biofloc technology application as a food source in a limited water exchange nursery system for pink...

Biofloc technology application as a food source in a

limited water exchange nursery system for pink shrimp

Farfantepenaeus brasiliensis (Latreille, 1817)

Maur|¤ cio Emerenciano1, Eduardo L C Ballester1,2, Ronaldo O Cavalli1,3 & WilsonWasielesky1

1Laborato¤ rio de Carcinicultura, Instituto de Oceanogra¢a, Universidade Federal do Rio Grande (FURG), Rio Grande, RS, Brazil2Laborato¤ rio de Carcinicultura, Universidade Federal do ParanaŁ (UFPR) ^ Campus Palotina, Palotina, PR, Brazil3Departamento de Pesca eAquicultura, Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE, Brazil

Correspondence: M Emerenciano, Posgrado en Ciencias del Mar y Limnolog|¤ a, Universidad Nacional Auto¤ noma de Me¤ xico (UNAM),

Unidad Multidisciplinaria de Docencia e Investigacio¤ n (UMDI), Puerto deAbrigo s/n, CP 97355, Sisal,YucataŁ n, Mexico.

E-mail: [email protected]

Abstract

In a 30-day experiment, Farfantepenaeus brasiliensisPL25 (25 � 10mg;17.9 � 1.6mm) were raised in ninecircular £oating cages with a stocking density of1000 shrimpm�3. Three treatments were evaluated:(1) culture in BFTsystem plus a commercial feed sup-ply (BFT1CF); (2) culture in BFT systemwithout feedsupply (BFT) and (3) culture in clear water with feedsupply (control). Post-larvae (PL) ¢nal weight (218.9,236.5 and176.0mg, for BFT1CF, BFT and control re-spectively), ¢nal biomass (17.9, 15.7 and 8.2 g) andweight gain (193.9, 211.5 and 151.0mg) were similarin the BFT regardless of whether theywere fed a com-mercial diet (P40.05), but were both signi¢cantlyhigher than the control (Po0.05). Survival (81.5%,67.0% and 84.8% respectively) and ¢nal length didnot di¡er between treatments (P40.05). The bio£ocanalysis identi¢ed ¢ve main microorganism groups:protozoa (ciliate and £agellate), rotifers, cyanobacter-ia (¢lamentous and unicellular) and pennate dia-toms. Free living bacteria and attached bacteria inbulk were 25.73 � 8.63 and 0.86 � 3.17 � 106mL�1

respectively. Proximate analysis in the bio£oc indi-cated high levels of crude protein (30.4%). Resultscon¢rmed favourable nutritional quality of bio£oc,and enhanced growth and production of F. brasilien-sis PL in bio£oc systems.

Keywords: bio£oc, environmental friendly, Far-fantepenaeus brasiliensis, nursery, nutrition

Introduction

The aquaculture industry is growing fast, at a rate of� 9% per year since the 1970s (Food and Agricul-ture Organization of the United States 2008).However, due to environmental concern, the require-ment for more ecologically sound management andculture practices is also growing fast. Moreover, theexpansion of aquaculture production is currently re-stricted due to the pressure it causes to the environ-ment by the discharge of waste products to naturalwater bodies and by its strong dependence on ¢sh-meal and ¢sh oil (Browdy, Bratvold, Stokes & Mcin-tosh 2001; De Schryver, Crab, Defoirdt, Boon &Verstraete 2008). Such ingredients are one of theprime constituents of feed for commercial aquacul-ture (Naylor, Goldburg, Primavera, Kautsk, Bever-idge, Clay, Folke, Lubchencoi, Mooney & Troell2000). Furthermore, feed costs represent at least50% of the total aquaculture production costs, whichis predominantly due to the cost of the protein com-ponent in commercial diets (Bender, Lee, Sheppard,Brinkley, Philips,Yeboah &Wah 2004).Another important concern is water use. In the

1980s, water exchange was identi¢ed as an impor-tant factor contributing to several epizootic diseasesin shrimp farms (Sandifer, Stokes & Hopkins 1991;Hopkins, Hamilton II, Sandifer, Browdy & Stokes1993). Inaddition, water discharge of nutrient-rich ef-£uent from intensive culture systems can contributeto eutrophication, potentially impacting both natur-

