Aquaculture 434 (2014) 442–448

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Effect of feed C/N ratio promoted bioflocs on water quality andproduction performance of bottom and filter feeder carp inminimum-water exchanged pond polyculture system

Zhigang Zhao, Qiyou Xu ⁎, Liang Luo, Chang'an Wang, Jinnan Li, Liansheng WangHeilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, People’s Republic of China

⁎ Corresponding author. Tel.: +86 451 8486 9493; fax:E-mail address: xuqiyou@sina.com (Q. Xu).

http://dx.doi.org/10.1016/j.aquaculture.2014.09.0060044-8486/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 February 2014Received in revised form 31 August 2014Accepted 4 September 2014Available online 16 September 2014

Keywords:Mirror carpCyprinus carpio specularisFeed C/N ratioProduction performanceWater qualityPolyculture

A 120-day experiment was conducted with the aim to examine the effects of feed C/N ratio promoted bioflocson water quality and production of bottom and filter feeder carp in land-based experimental mesocosm withminimum water exchange system. For the purpose four bioflocs treatments with carbohydrate feed referred as'C/N 11', 'C/N 15', 'C/N 19' and 'C/N 23', and control 'C/N 7' without carbohydrate addition consisted of threereplicate enclosures (7m × 7m x 1.5m)were arranged. Young bottom feedermirror carp, Cyprinus carpio, filterfeeders silver carp, (Hypophthalmichthysmolitrix) and bighead carp (Aristichtys nobilis)with initial mean individ-ual body weight of 194.72 (±4.42 g), 54.10 (±0.84 g) and 58.69 (±0.68 g) were stocked altogether inpolyculture at densities of 285 g.m−2, 45 g.m−2 and 15 g.m−2, respectively in control and treatments. Duringthe experiment water temperatures varied from 24.7 to 12.0 °C. The results showed a significant (P b 0.05) pos-itive correlation between bioflocs volumes and experimental water temperatures. The total ammonia nitrogen(TAN) concentrations in treatments from C/N 11 to C/N 23were significantly (P b 0.05) lower comparing to con-trol (C/N 7). Nitrite nitrogen (NO2-N), nitrate nitrogen (NO3-N), total inorganic nitrogen (TIN), orthophosphate(PO4-P) and total phosphorus (TP) concentrations decreased significantly (P b 0.05) with higher C/N ratios.There was no significant (P b 0.05) differences in concentrations of Chlorophyll a, chemical oxygen demand(COD) and dissolved organic carbon (DOC) with increasing C/N ratios in treatments. The total production (TP)of bottom and filter feeders fish reached up to 13895 kg.ha-1 in experimental treatments. The specific growthrates (SGR), protein efficiency ratios (PER) and net production (NP) of mirror carp increased gradually withincreases of C/N ratios. The feed conversion rates (FCR) of experimental fish declined significantly (P b 0.05)with increasing of C/N ratios in all control and treatments. With increasing C/N ratios, the total feed conversionrates (TFCR) of fish decreased significantly (P b 0.05), while total protein efficiency ratios (TPER) increased sig-nificantly (P b 0.05). The bioflocs influenced muscle compositions in terms of crude protein, crude fat and ash ofthe mirror carp significantly (P b 0.05). The present study revealed that improved production performance andfeed utilization inmirror, silver and bighead carp could be achievedwith improvedwater quality with increasingfeed C/N ratio.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Intensive pond aquaculture systems efficiently produce dense bio-masses of cultivable fish. An intrinsic feature of such systems could berapid accumulation of feed residues, organic matter and toxic inorganicnitrogen species. Fish in pond assimilate only 15-30% of nitrogen addedin the feed (Acosta-Nassar et al., 1994; Gross et al., 2000; Davenportet al., 2003), rests is lost as ammonia and organic N in feces and residues(Avnimelech and Ritvo, 2003). Intensive pond aquaculture systemfaces low feed use in high water flow system, water quality deteriora-tion and water discharge with serious environmental consequences

+86 451 8460 4803.

