodium bisulfate complex 25 CaP-dibasic 2Soy lecithin 7 Folic acid 26 Vitamin premix 1.8Limestone 8 Thiamine mononitrate 27 AquaGrow® Gold DHA3 1.4Dicalcium phosphate 9 Pyridoxine hydrochloride 28 Flax seed oil 1Fish oil 10 Biotin 29 AquaGrow ARA 1Magnesium oxide 11 Manganese proteinate 30 Soy oil 0.8Yeast extract 12 Zinc proteinate 31 Lecithin (soy refined) 0.5l-ascorbyl-2-polyphosphate 13 Calcium iodate 32 Betaine 0.5Xanthophylls 14 Iron proteinate 33 Trace mineral premix 0.5Di-methionine 15 Cobalt proteinate 34 Choline cloride 0.2Cholesterol 16 Calcium carbonate 35 Cholesterol 0.1Calcium propionate 17 Sodium selerite 36 Stay-C 0.1Vitamin A 18 Zinc oxide 37Vitamin D3 supplement 19

1Ingredients ranked in order of composition by weight, as listed on feed packaging label.2Percent composition by weight.3Advanced BioNutrition Corp., Columbia, MD, USA.

BIOFLOC REDUCTION AND MICROBIAL DYNAMICS 793

from two regenerative, 5-hp blowers (AmetekInc., Kent, OH, USA) delivered through eightceramic diffusers in each tank.

For each tank with SR, turbidity was mea-sured intermittently using a LaMotte Model2020e Turbidity Meter (LaMotte Co., Chester-town, MD, USA), and when it was found to beabove 30 Nephelometric turbidity units (NTU)the settling chamber was operated. Water fromthe culture tank was transported to a 200-L set-tling chamber by airlift at a flow rate of approxi-mately 6 L/min. The water velocity slowed as ittraveled down a central pipe, permitting solidsto settle while the water rose slowly and over-flowed back into the culture tank. The materialat the bottom of each settling chamber wasremoved weekly.

Water Quality

Total ammonia nitrogen, nitrite-nitrogen(NO2-N), nitrate-nitrogen (NO3-N), and alka-linity (mg CaCO3/L) concentrations were mon-itored weekly during the study. The threenitrogen species were measured using HachMethods 8155, 8507, and 8039, respectively(Hach Company, Loveland, CO, USA). Alka-linity was measured weekly following thePotentiometric Titration to Preselected pH pro-cedure, outlined in Section 2320 B by theAPHA (2005). Sodium bicarbonate was addedas needed in an attempt to maintain alkalinityat approximately 100 mg CaCO3/L throughoutthe study. Turbidity was measured weekly inall shrimp tanks using the LaMotte TurbidityMeter described above. Phosphate concentra-tion was measured in each tank the week aftershrimp were stocked and measured again thelast week of the experiment. Phosphate con-centration was measured using the PhosVer 3method outlined in Hach Method 8048 (HachCompany). DO, temperature, salinity, and pHwere monitored once daily between 0800 and0900 h. Beginning 52 d after shrimp werestocked, these parameters were also measuredbetween 1500 and 1600 h. These measurementswere collected using a YSI Handheld Multipa-rameter Instrument, model 556 (YSI Inc., Yel-low Springs, OH, USA). Total suspended solids(TSS) and volatile suspended solids (VSS) in

the shrimp tanks were measured using ESSMethod 340.2 (ESS 1993).