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al biota and adjacent culture operations (Browdyet al. 2001). Consequently, some shrimp producershave reduced water exchange, becoming more con-servative inwater use (Hargreaves 2006).Bio£oc technology (BFT) is an emerging alterna-

tive towards more environment friendly aquacultureproduction systems. This technology was developedto create economical and environment bene¢ts viareduced water use, e¥uent discharges, arti¢cial feedsupply and improved biosecurity (Wasielesky, At-wood, Stokes & Browdy 2006; Avnimelech 2007; Mis-hra, Samocha, Patnaik, Speed, Gandy & Ali 2008).Themicrobial community, mainlyheterotrophic bac-teria, can improve water quality (Asaduzzaman,Wa-hab, Verdegem, Huque, Salam & Azim 2008) andcontrol pathogenic bacteria, thereby reducing thepotential spread of diseases (Horowitz & Horowitz2001; Cohen, Samocha, Fox, Gandy & Lawrence2005). The bio£oc as a food source are available24 h day�1 (Avnimelech 2007), improving produc-tion per hectare and economic bene¢ts through low-er arti¢cial feed inputs (Hopkins, Sandifer & Browdy1995; Browdy et al. 2001). This kind of system can bede¢ned as one for which the maintenance of waterquality depends mainly on an active phytoplanktonbloom, free and attached bacteria, aggregates of liv-ing and dead particulate organic matter and micro-bial grazers (rotifers, ciliates and £agellates andprotozoa) maintained in suspension. Nitrogen com-pounds are controlled by a combination of phyto-plankton uptake, nitri¢cation and by heterotrophicbacteria immobilization inside the culture unit (Har-greaves 2006). Consumption of natural productivity(bio£oc, phytoplankton and associated grazers) bycultured organisms can increase the e⁄ciency offeed utilization by recovery of some fraction of ex-creted nutrients (Burford,Thompson, McIntosh, Bau-man & Pearson 2004; Wasielesky et al. 2006).Consumption of bio£oc can increase nitrogen reten-tion from added feed by 7^13% (Hari, Kurup,Vargh-ese, Schrama & Verdegem 2004; Schneider, Sereti,Eding & Verreth 2005; Hargreaves 2006).Nitrogenous by-products can be easily taken up by

microorganism biota and serve as a substrate for op-erating bio£oc systems and production of microbialprotein cells (Mishra et al. 2008). This approach issuccessfully performed by control of carbon and ni-trogen (C:N) ratio to promote ammonia for hetero-trophic microorganisms as a major pathway ofammonia control. E¡ective use of microbial biomasshas collateral bene¢ts for species that have a ¢lteringapparatus and/or digestive system that better assimi-

late bio£oc (Burford, Thompson, McIntosh, Bauman& Pearson 2003; Hari et al. 2004; Hargreaves 2006).High C:N ratio will help convert ammonium and or-ganic nitrogenous waste into bacterial protein bio-mass (Avnimelech, Kochva & Diab1994; Avnimelech1999; Schneider et al. 2005). There are many consid-erations for carbon source selection (i.e. local avail-ability, cost, biodegradability and e⁄cient bacteriaassimilation). Table 1 presents studies with di¡erentspecies and carbon source use on limited water ex-change systems.Intensive nursery systems present several bene¢ts

such as optimization of farm land, increased shrimpsurvival and enhanced growth performance in grow-out ponds (Apud, Primavera & Torres 1983; Sandiferet al. 1991; Arnold, Coman, Jackson & Groves 2009).This phase is de¢ned as an intermediate step betweenhatchery-reared, early postlarvae (PL) and the grow-out phase (Mishra et al. 2008). Previous studies,mainly with the Paci¢c white shrimp Litopenaeusvannamei, have reported several bene¢ts by the useof nursery phase (Moss & Moss 2004; Samocha et al.2007; Mishra et al. 2008). Information about rearingother penaeid species in a nursery environmentalfriendly system are scarce in recent literature.The pink shrimp F. brasiliensis (Latreille, 1817) is

naturally distributed from North Carolina, USA tothe north coast of Rio Grande do Sul, Brazil (291/S)(D’Incao 1995, 1999). This species is suitable for cul-ture in alternative structures like cages (Lopes,Wasielesky, Ballester & Peixoto 2009) and couldbe produced as live bait juveniles for sport ¢shing(Preto, Pissetti,Wasielesky, Poersch & Cavalli 2009).In terms of economics, F. brasiliensis is an importantresource for Brazilian ¢sheries (D’Incao 1999), hav-ing strong local demand and commanding a rela-tively higher price than white shrimp species(Valentini, D’Incao, Rodrigues, Rebelo & Rahn 1991).Thus, the aim of the present study was to evaluatethe potential of BFTas a food source for F. brasiliensisPL during the nursery phase under a limited waterexchange condition.

