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The biofloc technology (BFT) in indoor tanks:

Water quality, biofloc composition, and growth
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Article in Aquaculture · October 2008

DOI: 10.1016/j.aquaculture.2008.06.036

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 Aquaculture 283 (2008) 29–35

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The biofloc technology (BFT) in indoor tanks: Water quality, biofloc composition,
and growth and welfare of Nile tilapia (Oreochromis niloticus)
M.E. Azim ⁎, D.C. Little
Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, United Kingdom

A R T I C L E I N F O A B S T R A C T

Article history: The present study evaluates the biofloc technology (BFT) in light-limited tank culture of Nile tilapia
Received 13 April 2008 (Oreochromis niloticus). Two biofloc treatments and one control were managed in 250 l indoor tanks: BFT fed
Received in revised form 24 June 2008 a diet of 35% crude protein (CP), BFT fed a diet of 24% CP, and clean water control without biofloc with 35% CP.
Accepted 24 June 2008
BFT tanks were aerated and agitated using a dome diffuser. Three kg of Nile tilapia were stocked in each tank.
Feed was applied at 1.5% of the total fish biomass daily in each tank. Wheat flour was added in BFT tanks to
Keywords:
Biofloc
maintain an optimum C:N ratio for heterotrophic production. The total suspended solid (TSS) level was
Activated suspension technique maintained at around 500 mg l− 1 in BFT tanks.
Fish welfare The nutritional quality of biofloc was appropriate for tilapias. Fish survival was 100%. Net fish production was
Tank culture 45% higher in the BFT tanks than in the control tanks confirming the utilization of biofloc by fish as food.
Tilapia There was no difference in fish growth/production between 35% and 24% CP fed tanks under BFT. Welfare
indicators in terms of fin condition, gill histology, proximate composition, blood haematocrit and plasma
cortisol levels were compared and no significant differences between BFT and control tanks were recorded
indicating no increased fish stress due to the presence of biofloc. However, overall fish growth and
production was poor in terms of commercial feasibility. A modified system design that would allow enhanced
feed and biofloc utilization is proposed.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
The basic principle of the activated suspension technique (AST),
recently referred to as biofloc technology (BFT) is the retention of
waste and its conversion to biofloc as a natural food within the culture
system. This is done by constant aeration and agitation of the water
column and addition of carbon sources as organic matter substrate to
allow aerobic decomposition and maintain high levels of microbial
floc in suspension in fed and/or fertilized ponds (Avnimelech et al.,
1986; Hargreaves, 2006). Theoretically, increased C:N ratio through
carbon addition enhances conversion of toxic inorganic nitrogen
species to microbial biomass available as food for culture animals.
The optimum C:N ratio in an aquaculture system can be maintained
by adding different locally available cheap carbon sources and/or
reduction of protein content in feed (Avnimelech, 1999; Hargreaves,
2006).Goldman et al. (1987) found C:N ratios N10:1 were opti-
mal for optimizing biofloc production while minimizing ammonia
regeneration.
Typically only 20–25% of fed protein is retained in the fish raised in
intensive systems (Avnimelech, 2006), the remainder being lost to the
⁎ Corresponding author. Department of Physical and Environmental Sciences,
University of Toronto, 1265 Military Trail, Toronto M1C 1A4, Ontario, Canada.
Tel.: +1 416 287 7690; fax: +1 416 287 7279.
E-mail address: eazim@utsc.utoronto.ca (M.E. Azim).
system as ammonia and organic N in feces and feed residue. Microbial
breakdown of organic matter leads to the production of new bacteria,
amounting to 40–60% of the metabolized organic matter (Avnimelech,
1999). Under optimum C:N ratio, inorganic nitrogen is immobilized
into bacterial cell while organic substrates are metabolized. The
conversion of ammonium to microbial protein needs less dissolved
oxygen compared to oxygen requirement for nitrification (Avnime-
lech, 2006; Ebeling et al., 2006) suggesting the preference of
heterotrophic community rather than nitrifying bacteria in the BFT
system. In addition, the growth rate and microbial biomass yield per
unit substrate of heterotrophs are a factor 10 higher than that of
nitrifying bacteria (Hargreaves, 2006).
Although some commercial production based around the concept
of BFT has been used since the early eighties (Serfling, 2006), the
knowledge base concerning the technique is still undeveloped. The
benefits of this technology are documented mainly for shrimp culture
(e.g.Burford et al., 2004; Wasielsky et al., 2006) and to a lesser extent
for finfish culture (Milstein et al., 2001; Serfling, 2006). Preliminary
studies on tilapia culture in activated suspension ponds indicated that
the fish grew well on low protein feed and fed on suspended particles
leading to additional savings in feed costs; also the technology greatly
increased the water use efficiency (Avnimelech, 1999; Milstein et al.,
2001; Serfling, 2006). The technology was tested in aquaculture ponds
where both autotrophic and heterotrophic microorganisms interact.
Algae and bacteria have a range of stimulatory or inhibitory effects on

