Effect of feed C/N ratio promoted bioflocs on water quality and
production performance of bottom and filter feeder carp in
minimum-water exchanged pond polyculture system
Zhigang ZHAO（Ph.D）
Heilongjiang River Fisheries Research Institute, Chinese Academy
of Fishery Sciences, Harbin, China
29April 2016
Contents
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussions and conclusions
1. Introduction
With the more and more shortage of water, land and feed resources, and the
serious problem from the quality and safety of aquatic products
“Energy conservation, drainage reduction, health and high
efficiency” is the characteristics and directions of the pond
culture in China!
1. Introduction
 Intensive pond aquaculture systems efficiently produce dense
biomasses of fish. An intrinsic feature of these systems is the
rapid accumulation of feed residues, organic matter and toxic
inorganic nitrogen metabolites.
 It is reported that fish in ponds assimilate only 15-30 % of
the nitrogen added in the feed, the remainder being lost to the
system as ammonia and organic N through faecal and feed
residues.
 Therefore, intensive pond aquaculture industry faces major
problems of low feed use in high water exchange system, water
quality deterioration, and water discharge having serious
environmental consequences.
1. Introduction
 Biofloc technology (BFT) as a culture technique is described as that
accumulation of toxic inorganic nitrogen metabolites are prevented by
manipulating the carbon/nitrogen ratio (C/N ratio) and inducing the uptake
of ammonium by the microbial community in the zero or minimal water
exchange rate system.
Fig. The circulation and transform of carbon and nitrogen in the biofloc pond (Cited from Avnimelech, 2012)
1. Introduction
Biofloc technology (BFT)
Save
water
Improve
water quality
Increase
feed utilization
ratio
Prevent
disease
1. Introduction
 Mirror carp, as an important cultured species, is ideal for use in polyculture fish farming
because of broad diet spectrum. Similarly, silver carp and bighead carp have drawn much
attention worldwide as typical filter-feeding aquaculture species and their potential for
biomanipulation of plankton communities.
 The main objective of the present study was to determine the effects of feed C/N ratio on
growth performance of filter feeding planktivorous fish and water quality parameters in mirror
carp major polyculture systems with minimum zero-water exchange in pond.
2. Materials and methods
2.1. Experimental design and enclosure facilities
 A 120-days experiment was conducted in
land-based mesocosm enclosures (7 m×7 m)
in an earthen pond.
 Each enclosure was aerated continuously by the microporous aeration tubes. No
water was exchanged in the enclosures during the experiment period.
 The experiment consisted of five treatments of C/N 7 (control), C/N 11, C/N 15,
C/N 19 and C/N 23. Each treatment had three replicates.
2. Materials and methods
2.2. Fish stocking and management
 According to the factual culture, young fish of mirror carp (194.72±4.42 g), silver
carp (54.10±0.84 g) and bighead carp (58.69±0.68 g) were stocked into each
enclosure at the densities of 285 g·m−2, 45 g·m−2 and 15 g·m−2, respectively.
 The carp commercial pellet feed containing
34 % protein with C/N ratio 7 was applied.
 The feed was applied thrice daily at 07:30,
12:00 and 16:30 h.
2. Materials and methods
2.2. Fish stocking and management
 The corn starch was used as carbohydrate for
manipulating the C/N ratio.
 The amount of corn starch added was
calculated based on the C/N ratio of the daily
feeds input to the enclosures.
 In order to raise the C/N ratio from 7 (as control) to 11, 15, 19 and 23, the corn
starch was applied 0.66, 1.31, 2.03 and 2.80 kg for each kg of feed so as to provide
ratios of in the C/N 11, C/N 15, C/N 19 and C/N 23 treatment enclosures, respectively.