Materials and methods

Experimental design and culture conditions

This work was conducted at the MarineAquacultureStation (Rio Grande Federal University), located atCassino Beach (32112/S and 51150/W), Rio GrandeCity, Rio Grande do Sul, Brazil. Before starting the ex-periment, a round 7000 L (7 tons) outdoor ¢berglass

© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 447–457448

BFT as a food source for F. brasiliensis M Emerenciano et al. Aquaculture Research, 2012, 43, 447–457

‘macrocosm tank’was used for development of bio£oc(Emerenciano,Wasielesky, Soares, Ballester, Izeppi &Cavalli 2007). The tank was inoculated with diatom(Thalassiosira weiss£ogii, 5 � 104mL�1) to helpmaintainwater quality until a heterotrophic commu-nity was established. Water was vigorously aeratedusing air di¡users in the centre of the tank and eightsurrounding air lifts (2 cm diameter PVC pipe, 50 cmlength). With the goal of developing a conventionalBFT shrimp production system and to help developbio£oc, juveniles of F. paulensis (3.54 � 0.88 g) werestocked at a density of 40 shrimpm�2 and main-tained until the end of the experiment. Shrimp werefed three times per day (09:00,13:00 and18:00 hours)with 40% crude protein (CP) commercial diet (Car-gill’s Purina AgribrandsTM, Agribrands Purina doBrasil, Sa� o Lourenc� o da Mata, PE, Brazil) at 3% ofthe estimated biomass. The tank was totally coveredfor an approximately 80% reduction in light by shadecloth. Sugarcane molasses (90% of added carbonsource) and wheat bran (remaining10%) were addeddaily after the addition of the feed to maintain a highC:N ratio (20:1) to ensure optimal heterotrophic bac-teria growth (Avnimelech 1999; Asaduzzaman et al.2008; Crab et al. 2009). In the macrocosm tank, lim-ited water exchange (not exceeding 0.5% daily) wascarried out by a central drain to prevent accumula-tion of sludge throughout the experimental period.Dechlorinated freshwater and marine water (35 ppt),previously ¢ltered in a125 mm sand ¢lter were addedto compensate sludge removal and evaporationlosses.

The experiment was initiated after 25 days withbio£oc formation in the macrocosm tank [total sus-pended solids (TSS)4100mg L�1] (Ballester, Abreu,Cavalli, Emerenciano, Abreu &Wasielesky 2010). Far-fantepenaeus brasiliensis PL (PL25, 25 � 10mg;17.86 � 1.6mm) were obtained from routine larvi-culture carried out at the University station, usingprevious methods (Marchiori 1996). The PLwere dis-tributed in a completely randomized experimentaldesign with three treatments and three replicatesper treatment. Shrimp were stocked at a density of1000m�3 and reared for 30 days. The treatmentswere: (1) culture in BFT system plus a commercialfeed supply (BFT1CF); (2) culture in BFTsystemwithno feed supply (BFT); and (3) culture in clear waterwith commercial feed supply (control). The experi-mental units consisted of nine circular £oating cages(0.25m�2 bottom area, � 0.1m3 underwater vo-lume) made from 1.5mm mesh size plastic screenand placed directly inside the macrocosm tank forBFT treatments or in an 800 L cement tank for clearwater control treatment (with 100% water exchangedaily, covered with the same shade cloth). The PLwere fed at100% biomass with a 40% CPcommercialfeed twice a day (09:00 and18:00 hours).The pelletedfeed was screened at 2 � 2mm, sized larger thancage mesh and distributed to each cage. The water£owed freely between cages and tanks. Fifty shrimpswereweighed (Sartorius

s

, Sartorius, SantoAndre¤ , SP,Brazil) to the nearest 0.1mg at the beginning and theend of experiment. Survival (%), ¢nal weight (mg),weight gain (mg) and ¢nal total length (mm) were