0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2008.06.036
30 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35
each other (Cole, 1982) resulting in complexity of water quality
dynamics. Senescent algae or algal detritus are a major source of
organic substrate for bacterial growth whereas living algae provide
oxygen for decomposition. In return, bacteria regenerate inorganic
nutrients and vitamins that stimulate algal productivity. On the other
hand, both groups produce substances that are antagonistic to growth
of each other (Cole, 1982). Light limitation of autotrophic production
also likely restricts its economic viability to applications where
outdoor culture is possible whereas a heterotrophic production
could be applied in light-limited indoor systems. However, the
contribution of biofloc to fish production was not quantified so far
and the relative importance of bacteria within the system for both
maintaining water quality and as food was not distinguished. Our
preliminary study conducted in light-limited indoor tanks without
fish indicates that the biofloc quality and quantity were appropriate
for fish culture and water quality parameters can be managed properly
using the BFT (Azim et al., 2008). The nutritional value of biofloc to
aquatic animals is dependant on several factors: food preference, not
only the ability to both ingest and digest it but also the density of
suspended particles (Hargreaves, 2006). Tilapias being capable of both
filter feeding and detritivory are ideal candidates for such system
(Dempster et al., 1995; Azim et al., 2003). However, if successful, BFT
might be a promising alternative technology to conventional
recirculation aquaculture system (RAS) in which formulated diets
including high levels of protein and complex external filtration are
required.
The main objective of the present experiment was to test the
suitability of biofloc technology in light-limited indoor tanks for
rearing Nile tilapia Oreochromis niloticus. The specific objectives were
as follows: (1) to quantify the contribution of biofloc to fish growth
and production, (2) to evaluate fish welfare in BFT managed tanks,
(3) to test the effects of crude protein level in supplemental feed on
fish growth and productivity in BFT tanks (4) to investigate the effects
of protein level/C:N ratio of feed on proximate and taxonomic
composition of biofloc and (5) to compare dissolved inorganic
nitrogen dynamics. System management aspects have also been
discussed.
2. Materials and methods
2.1. Tank facilities and experimental design
The experiment was carried out in fiber glass indoor tanks (250 l
each) situated in the Tropical Aquarium Laboratory of the Institute of
Aquaculture, University of Stirling, UK. Two treatments (with four
replications) and one control (with two replications) were compared:
Biofloc tank fed with 35% crude protein (treatment BFT35), BFT fed
with 24% crude protein (treatment BFT24). As control, similar size
duplicate tanks were placed within a conventional recirculating
aquaculture system (RAS) and stocked with fish fed with similar
amount of a 35% crude protein diet (RAS35).
Biofloc was already developed in the treatment tanks during the
previous experiment (350 mg l− 1 TSS). The levels were initially
equilibrated through exchanging water between the tanks and adding
water for evaporation loss was done. Tanks were aerated and agitated
continuously using dome diffusers (AS9, 17.5 cm × 3.75 cm, Aquatic
Services, UK) connected to an air pump. Tanks were always covered
with lids.
2.2. Fish stocking and tank management
Mixed sex Nile tilapia O. niloticus, of individual weight ranging
from 80 to 120 g were stocked at 12 kg m− 3 in each tank. Pelleted feeds
(3 mm) were formulated using plant ingredients and made locally
using the technique developed in the Institute (Kim Jauncey, personal
communication). Percentage of raw ingredients and proximate
composition, energy content and C:N ratios of the experimental
diets are given in Table 1. Feeding rates were based on observation of
feeding behaviour of fish in the BFT treatments during the first several
days and fixed at 1.5% of the total stocked biomass daily, and adjusted
fortnightly after weighing a fish sample. The same amount of feed was
applied to all treatment and control tanks. Daily feed rations were split
into two equal amounts given at 0900 and 1800 h to all tanks. Wheat
flour was also added at a rate of 60% feed applied to BFT tanks to
maintain an optimum C:N ratio of bacteria (Avnimelech, 1999). Flour
was completely mixed with tank water and spread to the tank surfaces
in the afternoon. The fish were cultured for a period of 12 weeks
between June and September 2006.
Whenever floc levels exceeded 500 mg l− 1in any tank, a floc
separator was run for 8–12 h to settle and remove the floc from the
tanks. When the pH of water dropped below 6.5, NaHCO3 was added
to raise the pH to 7.5. Addition of water for evaporation loss and floc
removal was done on a weekly basis.
2.3. Assessment of water quality parameters
