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Aquaculture 264 (2007) 140 – 147

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Feeding with microbial flocs by tilapia in minimal discharge


bio-flocs technology ponds
Yoram Avnimelech
Department of Civil and Environmental Eng., Technion, Israel Inst. of Technology, 32000 Haifa, Israel
Received 7 August 2006; received in revised form 24 November 2006; accepted 25 November 2006

Abstract

The uptake of microbial flocs by tilapia was evaluated. Fish (tilapia Mozambique, 107 g) were stocked in 1 m3 tanks filled with
water from a limited exchange intensive tilapia producing pond (bio-floc technology, BFT system). Tagged ammonium nitrogen
(15NH4(SO4)2) and starch to ensure incorporation of the 15N into the bio-flocs, were added. Fish were held in the tanks for ca
2 weeks, not fed during a week period, when the only source of feed was microbial flocs. Floc volume, total suspended solids, as
well as total carbon and nitrogen in suspension were monitored.
The floc plug in settling cones contained 1.4% as dry matter.
Feed uptake, evaluated through the decrease with time in respect to 4 independently determined parameters, namely floc
volume, TSS, C and N in suspension, was found to be 10.3 ± 1.0 g kg fish− 1 day− 1, averaged for the computed values for these
parameters However, this preliminary evaluation was based on the assumption that fish harvesting is the only mechanism affecting
bio-flocs mass, neglecting biodegradation and production of flocs. Gross daily uptake of nitrogen as determined using 15N uptake
data was 0.25 gN kg− 1 (1.6 g protein), equivalent to the daily uptake of 6.2 g kg− 1 of dry bio-flocs, 60% of that computed by the
simplified mass balance approach. This difference may be attributed to microbial degradation of the bio-flocs.
Even the lower flux as evaluated through 15N uptake, constituted, in the specific case studied, almost 50% of conventional feed
ration.
© 2006 Elsevier B.V. All rights reserved.

15
Keywords: Microbial control; Intensive ponds; N uptake; Flocs; Bio-flocs technology

1. Introduction the water. As a result, intensive aquaculture industry


faces two major problems. The first is the water quality
Intensive aquaculture systems are used to efficiently deterioration caused by the high concentrations of me-
produce dense biomasses of fish or shrimp. An intrinsic tabolites and the second is the low feed utilization in
feature of these systems is the rapid accumulation of cases when high water exchange, within or outside the
feed residues, organic matter and toxic inorganic ni- pond system, is practiced.
trogen species. This cannot be avoided, since fish retain The principles of growing fish or shrimp in limited
only 20–30% of feed nutrients (e.g. Avnimelech and water exchange intensive ponds were developed simul-
Lacher, 1979; Avnimelech and Ritvo, 2003; Boyd, taneously for shrimp in the Waddel Mariculture Center in
1985). The rest is excreted and typically accumulates in the USA and for fish, mostly tilapia, in Israel (Avnimelech
et al., 1989; Avnimelech et al., 1994; Hopkins et al., 1993;
E-mail address: agyoram@tx.technion.ac.il. Chamberlain and Hopkins, 1994) and practiced in the
0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2006.11.025
Y. Avnimelech / Aquaculture 264 (2007) 140–147 141