Table 1 Summary of the carbon source and respective species used in limited or zero water exchange culture systems

Carbon source Culture species References

Acetate Macrobrachium rosenbergii Crab, Chielens, Wille, Bossier and Verstraete (2010)

Cassava meal Penaeus monodon Avnimelech and Mokady (1988)

Cellulose Tilapia Avnimelech, Mokady and Schoroder (1989)

Corn flour Hybrid bass and hybrid tilapia Milstein, Anvimelech, Zoran and Joseph (2001); Asaduzzaman,

Rahman, Azim, Islam, Wahab, Verdegem and Verreth (2010)

Dextrose Litopenaeus vannamei Suita (2009)

Glycerol and

Glycerol1Bacillus

M. rosenbergii Crab et al. (2010)

Glucose Penaeus monodon and M. rosenbergii respectively Avnimelech and Mokady (1988); Crab et al. (2010)

Molasses L. vannamei Samocha, Patnaik, Speed, Ali, Burger, Almeida, Ayub, Harisanto,

Horowitz and Brock (2007); Burford et al. (2004)

Sorghum meal Tilapia Avnimelech et al. (1989)

Tapioca M. rosenbergii and L. vannamei respectively Asaduzzaman et al. (2008) Hari et al. (2004)

Wheat flour Tilapia (Oreochromis niloticus) Azim and Little (2008)

Starch Tilapia (Mozambique) and tilapia O. niloticus �O reochromis aureus respectively

Avnimelech (2007); Crab, Kochva, Verstraete and

Avnimelech (2009)

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Aquaculture Research, 2012, 43, 447–457 BFT as a food source for F. brasiliensis M Emerenciano et al.

measured. Final biomass (g) was estimated on eachreplicate accord ¢nalweightmeans and survival rate.

Assessment of water quality parameters

Throughout the experimental period, temperature(mercury thermometer, precision � 0.5 1C), salinity(AtagoTM optical refractometer, Atago Co., Tokyo, Ja-pan; � 1g L�1), pHand dissolved oxygen concentra-tion (YSI Model 556,YSI Incorporated,YellowSprings,OH, USA) were measured daily at 08:00 hours inboth the bio£oc and clear water macrocosm tanks.Total ammonia nitrogen (TAN), nitrite (NO2-N) andreactive phosphorous (RP) (PO4) (UNESCO 1983)were measured three times per week. Total sus-pended solids were measured two times per week(¢rst 2 weeks) and after this period, once a week(Strickland & Parsons1972).

Assessment of microorganisms

Well-mixed water samples from the macrocosm tankwere collected at the end of the experiment in 30mLglass bottles and preserved with borate-bu¡ered for-malin (4% v/v) (Thompson, Abreu & Wasielesky2002). The identi¢cation and abundance of microor-ganisms were determined in 2.1mL samples pouredinto sedimentation chambers using an Olympus in-verted light microscope (Olympus, Tokyo, Japan)equipped with phase contrast (Uterm˛hl 1958). Forbacterial and £agellate counts, volumes of 0.1mLwere concentrated in darkened polycarbonate nucle-pore membrane ¢ltres (0.2 mm), stained with the£uorochrome 4 0,6-diamin-2-phenyl-indol (DAPI)(15 mgmL�1) (Ballester et al. 2010) and directlycounted using a Zeiss Axiolpan epi£uorescence mi-croscope (Zeiss, Oberkochen, Germany) equippedwith a 487701 light ¢lter set (BP365/11; FT 395; LP397) at � 1000 ¢nal magni¢cation (Porter & Feig1980). Counts were made in at least 30 ¢elds chosenat random. Free living bacteria in the water columnand attached bacteria on any substrate type (i.e. or-ganic and inorganic particles, shrimp faeces, etc.)were considered. Fifteen random £oc size measureswere made in the macrocosm tank water samples atleast once aweek.

Proximate analysis of carbon source,commercial feed and bio£oc

Proximate analysis was carried out from the carbonsources (sugarcane molasses and wheat bran), com-

mercial feed and bio£oc biomass (macrocosm tankwater ¢ltered in a 100 mm mesh every 10 days). Allsamples were dried in an oven at 102 1C (constantweight) and then stored in a refrigerator (�20 1C).At the end of the experiment, pooled samples wereground and processed according to AOAC (2000).The total carbohydrate was estimated by di¡erenceand gross energy content was calculated accordingtoTacon (1990).

