Biofloc technology. A practical guide book. The World Aquaculture Society

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Aquaculture 176 1999 227–235
Carbonrnitrogen ratio as a control element in
aquaculture systems
Yoram Avnimelech )
Faculty of Agricultural Engineering, Technion, Israel Institute of Technology, Haifa 32000, Israel
Accepted 15 February 1999
Abstract
Controlling the inorganic nitrogen by manipulating the carbonrnitrogen ratios is a potential
control method for aquaculture systems. This approach seems to be a practical and inexpensive
means of reducing the accumulation of inorganic nitrogen in the pond. Nitrogen control is induced
by feeding bacteria with carbohydrates, and through the subsequent uptake of nitrogen from the
water, by the synthesis of microbial proteins. The relationship among the addition of carbo-
hydrates, the reduction of ammonium and the production of microbial proteins depends on the
microbial conversion coefficient, the CrN ratio in the microbial biomass, and the carbon contents
of the added material. The addition of carbonaceous substrate was found to reduce inorganic
nitrogen in shrimp experimental tanks and in tilapia commercial-scale ponds. It was found in
tilapia ponds that the produced microbial proteins are taken up by the fish. Thus, part of the feed
protein is replaced and feeding costs are reduced. The addition of carbohydrates, or the equivalent
reduction of proteins in the feed, can be quantitatively calculated and optimised, as shown here.
Approximate parameters were used in this work. Additional research in this field should be
directed at gathering the precise data needed for the exact planning of feed composition. q1999
Elsevier Science B.V. All rights reserved.
Keywords: CrN; Ammonium; Inorganic nitrogen; Microbial proteins; Feeding
1. Introduction
1.1. General
One of the major water quality problems in intensive aquaculture systems is the
Žqy
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accumulation of toxic inorganic nitrogenous species NH4 and NO in the water
2
)Tel.: q972-4-8292-480; Fax: q972-4-8221-529; E-mail: agyoram@tx.technion.ac.il
0044-8486r99r$ - see front matter q1999 Elsevier Science B.V. All rights reserved.
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PII: S0044 - 8 4 8 6 99 00085 - X

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Y. AÕnimelechrAquaculture 176 1999 227–235228
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Colt and Armstrong, 1981 . Aquatic animals, such as fish and shrimp, excrete ammo-
nium, which may accumulate in the pond. A major source of ammonium is the typically
protein-rich feed. Aquatic animals need a high concentration of protein in the feed,
because their energy production pathway depends, to a large extent, on the oxidation and
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catabolism of proteins Hepher, 1988 . In highly aerated ponds, ammonium is oxidised
by bacteria to nitrite and nitrate species. Unlike carbon dioxide which is released to the
air by diffusion or forced aeration, there is no effective mechanism to release the
nitrogenous metabolites out of the pond. Thus, intensification of aquaculture systems is
inherently associated with the enrichment of the water with respect to ammonium and
other inorganic nitrogenous species. The management of such systems depends on the
developing methods to remove these compounds from the pond.
One of the common solutions used to remove the excessive nitrogen is to frequently
exchange and replace the pond water. This approach is limited for three reasons:
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a Environmental regulations prohibit the release of the nutrient rich water into the
environment;
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b The danger of introducing pathogens into the external water;
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c The high expense of pumping huge amounts of water.
Another approach is based upon means to encourage and enhance nitrification of the
ammonium and nitrites to the relatively inert nitrate species. This is often done by
employing biofilters, essentially immobile surfaces serving as substrates to the nitrifying
bacteria. A high surface area with immobilised nitrifying biomass enables a high
nitrifying capacity in a controlled environment. One problem associated with biofiltra-
tion is the high cost involved and the need to treat and digest a large mass of feed
residues. Effectively, about 50% of the feed material added to the pond needs to be
digested.
An additional strategy that is presently getting more attention is the removal of
ammonium from the water through its assimilation into microbial proteins by the
addition of carbonaceous materials to the system. If properly adjusted, added carbo-
hydrates can potentially eliminate the problem of inorganic nitrogen accumulation. A
further important aspect of this process is the potential utilization of microbial protein as
a source of feed protein for fish or shrimp.
Utilization of microbial protein depends upon the ability of the animal to harvest the
bacteria and its ability to digest and utilise the microbial protein. Neither are trivial. One
obvious problem is determined by the minimal size of particles that can be taken up by
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the fish. Schroeder 1978 reported that carp can filter out particles larger than 20–50
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mm. Odum 1968 reported that Mugil cephalus take up particles as small as 10 mm.
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An interesting observation was made by Taghon 1982 , who found that benthic
invertebrates were able to take up microscopic glass beads when they were coated with
proteins. This demonstrates that the chemical nature of the particle may favor its
harvesting by fish. The fact that relatively large microbial cell clusters are formed due to
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flocculation of the cells, alone or in combination with clay or feed particles Harris and
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Mitchell, 1973; Avnimelech et al., 1982 , additionally favors cell uptake by fish.
The adjustment of the CrN ratio in the feed, as a means to control the pond water
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quality, is presently under active research in many research centers e.g., presentations in
the last WAS meeting: McGoogan and Galtin, 1998; Rudacille and Kohler, 1998;