USA (Serfling, 2000), in the beginning of the 1990's. Bio- Where d[BF] / dt is the bio-flocs concentration
floc technology, BFT, (called also active suspension change with time, as affected by production, harvesting
ponds, heterotrophic ponds, green soup and other terms) by fish and biodegradation. Each of the processes given
was first developed to solve water quality problems. in Eq. (1) is complex and depends on a variety of
Water quality management is based upon developing and factors:
controlling dense heterotrophic bacteria within the culture
component. This goes in tandem with zero or minimal a. Production of bio-flocs depends on the supply of
water exchange rate. A microbial community develops, organic substrates to the microbial community, both
reaching a density in the order of 107 colonies forming external sources (feed supply, algal activity) or by the
units, CFU, ml− 1 (Burford et al., 2003), forming bio-flocs excretion of un-utilized feed components by fish. In
that contain bacteria, other micro-organisms, protozoa addition bio-flocs production most probably depends
and zooplankton. on the quality of the added substrates, its C/N ratio,
Accumulation of toxic inorganic nitrogen species bio-availability and other factors.
(NH4, NO2) is prevented in bio-flocs system by main- b. Uptake of the bio-flocs by fish depends most
taining a high C/N ratio and inducing the uptake of probably on the fish species and feeding traits, fish
ammonium by the microbial community (Avnimelech size, floc size and floc density. It is possible that bio-
et al., 1994; McIntosh, 2000). This process was quan- flocs harvesting depends also on the presence and
titatively formulated (Avnimelech, 1999), verified and rate of formulated feed added to the pond. In
practiced by farmers world-wide (Browdy et al., 2001; addition, feed eaten by the fish may be utilized and
Panjaitan, 2004). accumulate in the fish, or excreted and serve as a
A by-product of this is the growth of the microbial substrate for the production of more bio-flocs.
community and the production of microbial protein. c. Biodegradation of the floc depends on the microbial
Harvesting of the bio-flocs by fish serves as an addition community associated with the bio-flocs, be it
of high value feed, recycling of the non-utilized fraction bacteria, protozoa or others.
of the feed and was shown to double the utilization of d. Finally, all of these processes may be affected by
protein and feed by fish or shrimp (Avnimelech et al., environmental and operational conditions such as
1989; McIntosh, 2000; Velasco et al., 1998). temperature, water salinity, water exchange rate
The fast spread and the large number of BFT shrimp (affecting floc mean residence time, FMRT), mixing
farms induced significant research effort of processes in intensity and many other.
BFT shrimp production systems (e.g Browdy et al.,
2001; Burford et al., 2003; Taw, 2005). Yet, practically The study of this complex system is difficult, since
no work was done in BFT fish production units. it is practically impossible to study all factors
The biomass in fish producing BFT is in the range of simultaneously and since the monitoring of any of
10–40 kg m− 3, as compared to a range of 1–2 kg m− 3 the processes given in Eq. (1) is technically difficult.
for shrimp. It is expected that process rates and We consider this work as a preliminary work aimed at
concentrations in the fish ponds will be higher than in the development of methodology and conceptual
shrimp ponds and easier to evaluate. Thus, besides its approach toward the quantitative evaluation of bio-
importance for fish culture, research in such ponds can flocs technology.
reveal processes that are more difficult to evaluate in The goal of this work was to reveal some of the
shrimp ponds. processes involved in the bio-floc system, to get some
It is clear that bacteria, metabolizing carbohydrates, quantitative data on harvest of flocs by tilapia and to
take up dissolved inorganic nitrogen (mostly NH4) and evaluate some methods that can be used in future
produce protein. Microbial flocs of different sizes can be research and monitoring. This work is based, on one
taken up by fish or shrimp and serve as a feed source hand, upon the evaluation of the material balance of
(Avnimelech et al., 1989; Burford et al., 2004; Tacon suspended components along a two weeks experimen-
et al., 2002) However, a quantitative and predictive study tal period. The second approach was to evaluate
of this is very complicated. A number of processes are nutrients uptake from flocs through the evaluation of
15
taking place simultaneously, formulated schematically as: N tagging of the flocs and its uptake by fish. The 15 N
uptake was used as a means to evaluate the
contribution of microbial protein to the nutrition of
d½BF=dt ¼ BFproduction −ðBFharvesting þ BFdegradation Þ
shrimp by Parker and Anderson (1989), Cam et al.
ð1Þ (1991) and Burford et al. (2004). Avnimelech et al.
142 Y. Avnimelech / Aquaculture 264 (2007) 140–147