PAR Extinction

Photosynthetically active radiation (PAR)refers to light with wavelengths between 400and 700 nm; the range utilized during photo-synthesis. Once a week, beginning the secondweek after stocking shrimp, PAR extinctioncoefficients were calculated in each of theexperimental tanks. A hand-held probe wasconstructed using 3-cm diameter polyvinylchloride pipe. The probe had two LI-192 Under-water Quantum PAR sensors (LI-COR® Bio-sciences, Lincoln, NE, USA): an upper sensorand a lower sensor. When submerged to a par-ticular depth, labeled on the probe, the lowerlight sensor was 20 cm below the water’s sur-face, while the upper sensor was above thewater surface. The sensors were connected toan LI-1400 Data Logger (LI-COR Biosciences),which recorded data from each sensor. Usingirradiance values from the two sensors, PARextinction coefficients were calculated and usedto infer light availability for algae. Irradi-ance was measured in μmol/sec/m2 and extinc-tion coefficients, similar to those describedby Armengol et al. (2003), were calculatedusing the following equation: EC = 1

D× ln

(sb

),

where EC = extinction coefficient, D = depthin meters, s = surface irradiance, and b =irradiance “D” meters (20 cm below the watersurface).

Photosynthetic Oxygen Production

The methods used to measure photosyntheticoxygen production were those described byVinatea et al. (2010). During the ninth andtenth weeks of the experiment, water was col-lected from each tank and placed into four300-mL biochemical oxygen demand (BOD)bottles. Two replicate BOD bottles were clearand two were wrapped tightly in black vinyl toprevent light penetration. Initial and final DOconcentrations were measured using a YSI 5000Digital Oxygen Meter equipped with a stir-ring BOD probe (YSI). After measuring initialDO, the bottles were sealed and attached to a

794 RAY ET AL.

slowly rotating wheel contained within a 250-Lpolycarbonate aquarium and incubated for 2 h.The aquarium contained water that was heldat 28 C, similar to that of the experimentaltanks, and had a metal halide light above itproviding a constant light source (150 μmolphotons/m2/sec). The movement of the rotat-ing wheel kept particles within the bottles insuspension. By using constant light, tempera-ture, and turbulence levels, the bottles could beincubated in standard conditions regardless ofthe time of day or outdoor weather conditions.Net photosynthetic oxygen production was cal-culated using the following equation (Strickland1960): mg O2/L/h = (final O2 of light bottle −final O2 of dark bottle)/time (h).

Chlorophyl-a and Pheophytin-a Concentrations

On the second, fourth, ninth, and final weeksof the experiment, chl-a and pheo-a concen-trations were measured in each tank. Pig-ment extraction was performed according to themethods described by DeLorenzo et al. (2004),using a combination of acetone submersionand freezing. Absorbances were measured ina Hach DR/4000V spectrophotometer (HachCompany). Absorbances at 664, 665, and750 nm were measured, 100 μL of 0.1 Nhydrochloric acid was then added to degradechl to pheo, and absorbance was measuredagain at 665 and 750 nm. Concentrations ofchl-a and pheo-a, a degradation product of chl-a, were calculated using equations given byAPHA (2005).

Bacterial FA Assessment

FA composition of lipid extracted from themicrobial community was determined at 3-wkintervals throughout the study. The concentra-tion of branched and odd chain FAs was deter-mined, as described by Ray et al. (2010b), toassess changes in overall abundance of bac-teria. The use of odd and branched chainFA sums as BAIs is well established (Par-rish et al. 2000; Budge et al. 2001; Alfaroet al. 2006). The odd and branched chain FAsare iso13:0, aiso13:0, C13:0, iso14:0, iso15:0,iso15:1, aiso15:0, aiso15:1, C15:0, C15:1n-6,

iso16:0, C17br(1), iso18:0, C19br(1), C19:0,and C19:1n-8. These basic FAs are character-istic of heterotrophic and autotrophic bacteria.This method is effective for tracking changesover time and among treatments within a study,although it does not provide a quantitativeassessment of the absolute levels of bacteria.