(Avnimelech, 2007). To overcome such constraints biofloc technology(BFT) is known to prevent accumulation of toxic inorganic nitrogenmetabolites by inducing and manipulating carbon/nitrogen ratio (C/Nratio) and uptake of ammonium by the microbial community even inzero water exchange system (Avnimelech et al., 1994; McIntosh,2000). Where, nutrients from excretion and remnant feed are recycledinto microbial community forming bioflocs containing bacteria, phyto-plankton, protozoa and zooplankton kind of high valued food by fishor shrimp (McIntosh, 2000; Velasco et al., 1998; Avnimelech, 2007).This technology reduces the potential spread of pathogenic bacteriaand feed conversion ratio (Burford et al., 2003; Crab et al., 2010;Xu and Pan, 2013). Currently, biofloc technology has been receivingattention for closed-water carp, shrimp and tilapia cultivation (Hariet al., 2006; Zhao et al., 2013; Anand et al., 2014; Liu et al., 2014).

443Z. Zhao et al. / Aquaculture 434 (2014) 442–448

Relatively high C/N ratio feed (10 to 20) are recommended for bioflocs(Hargreaves, 2006; Asaduzzaman et al., 2008; Ballester et al., 2010).The C/N ratio of most artificial feeds used in semi-intensive pond aqua-culture is around 10, but bacteria require about 20 units of carbon perunit of nitrogen assimilated (Avnimelech, 1999). Therefore, addingcarbohydrates can be a practical way to increase the C/N ratio forbioflocs promotion (De Schryver et al., 2008; Anand et al., 2013). Ithas been suggested that higher feed C/N ratio could increase bioflocsvolume without compromising nutritional quality (Azim and Little,2008; Asaduzzaman et al., 2010; Crab et al., 2012). The optimum feedC/N ratio in carp aquaculture system can be maintained by addinglocally available inexpensive carbon sources and/or reduction of proteincontent in feeds (Avnimelech, 1999; Hargreaves, 2006). Generally, carpaquaculture includes bottom feeder mirror, typical filter feeding speciessubsist on plankton such as silver (Hypophthalmichthys molitrix), big-head (Aristichthys nobilis) and other carp. Thus, bioflocs technologycombined with polyculture might enhance production performanceof carp, water quality, natural food availability (Rahman et al., 2008;Fukushima et al., 1999; Ke et al., 2007; Yan et al., 2009). However,there is only little documentation on practicability of biofloc technologyto bottom and filter feeders carp in minimum water exchange pondpolyculture system. Thus, the main objective of the present study wasto examine the effects of feed C/N ratio onwater quality and productionperformance of bottom and filter feeder carp in minimum water ex-change outdoor ponds.

2. Materials and Methods

2.1. Experimental design

A 120-day experiment was conducted in land-based experimental15 mesocosm having a dimensions of 7 m long × 7 m wide × 1.5 mdeep in an earthen pond made of impervious polyethylene wovencloth aerated continuously bymicroporous aeration tubes experimentalstation of Heilongjiang Fisheries Research Institute, Hulan, ChineseAcademy of Fishery Sciences from June 15 to October 12, 2012. Theearthen pond holding mesocosm for experiment had a surface area of4,000 m2 and an average depth of 1.5 m. The structure of the enclosurein detail was described by Li et al. (1998) and Wang et al. (1998). Nowater was exchanged in the enclosures throughout experiment period;except the rainwater compensated evaporation and some leakagelosses. The experiment consisted of one control (C/N 7) and four treat-ments (C/N 11, C/N 15, C/N 19 and C/N 23). All control and treatmentshad three randomly assigned replicates.

2.2. Fish stocking and management

Young bottom feeder mirror carp (Cyprinus carpio specularis),filter feeders silver carp (Hypophthalmichthys molitrix) and big carp(Aristichthys nobilis) having initial individual body weight 194.72(±4.42) g, 54.10 (±0.84) g, and 58.69 (±0.68) g, respectively werestocked into each enclosure on June 15. The stocking number and bio-mass of carp in control and treatments were 135 and 285 g · m−2 formirror carp; 45 g · m−2 for bighead and 15 g · m−2 for silver carp,respectively. These carp were fed with commercial pellet containing34% protein with C/N ratio approximately 7. The daily feeding ratewas 5% body weight of mirror carp at beginning, and but graduallyreduced to 3% at the end. The carps were feed thrice daily at 07:30,12:00 and 16:30 h. The corn starchwas used as carbohydrate formanip-ulating the feed C/N ratio. The amount of corn starch to be added wascalculated based on the C/N ratio of the daily feed input. In order toraise the C/N ratio from 7 (control) to 11, 15, 19 and 23; corn starch0.66, 1.31, 2.03 and 2.80 kg were applied for each kg of formulatedfeed in the C/N 11, C/N 15, C/N 19 and C/N 23 treatments, respectively.The pre-weighed corn starch was mixed in bucket with enclosure'swater before uniformly distributing over surface directly after the feed

application daily at 09:30 h. The starch was added daily from 8 Augustto 10 October.