(1989) used the distribution of the naturally occurring Five g 15NH4Cl (60% 15N enrichment), dissolved in
13
C in feeds and fish to evaluate the contribution of water was distributed to the 3 tanks. Nitrogen addition
microbial proteins to fish nutrition. amounted to about 0.5 mg l− 1. One hundred g starch in
suspension was added to each tank to enhance the
2. Materials and methods assimilation of the background and the added ammoni-
um into the microbial biomass. Two days later an
2.1. Facility additional portion of 50 g starch was added to each tank.
The amount of starch added was in excess of theoretical
The experiment was conducted in Pacific Aquafarms amount needed to immobilize the added ammonium, to
in summer 2004, using water from a pond in that farm. ensure a complete immobilization into the microbial
Pacific Aquafarms is a commercial farm located near biomass. TAN concentration following the addition of
Salton Sea, in the Imperial Valley, CA. The farm consists the starch was maintained at low levels (0.1–0.2 mgN
of about 20 grow-out ponds (ca 1000 m2 each). The daily l− 1) all along the experimental period. Twenty fish at an
water exchange rates used in most ponds was 20%. average weight of 107 g were introduced to each tank on
However, limited exchange BFT ponds (daily exchange September 17. The experiment lasted for 2 weeks. No
ca 5%) were initiated and the tendency is to change all fish mortality was detected.
production ponds toward BFT systems. Water for the Fish were not fed during a 6 days period along which,
ponds is extracted from a saline thermal water aquifer. the only feed source was the microbial suspension. A
Water temperature at the source is 60 °C and its salinity is portion of 100 g feed was added on September 23,
3 ppt. All ponds are fully lined with soil cement mixture, followed by daily additions of 30 g (ca 2% of fish body
and have an average water depth of 100 cm. weight), dropping to 25 g according to the decrease of
The research was conducted using water from pond fish biomass due to sampling. Feed, as 20% protein
T2, a pond that also served as a reference pond. The floating pellets, was distributed 3 times a day.
pond was stocked on May 11, 2004 with 41,000 tilapia Oxygen and temperature were determined daily.
Mozambique with an average weight of 47 g each. Oxygen level was on the average 8.2 mg l− 1 and did not
Limited water exchange of about 6% per day was drop to values below 6 mg l − 1 . Average water
practiced. The pond was aerated with paddlewheels temperature was 26.2 °C. Tanks were covered with a
(13 HP) creating a circular current. Sludge that shade net to prevent excessive heating. Floc volume
accumulated in the pond centre, as a result of deposition (FV) was determined, in most cases, daily. Fish were
of waste products of feeding and other detritus, was sampled at stocking and then every 3 days, 2 per tank, 6
removed daily via a central drain. replicates in total. Fish were weighed and chilled. Head
A 20% protein feed was used in the pond, to and fins were removed prior to grinding (guts were not
minimize ammonia accumulation and maximize protein removed). Ground fish samples were then dried to
utilization (Avnimelech, 1999). Total ammonium nitro- constant weight and kept frozen. Samples of fish were
gen (TAN) concentration in the water was determined placed at 60 °C for 2 days to determine dry weight.
twice a week. Starch, (pre-gelatinized wheat starch), at a Water was also sampled at the same days to
rate of 45 kg was added daily whenever TAN determine soluble nitrogen species, total suspended
concentration rose to values of above 7.0 mg l− 1. matter, suspended nitrogen and carbon and 15 N enrich-
ment in the suspended matter.
2.2. Experimental system and details
2.3. Analyses
Three 1 m3 fiber-glass tanks were placed near pond
T2 and filled on September 14, 2004, by pumping pond FV was determined by sampling 1000 ml pond water
water (800 l) into the tanks. Pond water con- into a series of Imhoff cones (Eaton et al., 1995). The
tained ammonium and nitrate-N at levels of about 5 volume of the floc plug accumulating on the bottom of
and 40 mgN l− 1, respectively. Air stones were placed to the cone was determined 15 min following sampling. A
ensure proper aeration and mixing of the water. In ad- period of 10–15 min was long enough to get a stable FV.
dition, tanks were manually mixed twice daily, bottom Keeping the sample for longer periods of time led to the
sediments resuspended and walls cleaned, using a mop, formation of gas bubbles in the floc plug and floatation
to prevent the accumulation of organic-rich and oxygen- phenomenon.
poor environments and to ensure mixed and uniform The apparent dry density of the plug was evaluated
water body before sampling. following a careful siphoning of the overlying water
Y. Avnimelech / Aquaculture 264 (2007) 140–147 143