Water temperature, dissolved oxygen (portable DO meter WTW
Oxi 340i) and pH (portable HANNA pH/ORP/Temperature meter HI
991002) were determined twice weekly. Total alkalinity was mea-
sured by acid titration on a weekly basis followingStirling (1985). Total
ammonia nitrogen (TAN), nitrite (NO2–N) and nitrate (NO3–N) were
analyzed spectrophotometrically with an autoanalyzer (BRAN LUBBE
AutoAnalyzer 3) according to standard methods (APHA, 1998). Water
samples (50 ml) were collected weekly at around 12:00 h from each
tank and filtered under vacuum pressure through pre-dried and pre-
weighed GF/C filter paper. The filtered water was used for nutrient
analysis and the filter paper for total suspended solid (TSS)
determination.
Total suspended solid (TSS) and biochemical oxygen demand
(BOD5) of water were measured on a weekly basis followingStirling
(1985). After filtering water for nutrient analysis, the pre-dried and
weighed filter paper containing suspended materials was dried in an
oven until constant weight. Dried samples were weighed to 0.01 mg
using a Mettler AC 100 balance. The TSS was calculated from the
weight differences. For BOD determination, water samples were
collected in plastic bottles from each tank. Two diluted samples in
each tank and two controls (with only distilled water) were used in
170 ml BOD bottles. Samples were diluted 10 times initially and 20
times at the end of the experiment with distilled water. The initial
oxygen concentrations in the samples were determined using a BOD
YSI probe and DO meter (YSI model 57). The necessary chemicals were
added to each bottle in case of diluted samples according toStirling
(1985). The samples were firmly closed with stopper and placed in an
incubator (Gallenkamp Cooled Incubator) in the dark at 22 °C for
5 days. At the end of this time, the dissolved oxygen level was
determined again and BOD5 was calculated in mg l− 1 (Stirling, 1985).
Table 1
Diet formula and biochemical composition of the experimental diet
High protein diet Low protein diet
Feed ingredients (%)
Soy meal 60 30
Wheat meal 30 60
Vegetable oil 5 5
Molasses 5 5
Biochemical composition (%DM)
Moisture 8.51 4.51
Crude protein 35.13 23.88
Crude lipid 6.23 6.64
Ash 5.23 3.83
Crude fibre 2.63 2.13
Energy (kJ g− 1) 19.6 19.1
C:N ratio 8.4 11.2
 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35
2.4. Imaging and identification of microorganisms in biofloc
The 3-D images of bacterial floc were taken using a confocal
microscope (Leica TCS 4PI) on a biweekly basis. Water samples were
collected from one tank in each treatment and processed immediately
for microscopy. Each sample was diluted with pre-filtered water of the
same tank, stained with fluorescence 4′-6-Diamidino-2-phenylindole
(DAPI), and observed and photographed at magnification 10.
Taxonomic composition of other organisms in the water was
studied in three consecutive weeks in the middle of experimental
period. Well-mixed water samples were collected in 30 ml plastic
bottles and the organisms were killed by adding several drops of
Lugol's iodine. One ml sample was placed on the counting chamber of
the Sedgewick Rafter counting cell (S-R cell) and left for several
minutes to allow organisms to settle. Then the organisms on 10
randomly selected fields of the chamber were identified and counted
using magnification of 20–40 of a binocular microscope (Olympus
LH50A). The number was multiplied by 100 to give the results in total
number of organisms per ml water. This counting was repeated three
times in each sample in each tank. The keys used for taxonomic
identification of the microorganisms were Donner (1966), Patterson
and Hedley (1992) and Nuffield Advanced Science (1970).
2.5. Determination of biochemical composition of biofloc
Biofloc samples for biochemical analysis were collected on a
fortnightly basis. Concentrated floc samples collected from each tank
using a floc separator were dried in an oven at 102°C until constant
weight and then preserved in a refrigerator. At the end of the
experiment, tank-wise pooled samples were ground and processed for
proximate analysis followingAOAC (1990). For ash contents, a known
amount of dry sample was burnt in a muffle furnace at 550 °C for 4 h
and the ash cooled and weighed. The crude protein content was
determined by the Kjeldahl method. Lipid content was determined
with Soxhlet apparatus. The energy content was determined by a
bomb calorimeter (Gallenkamp Autobomb). The C:N ratios were
determined using a CHN analyzer (Perkin Elmer 2400 Series II). Biofloc
samples for fatty acid analysis were collected at the end of the
experiment and freeze-dried. Fatty acid analysis was undertaken by a
modified direct methylation method (Christie, 2003) using an N-Evap
112 Liquid Gas Chromatograph (Organomation Associates Inc.).
2.6. Determination of fish welfare indicators
More than 50% of fish in each tank were sampled fortnightly,
anaesthetized and length and weight measured individually. These
fish were also checked for any skin or fin damage. At the end of the
experiment, one fish in each BFT treatment tank and two fish in each