2. Materials and methods
2.3. Water quality parameters
 The water temperature, dissolved oxygen, pH, total ammonia nitrogen (TAN), nitrate
nitrogen (NO3-N), chemical oxygen demand (COD), total phosphorus (TP), total alkalinity,
chlorophyll a, nitrite nitrogen (NO2-N), orthophosphate (PO4-P), dissolved organic carbon
(DOC) and bioflocs volumes (BFV) were determined.
 The particle sizes of bioflocs were measured under a microscope.
2. Materials and methods
2.4. Growth parameters and body compositions
 Survival rate (SR), specific growth rate (SGR),
feed
conversion
rate
(FCR),
total
feed
conversion rate (TFCR), protein efficiency
ratio (PER), total protein efficiency ratio
(TPER), net yield (NY), total net yield (TNY)
and total yield (TY) were calculated.
 At the end of the trial, the body muscle compositions for crude protein, lipids,
moisture and ash in mirror carp were determined from each treatment using the
standard methods.
3. Results
Table 1 Initial mean individual weight and growth performance of mirror carp, Cyprinus carpio specularis in
control and treatments
Parameters
Initial mean
individual weight (g)
Final mean
individual weight (g)
Specific growth rate
(SGR) (%·d-1)
Feed conversion rate
(FCR)
Protein efficiency
ratio (PER)
Survival rate
(SR)(%)
Net yield
(NY) (kg·ha-1)
C/N 7 (control)
C/N 11
C/N 15
C/N 19
C/N 23
196.21±7.67
189.58±7.99
196.99±4.75
191.90±5.22
200.93±1.52
736.70±10.44
734.22±28.28
759.86±25.65
767.66±2.23
764.32±13.32
1.20±0.02
1.21±0.01
1.23±0.05
1.24±0.02
1.21±0.02
1.84±0.02a
1.79±0.03ab
1.76±0.09ab
1.73±0.02ab
1.65±0.03b
1.59±0.01b
1.65±0.02ab
1.68±0.09ab
1.70±0.01ab
1.78±0.02a
99.07±0.46a
97.69±0.93a
100.00±0.00a
99.07±0.93a
71.30±8.07b
7730.35±62.16a
7731.30±172.12a
8270.68±446.46a
8392.15±82.74a
5288.44±502.23b
Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 The SGR, PER and NY of mirror carp increased gradually as the C/N ratios increased from 7 to 19, while
these parameters did not show significant (P>0.05) differences during C/N ratios 7-19.
 The SR and NY of the carp in the treatment of C/N 23 were significantly (P<0.05) lower than those in other
treatments.
 The FCR of the fish declined significantly (P<0.05) with increasing of C/N ratios.
3. Results
Table 2 Initial mean individual weight and growth performance of silver carp, Hypophthalmichthys molitrix in
control and treatments
Parameters
Initial mean
individual weight
(g)
Final mean
individual weight
(g)
Specific growth rate
(SGR) (%·d-1)
Survival rate
(SR) (%)
Net yield (NY)
(kg·ha-1)
C/N 7 (control)
C/N 11
C/N 15
C/N 19
C/N 23
54.06±0.59
55.46±0.38
53.27±0.96
54.13±0.98
53.57±0.39
163.21±8.08c
166.48±3.78c
196.66±23.63bc
232.82±10.07ab
269.72±40.60a
1.00±0.06c
1.00±0.02c
1.18±0.12bc
1.32±0.05ab
1.46±0.14a
96.83±3.17a
100.00±0.00a
99.21±0.79a
98.41±1.59a
76.19±13.75b
903.70±65.32b
951.62±33.64b
1220.99±213.02b
1507.44±95.66a
1243.96±404.27b
Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 The SGR and NY of silver carp increase significantly (P<0.05) while C/N ratios increased
from 19 to 23.
 The SR of silver carp in the treatment of C/N 23 was significantly (P<0.05) lower than those
in other treatments.