with minimal disturbance of the plug. The remaining ly, there was a clear diminution in the FV in the first
suspension was transferred to a bottle wherein the 6 days, when feed was not given, from about 30 ml l− 1 to
volume was adjusted to 100 ml. Total Suspended Solids 20 ml l− 1. The daily change of FV (omitting the probably
(TSS) in the suspension were determined and the erroneous point of day zero) was:
original volume of the floc plug and its dilution were
considered to calculate dry matter concentration in the FV ðml l−1 Þ ¼ 31−1:714 t; R2 ¼ 0:686 ð2Þ
original plug. TSS were determined by filtration of a
sample of pond water or floc plug suspension through a Where t is the time in days.
pre-weighed glass filter (GFA). The filter was re- From day 6 onward, FV rose up to an average level of
weighed following oven drying at 100 °C up to a about 27 ml l− 1.
constant weight. Ten replicated samples of settled flocs were collected
Filtered water samples were frozen and later used for on days 6 and 12 of the experimental period. The
a colorimetric determination of TAN, NO2 and NO3, average concentration of dry solids in the floc plug was
using an auto-analyzer, following standard methods 14.2 ± 4.3 mg l− 1, i.e. the floc plug contained 1.4% dry
(Eaton et al., 1995). Homogenized water samples were solids. Using these results, the daily change of FV can
filtered on 2.3 cm diameter GFA, dried and sent to a be given as a change in the dry weight of the flocs, a
laboratory (UC Santa Barbara Marine Science Inst. daily drop of 24.3 mg l− 1.
Analytical Laboratory) to determine total suspended
carbon and nitrogen, using a CHN analyzer. Similar 3.3. TSS, suspended carbon and suspended nitrogen
samples were sent to UC Davis Stable Isotope Facility
for 15N enrichment determination. Total suspended matter, suspended carbon and sus-
pended nitrogen concentrations along the experimental
3. Results and discussion period are given in Figs. 2, 3 and 4, respectively and in
Table 1. Both TSS, carbon and nitrogen concentrations
3.1. Fish growth and feeding in pond T2 decreased during the non-fed period. An increase of these
components started slowly from day 9 onward.
Fish growth was followed at fortnight intervals in TSS dropped during the no-feed part of the experiment
pond T2, the commercial reference pond. Average from an initial value of 582 mg l− 1 to 460 mg l− 1 in day 6,
specific daily fish growth along the growing season was i.e. a daily decrease of about 20 mg l− 1. Later on, when
1.39%, better than that in control ponds. Fish in the feed was applied, TSS increased up to 643 mg l− 1.
control pond responded to feed application by jumping The decrease of suspended carbon and nitrogen
toward the floating pellets. The floating pellets dis- during the non-fed period followed a linear pattern:
appeared within a period of less than 10 min. Feed
application to the bio-flocs technology pond (BFT) Suspended C ðmgl−1 Þ ¼ 168−6:61 t R2 ¼ 0:986
(with fish of about the same size and density), did not
ð3Þ
lead to a dramatic response. Fish slowly nibbled at the
feed that remained floating on the water for an average
period of about 30 min. Fish in BFT pond T2 grew Suspended N ðmgl−1 Þ ¼ 24:8−0:868 t R2 ¼ 0:987
better than those in the control ponds, thus the slow ð4Þ
feeding does not seem to be due to stressed fish but
probably stemmed from the fact that fish ate perma- Where t is the time in days.
nently, harvesting the microbial flocs. The average carbon content of the suspended solids
was 24.6(± 4)% and that of suspended nitrogen was 3.7
3.2. Floc volume (± 0.55)%. The average C/N ratio in the suspended
matter was 6.6, a rather uniform value all along, with a
The volume of flocs settled in the Imhoff cones (FV) as coefficient of variation (CV) of only 2.3%.
a function of time, averaged for the 3 replicated tanks, is The drop of suspended carbon and nitrogen can be
shown in Fig. 1. The FV increased during the 2 days prior expressed as equivalent TSS units by dividing the carbon
to fish stocking, a period when rather high levels of starch or nitrogen daily change by their appropriate concentra-
were added to the tanks. (The value in day 0, when fish tions in the TSS. The daily TSS decrease, calculated using
were stocked was probably too high, since the FV was the daily rates of carbon and nitrogen decrease is equal to
evaluated shortly following starch addition). Subsequent- −26.9 or −23.5 mg l− 1, respectively.
144 Y. Avnimelech / Aquaculture 264 (2007) 140–147

Fig. 3. Suspended carbon, mg/g, in the experimental tanks as a


Fig. 1. Floc volume (FV, ml/l) in the experimental tanks as a function of function of time from fish stocking. [Error bars represent standard
time from fish stocking. [Error bars represent standard deviation (n=3)]. deviation (n = 3)].