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Y. AÕnimelechrAquaculture 176 1999 227–235 229
.
Conquest et al., 1998 . The objectives of this paper are to formulate the basic reactions
and mechanisms affecting this process; to demonstrate its potential; to develop the
quantitative means needed to adjust the CrN ratio; and to control inorganic nitrogen
accumulation in ponds.
1.2. Theory
The control of inorganic nitrogen accumulation in ponds is based upon carbon
metabolism and nitrogen-immobilizing microbial processes. Bacteria and other microor-
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ganisms use carbohydrates sugars, starch and cellulose as a food, to generate energy
and to grow, i.e., to produce proteins and new cells:
organicC™CO qenergy qCassimilated in microbial cells. 1
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2
The percentage of the assimilated carbon with respect to the metabolised feed carbon, is
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defined as the microbial conversion efficiency Eand is in the range of 40–60% Paul
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and van Veen, 1978; Gaudy and Gaudy, 1980 . Nitrogen is also required since the major
component of the new cell material is protein. Thus, microbial utilization of carbo-
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hydrate or any other low nitrogen feed is accompanied by the immobilization of
inorganic nitrogen. This process is a basic microbial process and practically every
microbial assemblage performs it.
The addition of carbohydrates is a potential means to reduce the concentration of
inorganic nitrogen in intensive aquaculture systems. The amount of carbohydrate
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addition DCH needed to reduce the ammonium can easily be evaluated.
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According to Eq. 1 and to the definition of the microbial conversion coefficient, E,
the potential amount of microbial carbon assimilation, when a given amount of
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carbohydrate is metabolised DCH , is:
DCsDCH=%C=E,2
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mic
where DC is the amount of carbon assimilated by microorganisms and %C is the
mic Ž.
carbon content of the added carbohydrate roughly 50% for most substrates .
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The amount of nitrogen needed for the production of new cell material DN depends
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on the CrN ratio in the microbial biomass which is about 4 Gaudy and Gaudy, 1980 :
wx wx
DNsDCrCrNsDCH=%C=ErCrN, 3
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mic mic
mic
Žwx .
and using approximate values of %C, Eand CrN as 0.5, 0.4 and 4, respectively :
mic
DCHsDNr0.5=0.4r4sDNr0.05. 4
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According to Eq. 4 , and assuming that the added carbohydrate contains 50% C, the CH
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addition needed to reduce total ammonia nitrogen TAN concentration by 1 ppm N i.e.,
3.3
1gNrm is20grm.
A different approach is to estimate the amount of carbohydrate that has to be added in
order to immobilise the ammonium excreted by the fish or shrimp. It was found that fish
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or shrimp in a pond Avnimelech and Lacher, 1979; Boyd, 1985; Muthuwani and Lin,
.
1996 assimilate only about 25% of the nitrogen added in the feed. The rest is excreted
as NH or as organic N in feces or feed residue. It can be assumed that the ammonium
4