During the first and third weeks of the exper-iment, 30-mL composite samples, consisting ofequal parts of water from each of the fourtanks belonging to each treatment, were col-lected. On the sixth, ninth, and final weeksof the experiment, 30-mL samples were col-lected from individual tanks. Samples werecentrifuged at 2640 g for 1 h. After remov-ing the supernatant, 10 mL of a 2:1 chloro-form: methanol mixture was added to eachpellet. FA methyl esters (FAMEs) were thenextracted by a modification of the Folch method(Folch et al. 1957) as modified by Budge andParrish (2003) and further modified to accom-modate sample size and the use of an inter-nal standard (IS). The FAMEs were analyzedby gas chromatography with splitless injec-tion. With the use of dual injection modules,samples were simultaneously analyzed on dualDB225ms columns (50%-cyanopropylphenyl-methylpolysiloxane 30 m × 0.25 mm; J&WScientific, Folsom, CA, USA) with detection byflame ionization (FID) and mass spectrometry(MS). Scans from the MS detector were used inconjunction with comparison of retention timesto those of known standards for peak identifica-tion. Correction factors, determined by analysisof quantitative standards: NuChek 85 and 411(NuChek Prep., Elysian, MN, USA) and ver-ification against theoretical correction factors,were applied to FA peak areas from the FID.The amount of each FA was calculated usingthe IS. The sum of branched and odd chain FAs(reported as μg analyte/L of shrimp tank water)was used as a BAI.

Statistical Analyses

The statistical software used for this studywas SigmaPlot Version 11 (Systat SoftwareIncorporated, San Jose, CA, USA). The lightextinction coefficients, chl-a, pheo-a, and BAI

BIOFLOC REDUCTION AND MICROBIAL DYNAMICS 795

concentrations from the final sample date wereanalyzed using a fixed model two-way analysisof variance (ANOVA) to assess the end effectof each treatment. The photosynthesis data werealso analyzed using a fixed model two-wayANOVA. In all cases, the two factors in theANOVA models were diet and settling cham-bers. Differences are considered significant atthe P ≤ 0.01 level, unless indicated otherwise.Water quality and shrimp production data wereanalyzed by Ray et al. (2010a).

Results

The mean volume removed from each SRtank through settling chambers was 951 L (15%of the volume of the shrimp culture tanks);315 L of that was replaced with clean water,constituting 5% water exchange. A mean of5.8 kg of TSS was removed from each SRtank, and 4.6 kg (79%) of that was VSS. The

TSS removed from SR tanks represents 15%of the feed loading in these systems; eachexperimental tank received 37.8 kg of feed (dryweight).

As reported by Ray et al. (2010a), water inthe tanks with SR contained 41% lesser meanTSS concentration, 42% lesser nitrate-nitrogenconcentration, 62% lesser phosphate concentra-tion, and a 32% greater alkalinity concentra-tion compared to tanks without SR (Table 3).Shrimp in tanks with SR had significantlyimproved feed conversion ratios, growth rates,and final individual weights (Table 4). Totalshrimp biomass (kg/m3) was 40% greater inthe two treatments with SR than those with-out. Each of these differences in water qualityand shrimp production was significant (P <

0.001). There were no significant differencesin shrimp production or water quality betweenthe two diets, with the exception of phosphate

Table 3. Water quality values in the shrimp culture tanks during the experiment.1,2

Treatment

CF CF-SR FF FF-SR

Temperature (C)AM 26.5 ± 1.5 26.5 ± 1.4 26.5 ± 1.4 26.6 ± 1.4PM 27.4 ± 2.0 27.5 ± 1.9 27.4 ± 2.0 27.6 ± 1.9

Dissoved oxygen (mg/L)AM 5.5 ± 0.8 5.7 ± 0.8 5.6 ± 0.8 5.7 ± 0.7PM 5.8 ± 0.6 6.1 ± 0.7 6.1 ± 0.5 6.2 ± 0.7

pHAM 7.2 ± 0.4 7.5 ± 0.3 7.3 ± 0.4 7.6 ± 0.3PM 7.1 ± 0.2 7.6 ± 0.2 7.2 ± 0.2 7.7 ± 0.2

Salinity (g/L)AM 19.5 ± 0.8 18.6 ± 1.3 19.4 ± 0.8 18.9 ± 1.0PM 19.0 ± 0.6 18.2 ± 1.2 18.8 ± 0.7 18.0 ± 0.6