RAS35 tanks were sacrificed, the 1st and second gills from one side
were removed and preserved in 10% buffered formalin. The samples
were then processed for histology. Unpreserved gills were examined
for parasites using a binocular microscope. Samples of skin mucus
were also checked under microscope.
At the end of the experiment, 10 fish were netted randomly from
each BFT35 and RAS35 tank. They were mildly anaesthetized with
benzocaine in a bucket, weighed and measured. Blood samples were
collected from the dorsal caudal aorta using heparinised syringes and
placed in microcentrifuge tubes. After blood sampling, fish were
individually tagged (internally) and recovered in another bucket with
clean water and air supply. Then they were transferred to another fish
tank within a RAS until they were blood sampled for second time after
1 h following the same procedure mentioned above.
Haematocrit (% packed red cell volume) was measured after
centrifuging sub-samples of whole blood collected the first time using
heparinised micro-haematocrit tubes for 5 min at 5000 g. The
remaining blood was centrifuged at 1200 g for 15 min and the plasma
31
was separated and stored at −70 °C. Concentrations of plasma cortisol
were determined using the radioimmunoassay described by Ellis et al.
(2004) with the modifications to the steroid extraction. Aliquots
(200 μl) of plasma were extracted with 1 ml ethyl acetate and after
thorough mixing and centrifugation (430 g for 10 min), 200 μl of
chilled (4 °C) supernatant were pipetted into duplicate polypropylene
tubes, which were evaporated to dryness under vacuum at 35 °C and
before cooling to 4 °C. Proximate composition and energy content of
fish were also compared. Ten randomly selected fish at stocking and
three fish in each tank at harvest were analyzed using the similar
methods described above for biofloc.
2.7. Statistical analysis
Water quality parameters were compared by two-way repeated
measures ANOVA with treatment (system type) as main factor and
sampling date as repeated measures factor (Gomez and Gomez, 1984).
The fish yield and blood parameters and biochemical composition of
floc and fish were compared using one-way ANOVA. If main effects
were significant, differences among the treatments were tested with
Tukey's multi-comparison test of means. The analyses were run at 5%
significance level using Statistica package.
3. Results and discussion
3.1. Water quality
Average water temperature (28 °C, range 26–30 °C), DO concen-
trations (6 mg l− 1, range 3.0–7.5 mg l− 1) and pH (6.7, range 5.0–8.5)
were within the range for tropical fish culture except for low pH levels
observed, and then corrected for, on several occasions. While total
alkalinity was very stable in the control tanks (18–27 mg l− 1) it
fluctuated very much in the tanks with biofloc (8–250 mg l− 1). This
indicated that the biofloc systems lose buffering capacity and
therefore required frequent additions of NaHCO3. High rates of
nitrification, indicated by constant nitrate accumulation (discussed
later) coupled with ammonia immobilization into bacteria (Azim et al.,
2008) were observed in BFT treatments. It is reported that nitrification
requires approximately 4 mg O2 and 8 mg HCO−3 for oxidizing 1 mg
TAN (Gujer and Jenkins, 1974; Sharma and Ahlert, 1977). However,
continuous vigorous aeration ensured that dissolved oxygen was not
limiting.
The average TSS levels were 16, 597 and 560 mg l− 1 in the control,
BFT35 and BFT24, respectively. Efforts were made to keep the TSS level
at 500 mg l− 1 using a floc separator but the levels became
uncontrollable using this regime at the last two sampling dates
reaching up to 1000 mg l− 1. Sometimes floc settled to the bottom of
the separator and sometimes floated on the surface, suggesting
qualitative changes in flocs. This characteristic as also observed by
Murray and Little (submitted for publication) in light-limited BFT
systems. Therefore, characterization of floc and improved approaches
to its removal are a pre-requisite for effective management of this
system. Optimum floc levels in terms of TSS level in relation to the fish
stocking density needs further investigation. The BOD level corre-
sponded to the TSS level with mean values of 7, 87 and 88 in the
control, BFT35 and BFT24, respectively with a peak of 290 mg l− 1 at the
last sampling date in the biofloc treatments.
Dissolved inorganic nitrogen (TAN, NO2–N and NO3–N) concentra-
tions throughout the experimental period are shown in Fig. 1. A high
degree of fluctuation in TAN and NO2–N levels was observed in biofloc
treatments (high standard deviation) during the entire experimental
period. TAN concentrations varied significantly between treatments
and sampling dates with higher mean values in the tanks fed 35% CP
with biofloc as compared to the control tanks without biofloc (Fig. 1A).
The NO2–N concentrations in both biofloc treatments were signifi-
cantly higher than in the control (Fig. 1B). There were also significant
32 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35