2.3. Assessment of water quality

The water temperature, dissolved oxygen, pH, total ammonia nitro-gen (TAN) and nitrate nitrogen (NO3–N) were monitored daily with amultiparameter water quality instrument (YSI Professional Plus, YSIIncorporated, Yellow Springs, USA) in-situ at 10:00 am. Water sampleswere collected at 10:00 am at every 14-day intervals. Parts of thewater sample were analyzed for chemical oxygen demand (COD),total phosphorus (TP), total alkalinity and Chlorophyll a. The remainingwas filtered under vacuum pressure through 0.45 μm fiber filter paperfor nitrite nitrogen (NO2-N) and orthophosphate (PO4-P) analysisin the filtrate using the methods described in APHA (1998). Dissolvedorganic carbon (DOC) was measured by the TOC/TNb Analyzer (multiN/C 2100, Germany). Bioflocs volumes (BFV) were determined on siteonce in a week using Imhoff cones, registering the volume taken bythe bioflocs in 1000mL volumetric cylinder after 20 min sedimentationof the enclosure water (Avnimelech and Kochba, 2009). The particlesizes of bioflocs were measured by simple microscope.

At the end of the trial, the body muscle compositions of crude pro-tein, lipids, moisture and ash in mirror carp were determined fromeach treatment using standard methods (AOAC, 1990). The fish wereharvested and total biomass for estimating production was performedby weighing the fish using simple balance.

2.4. Calculations, statistics and analysis

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 production (NP), total netproduction (TNP) and total production (TP) were calculated using thefollowing equations:

Survival rate %ð Þ ¼ 100� final fish count=initial fish countð Þ;

Specific growth rate % � d−1� �

¼ 100� ½Ln final body weightð Þ– Ln initial body weightð Þ�=experimental duration daysð Þ;

Feed conversion rate ¼ total dry weight of feed offered

= total mirror carp wet weight gained;

Total feed conversion rate ¼ total dry weight of feed offered

= total fish wet weight gained;

Protein efficiency ratio ¼ total mirror carp wet weight gained

= total dry weight of feed protein offered;

Total protein efficiency ratio ¼ total fish wet weight gained

= total dry weight of feed protein offered;

Net production kg � ha−1ð Þ ¼ 10000

� ðfinal body weight� final fish count−initial body weight

� initial fish countÞ=49;

Total production kg � ha−1ð Þ ¼ 10000

� final body weight� final fish countð Þ=49;

Total net production kg � ha−1ð Þ ¼ the summation of net productionof mirror carp; silver carp andbighead carp:

Table 1Initial mean individual weight and growth performance of mirror carp, Cyprinus carpio specularis in control and treatments.

Parameters C/N 7 (control) C/N 11 C/N 15 C/N 19 C/N 23

Initial mean individual weight (g) 196.21 ± 7.67 189.58 ± 7.99 196.99 ± 4.75 191.90 ± 5.22 200.93 ± 1.52Final mean individual weight (g) 736.70 ± 10.44 734.22 ± 28.28 759.86 ± 25.65 767.66 ± 2.23 764.32 ± 13.32Specific growth rate (% · d-1) 1.20 ± 0.02 1.21 ± 0.01 1.23 ± 0.05 1.24 ± 0.02 1.21 ± 0.02Feed conversion rate 1.84 ± 0.02a 1.79 ± 0.03ab 1.76 ± 0.09ab 1.73 ± 0.02ab 1.65 ± 0.03b

Protein efficiency ratio 1.59 ± 0.01b 1.65 ± 0.02ab 1.68 ± 0.09ab 1.70 ± 0.01ab 1.78 ± 0.02a

Survival rate (%) 99.07 ± 0.46a 97.69 ± 0.93a 100.00 ± 0.00a 99.07 ± 0.93a 71.30 ± 8.07b

Net production (kg · ha-1) 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 b 0.05).

Table 2Initial mean individual weight and growth performance of silver carp, Hypophthalmichthys molitrix in control and treatments.