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Y. AÕnimelechrAquaculture 176 1999 227–235230
flux into the water, DNH , directly by excretion or indirectly by microbial degradation
4
of the organic N residues, is roughly 50% of the feed nitrogen flux:
DNsfeed=%N feed=%N excretion. 5
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A partial water exchange or removal of sludge reduces the ammonium flux in a
manner that can be calculated or estimated. In zero exchange ponds, all the ammonium
remain in the pond. The amount of carbohydrate addition needed to assimilate the
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ammonium flux into microbial proteins is calculated using Eqs. 4 and 5 :
DCHsfeed=%N feed=%N excretionr0.05. 6
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The CrN ratio, or the equivalent protein concentration of the feed, can be calculated
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using the derived Eq. 6 . Assuming 30% protein feed pellets 4.65% N and 50% of the
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feed nitrogen are excreted %N excretion , we get:
DCHsfeed=0.0465=0.5r0.05 s0.465=feed. 7
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According to Eq. 7 , the feed having 30% protein should be amended by an
additional portion of 46.5% made of carbohydrates with no protein. The corrected
protein percentage should accordingly be:
corrected protein percentages30%r1.465 s20.48%, 8
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and the original CrN ratio 10.75 in the 30% protein feed should be raised to 15.75.
2. Materials and methods
Several experimental results are presented, in order to demonstrate and substantiate
the theoretical approach developed.
The basic process of microbial ammonium immobilization was demonstrated in a
laboratory experiment where pond sediment suspension was enriched with ammonium
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salt. Twenty-gram samples of pond bottom clay soil, from commercial tilapia pond
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were shaken for 12 h with 1000 ml tap-water enriched with NH SO , at an initial
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concentration of about 10 mgrl, and 200 mgrl glucose. Samples were taken periodi-
cally and filtered. Ammonium concentrations were determined according to standard
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methods using an auto-analyzer EPA, 1974 .
The effects of carbohydrates addition on ammonium accumulation in a dense shrimp
culture were tested in 25 m2indoor tanks stocked with 0.8 kgrm2Penaeus monodon.
Shrimp were fed with pellets containing 40% protein at a daily rate of 2% body weight
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i.e., 16 g feed, 6.4 g protein or daily 0.96 g Nrm . It was assumed that 33% of the feed
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nitrogen is excreted. Sugar glucose or cassava meal were added at a rate of seven times
the expected ammonium excretion, i.e., daily 2.2 grm2. The experiment was conducted
in triplicates.
The effects of changing the CrN ratio in the feed on the growth and feed utilization
in tilapia are shown in data adapted in part from previous research. In the first
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experiment Avnimelech et al., 1989 , tilapia grown in tanks were fed by either:
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I Conventional feed pellets with 30% protein;
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II Pellets made of wheat meal 10% protein ; and

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Y. AÕnimelechrAquaculture 176 1999 227–235 231
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III Feeding with 10% protein pellets at one-half the daily ratios as compared to
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treatment II , amended by daily additions of cellulose powder and NH SO .
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Protein, fat and stable carbon isotopes were determined in fish tissue at the end of the
experimental period.
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The second experiment Avnimelech et al., 1994 was a pond experiment where
tilapia were grown in circular 50 m2ponds at a density of about 10 kgrm2. Fish were
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fed using conventional 30% protein pellets CrNs11.1 or a tested formulation of low
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protein diet of 20% protein CrNs16.7 . The daily feed addition was 2% of body
weight for the conventional feed and 2.6%, to include carbohydrates needed for the
microbial ammonium conversion, with the low protein pellets. The results presented
here, partially recalculated from the original report, summarise two triplicated experi-
ments.
3. Results and discussion
The effect of addition of carbohydrates on the immobilization of TAN was demon-
strated in a laboratory experiment consisting of a sediment suspension amended with
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ammonium about 10 mgrl and glucose at a concentration 20 times higher than that of
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the TAN. It was found Fig. 1 that almost all the added ammonium disappeared over a
period of about 2 h, following a short lag period, with no concomitant production of
yy
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NO or NO not shown .
23
The addition of carbohydrates to control nitrogen concentrations in shrimp ponds was
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tested in tanks containing dense 0.8 kgrm shrimp biomass. Sugar glucose and
cassava meal were added to reduce TAN accumulation. The carbonaceous substrates
additions were calculated assuming an ammonium excretion equivalent to 33% of the
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feed a significant underestimation of ammonium excretion . The carbonaceous sub-
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Fig. 1. Changes in TAN concentration in a suspension of pond bottom soil 2% dry soil following the addition
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of glucose TANrglucose ratio of 1r20 .