Ammonia (mg TAN/L) 0.1 ± 0.4 0.3 ± 0.2 0.4 ± 0.8 0.4 ± 0.6Nitrite (mg NO2-N/L) 0.8 ± 1.5 1.1 ± 1.3 0.9 ± 1.6 1.0 ± 1.3Nitrate (mg NO3-N/L) 85 ± 47a 48 ± 18b 86 ± 46a 51 ± 21b

Phosphate (mg PO4/L)3 124 ± 2a 50 ± 6b 85 ± 3c 30 ± 1d

TSS (mg/L) 820 ± 135a 453 ± 106b 745 ± 165a 465 ± 93b

VSS (mg/L) 509 ± 99a 256 ± 50b 485 ± 114a 266 ± 44b

Turbidity (NTU) 63 ± 9a 27 ± 6b 56 ± 13a 34 ± 7b

Alkalinity (mg CaCO3/L) 58 ± 5a 85 ± 4b 61 ± 5a 91 ± 4b

CF = conventional feed; CF-SR = CF, with solids reduction; FF = fish-free feed; FF-SR = FF, with solids reduction;NTU = Nephelometric turbidity units; TAN = total ammonia nitrogen; TSS = total suspended solids; VSS = volatilesuspended solids.

1Adapted from Ray et al. (2010a).2Values are mean ± SEM. Within the same row, significant differences are indicated by different superscripts.3Only mean phosphate concentrations from the final sample date are reported.

796 RAY ET AL.

Table 4. Shrimp production parameters from the experiment.

Treatment

CF CF-SR FF FF-SR

Growth rate (g/wk) 0.7 ± 0.0a 0.9 ± 0.0b 0.6 ± 0.0a 0.9 ± 0.1b

Final weight (g) 9.2 ± 0.1a 12.0 ± 0.3b 8.4 ± 0.3a 11.6 ± 1.1b

Net biomass (kg/m3) 2.2 ± 0.1a 3.2 ± 0.4b 2.1 ± 0.1a 2.8 ± 0.2b

Survival (%) 76.0 ± 2.6 79.8 ± 7.4 79.7 ± 1.4 75.9 ± 5.2FCR 2.6 ± 0.1a 1.9 ± 0.2b 2.7 ± 0.1a 2.1 ± 0.1b

CF = conventional feed; CF-SR = CF, with solids reduction; FCR = feed conversion ratio (dry weight of feed/wetweight of shrimp); FF = fish-free feed; FF-SR = FF, with solids reduction.

1Adapted from Ray et al. (2010a).2Values are mean ± SEM. Within the same row, significant differences (P ≤ 0.006) are indicated by different

superscripts.

Table 5. Final microbial community characteristics during the experiment.1

Treatment

CF CF-SR FF FF-SR

PAR extinction coefficient (/m) 31.9 ± 1.5a 16.7 ± 1.2b 29.6 ± 1.4a 17.8 ± 1.1b

Photosynthetic oxygen production (mg/L/h) 0.5 ± 0.1a 2.2 ± 0.2b 0.8 ± 0.1a 2.1 ± 0.2b

Chlorophyll-a concentration (μg/L) 608 ± 57a 429 ± 69b 584 ± 47a 430 ± 74b

Pheophytin-a concentration (μg/L) 318 ± 41a 265 ± 64b 383 ± 63a 179 ± 51b

Branched and odd chain fatty acids (μg/L) 637 ± 114a 183 ± 37b 861 ± 145a 254 ± 44b

CF = conventional feed; CF-SR = CF, with solids reduction; FF = fish-free feed; FF-SR = FF, with solids reduction;PAR = photosynthetically active radiation.

1Values are mean ± SEM. Within the same row, significant differences are indicated by different superscripts.

concentration which was significantly lower inthe FF diet treatments (P < 0.001).