Parameters C/N 7 (control) C/N 11 C/N 15 C/N 19 C/N 23

Initial mean individual weight (g) 54.06 ± 0.59 55.46 ± 0.38 53.27 ± 0.96 54.13 ± 0.98 53.57 ± 0.39Final mean individual weight (g) 163.21 ± 8.08c 166.48 ± 3.78c 196.66 ± 23.63bc 232.82 ± 10.07ab 269.72 ± 40.60a

Specific growth rate (% · d-1) 1.00 ± 0.06c 1.00 ± 0.02c 1.18 ± 0.12bc 1.32 ± 0.05ab 1.46 ± 0.14a

Survival rate (%) 96.83 ± 3.17a 100.00 ± 0.00a 99.21 ± 0.79a 98.41 ± 1.59a 76.19 ± 13.75b

Net production (kg · ha-1) 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 b 0.05).

444 Z. Zhao et al. / Aquaculture 434 (2014) 442–448

In the present study, the efficiency parameters (FCR, TFCR, PER andTPER) are referred to as “apparent” efficiency, more practical than ofbiological significance, because actual consumption of the diets couldnot be monitored in BFT enclosures. All statistical analyses were per-formed using SPSS 17.0 for Windows. Data were analyzed by one-wayANOVA after homogeneity of variance test.When significant differenceswere found, Duncan's multiple range tests were used to identify differ-ences among experimental groups. Differences were considered signif-icant at level P b 0.05.

3. Results

3.1. Growth and productions

The specific growth rates (SGR) andprotein efficiency ratios (PER) ofmirror carp increased gradually as feed C/N ratios increased from7 to 23in control and all treatments. These parameters did not show significant(P N 0.05) differences in treatments having feed C/N ratios from 7 to 19(Table 1). The survival rates (SR) and net production (NP) of mirror andsilver carp in the treatment of C/N23were significantly (P b 0.05) lowerthan those in other treatments because of fish mortality in C/N 23.The feed conversion rates (FCR) of mirror carp declined significantly(P b 0.05) with increasing of the feed C/N ratios from 7 to 23 in control

Table 3Initial mean individual weight and growth performance of bighead carp, Aristichthys nobilis in

Parameters C/N 7 (control) C/N 11

Initial individual weight (g · ind-1) 58.26 ± 0.41 59.04 ± 0.31Final individual weight (g · ind-1) 159.76 ± 24.64b 162.01 ± 12.26Specific growth rate (% · d-1) 0.89 ± 0.14b 0.91 ± 0.08b

Survival rate (%) 100.00 ± 0.00 100.00 ± 0.00Net production (kg · ha-1) 290.01 ± 69.53b 294.21 ± 35.87

Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript

Table 4Production and feed utilization in control and treatments.

Parameters C/N 7 (control) C/N 11

Total production (kg · ha-1) 12522.25 ± 105.43a 12382.65 ± 322.79a

Total net production (kg · ha-1) 9046.24 ± 45.21a 8978.93 ± 218.90a

Total feed conversion rate 1.60 ± 0.01a 1.54 ± 0.03a

Total protein efficiency ratio 1.84 ± 0.01d 1.92 ± 0.02cd

Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript

and all treatments. The SGR and NP of silver carp and bighead carpincreased significantly (P b 0.05) in treatments from C/N 19 to 23(Tables 2 and 3). The SR of silver carp in treatment C/N 23 was signifi-cantly (P b 0.05) lower comparing to other treatments, while the SRof bighead carp in all treatments were always 100%. The total produc-tion (TP) and total net production (TNP) of fish increased from12522 kg · ha-1 and 9046 kg · ha-1, respectively in control (C/N 7).In treatment C/N 19, the TP and TNP was 13895 kg · ha-1 and10385 kg · ha-1, respectively. There was no significant (P N 0.05) differ-ence in TP and TNP parameters in control (C/N 7) and treatments C/N11, C/N 15 and C/N 19 (Table 4). But, in treatment C/N 23, the fish TPand TNP decreased significantly (P b 0.05). With increasing of C/N ra-tios, the total feed conversion rates (TFCR) of fish declined significantly(P b 0.05), while the total protein efficiency ratios (TPER) of fishincreased significantly (P b 0.05).

3.2. Tissue composition

Therewere no significant (P N 0.05) differences inmoisture contentsof mirror carp muscle comparing to control and treatments (Table 5).With increasing feed C/N ratios, the crude protein and ash contents inmirror carp increased significantly (P b 0.05) with highest value at C/N 15, but declined significantly (P b 0.05) with further increase of feed

control and treatments.

C/N 15 C/N 19 C/N 23

58.13 ± 1.26 58.27 ± 1.54 59.74 ± 0.99b 181.19 ± 14.85ab 232.14 ± 26.80a 240.17 ± 5.58a

1.03 ± 0.05ab 1.24 ± 0.09a 1.26 ± 0.04a

100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00b 351.60 ± 38.98ab 496.76 ± 72.28a 513.94 ± 19.28a

letters are significantly different (P b 0.05).