By using a common denominator, TSS, to relate the 3.4. Changes with time of 15N enrichment in suspended
concentration changes of the 4 components it was solids and fish
possible to compare the 4 fluxes. The daily changes in
concentration of suspended matter (dry weight) calculated The enrichment of 15N (%) in the suspended solids,
from the change of FV, TSS, C and N, attributed to the and its dependence on time, are given in Fig. 5. A
uptake by the fish are given in Table 2. The daily loss of logarithmic decrease with time (t, days) of the 15N
material was calculated by multiplying the daily concen- enrichment in the suspended matter, was detected:
tration change (mg l− 1 day− 1) by the volume of water in
the tank (800 l). The uptake attributed to fish was %15 N ðflocsÞ ¼ 1:614−0:2033 ln t R2 ¼ 0:9341
calculated by the dividing these values by fish weights in ð6Þ
the tanks for the non-fed period:
15
The rate of increase with time of N in the fish
−1
Feed retained by fish ðg  kgfish Þ tissues (Fig. 5), followed:
¼ DS  V  FW−1 ð5Þ
%15 NðfishÞ ¼ 0:3728 þ 0:011 lnðtÞ R2 ¼ 0:9588 ð7Þ
Where ΔS is the daily change in concentration of FV,
TSS, C or N (normalized to TSS equivalents), V is the The 15N enrichment in the first day of the experiment,
volume of the tank (800 l) and FW is the weight of fish in 0.37%, represents the natural 15N enrichment of fish
each tank. before the exposure to the tagged suspension.
The four different estimates were found to be very The measured, net uptake, of 15N by the fish, (Fig. 6),
similar. The daily uptake by 1 kg fish was, on the average, was calculated by multiplying the change in %15N by the
10.28 + 1.03 g, as dry suspended matter. nitrogen concentration in the fish (40 g N kg− 1). The net

Fig. 2. Total suspended solids (TSS, mg/g)) in the experimental tanks Fig. 4. Suspended nitrogen, mg/g, in the experimental tanks as a
as a function of time from fish stocking. [Error bars represent standard function of time from fish stocking. [Error bars represent standard
deviation (n = 3)]. deviation (n = 3)].
Y. Avnimelech / Aquaculture 264 (2007) 140–147 145

Table 1
Suspended solids, suspended carbon and nitrogen concentrations, averages of data along the experiment
Day TSS Carbon Nitrogen Carbon in TSS Nitrogen in TSS C/N
mg/l, (SD) mg/l, (SD) mg/l, (SD) % %
0 582 (13) 163 (7) 24 (0.8) 28.0 4.1 6.75
3 555 (22) 146 (6) 22 (0.8) 26.3 3.9 6.67
6 460 (53) 129 (2) 20 (0.3) 28.1 4.3 6.58
9 612 (40) 119 (25) 19 (3) 19.5 3.1 6.34
12 643 (20) 137 (20) 21 (2.8) 21.3 3.2 6.68
Average 24.6 3.7 6.6
Standard deviation 4 0.55 0.15
CV % 16.2 14.8 2.3