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Y. AÕnimelechrAquaculture 176 1999 227–235232
Fig. 2. Changes with time of TAN concentration in shrimp tank experiment. The shrimp biomass was 0.8
2Ž.
kgrm . The control tank was supplied with 40% protein pellets. Carbohydrates glucose, cassava meal were
added daily at a rate of seven times the assumed TAN excretion by the shrimps.
strates addition led to a significant reduction in the accumulation of ammonium in the
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tanks Fig. 2 . Nitrates and nitrites were also reduced, from 1.97 mg NO qNO yNrl
32
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in the control treatment to 1.13 mg NO qNO yNrl in the treatment tank.
32
Results of tank experiment comparing growth and body composition of tilapia fed
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with I conventional pellets, II control, 10% protein pellets, made from wheat flour,
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and III 10% protein pelletsqcellulose powderqNH SO are given in Table 1.
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Detailed results were published elsewhere Avnimelech and Mokady, 1988; Avnimelech
.
et al., 1989 . Bacterial flocculation was observed, probably supporting filtering out by
the fish, and thus supplying protein that was available and suitable to fish nutrition.
Though tilapia do not digest cellulose, it was found that the addition of cellulose
supported fish growth, obviously through the ingestion and digestion of bacteria growing
on the cellulose. Daily growth of tilapia fingerlings was 0.5, 0.12 and 0.33% for diets I,
Table 1
Tilapia feeding with microbial protein
Treatment
I. Conventional II. Control III. Test
pellets pellets pellets
Daily growth % 0.5 0.12 0.33
Protein % in muscle 15.5 14.2 15.9
Fat % in muscle 4.2 4.3 2.6
13 Ž.
dC in feed % y13.8 y14.8 y23.5 cellulose
13
dC in fish muscle % y20.8 y20.5 y23.0
Growth and feed utilization data for fish fed with I. conventional 30% protein pellets; II. 10% protein pellets;
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and III. test treatments of 10% protein pelletsqcellulose powderqNH SO , as a substrate for the
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production of microbial proteins.

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Y. AÕnimelechrAquaculture 176 1999 227–235 233
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II and III, respectively. Protein content of the fish in the control treatment II was low,
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yet that in fish fed with microbial protein III was as high as with conventional feeding
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II . Carbon isotopes distribution in the feed materials and fish tissues indicated that the
fish digested and ingested carbon derived from the cellulose, most probably by uptake of
the microbial proteins.
Following the pilot scale work, a series of pond scale experiments was conducted.
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Results presented here are adapted from Avnimelech et al., 1992, 1994. It was found
that the addition of carbohydrates, essentially changing the 30% protein feed material to
20% protein feed, led to:
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a a significant reduction of inorganic nitrogen accumulation;
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b increased utilization of protein feed;
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c a significant reduction of feed expenditure.
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Fish growth and feed utilization data in two triplicated pond experiments 50 m
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ponds stocked with tilapia hybrids at a density of 80 fishrm are given in Table 2. It
can be seen that fish growth was better in the 20% protein treatment, most likely due to
the lower concentrations of toxic inorganic nitrogen species. In addition to a lower feed
Ž. Ž.
conversion ratio FCR , the protein conversion ratio PCR was markedly reduced in the
20% protein treatment. The PCR in the conventional 30% protein feed treatment was
4.35–4.38, meaning that only 23% of the feed protein was recovered by the fish. The
Table 2
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Fish growth and yield coefficients of tilapia fed with conventional pellets 30% protein, CrNs11.1 and low
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protein pellets 20% protein, CrNs16.6 in two pond experiments
Treatment
Conventional feeding C-enriched
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30% protein 20% protein
Experiment no. 1: 51 days, aÕerage of three replicates
Feed CrN ratio 11.1 16.6
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Fish weight grfish
Initial weight 112 112
Final weight 193 218
Uab
Daily gain 1.59 2.0
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Mortality % 14.6 10.3
Feed conversion coefficient 2.62 2.17
Protein conversion coefficient 4.38 2.42
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Feed cost coefficient US$rkg fish 0.848 0.583
Experiment no. 2: 30 days, aÕerage of three replicates
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Fish weight grfish
Initial weight 205 205
Final weight 254 272
Uab
Daily gain 1.63 2.22
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Mortality % 3.4 0
Feed conversion coefficient 2.62 2.02
Protein conversion coefficient 4.35 2.18
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Feed cost coefficient US$rkg fish 0.848 0.543
UŽ.
Values not sharing a common letter differ significantly p-0.05 .