C/N 15 C/N 19 C/N 23

13356.91 ± 301.34a 13895.72 ± 180.95a 9411.65 ± 1066.96b

9843.27 ± 367.37a 10385.84 ± 132.10a 7046.33 ± 887.22b

1.48 ± 0.06ab 1.40 ± 0.02b 1.25 ± 0.02c

2.00 ± 0.07bc 2.11 ± 0.02b 2.36 ± 0.02a

letters are significantly different (P b 0.05).

Table 5Proximate 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 ± 0.61Crude 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 rowwith different superscript letters are significantly different (P b 0.05). The contents of crude protein, crude lipid andash were obtained from the dry matter.

445Z. Zhao et al. / Aquaculture 434 (2014) 442–448

C/N ratios. The crude lipid contents increased significantly (P b 0.05)with raising the feed C/N ratios, and reached the high value at C/N 23.

3.3. Water quality

Water temperature changed from higher 24.7 °C to lower 12.0 °Cin all experimental enclosures (Fig. 1). The bioflocs volume in the treat-ments from C/N 15 to 23 increased gradually depending upon theamount of corn starch addition, but later decreased with loweringtemperature (Fig. 2-A). Therewas a significant (P b 0.05) positive corre-lation between the bioflocs volume andwater temperature (Fig. 3). Thebioflocs biovolume ranged from 100-2000 μm.

The total ammonia nitrogen (TAN) (Fig. 2-B) and nitrite nitrogen(NO2-N) (Fig. 2-C) concentrations decreased significantly (P b 0.05) intreatments C/N 15, C/N 19 and C/N 23, respectively; while the nitratenitrogen (NO3-N) (Fig. 4) and total inorganic nitrogen (TIN) (Fig. 5)increased significantly (P b 0.05) in all treatments. Phosphate (PO4-P)(Fig. 2-D) and total phosphorus (TP) (Fig. 2-E) in control (C/N 7)increased significantly (P b 0.05), while showed fluctuation fromincreases to declines in C/N 15, C/N 19 and C/N 23 treatments. Thechemical oxygen demand (COD) (Fig. 2-F) and dissolved organic carbon(DOC) (Fig. 2-H) increased significantly (P b 0.05), while the total alka-linity (Fig. 2-G) and chlorophyll a concentration (Fig. 2-I) did not showsignificant (P N 0.05) difference in control and treatments.

Water quality parameters in control and treatments during theexperiment were showed in Table 6. The TAN concentrations in treat-ments of C/N 11-23 were significantly (P b 0.05) lower than those inthe control of C/N 7. The NO2-N, PO4-P and TP concentrations decreasedsignificantly (P b 0.05) with raising C/N ratios. The NO3-N and TIN con-centrations decreased gradually with increases of C/N ratios, but theydid not show significant (P N 0.05) differences among 11-23 of C/Nratios. With increasing of C/N ratios from 7 to 23, there were no signif-icant differences (P N 0.05) in the concentrations of chlorophyll a, CODand DOC. In addition, there were slight changes in total alkalinity andpH values during C/N 7-23 (P N 0.05).

4. Discussions

The present study confirmed beneficial effects of promoted bioflocson fish production performance and water quality in mirror, silver andbighead carp polyculture with minimum water exchange system. It is

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25/Jul 9/Aug 24/Aug 8/Sep 23/Sep 8/Oct 23/Oct

Tem

pera

ture

(˚C

)

Date

Fig. 1. Changes of water temperature during the experiment.

clear that the key of bioflocs technology is the transformation of micro-bial proteins resulted in increased fish production achievable by addingorganic carbon (Zhao et al., 2013). In earthen pond, heterotrophicmicroorganisms synthesize protein from organic carbon and inorganicnutrients (Avnimelech, 2012). The efficiency ofmicrobial protein assim-ilation could depend on food particle size for ingestion, digestion andabsorption ability in fish. The silver carp and bighead carp are typicalfilter-feeding pelagic fishes having gill racker gap ranging 11-19 μmand 34-41 μm, respectively, for ingestion (Liu and Haung, 2008; Zhaoet al., 2011, 2014). Silver and bighead carp exhibit food particle size-selection, but cannot select preferred plankton species actively fromevenly distributed food particles in the water (Dong et al., 1992). Theparticle sizes of bioflocs (100-2000 μm) were in their ingested rangein current experiment.