uptake of 15N is assumed to be equal to the gross uptake N were determined in the same samples), and thus each
minus the subsequent excretion of 15N. The measured represents an experimentally independent estimate. The
uptake as above represents the net change. To evaluate the number of points to determine the rate of decrease with
gross 15N uptake, it was assumed as an approximation, time of these parameters was limited, yet, the consis-
that there was no excretion of newly consumed 15N tency of all independent determinations adds strength to
during the first day. Since there was probably some the results.
excretion during day 1, our calculated gross uptake is a The decrease of these parameters, during a week
low estimate. Further, it was assumed that the 15N uptake without feeding, when the sole source of feed was the bio-
is proportional to the TSS, the concentration of flocs in the flocs suspended material, was considered as a preliminary
water, multiplied by the 15N enrichment of the suspended means to estimate bio-flocs uptake by fish.. At this stage it
solids, both lowered with time. The product of the two was assumed that the bio-flocs are not bio-degraded or
parameters, TSS and % 15N, defining the 15N stock in the produced. This certainly is just a first approximation.
water, was calculated for each day. Multiplying the 15N A different approach to evaluate uptake of microbial
uptake in day 1 (GU1) by the 15N stock in day n divided proteins by the fish was through the accumulation of 15N
by the stock at day 1, gave the assumed gross 15N uptake, in the fish. Since the measured accumulation is a function
GU(n): of both uptake and excretion, we used here the 15N uptake
during the first day, 0.455 mg kg− 1, as the best estimate of
Assumed GU ðnÞ ¼ GU1  ð15 N stockday nÞ
daily 15N uptake. This is probably a low estimate of the
=ð15 N stockday 1Þ ð8Þ true uptake flux, since some excretion could take place
even in the first day. The total computed N uptake by fish
The difference between the net and the assumed for the first day, derived from the 15N uptake data, was
gross uptake represents the 15N excretion. The excretion 254 mgN kg− 1. By dividing this value by the nitrogen
of 15N varied only slightly with time (Fig. 6). concentration in the suspended matter at that period,
4.1%, we find that daily bio-floc uptake by fish was 6.2 g
3.5. Combining the different uptake indicators kg− 1. This value is about 60% of the attributed TSS
uptake by fish, 10.3 ± 1.03, computed from the average for
The changes in concentrations of several components the diminution of FV, TSS, N and C.
suspended in the water-FV, TSS, suspended carbon (C) The fact that we got a smaller value using the 15N
and suspended nitrogen (N), were determined. These uptake is reasonable, both due to the fact that the 15N
four components were determined independently (C and uptake was a low estimate and that diminution of bio-

Table 2
Daily decrease of TSS, FV, suspended carbon and nitrogen, during the no-feed period
TSS Floc volume Carbon Nitrogen
Daily measured change 20 mg/l 1.74 ml/l 6.61 mg/l 0.87 mg/l
Equivalent dry SS change (mg/l) 20 24.3 26.9 23.5
Daily uptake by fish as equivalent SS (g/kg fish) 8.91 10.79 11.03 9.66
Changes in equivalent suspended solids (SS) concentration (as dry weight) and daily uptake attributed to 1 kg fish (107 g average fish weight).
146 Y. Avnimelech / Aquaculture 264 (2007) 140–147

4. Conclusions

Microbial flocs developing in bio-flocs technology


(BFT) ponds are demonstrated to be an effective potential
food source for tilapia, and possibly other fish. The feed
contribution of microbial flocs in the tested ponds
contributed close to 50% of fish protein requirement.
The potential feed storage and dynamics of the pond water
can be easily determined in the field through the
monitoring of FV. Fish growing in the BFT pond did
not rush onto the added feed pellets, since the pond
contained flocs as a potential feed, feed that is available
Fig. 5. 15N enrichment (%) of suspended matter and fish as a function 24 h per day.
of time from fish stocking. [Error bars represent standard deviation An important potential conclusion of this work is
(n = 3)].
the possibility to reduce feed rations in BFT systems.
Avnimelech et al. (1994) estimated that feed utilization
flocs was attributed only to fish harvesting, disregarding is higher in BFT systems, while tilapia in such ponds is
microbial degradation. Considering these un-certainties, fed a ration 20% less than conventional one. Fish yields
the differences between the values obtained using the in these ponds were high, yet the authors did not check
different methods are not too far apart. the possibility of feed reduction in a controlled,
However, even the low estimate of fish bio-flocs quantitative way. The feed requirement of shrimp
harvesting may represent a very significant feed source, growing in BFT models was studied recently by
constituting about 50% of the regular feed ration of fish Panjaitan (2004). It was found that lowering feed
this size (assuming daily feeding of 2% body weight, application by up to 30% of conventional feeding
i.e. 20 g kg− 1). The daily uptake of nitrogen was about ration, did not lower shrimp growth, probably due to
0.3 g N per kg fish, an amount equivalent to 2 g protein, the partial replacement of feed by the microbial flocs.
a significant portion (close to 50%) of protein feed The recycling of protein and the lower demand for
requirements of the fish. The estimated contribution of external protein sources is an important environmental
the daily nitrogen retention from natural biota in shrimp advantage, lowering the fishing pressure on marine
(1 to 9 g) was 18–29% of the total nitrogen uptake resources.
(Burford et al., 2004). However, the suspended nitrogen The determination of FV can be practiced in the
in the shrimp ponds studied was in the range of farm. It is very easy to conduct and does not demand
7–11 mgN l− 1 , less than a half of suspended N sophisticated laboratory or expensive instruments. Yet,
concentrations in the present system. Considering this it can be used to estimate the storage of feed equivalent
difference, the results reported by Burford et al. (2004) in the water. According to data obtained in the present
are in line with findings of this experiment. work, each cm3 of settled flocs contains about 14 mg
The accumulation of 15 N in the fish, as a function of flocs as dry weight. A relatively low reading of 5 ml l− 1
time represents its uptake minus its excretion. The ex- FV is equivalent to an amount of 700 kg dry matter per
cretion of TAN by the fish was demonstrated through
the decrease of the 15 N enrichment in the water. The
uptake of tagged bio-flocs should result in the
diminution of the amount of 15 N in the water, not its
concentration. The lowering of 15 N enrichment was
most probably due to the fact that low enriched TAN
was excreted, diluting the 15 N concentration in the
water. Unfortunately, this process could not be followed
quantitatively, due to the high nitrate concentrations in
the water and the relatively high variability of its
concentrations, not enabling to clearly define and
compute nitrogen balances in the water. (Experiments
like the one reported here should be conducted keeping Fig. 6. Actual 15N uptake, computed gross uptake and excretion of 15N
as low as possible NO3 concentrations). tagged bio-flocs.
Y. Avnimelech / Aquaculture 264 (2007) 140–147 147