Statistical analysis

Water quality parameters and growth performancewere analyzed by one-wayANOVA. If main e¡ects weresigni¢cant, di¡erences among the treatments weretested with Tukey’s multi-comparison test of meansat 5% signi¢cance level. Survival was arcsine trans-formed for analysis, but only original values are pre-sented.

Results

Water quality values are shown in Table 2. Therewere signi¢cant di¡erences (Po0.05) between treat-ments in terms of some water quality parameters(salinity, dissolved oxygen and pH). Total ammonianitrogen, nitrite (NO2

� -N) and reactive phosphorous(PO4) in the BFT macrocosm tank are given in Fig.1.Total suspended solids results in themacrocosm tankdescribedmaximumandminimumvalues of123 and414mg L�1, respectively, with an average (� SD) of257.88 � 105.21mg L�1. Fluctuations of TSS and pHare shown in Fig. 2.Shrimp growth performance is presented in Table

3. Post-larvae ¢nal weight, ¢nal biomass and weightgain in BFT1CF versus BFT treatments were not sig-ni¢cantly di¡erent (P40.05) but were higher thanthe control (Po0.05). Survival and ¢nal length re-sults did not di¡er between all treatments (P40.05).Proximate analysis of commercial feed, sugarcane

molasses, wheat branand bio£oc are given inTable 4.Levels of CP in arti¢cial feed and bio£oc were 39.7%and 30.4% respectively. Ash content in bio£oc was39.2% and crude lipid level was 0.47%.During the experimental period, bio£oc varied in

size ranging from 50 to1000 mm.The bio£oc samplesindicated mainly presence of microorganisms suchas protozoa (ciliate and £agellate), rotifers, cyanobac-teria (¢lamentous and single cell) and pennate dia-toms (Fig. 3). Free living bacteria and attachedbacteria in bulk were 25. 73 � 8. 63 and 0.86 �3.17 � 106mL�1respectively (Fig. 4).



Discussion

Water quality parameters

The pink shrimp F. brasiliensis is a new species inaquaculture literature. No datawere found about op-timal water quality for F. brasiliensis. Some para-meters showed signi¢cant di¡erences between themacrocosm tank and the clear water tank. Salinitywas slightly higher in the bio£oc treatments com-pared with the control. This possibly occurred due toevaporation in BFT limited water exchange systems.Dissolved oxygen and pH values were low in BFTtreatments. This was likely a result of higher respira-tion rates due to the presence of a heterotrophiccommunity that increased the carbon dioxide con-centration in limitedwater exchange systems (Tacon,Cody, Conquest, Divakaran, Forster & Decamp 2002;Wasielesky et al. 2006).In limited water exchange bio£oc-based systems

the accumulation of toxic inorganic nitrogen species

can be prevented bymaintaining a highC:N ratio (Av-nimelech 1999) and inducing the uptake of ammo-nium by the microbial community (Avnimelech et al.1994). In this study, the concentration of inorganicnitrogen by-products (TAN and nitrite) and RP pre-sented no signi¢cant di¡erences between the macro-cosm tank and the clear water tank (P40.05). TheC:N ratio of 20:1 by addition of a carbon source (mo-lasses and wheat bran) seems to be a helpful tool toreduce and maintain optimal levels of inorganic Nconcentrations (Asaduzzaman et al. 2008). Reactivephosphorous levels decreased sharply from 10th to20th days and then increased. This trend could indi-cate a period of phytoplankton uptake and conse-quently growth. Hargreaves (2006) showed thatalgal assimilation could temporarily reduce RP le-vels, but these would increase again following algalcrashes. Despite di¡erences in water quality para-meters among treatments, all of them were withinacceptable ranges for most penaeid species (Wickins1976;VanWyk & Scarpa1999).The control of TSS in the BFT tanks is an important

issue closely related to optimum levels of dissolvedoxygen and inorganic N compounds (Ray, Lewis,Browdy & Le¥er 2010), as well as the prevention ofgill clogging. Total suspended solids in the macro-cosm tank £uctuated greatly (123^414mg L�1), witha mean of 257.88 � 105.21mg L�1. As observed withRP, TSS levels decreased quickly from 15th to 20thdays. This trend could indicate that shrimp juvenilesconsumed a large fraction of the bio£ocs present inthe macrocosm tank or shifts in the compositionand abundance of the microbiota due to ecologicalsuccession in the food web (Moriarty1997). Samochaet al. (2007) recommended TSS values below500mg L�1 for shrimp culture. Mishra et al. (2008)