3. Results
Table 3 Initial mean individual weight and growth performance of bighead carp, Aristichthys nobilis in control
and treatments
Parameters
C/N 7 (control)
C/N 11
C/N 15
C/N 19
C/N 23
Initial individual
weight (g·ind-1)
58.26±0.41
59.04±0.31
58.13±1.26
58.27±1.54
59.74±0.99
159.76±24.64b
162.01±12.26b
181.19±14.85ab
232.14±26.80a
240.17±5.58a
0.89±0.14b
0.91±0.08b
1.03±0.05ab
1.24±0.09a
1.26±0.04a
100.00±0.00
100.00±0.00
100.00±0.00
100.00±0.00
100.00±0.00
290.01±69.53b
294.21±35.87b
351.60±38.98ab
496.76±72.28a
513.94±19.28a
Final individual
weight (g·ind-1)
Specific growth
rate (SGR) (%·d-1)
Survival rate
(SR) (%)
Net yield (NY)
(kg·ha-1)
Each value represents mean ± S.E. ( n= 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 The SGR and NY of bighead carp increase significantly (P<0.05) while C/N ratios increased
from 19 to 23.
 The SR of the fish in all treatments and control were always 100 %.
3. Results
Table 4 Yields and feed utilization in control and treatments
Parameters
C/N 7 (control)
C/N 11
C/N 15
C/N 19
C/N 23
Total yield
(TY) (kg·ha-1)
12522.25±105.43a
12382.65±322.79a
13356.91±301.34a
13895.72±180.95a
9411.65±1066.96b
Total net yield
(TNY) (kg·ha-1)
9046.24±45.21a
8978.93±218.90a
9843.27±367.37a
10385.84±132.10a
7046.33±887.22b
1.60±0.01a
1.54±0.03a
1.48±0.06ab
1.40±0.02b
1.25±0.02c
1.84±0.01d
1.92±0.02cd
2.00±0.07bc
2.11±0.02b
2.36±0.02a
Total feed
conversion rate
(TFCR)
Total protein
efficiency ratio
(TPER)
Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 The TY and TNY of fish gradually increased, while there were no significant differences
(P>0.05) among control and treatments. When C/N ratios rose to 23, the TY and TNY of
fish decreased significantly (P<0.05).
 With increasing of C/N ratios, the TFCR of fish declined significantly (P<0.05), while the
TPER of fish increased significantly (P<0.05).
3. Results
Table 5 Proximate muscle composition of mirror carp, Cyprinus carpio specularis in control and treatments at
the end of experiment
Parameters
C/N 7 (control)
C/N 11
C/N 15
C/N 19
C/N 23
Moisture (%)
77.34±0.62
77.00±0.87
78.50±1.03
77.52±1.04
78.20±0.61
Crude protein (%)
77.14±0.97b
81.79±0.39a
82.43±1.07a
78.91±1.23ab
78.87±1.66ab
Crude lipid (%)
10.51±1.07b
10.91±0.61b
11.96±0.13b
14.09±0.67a
14.29±0.36a
Ash (%)
4.75±0.18b
5.12±0.10a
4.81±0.08ab
4.78±0.06ab
4.56±0.03b
Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 With increasing C/N ratios, the crude protein and ash contents increased significantly
(P<0.05), and reached the peak at C/N 15 and C/N 11, but declined significantly with further
increases of C/N ratios.
 The crude lipid contents increased significantly (P<0.05) with raising C/N ratios, and reached
the high value at C/N 23.