Table 2
Mean (±SD) proximate composition, energy content and C:N ratio of biofloc in high and
low protein fed tanks

Composition (%DM) 35% CP Feed (C:N = 8) 24% CP Feed (C:N = 11)

Crude protein 37.93 ± 2.38 38.41 ± 3.62
Crude lipid 3.16 ± 0.31 3.23 ± 0.21
Ash 13.38 ± 1.35 11.83 ± 0.80
Crude fibre 6.27 ± 0.44 5.71 ± 1.86
Energy ((kJ g− 1) 18.62 ± 0.43 19.04 ± 0.22
C:N ratio 7.2 ± 0.43 7.3 ± 0.22

(Ebeling et al., 2006). The relative importance of each varies with

system type and production intensity. In the present experiment,
although immobilization of heterotrophic bacteria was encouraged,
the system was dominated by nitrifying bacteria. Further research on
how to minimize nitrification in BFT systems is needed.
Despite the fact that carbohydrate was added based on TAN: CHO
of 1: 20 (Avnimelech, 1999), TAN and NO2–N concentrations were
elevated, sometimes reaching critical levels in BFT tanks during the
experiment. The CHO requirement to minimize TAN needs further
evaluation. The calculation might include NO2–N concentration along
with TAN concentration as also suggested byMurray and Little
(submitted for publication).

3.2. Nutritional quality of biofloc as fish food

The biochemical compositions and energy contents of biofloc are

given in Table 2. There were no significant differences in any
nutritional parameters between the 35% and 24% CP fed treatments,
indicating that the biofloc quality was independent of feed quality

Table 3
Fatty acid composition (% lipid) of biofloc in high and low protein fed tanks

Fatty acid 35% CP Feed 24% CP Feed

C14:0 2.48 2.02
C15:0 0.77 0.70
C16:0 19.10 17.88
C18:0 6.24 7.27
C20:0 1.44 0.87
C22:0 1.31 1.06
C24:0 0.33 0.40
Total saturated 34.92 30.20
Fig. 1. Dissolved inorganic nitrogen in different treatments throughout the experi-
16:1n-9/n-7 7.74 7.15
mental period. (A) TAN, (B) NO2–N and (C) NO3–N. Values are means (± standard
18:1n-9 8.51 10.08
deviation) of four and two replications in each sampling date respectively for treatment
18:1n-7 11.05 11.28
and control.
20:1n-11/n-9 0.80 0.74
20:1n-7 0.00 0.13
Total monounsaturated 28.10 29.38
time effects and treatment–time interactions indicating that the
18:2n-6 15.38 16.68
concentrations in different treatments behaved differently over time. 20:2n-6 0.55 0.50
The NO3–N accumulation in the systems started on the 3rd 20:3n-6 0.40 0.46
sampling date (Fig. 1C). Similar to TAN, the NO3–N concentrations in 20:4n-6 3.55 3.11
22:4n-6 0.34 0.59
the treatment BFT35 were unstable throughout the experimental
22:5n-6 3.28 4.47
period. In contrast to TAN levels, NO3–N concentrations increased Total n-6 PUFA 23.50 25.81
gradually in BFT24 tanks throughout the experiment indicating better 18:3n-3 0.65 0.73
nitrification in low protein fed tanks. While TAN and NO2–N 18:4n-3 0.06 0.05
concentrations were very unstable, NO3–N concentrations were 20:3n-3 0.00 0.02
20:4n-3 0.00 0.06
more stable and tended to increase in the biofloc treatments
20:5n-3 0.46 0.39
throughout the experiment. The concentrations were significantly 22:5n-3 0.00 0.09
higher in the high protein fed tanks followed by low protein fed tanks 22:6n-3 0.74 0.77
and control tanks. There were significant time effects and treatment– Total n-3 PUFA 1.91 1.38
16:2 0.00 0.02
time interactions as well indicating the different trends of the
16:3 1.32 0.80
concentrations irrespective of the treatments over time. There are 16:4 0.03 0.08
three principal pathways to remove hazardous N species in aqua- Total PUFA 26.76 28.09
culture: (1) photoautotrophic removal by algae, (2) immobilization by Unknown 10.22 12.33
heterotrophic bacteria as proteinacious microbial biomass and Total 100.00 100.00
Total lipid % 3.16 3.23
(3) chemo-autotrophic oxidation to nitrate by nitrifying bacteria
 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35 33