Each of the microbial characteristics mea-sured for this study was significantly (P ≤0.003) affected by SR (Table 5). The finalmean ± SEM PAR extinction coefficient fortanks without SR was 36.0 ± 5.4/m and was19.2 ± 3.1/m for tanks in which solids werereduced. This represents a significant (P <

0.001) 47% reduction in PAR extinction coef-ficient as a result of reducing solids (Fig. 1).A reduced extinction coefficient implies that agreater amount of light was able to penetratethe water column in tanks with SR.

The mean photosynthetic oxygen productionin tanks without SR was 0.7 ± 0.1 mg/L/h andwas 2.1 ± 0.1 mg/L/h in tanks that had solidsreduced (Table 5). This tripling of photosyntheticoxygen production in tanks with SR constituteda significant increase (P < 0.001).

On the final sample date of the experiment,the mean chl-a concentration in tanks withoutSR was 550 ± 57 μg/L, while in the tanks with

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Figure 1. Photosynthetically active radiation extinctioncoefficients in the four treatments. Error bars representone standard error around the mean.

SR it was 195 ± 29 μg/L. This was a significant(P < 0.001) 65% reduction in chl-a (Fig. 2A).

The mean final concentration of pheo-a was234 ± 32 μg/L for tanks without SR and 65 ±18 μg/L in tanks with SR. This constitutes asignificant (P < 0.001) 72% reduction in theconcentration of these chl degradation pigments(Fig. 2B).

The final concentration of BAI FAs (Table 5)was 80% higher in tanks without SR (1219 ±172 μg/L) than in tanks with SR (238 ±

BIOFLOC REDUCTION AND MICROBIAL DYNAMICS 797

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Figure 2. The concentrations of chlorophyll-a, pheophytin-a, and branched and odd chain fatty acids used as bacterialabundance indicators. Error bars represent one standard error around the mean.

32 μg/L), constituting a significant difference(P = 0.003). This indicates that bacterial abun-dance increased to a greater extent in the tankswhere solids were not reduced than in thosewith SR (Fig. 2C).

There were no significant differences amongany of the microbial characteristics with respectto diet.

Discussion

Extinction Coefficients and Algal Productivity

By removing a portion of the biofloc particlesfrom these superintensive systems, a greateramount of light was able to penetrate the water

column. However, the extinction coefficientsreported here are higher than those reported bysome researchers. In an Alaskan lake, whereturbidity was caused mostly by glacial sedi-ments, the highest extinction coefficient valuethat Lloyd et al. (1987) reported was 3.1/m.Giovannini and Piedrahita (1994) predictedthat to optimize photosynthetic oxygen pro-duction, extinction coefficients in aquacultureponds should be maintained between 2.7 and11.2/m. The overall mean extinction coefficientin the treatments with SR in this study (19.2/m)was 71% greater than the highest value rec-ommended by these authors. Algal productiv-ity may have been high in the SR treatments,

798 RAY ET AL.

despite elevated extinction coefficients, becausethe constant aeration from diffusers on the tankbottoms thoroughly mixed the water. This mix-ing likely allowed greater exposure of algae tolight than if contained in a relatively stagnantlake or pond.

High chl-a concentrations in the tanks with-out SR may indicate greater algal abundance.However, with greater algal abundance self-shading by algae can become a limiting fac-tor for photosynthesis (Agustí 1991; Giovanniniand Piedrahita 1994). The lower availability oflight, indicated by high PAR extinction coeffi-cients, likely led to the significantly lower pho-tosynthetic oxygen production in non-SR tanks.Light was probably shaded by the greater con-centration of particles, including algae and othermicrobes, as indicated by higher TSS, VSS, andturbidity (Table 3).