ha. This is a very significant potential feed equivalent, Browdy, C.L., Bratford, D., Stokes, A.d., McIntosh, R.P., 2001.
as compared to the amount of feed rations in ponds. Perspectives on the application of closed shrimp culture systems.
In: Browdy, C.L., Jory, D.E. (Eds.), The New Wave, Proceedings
The results obtained in this work may be site of the Special Session on Sustainable Shrimp Farming. World
specific or may vary with the change of fish, pond and aqua. Soc., Baton Rough. LA, pp. 20–34.
other variables. The potential importance of the results Burford, M.A., Thompson, P.J., McIntosh, P.R., Bauman, R.H.,
obtained here fully justify further research and Pearson, D.C., 2003. Nutrient and microbial dynamics in high-
intensity, zero-exchange shrimp ponds in Belize. Aquaculture 219,
widening of the knowledge base to help utilize the
393–411.
natural feed recycling potential of bio-floc systems. In Burford, M.A., Thompson, P.J., McIntosh, P.R., Bauman, R.H.,
addition, methods used to quantitatively evaluate the Pearson, D.C., 2004. The contribution of flocculated material to
different fluxes taking place in bio-floc technology shrimp (Litopenaeus vannami) nutrition in a high-intensity, zero-
systems should be improved and refined. exchange system. Aquaculture 232, 525–537.
Cam, D., Rollet, P.E., Mariotti, A., Guillaume, J., 1991. The relative
contribution of natural productivity and formulated food in the
Acknowledgments nutrition of Panaeus japonicum reared in semi-intensive condi-
tions. Aquatic Living Resources 4, 175–180.
The execution of this work was possible only due to Chamberlain, G.W., Hopkins, S.J., 1994. Reducing water use and feed
the help of Pacific Aquafarm, headed by the president cost in intensive ponds. World Aquaculture 25, 29–32.
Mr. Bill Engler and the manager Mr. Colin Bornia. The Eaton, A.D., Cleserci, L.S., Greenberg, A.E. (Eds.), 1995. Standard
Methods for the Examination of Water and Waste Water, 10th
help of Kent SeaTech farm, enabling the use of their edition. Amer. Pub. Health Assoc., Washington D.C.
laboratory facilities is appreciated. The help of the UC Hopkins, J.S., Hamilton II, R.D., Sandifer, P.A., Browdy, C.L., Stokes,
Santa Barbara Marine Science Inst. Analytical Labora- A.D., 1993. Effect of water exchange rate on production, water
tory in determining carbon and nitrogen is greatly quality, effluent characteristics and nitrogen budgets of intensive
appreciated. Finally I want to thank Mr. Dean Farrell for shrimp ponds. Journal of the World Aquaculture Society 24,
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