Table 2 Water-quality parameters of 30 days experimental period for Farfantepenaeus brasiliensis reared from PL25 andstocked at a density of1000 shrimpm�3

Parameter BFT1CFand BFT (macrocosm tank) Control (clear-water tank) P-value

Temperature ( 1C) 23.7 (� 1.7) 24.3 (� 1.5) 40.05

Salinity (g L� 1) 35.9A (� 1.0) 34.5B (� 0.9) o0.001

Dissolved oxygen (mg L� 1) 5.9A (� 1.0) 7.1B (� 0.9) o0.001

pH 6.5A (� 0.2) 8.3B (� 0.3) o0.001

TAN (mg L�1) 0.4 (� 0.5) 0.1 (� 0.1) 40.05

NO2–N (mg L�1) 0.9 (� 0.6) 0.5 (� 0.7) 40.05

PO41 (mg L� 1) 1.3 (� 0.7) 1.1 (� 1.0) 40.05

Data are means (� standard error) and signi¢cance level, along experiment. Di¡erent letters indicate means are signi¢cantly di¡erent(Po0.05).Treatments: BFT1CF, BFT culture system plus a commercial feed supply; BFT, BFT with no feed supply; control, clear water conditionwith commercial feed supply.TAN, total ammonia nitrogen.

0

0,5

1

1,5

2

2,5

1 4 7 10 15 20 25 30

Time (days)

mg

L−1

TANNO2PO4

Figure 1 Total ammonia nitrogen (TAN), nitrite andreactive phosphorous concentrations in a bio£oc technol-ogy (BFT)macrocosm tank during a 30 days experimentalperiod.



recorded TSS values below 300mg L�1 in an inten-sive shrimp nursery system when evaluating foamfractionators as a solids removal device. In a recentstudy, Ray et al. (2010) demonstrated that controllingthe abundance of solids by a simple settling low-techsolids removal device can bene¢t shrimp yields. In ti-lapia culture, Avnimelech (2007) and Azimand Little(2008) recorded values ranging from 460 to643mg L�1and 597 and 560mg L�1respectively.

Growth performance

The BFT limitedwater exchange system is consideredto be a suitable approach for sustainable and e⁄cientaquaculture production of high ¢sh or shrimp bio-mass. In our study, ¢nal weight, ¢nal biomass andweight gain results were better in BFT treatments ascompared with clear water (Po0.05). Higher growthrates of PLs were often associated with higher grow-out performance or at least during PL pond stocking(Aquacop, LeMoullac & Damez1991; Fegan1992; Cas-tille, Samocha, Lawrence, He, Frelier & Jaenike1993).Also an interesting ¢nding was that pelletized feedsupply did not change shrimp performance. The PLin the BFT tank had similar growth performance re-gardless of whether they were fed a commercial feed.Some nutrients (as vitamins and minerals) derivedfrom the commercial feed used to feed shrimp juve-niles in the macrocosm tank could have ‘enriched’the bio£oc, enlarging the nutrient supply to the PLs.In this study, survival rates (67^84%) were similar

to F. paulensis results. Survival rates were 54^82% forF. paulensis PL reared in a clear-water nursery (Hen-nig & Andreatta 1998), 90^95% in estuarine cages

Table 3 Performance parameters of 30 days experimental period for Farfantepenaeus brasiliensis reared from PL25 andstocked at a density of1000 shrimpm�3

Parameter BFT1CF BFT Control P-value

Mean final weight (mg) 218.9A (� 13.9) 236.5A (� 13.5) 176.0B (� 4.7) o0.001

Final length (mm) 27.6 (� 1.3) 27.7 (� 1.1) 27.2 (� 0.4) 40.05

Mean weight gain (mg) 193.9A (� 13.9) 211.5A (� 13.5) 151.0B (� 4.7) o0.001

Final biomass (g) 17.9A (� 0.8) 15.7A (� 1.3) 8.2B (� 0.1) o0.001

Survival (%) 81.5 (� 3.5) 67.0 (� 7.0) 84.8 (� 4.2) 40.05

Data are means (� standard error) and signi¢cance level, along experiment. Di¡erent letters indicate means are signi¢cantly di¡erent(Po0.05).Treatments: BFT1CF, BFT culture system plus a commercial feed supply; BFT, BFT with no feed supply; control, clear water conditionwith commercial feed supply.