Table 4
Growth performance of Nile tilapia in activated suspension and clean water tanks fed
different levels of protein feed

Parameters 35% CP without 35% CP with 24% CP with

biofloc biofloc biofloc
Initial individual weight (g) 99.61 ± 13.74 100.69 ± 13.61 98.45 ± 12.71
Final individual weight (g) 127.51 ± 28.17b 140.72 ± 27.26a 138.58 ± 24.99a
Survival (%) 100 100 100
Individual weight gain (g) 27.9 ± 0.69b 40.04 ± 3.04a 40.08 ± 4.34a
Net yield (kg m− 3) 3.35 ± 0.08b 4.80 ± 0.60a 4.90 ± 0.59a
FCR 4.97 ± 0.12a 3.51 ± 0.44b 3.44 ± 0.45b

Data shown are Means (±SD) for m3 water and 12 weeks culture period.
In each row, different superscript letters indicate significant difference at 0.05 level
(ANOVA and Tukey test).

Fig. 2. A 3-D image of six weeks old bacterial flocs stained with 4–6-diamidino-2-
phenylindole (DAPI) using a confocal microscope.
fatty acid (15%) than in the present study.Jauncey (2000) reviewed that
about 0.5–1% n-6 fatty acid of dietary lipid required for Nile tilapia.
However, a better quality biofloc was reported in the similar system
without fish (Azim et al., 2008) indicating that in situ floc utilization by
fish could have an effect on the biochemical composition of floc.
3.3. Taxonomic composition of biofloc

A 3-D picture of two individual bacterial flocs using a confocal

applied in this experiment. A similar observation was made byAzim et
al. (2008) using the similar feeds and system but without any fish.
Goldman et al. (1987) also reported that the C:N ratio of bacteria
varied little (5–6:1) despite a wide variation in substrate C:N ratio
(1.5–10:1).
Jauncey (2000) suggested 25–30% crude protein and 6–8% crude
lipid in tilapia diets for larger fish.Chou and Shiau (1996) reported that
5% dietary lipid appeared to be sufficient to meet the minimal
requirement of juvenile hybrid tilapia (O. niloticus ×O. aureus), but a
level of 12% was needed for maximal growth. It is also not desirable to
have a fibre content exceeding 8–12%, and ash content 12% in diets for
fish, as the increase in fibre content would consequently result in the
decrease of the quantity of a usable nutrient in the diet (De Silva and
Anderson, 1995). The biofloc quality in terms of fish nutrition containing
38% protein, 3% lipid, 6% fiber, 12% ash and 19 kJ g− 1 energy (on dry
matter basis) in the present study was appropriate for tilapias except for
the very low crude lipid levels. The biofloc was further analysed to see
whether it contained essential fatty acids for the fish (Table 3). There
were 27–28% polyunsaturated, 28–29% monounsaturated, 30–35%
saturated fatty acids and 10–12% unknown peaks. Tacon et al. (200)
estimated 35–38% crude protein, 5–9% crude lipid, 7–10% ash, and 18–
19 kJ g− 1 energy in the biofloc collected from zero-exchange outdoor
system cultured with shrimp and fed different formulated and
commercial diets. They also reported a lower level of polyunsaturated
microscope is shown in Fig. 2. The blue channel dominating the picture
indicates the clusters of bacterial nuclei. There are trace amounts of
mucopolysaccharides, typically secreted by bacteria (green channel).
The flocs were more or less similar in shape but varied in size ranging
from 50 to 200 μm during the experiment.
The abundance and taxonomic composition of different floc-
associated organisms are given by treatment in Fig. 3. Three groups of
organisms were identified: Protozoa, Rotifera and Oligochaeta. Among
protozoans, three genera, namely, Paramecium, Tetrahymena and Pe-
talomonas dominated. Four genera of rotifers were identified, namely,
Lecane, Trichocerca, Polyarthra and Asplanchna. Only Tubifex was
found in the group Oligochaeta. The total numbers of organisms
were significantly higher in the low protein fed tanks followed by the
high protein fed tanks and control (Fig. 3).These microorganisms were
observed grazing on the flocs using fresh samples under microscope.
In natural system, coupling between bacteria and heterotrophic
nanoflagellates was such that about 10–70% of the water column
was daily cleared of bacteria (Bloem et al., 1989).
3.4. Fish yield parameters
The fish yield parameters are given in Table 4. Tilapia survival was
100% in all treatment and control tanks. Individual fish weight at
harvest was 9–10% higher in the BFT treatments than in the control.
The BFT treatments contributed 44–46% greater individual weight
gain and net fish production than those in the control confirming the