In the present study, treatment C/N 23 suffered a serious hypoxia in-cidents causing mortality in mirror and silver carp. Therefore, survivalrates (SR) and net production (NP) of mirror and silver carp, total pro-duction (TP) and total net production (TNP) in C/N 23 treatment waslower comparing to other treatments (Table 4). These indicating thatattention should be paid to hypoxia in treatments with high feed C/Nratio culture systems due to increased heterotrophic microorganisms'activities. The bioflocs could not only provide supplemental microbialnutrition in- situ for mirror carp, silver carp and bighead carp, but alsoexert a positive effect on controlling levels of nitrogen and phosphorouscompounds. In the mirror carp polyculture system, improved growthperformance and feed utilization could be achieved and good waterquality could be maintained.

However, high protein diets usually employed in intensive aquacul-ture systems containing C/N ratios of less than 10might slow down therate ofmicrobial nitrogen assimilation causing accumulationof inorgan-ic nitrogen (ammonia, nitrite and nitrate) in culture system (Mcintosh,2000). Chamberlain et al. (2001) recommended the use of balancedmixtures of carbonaceous and nitrogenous materials with approxi-mately 20 C/N ratios to stimulate microbial nitrogen assimilation.Avnimelech (1999) determined that the use of a carbon source toraise the C/N ratio within the culture system was a practical and inex-pensive way to reduce inorganic nitrogen accumulation. In the presentstudy, the use of corn starch to raise the C/N ratio in treatments C/N 15to C/N23was efficient inmaintaining low levels of nitrogen compounds(TAN, NO2-N and NO3-N) and orthophosphate (PO4-P) withoutsubstantial increase in chemical oxygen demand (COD) and dissolvedorganic carbon (DOC). This may be the result of high density of hetero-trophic bacteria algae, protozoan, ciliates, rotifers, zooplankton andorganic matter attached to the flocs (Fig. 2-A) due to nitrogen, phos-phorus and organic carbon available to increase their biomass. Thereduction in TAN observed in our study is probably also due to the bac-terial load as has been previously reported (Avnimelech et al., 1994;McIntosh, 2000; Anand et al., 2014). A detailed investigation into thebacterial dynamics in such system can help in understanding theother dimensions of biofloc based aquaculture.

Themuscle compositions showed that bioflocs could influence qual-ity ofmirror carp. In general, with increasing feed C/N, the crude proteinand crude lipid contents of the muscle tend to increase comparing tocontrol (Table 5). Similar trend of whole body lipid contents havebeen observed by Xu and Pan (2012). Izquierdo et al. (2006) showedthat whole lipid content of Litopenaeus vannamei reared in mesocosm

Fig. 2. Changes of water quality parameters in control (C/N 7) and treatments during the experiment. Values aremeans (±S.E.) of three replicate enclosures per sampling time in each group. These parameters include bioflocs volume (BFV, A), totalammonia nitrogen (TAN, B), nitrite nitrogen (NO2-N, C), orthophosphate (PO4-P, D), total phosphorus (TP, E), chemical oxygen demand (COD, F), total alkalinity (G), dissolved organic carbon (DOC, H), chlorophyll a (I).

446Z.Zhao

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y = 0.0106x - 0.0726(R2 = 0.5659, P<0.01)

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V (

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Fig. 3. The relationships between the bioflocs volumes (BFV) and temperature during theexperiment.

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Fig. 4. Changes of nitrate nitrogen (NO3–N) in control (C/N 7) and treatments during theexperiment. Values are means (±S.E.) of three replicate enclosures per sampling time ineach group.

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C/N 11

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C/N 23

Fig. 5. Changes of total inorganic nitrogen (TIN) in control (C/N 7) and treatments duringthe experiment. Values aremeans (±S.E.) of three replicate enclosures per sampling timein each group.

447Z. Zhao et al. / Aquaculture 434 (2014) 442–448

with bioflocs had higher values than in clear water systems, attributingto the effective assimilation of bioflocs. In present study increasedmus-cle crude protein contents in mirror carp might be due to the artificial

Table 6Water quality parameters in control and treatments during the experiment.