3. Results
Table 6 Water quality parameters in control and treatments during the experiment
Parameters
TAN (mg·L-1)
C/N 7 (control)
1.06±0.15a
C/N 11
0.68±0.16b
C/N 15
0.58±0.11b
C/N 19
0.49±0.10b
C/N 23
0.45±0.12b
NO2-N(mg·L-1)
0.19±0.02a
0.15±0.02ab
0.08±0.02bc
0.05±0.02c
0.05±0.03c
NO3-N(mg·L-1)
13.13±1.41a
9.46±2.03ab
7.90±1.59b
7.81±1.55b
7.66±1.68b
TIN(mg·L-1)
pH
PO4-P(mg·L-1)
14.49±1.46a
8.13±0.04
0.25±0.04a
10.35±2.06ab
8.17±0.06
0.19±0.03ab
8.63±1.54b
8.16±0.05
0.11±0.02bc
8.41±1.52b
8.09±0.07
0.07±0.03c
8.13±1.63b
8.06±0.06
0.06±0.03c
TP(mg·L-1)
Chlorophyll a
(μg·L-1)
COD(mg·L-1)
Total alkalinity
(mmol·L-1)
DOC (mg·L-1)
1.13±0.14a
0.95±0.14ab
0.69±0.04bc
0.58±0.06c
0.55±0.07c
0.06±0.01
0.08±0.01
0.09±0.02
0.08±0.01
0.07±0.01
11.53±0.74
11.43±0.66
11.12±0.68
11.41±0.46
11.42±0.56
6.03±0.11
6.33±0.13
6.12±0.16
6.78±0.16
6.41±0.14
12.22±0.64
15.13±1.57
15.86±2.38
16.30±1.74
16.43±2.60
Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript letters are significantly different (P<0.05).
 The TAN, NO2-N, NO3-N, TIN, PO4-P and TP concentrations decreased significantly (P<0.05)
with raising C/N ratios.
 With increasing of C/N ratios from 7 to 23, there were no significant differences (P>0.05) in pH,
total alkalinity and the concentrations of chlorophyll a, COD and DOC.
3. Results
Fig. 1 Changes of water quality parameters in control (C/N 7) and treatments during the experiment
 The TAN and NO2-N concentrations decreased significantly (P<0.05) in treatments of C/N 15, C/N 19 and
C/N 23, respectively, as corn starch was added continuously.
 The PO4-P, TP, chlorophyll a concentrations and total alkalinity remained stable showing fluctuation with
small range in treatments of C/N 15, C/N 19 and C/N 23 (P>0.05) .
 The COD and DOC concentrations increased significantly in the treatments of C/N 15-23 (P<0.05).
3. Results
0.25
y = 0.0106x - 0.0726
(R2 = 0.5659, P<0.01)
BFV (mg•L-1)
0.2
0.15
0.1
0.05
0
10
13
16
19
22
25
Temperature (℃)
Fig. 2 The relationships between the bioflocs volumes (BFV) and temperature during the experiment
 The bioflocs volumes in treatments of C/N from 15 to 23 increased gradually as the
carbohydrate was added, and then decreased with decline in temperature.
 It showed a significant (P<0.05) positive correlation between the bioflocs volumes and
temperature during the experiment.
4. Discussions and conclusions
4.1. Effects of C/N ratios on water quality
 1. In the present study, the use of corn starch to raise the C/N ratio in treatments 15-23 were
found to be efficient in maintaining low levels of nitrogen compounds (TAN, NO2-N and NO3N) and PO4-P throughout the experimental period.
 2. High density of heterotrophic bacteria in the water as well as algae, protozaons,
zooplankton and other organic matter attached the flocs, using the nitrogen, phosphorus and
carbon available.
 3. All of three principal pathways (algae, heterotrophic bacteria and nitrifying bacteria) to
remove hazardous nitrogen were in the present polyculture system.
4. Discussions and conclusions
4.2. Effects of C/N ratios on growth of fish
 1. The SGR of silver carp and bighead carp increased significantly while the C/N ratios
increased from 7 to 23, indicating more biofloc particles were ingested and assimilated by both
of the species with increasing of C/N ratios in the culture system.
 2. The attention should be paid to hypoxia in high C/N ratios culture systems because of the
significant mortality in mirror carp and silver carp in C/N 23 treatment.
 3. The increased muscle crude protein contents of mirror carp might be explained that the
carp fed on the artificial feed and the bioflocs might have better nutrient assimilation when
compared to those fed only the artificial feed.
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