Burford et al. (2003) reported photosyn-thetic oxygen production of between 1.4 and5.1 mg/L/h in high-intensity shrimp ponds. Themean oxygen production of 2.2 mg/L/h in theSR treatments falls into this range. However,the mean oxygen production in non-SR treat-ments of 0.7 mg/L/h was half the lowest ratereported by those authors. In relatively low-intensity ponds, Bratvold and Browdy (1998)reported photosynthetic rates of between 2.9and 9.2 mg/L/h. Brune et al. (2003) suggestedthat low-exchange, low-intensity culture sys-tems often contain algae as the primary micro-bial taxa. These authors noted that algae werelimited in the amount of nutrients they couldassimilate and as systems become more inten-sive, due to increased nutrient inputs, bacte-rial processes begin to dominate. Brune et al.(2003) went on to describe the removal of algaeas a mechanism for increasing algal productiv-ity in culture systems. As algae are removed,opportunity is created for new, more productivealgal cells to proliferate (Turker et al. 2003).Giovannini and Piedrahita (1994) also demon-strated that removing a portion of the algalpopulation can increase the photosynthetic pro-duction of oxygen in aquaculture ponds. Thisappears to be the case in this study; in the treat-ments where chl-a concentration was reducedthrough SR, photosynthetic oxygen production

was enhanced. Increased oxygen productionmay be beneficial in superintensive culture sys-tems, especially during the hottest times of daywhen gas solubility in water is lowest.

Reduced Chl-a and Enhanced AlgalProductivity

Because chl-a concentration is generally usedas an indicator of algal abundance (Dolan et al.1978), it may seem contradictory that lowerchl-a concentration corresponded with greateralgal productivity in the SR treatments. Fourpotential explanations for this observation aresuggested here. First, in the non-SR treatments,there may have been an increase of chl-a con-centration in response to lower light levels. Sec-ond, the composition of the algal communitymay have been changed by removing solids,thereby contributing to the differences in chl-a concentration and productivity. Third, theremay have been a relatively greater proportionof living, healthy algae in the SR treatments,thereby contributing to a relatively more pro-ductive algal community. Last, related to thethird point, a decreased relative abundance ofbacteria and possibly zooplankton in SR sys-tems may have favored algal health and nutrientacquisition.

In response to low light conditions, manyalgae can increase the concentration of chlin their cells in an attempt to capture morelight (Nielsen and Jørgensen 1968; Agustí et al.1994). However, the higher chl density oftenleads to inefficient light absorption due to self-shading, and can contribute to lower oxygenproduction overall (Agustí et al. 1994). Thismay have been the case in the non-SR treat-ments in which less light penetrated the watercolumn, chl-a concentration was higher, butthere was a lower rate of photosynthetic oxygenproduction compared to the SR treatments.

The composition of the algal communitytaxa may have been changed through SR inthis study. Ray et al. (2010b) showed thatreducing solids in superintensive shrimp culturesystems can lead to a significant reduction inthe abundance of cyanobacteria. Cyanobacteriacontain chl-a, but exhibit low photosynthesis

BIOFLOC REDUCTION AND MICROBIAL DYNAMICS 799

rates compared to algae of similar size (Agustí1991). If the non-SR treatments in this studycontained a greater abundance of cyanobacteriacompared to the SR treatments, this couldhelp explain the higher chl-a concentration andlower photosynthetic oxygen production in non-SR treatments. Also, the size of algal taxa canhelp to dictate the concentration of chl-a in asystem because larger algae typically contain arelatively lower concentration of chl-a (Agustí1991). If species composition was affected bysolids management and relatively larger algaewere present in the SR treatments, this mayhelp to explain the lower chl-a concentrationin those treatments.

There were no significant differencesbetween treatments with respect to chl-a/pheo-a ratios and it was not possible to accuratelycalculate chl-a/BAI ratios (chl-a/BAI) becausesamples for the two analyses were not collectedon the same days. However, calculations madefrom the mean values presented in Table 5 mayprovide some indication of trends with respectto these ratios. According to these calculations,the non-SR treatments had a chl-a/pheo of 2.4,while the SR treatments had a chl-a/pheo of 3.0.Although this difference was not significant, ahigher chl-a/pheo can indicate that a greaterproportion of living algal cells are present, lead-ing to relatively enhanced primary productivity(Conover and Siferd 1993). Turker et al. (2003)suggested that by regularly removing a portionof the algal community, a younger, healthierpopulation is sustained, and this younger algalcommunity can contribute greater oxygen pro-duction and can potentially be more nutritiousto cultured animals.