Parameters C/N 7 (control) C/N 11

TAN (mg · L-1) 1.06 ± 0.15a 0.68 ± 0.16b

NO2-N (mg · L-1) 0.19 ± 0.02a 0.15 ± 0.02ab

NO3-N (mg · L-1) 13.13 ± 1.41a 9.46 ± 2.03ab

TIN (mg · L-1) 14.49 ± 1.46a 10.35 ± 2.06ab

pH 8.13 ± 0.04 8.17 ± 0.06PO4-P (mg · L-1) 0.25 ± 0.04a 0.19 ± 0.03ab

TP (mg · L-1) 1.13 ± 0.14a 0.95 ± 0.14ab

Chlorophyll a (μg · L-1) 0.06 ± 0.01 0.08 ± 0.01COD (mg · L-1) 11.53 ± 0.74 11.43 ± 0.66Total alkalinity (mmol · L-1) 6.03 ± 0.11 6.33 ± 0.13DOC (mg · L-1) 12.22 ± 0.64 15.13 ± 1.57

Each value represents mean ± S.E. (n = 3). Values in the same row with different superscript

feed as regular diet, further the ingestion of bioflocs might facilitatedbetter nutrient assimilation.

Temperature is major important environmental factor to affectingmicrobial metabolism (Yang and Li, 2009). Several studies showedthat optimum temperature for bioflocs formation ranged from 18 to25 °C (Wilen and Balmer, 1999; Yang and Li, 2009). Corresponding todecline in water temperature the bioflocs volumes decreased graduallyin treatments from C/N 15 to C/N 23. This was further demonstratedthat there was significant (P b 0.05) positive correlation between bioflocvolumes and water temperature (Fig. 3) suggesting that heterotrophicmicroorganism and plankton increased during high temperatures.

A potential obstacle in maintenance of dense fish biomass withouthigh water replacement is the accumulation of toxic ammonia andnitrite (Avnimelech, 2007). A number of studies have suggested waysto promote bio-flocs using the accumulated hazardous inorganic nitro-gen (Burford et al., 2003; Asaduzzaman et al., 2010; Xu and Pan, 2012).Ebeling et al. (2006) showed three principal pathways of removinghazardous nitrogen by aquaculture: (1) photo-autotrophic removal byalgae (2) immobilization by heterotrophic bacteria and (3) chemo-autotrophic oxidation by nitrifying bacteria. In the present study, theconcentrations of NO3-N in treatments C/N ratios of 11-23 significantlyincreased as starch was added continuously (Fig. 4) indicating the mas-sive nitrifying bacteria were present in the system. In addition, therewere no significant differences in the concentrations of Chlorophyll aduring in control and treatments, suggestingprobably all three principalpathways were functional for removing hazardous nitrogen in presentexperimental system. These implying that immobilization of heterotro-phic bacteria should be encouraged in such high C/N system.

In the present study, the total alkalinity and pH values in controland treatment C/N ratios did not show significant (P b 0.05) differences,indicating biofloc system in outdoor earthen pond had strong bufferingcapacity for total alkalinity and pH value. Further research is needed tobetter understand pathways and mechanisms of bioflocs effects on thenutrition physiology of fish in polyculture system.

Acknowledgements

This work was financially supported by the Central-level Non-profitScientific Research Institutes Special Funds (HSY201203), earmarkedfund for China Agriculture Research System (CARS-46) and NationalScience and Technology Support Program (2012BAD25B00).

References

Acosta-Nassar, M.V., Morell, J.M., Corredor, J.E., 1994. The nitrogen budget of a tropi-cal semi-intensive fresh water fish culture pond. J. World Aquacult. Soc. 25 (2),261–270.

Anand, P.S.S., Kumar, S., Panigrahi, A., Ghoshal, T.K., Dayal, J.S., Biswas, G., Sundaray, J.K.,De, D., Raja, R.A., Deo, A.D., Pillai, S.M., Ravichandran, P., 2013. Effects of C:N ratioand substrate integration on periphyton biomass, microbial dynamics and growthof Penaeus monodon juveniles. Aquacult. Int. 21, 511–524.

Anand, P.S.S., Kohli, M.P.S., Kumar, S., Sundaray, J.K., Roy, S.D., Venkateshwarlu, G., Sinha,A., Pailan, G.H., 2014. Effect of dietary supplementation of biofloc on growth

C/N 15 C/N 19 C/N 23

0.58 ± 0.11b 0.49 ± 0.10b 0.45 ± 0.12b

0.08 ± 0.02bc 0.05 ± 0.02c 0.05 ± 0.03c

7.90 ± 1.59b 7.81 ± 1.55b 7.66 ± 1.68b

8.63 ± 1.54b 8.41 ± 1.52b 8.13 ± 1.63b

8.16 ± 0.05 8.09 ± 0.07 8.06 ± 0.060.11 ± 0.02bc 0.07 ± 0.03c 0.06 ± 0.03c

0.69 ± 0.04bc 0.58 ± 0.06c 0.55 ± 0.07c

0.09 ± 0.02 0.08 ± 0.01 0.07 ± 0.0111.12 ± 0.68 11.41 ± 0.46 11.42 ± 0.566.12 ± 0.16 6.78 ± 0.16 6.41 ± 0.14

15.86 ± 2.38 16.30 ± 1.74 16.43 ± 2.60

letters are significantly different (P b 0.05).