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Y. AÕnimelechrAquaculture 176 1999 227–235234
PCR in the tested treatment was 2.2–2.4, i.e., protein utilization was twice as high. The
increased protein utilization is due to its recycling by the microorganisms. It may be said
that the proteins are eaten by the fish twice, first in the feed and then harvested again as
microbial proteins. It is possible that protein recycling and utilization can be further
increased.
Due to the fact that proteins are the expensive component of the feed, its reduction
was reflected in the feed price which decreased from US$0.85rkg fish to about
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US$0.55rkg Table 2 . Similar results were obtained recently in the desert aquaculture
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farms that are operated following principles presented here Avnimelech et al., unpub-
.
lished data .
4. Conclusions
Controlling the inorganic nitrogen by manipulating the carbonrnitrogen ratios is a
potential control method for aquaculture systems. This approach seems to be a practical
and inexpensive means to reduce the accumulation of inorganic nitrogen in the pond.
Such a strategy can be practiced as an emergency response, i.e., addition of a
carbonaceous substrate in case of increased ammonium concentration. It is possible to
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add cheap sources of carbohydrates e.g., cassava meal, flor in cases such as a series of
cloudy days slowing down algae growth, or severe algae crash. However, additional
pond aeration may be required to compensate for the additional oxygen consumption.
The conventional control means for ponds are to intensively exchange the water, a
strategy that is not always practical, and to stop feeding to slow down TAN build up.
The proposed method enables keeping a high biomass and to have a corrective means in
case of a failure of conventional controls.
A more advanced approach is to adjust the protein level in the feed so as to avoid the
build up of inorganic nitrogen in the water. This approach was tested and proven
successful in intensive ponds that are continually mixed and aerated. The intensive
culture of fish in these ponds is based on a system that is similar to biotechnological
reactors. Such systems are amendable to a set of controls similar to biotechnological
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controls Avnimelech, 1998 . The ability to control inorganic nitrogen concentrations
through the manipulation of CrN ratios in the system is one example of such a control.
The addition of carbohydrates was done as a part of the feeding scheme. In this case, the
addition of carbonaceous substrate leads to the recycling and increased utilization of
proteins through the utilization of the microbial proteins. Production and utilization of
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microbial proteins SCP, single-cell protein have been studied extensively during the
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last few decades e.g., Tannenbaum and Wang, 1975 . The major problem involved in
economically sound utilization of SCP cultures is the harvesting, dehydration and
packaging of the material. In contrast, for in situ microbial protein culturing in the pond,
all of these expensive processing stages are not needed since harvesting is done by the
fish, as part of the system.
The applicability of the same approach in earthen stagnant ponds is not trivial and has
to be further studied in conventional fish and shrimp ponds. The addition of carbo-
hydrates to the feed may result in an accelerated sedimentation of organic matter to the

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Y. AÕnimelechrAquaculture 176 1999 227–235 235
pond bottom, where the microbial biomass will not be utilised by the fish and will
increase the organic load in the pond.
The addition of carbohydrates, or the equivalent reduction of proteins in the feed, can
Ž. Ž.
be quantitatively calculated and optimised, as shown in Eqs. 6 – 8 . However, approxi-
mate parameters were used in this work. Additional research in this field should be
directed at gathering precise data needed for the exact planning of feed composition and
feeding rate.
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