Fig. 3. Abundance of biofloc associated organisms in different treatments. Values are Fig. 4. Blood cortisol level in fish cultured in tanks with and without biofloc and under
means (± standard deviation) of four replications and three sampling dates for pre-stressed and post-stressed conditions. Values are means (±standard deviation) of
treatment (N = 12) and two replications and three sampling dates (N = 6) for control. 10 fish for both treatment and control.
34 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35

Table 5
Mean (±SD) proximate composition and energy content of fish at stocking and harvest
in BFT and RAS tanks fed different levels of protein feed
Composition Stock Harvest
(%DM)
35% CP without 35% CP with
biofloc biofloc
Moisture 77.50 71.25 ± 1.06 69.03 ± 1.34
Crude protein 55.13 55.28 ± 2.12 53.41 ± 2.74
Crude lipid 19.15 25.88 ± 2.84 27.83 ± 2.84
Ash 20.37 16.61 ± 0.54 17.05 ± 0.78
Energy ((kJ g− 1) 21.23 23.06 ± 0.69 23.34 ± 0.55
utilization of biofloc by fish as food. Most of the tilapias are known to
utilize in situ produced food particles including suspended bacteria
(Beveridge et al., 1989; Beveridge and Baird, 2000).Avnimelech (2007)
also confirmed the biofloc uptake by Mozambique tilapia using stable
nitrogen isotope labelling technique. However, there was no sig-
nificant difference in fish growth/production between 35% and 24% CP
fed tanks under the BFT. The FCR value was significantly higher in the
control than in the BFT treatments.
Although there was clear evidence that the biofloc significantly
contributed to the growth and production of fish, the poor FCR and
level of production was well below commercially viable levels.Little
et al. (2008) reviewed final biomass levels of 10–28 kg fish m− 3
achieved in indoor and outdoor BFT systems. These productions are
even far below its counterpart traditional recirculating aquaculture
systems in which standing biomass exceeding 100 kg fish m− 3 with
oxygenation and 70–80 kg fish m− 3 with aeration (Timmons et al.,
2002). However, there might be several reasons attributed to the poor
fish growth and production. Increased turbidity due to biofloc reduces
the visibility and hence artificial feed intake. Although floc separator
was used, it was not easy to maintain 500 mg l− 1 TSS level, and very
often the level reached to 1000 mg l− 1 TSS especially during the
second half of the experiment. Maintaining optimum floc levels was
also identified as a critical issue in managing BFT systems byLittle et al.
(2008). Secondly, water quality parameters were not stable: high
fluctuation of pH and alkalinity, high concentrations of inorganic
nitrogen species might have chronic effects on fish health. Thirdly,
some levels of fish reproduction occurred in the tanks inevitably
reducing their growth. However, most of the fish seeds did not survive
until the end of the experiment, might be due to adverse water quality
on some occasions, rapid agitation of water and also cannibalism.
3.5. Fish welfare indicators
Visual and microscopic observations of skin, fin and gill indicated
no physical damage in any fish. Out of eight gills examined by
histology, four were perfectly normal in the treatment BFT24. Some
primary filaments of the remaining four gills had minor distal
extremity thickenings. Also one gill had couple of very minor nodular
focal thickenings lower down on two filaments. In treatment BFT35,
six gills looked perfectly normal, one had thickening of distal
24% CP with
extremities of some primary filaments and the remaining gill
biofloc
displayed telangiectasis in five adjacent filaments near distal
67.83 ± 0.76
49.55 ± 1.75 extremities. In the control tanks, out of eight gills, four were perfectly
32.85 ± 2.00 normal, the remaining had minor distal extremity thickenings, also
15.92 ± 0.39 one with telangiectasis. Generally, high levels of suspended solids are
24.20 ± 0.40 related to poor fish welfare as indicated by poor growth, fusion of gill
lamellae (Mettam, 2005) and susceptibility to bacterial or parasite
infections (Noble and Summerfelt, 1996). However, since minor
abnormalities in some fish gills from both treatment and control
tanks were affected, there is no evidence of potential gill damage due
to presence of biofloc in this experiment. The similar observation was
made byVincent (2006) who compared fish raised over extended
periods within BFT and RAS systems in a commercial farm.
The proportion of blood haematocrit in both BFT35 and RAS35 fish
was 27%. Blood cortisol concentrations also did not vary significantly
between the treatment and control tanks. However, after 1 h stress,
the concentrations increased by 8 times in the treatment BFT35 and by
fives times in the control RAS35 (Fig. 4). This difference might be due
to an increased initial load of, and challenge from, bacteria entering
the fish at the tagging site in the biofloc treatment.
The proximate composition and energy content of fish in different
treatment and control tanks are also compared and presented in
Table 5. There were no significant differences in any fish nutritional
parameters among the tanks under biofloc treatments and control.
4. Conclusion and future research directions
Biofloc clearly contributed to the growth and production of fish in
light-limited indoor systems. The nutritional quality of biofloc was
appropriate at least for herbivorous and omnivorous fish species
including tilapias. Although there was no evidence that the prevailing
biofloc levels affected fish welfare, observed lower feed intake, slower
growth rate and sometime chronic mortalities (Murray and Little,
submitted for publication) suggest that the growth and production of
fish in indoor BFT are not comparable to its counterpart indoor RAS
hence unsecured economic sustainability. Uptake of biofloc by the fish
was insufficient to prevent its build-up and the need to remove it
regularly from the system was clear. Water quality parameters were not
stable. The biofloc technology seems not appropriate to those species
that do not graze on biofloc directly. Therefore, further development of
mixed systems has been advocated in which culture units are
partitioned with algae, microbial floc and/or periphyton (e.g.Azim and
Little, 2006; Avnimelech, 2006, 2007). Research efforts should be made