Table 4 Proximate analysis of commercial diet (Cargill’s Purina AgribrandsTM), sugarcanemolasses andwheat bran (carbonsource) and bio£oc

Composition Commercial feed Molasses Wheat bran Biofloc

Dry matter (%) 90.4 61.1 88.1 87.1

Crude protein (% DW) 39.7 5.6 18.9 30.4

Crude lipid (% DW) 13.1 0.2 2.7 0.47

Crude fibre (% DW) 2.6 0.2 11.3 0.8

Carbohydrate (% DW) 35.6 81.6 61.1 29.4

Ash (% DW) 9.0 12.4 6.0 39.2

Gross energy (kJ g�1 DW) 20.2 15.4 15.9 12.2

Carbohydrates estimates by di¡erence. Gross energy content were calculating according to Tacon (1990).DW, dry weight.

0

100

200

300

400

500

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Time (days)

TS

S (

mg

L−1

)

5,2

5,6

6

6,4

6,8

7,2

pH

TSSpH

Figure 2 Total suspended solids and pHmeasurement ina bio£oc technology (BFT) macrocosm tank during a 30days experimental period.



(Ballester,Wasielesky, Cavalli & Abreu 2007), and 89^95% in a similar bio£oc device (Ballester et al. 2010).For L. vannamei reared in an intensive raceway nur-sery system, Mishra et al. (2008) and Cohen et al.(2005) reported survival rates ranging from 55.9% to100% and 97% and 100% respectively. Even thoughsurvival rates and ¢nal biomass were not signi¢-cantly di¡erent between BFT and BFT1CF (67% and15.7 g and 81.5% and 17.9 g respectively), the lowervalues of BFTcould indicate cannibalism, increasingthe nutrient supply to the survivors. Further studiesare needed to better understand the bio£oc as a nu-tritional resource in F. brasiliensis nurseries, aimingto improve the percentage survival. Mineral contentand lipid pro¢le in bio£oc seems to need further in-vestigations.In relation to improved growth performance in

BFT treatments, the cage mesh possibly provided anadded substrate for colonization of biota (bio¢lm),thereby enhancing the amount of microorganismsavailable for shrimp intake as compared with theclear-water tank. Moreover, no bio¢lm formationwas observed in control cages. Many authors reports

that bio¢lm enhances shrimp performance (Moss &Moss 2004; Ballester et al. 2007), decreases feed con-version ratio (FCR) (Bratvold & Browdy 2001;Tidwell,Coyle, Arnum & Weibel 2007) and improves waterquality (Asaduzzaman et al. 2008; Arnold et al.2009). Further investigations are needed to betterunderstand the role of substrate for F. brasiliensis PLas substrate type, size and display.The role of particulate organic matter and micro-

organisms in the microbial food web as a potentialfood source for cultured aquatic animals has beendiscussed since the early 1960s (Baylor & Sutcli¡e1963) and more recently by Moriarty (1997). Naturalproductivity is a likely avenue to decrease feed inputin aquaculture systems (Browdy et al. 2001). High-in-tensity production has been most successful with L.vannamei (McIntosh, Samocha, Jones, Lawrence,McKee, Horowitz & Horowitz 2000; Moss 2002; Bur-ford et al. 2003) and more recently with Penaeusmonodon (Arnold et al. 2009). Burford et al. (2004) re-ported that more than 29% of daily food consumedfor L. vannamei could be bio£oc. Wasielesky et al.(2006) described the e¡ect of natural productivity ina heterotrophic super-intensive system for L. vanna-mei juveniles. The authors showed a decrease in FCR(1.54^1.03) and increased growth rate (0.39^1.25 gweek�1) derivate from bio£oc consumptionwhen compared with clear-water conditions.