SooHoo and Kiefer (1982) discussed twoprincipal means of pheo generation in themarine environment: grazing by microzoo-plankton and chl breakdown through photoox-idation. In the non-SR tanks, it is unlikelythat increased photooxidation took place, basedon the higher PAR extinction coefficients inthose tanks. However, a greater abundance ofzooplankton grazing on algae in non-SR tanksmay have generated more pheo, contributing tothe relatively lower chl-a/pheo in those tanks. Ina similar experiment, Ray et al. (2010b) found

that SR caused a significant decrease in theabundance of rotifers and nematodes, support-ing the suggestion that non-SR tanks may havecontained a higher abundance of zooplankton inthis study. Also, based on the data presented inTable 5, the mean chl-a/BAI in non-SR treat-ments was 0.5, while it was 0.8 in SR treat-ments. This indicates that there may have beena higher proportion of bacteria relative to algaein the non-SR tanks. Increased grazing pres-sure from zooplankton and increased competi-tion for nutrients from bacteria can be reasonsfor decreased photosynthetic oxygen production(Rhee 1972; Carpenter et al. 1985), potentiallyhelping to explain the differences in oxygenproduction between the SR and non-SR treat-ments in this study.

Reducing the concentration of biofloc parti-cles increased the availability of light, increasedphotosynthetic oxygen production, decreasedthe abundance of bacteria, and likely decreasedthe abundance of algae. These changes cor-responded to improved shrimp production,lower phosphate and nitrate concentrations, andhigher alkalinity. In systems such as these, themicrobial community plays an essential rolein nutrient cycling, and potentially the provi-sion of supplemental nutrition. Understandingthe effects of management decisions on waterquality and microbial dynamics may be help-ful in making informed choices. In this case,biofloc concentration reduction caused signifi-cant changes in the structure and function ofthe microbial community. Although a directrelationship is impossible to establish, thesechanges in microbial dynamics correspond tosignificantly improved water quality and shrimpproduction metrics, supporting the idea thatmanaging the microbial community may be animportant consideration for proper overall sys-tem management.

Diets

Shrimp production did not differ significantlybetween systems fed the two diets, a finding thatmay help to support the use of fish-free diets forthe culture of Litopenaeus vannamei. The onlysignificant difference in water quality between

800 RAY ET AL.

systems fed the two diets was phosphate con-centration. Regardless of the lower phosphateconcentration in FF diet treatments, there wereno significant differences in any of the mea-sured microbial characteristics with respect todiet. A 20-g/L salinity environment may be con-sidered brackish, where many marine organismscan be found; however, the lack of marine pro-teins, aside from 2% squid meal content, inthe FF diet did not lead to significant changesin the measured parameters. This may indicatethat the microbial communities in these sys-tems were resistant to changes caused by dietformulation. Based on the water quality andmicrobial characteristics measured during thisstudy, microbial function and structure seem tohave been appropriate for proper system func-tion independent of diet type, supporting theequivalency of the FF diet to the CF diet insuperintensive systems.

Acknowledgments

This work was supported by grants fromthe US Department of Agriculture (USDA)Integrated Organic Program and the USDA-National Institute of Food and Agriculture(NIFA) US Marine Shrimp Farming Program.Mention of a trademark or proprietary prod-uct is not an endorsement of that product or asuggestion of its superiority over other prod-ucts. This is contribution number 667 fromthe South Carolina Department of NaturalResources Marine Resources Research Institute.The authors would like to thank David Kuhn,Beth Lewis, Janet Moore, Paul Pennington, JoeWade, Emmet Wright, the staff of the Wad-dell Mariculture Center, and two anonymousreviewers.

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