Fig. 5. Schematic diagram of a biofloc-based recirculation aquaculture system.

 M.E. Azim, D.C. Little / Aquaculture 283 (2008) 29–35
to develop a hybrid technology of BFT and RAS. A simplified diagram of
such technology is depicted in Fig. 5. All biofiltration units of an RAS are
replaced with a biofloc reactor or activated suspension unit. Fish tank
effluents are continuously pumped into the biofloc reactor in which
bacterial flocs are produced by vigorous agitation and aeration with a
dome diffuser and carbon supplementation. Then the flocs are passed
through a floc separator in which flocs are settled and removed
periodically and processed for formulated feed. The clean water along
with controlled amount of bioflocs are pumped to the fish tanks again.
The production and potential use of heterotrophic bacteria using fish
waste concurrently improving water quality in the proposed indoor
system would be a prospective technology to lower nutrient discharge
and to increase nutrient retention thereby ensuring its future sustain-
ability. A preliminary study indicated that microbial flocs generated in
bioreactors using fish waste and offered as supplemental feed improved
shrimp growth (Kuhn et al., 2007). However, clear understanding on the
microbiological aspects particularly bacteria growth patterns, charac-
terization of biofloc, possible manipulation of microbial community is
necessary for the successful design and operation of such technology.
Acknowledgements
The research is funded through the Marie Curie Incoming Interna-
tional Fellowship (IIF) of the European Commission (ASPECT: MIFT-CT-
2005-008965). Special thanks are due to our colleagues, Dr. James Bron
for Confocal Microscopy, Mr. James Dickson for fatty acid analysis, Dr. Ben
North for Radioimmunoassay, Mr. Keith Ranson for preparing experi-
mental set up and providing experimental fish, Mr. William Struthers for
the water quality analysis, Mr. Allan Porter for proximate analysis and
Nuffield Fellow Miss Erin Matthews for taxonomic identification.
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