Proximate analysis and assessment of bio£oc

The nutritional bene¢ts of bio£oc as a natural foodsource for F. brasiliensis is still under investigation.The bio£oc can contain high levels of CP and othernutrients such as essential fatty acids and aminoacids (Azim & Little 2008; Ju, Forster, Conquest,Dominy, Kuo & Horgen 2008). Proximal analysis ofbio£oc from the current study indicates the presenceof a highCP level (30.4%). However, Froe¤ s, Abe,Wasie-lesky, Prentice and Cavalli (2006) demonstrated thatwhen reared in clear water, the optimal dietary pro-tein level for F. paulensis pink shrimp juveniles is 45%CP. In contrast, Ballester et al. (2010) showed that mi-croorganisms present in BFT system played an im-portant role for provision of essential nutrients for F.paulensis, thereby enabling the CP content in practi-cal diets to be reduced from 45% to 35% withoutany reduction in growth. The CP level of 31% foundin our study is consistent with levels reported by Ta-con et al. (2002) andWasielesky et al. (2006), but lessthan the 43% and 49% CP described by Jory (2001)

Figure 4 The number of free living and attached bacter-ia (mean � SD) in a bio£oc technology (BFT) macrocosmtank at the end of a 30 days experimental period.

Figure 3 The number of associated microorganisms(mean � SD) in water from a bio£oc technology (BFT)macrocosm tank at the end of a 30 days experimentalperiod.



0

400

800

1200

1600

2000

Protozoa Cyanobacteria Rotifers Pennatediatoms

Type of microorganism

Nu

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er (m

L−1

)

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20

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Free living bacteria Attached bacteria

Type of microorganism

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er (

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−1)

and Kuhn, Boardman, Lawrence, Marsh and Flick(2009) respectively. In addition, Crab et al. (2010)found that using glucose or a combination of glyceroland Bacillus as a carbon source in‘bioreactors’ led tohigher bio£oc protein content, higher n-6 fatty acids,which resulted in improved survival rates of fresh-water prawns PLs. Further investigations are neededto compare di¡erent carbon sources and externalbioreactor devices versus in situ bio£oc tank produc-tion in terms of bio£oc nutritional quality and eco-nomical bene¢ts.The bio£oc samples showed a large quantityof gra-

zers. Protozoa appeared to be the most abundant bio-£oc microorganism group (Fig. 3). The same trendwas observed byAzim and Little (2008) and Ballesteret al. (2010).The great number of protozoaand rotifersmay contribute to better shrimp performance in BFTtreatments compared to the control (Thompson et al.2002). In terms of bacteria counts, the number of freeliving bacterial cells was almost 20 times higher thanattached bacteria. This may be a satisfactory free liv-ing/attached bacteria ratio for good bio£oc aggrega-tion as corroborated with TSS levels. These resultsmake a few assumptions and further studies areneeded to better understand the di¡erences in micro-organism pro¢les and their bene¢ts for shrimp nutri-tion. Microbial ecology of bio£ocs (i.e. using di¡erentcarbon source) certainly merits further investigation.

Conclusion

In summary, the BFT limited water exchange systembene¢ted F. brasiliensis by improving growth perfor-mance and also maintenance of water quality. Post-larvae that did not receive a commercial feed hadthe same growth as those that received feed supply.The microorganism community, mainly representedby protozoa grazers, rotifers, cyanobacteria and dia-toms, provided a continuous natural food source.The bio£oc could also provide ‘extra components’,such as growth factors, that enhance the growthrates. Further research e¡orts are highly recom-mended to detect these extra components in bio£ocand also to compare the economical bene¢ts withother alternative culture systems.

Acknowledgments

This work was supported by the Brazilian Council forScienti¢c and Technological Development (CNPq).The authors would like to thank Roberta Soares, Pau-

loAbreu and the sta¡ of the EMAMariculture Centerfor their contribution towards this study. Advice andtechnical assistance from Ligia Abreu and SandroDias is greatly appreciated. Thanks are also due toAndrew Ray and Stuart Arnold for critical readingof the manuscript, CAPES-Brazil for the M.Sc. grantto the primary author and the anonymous refereesfor their valuable suggestion on this manuscript.W.Wasielesky and R. O. Cavalli are research fellowsof CNPq.

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