448 Z. Zhao et al. / Aquaculture 434 (2014) 442–448

performance and digestive enzyme activities in Penaeus monodon. Aquaculture418–419, 108–115.

AOAC, 1990. Official methods of analysis. In: Horwitz, W. (Ed.), Association of OfficialAnalytical Chemists. AOAC International, OMA, Washington, DC.

APHA, 1998. Standard Methods for the Examination of the Water and Wastewater,22nd edn. American Public Health Association, Washington, DC.

Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M.E.,2008. C/N ratio control and substrate addition for periphyton development jointlyenhance freshwaterprawnMacrobrachiumrosenbergiiproduction inponds.Aquaculture280, 117–123.

Asaduzzaman, M., Rahman, M.M., Azim, M.E., Islam, M.A., Wahab, M.A., Verdegem, M.C.J.,Verreth, J.A.J., 2010. Effects of C/N ratio and substrate addition on natural foodcommunities in freshwater prawn monoculture ponds. Aquaculture 306, 127–136.

Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture systems.Aquaculture 176, 227–235.

Avnimelech, Y., 2007. Feeding withmicrobial flocs by tilapia in minimal discharge bio-flocstechnology ponds. Aquaculture 264, 140–147.

Avnimelech, Y., 2012. Biofloc Technology - A Practical Guide Book, 2nd edition. TheWorldAquaculture Society, Baton Rouge, Louisiana, United States.

Avnimelech, Y., Kochba, M., 2009. Evaluation of nitrogen uptake and excretion by tilapiain bio floc tanks, using N-15 tracing. Aquaculture 287, 163–168.

Avnimelech, Y., Ritvo, G., 2003. Shrimp and fish pond soils: processes and management.Aquaculture 220, 549–567.

Avnimelech, Y., Kochva, M., Diab, S., 1994. Development of controlled intensive aquacul-ture systemswith a limitedwater exchange and adjusted carbon to nitrogen ratio. Isr.J. Aquacult. - Bamidgeh 46 (3), 119–131.

Azim, M.E., Little, D.C., 2008. The biofloc technology (BFT) in indoor tanks: water quality,biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus).Aquaculture 283, 29–35.

Ballester, E.L.C., Abreu, P.C., Cavalli, R.O., Emerenciano, M., Abreu, L., Wasielesky, W., 2010.Effect of practical diets with different protein levels on the performance ofFarfantepenaeus paulensis juveniles nursed in a zero exchange suspended microbialflocs intensive system. Aquac. Nutr. 16, 163–172.

Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., 2003. Nutrientand microbial dynamics in highintensity, zero-exchange shrimp ponds in Belize.Aquaculture 219, 393–411.

Chamberlain, G., Avnimelech, Y., McIntosh, R.P., Velasco, M., 2001. Advantages of aeratedmicrobial reuse systems with balanced C:N. II: composition and nutritional value oforganic detritus. Glob. Aquacult. Advocate 4, 22–24.

Crab, R., Chielens, B., Wille, M., Bossier, P., Verstraete, W., 2010. The effect of differentcarbon sources on the nutritional value of bioflocs, a feed forMacrobrachium rosenbergiipostlarvae. Aquac. Res. 41, 559–567.

Crab, R., Defoirdt, T., Bossier, P., Verstraete, W., 2012. Biofloc technology in aquaculture:beneficial effects and future challenges. Aquaculture 356, 351–356.

Davenport, J., Black, K., Burnell, G., Cross, T., Culloty, S., Ekaratne, S., Furness, B., Mulcahy,M., Thetmeyer, H., 2003. Aquaculture, the Ecological Issues. Blackwell Publishing,Oxford.

De Schryver, P., Crab, R., Defoirdt, T., Boon, N., Verstraete, W., 2008. The basics of bioflocstechnology: the added value for aquaculture. Aquaculture 277, 125–137.

Dong, S.L., Li, D.S., Bing, X.W., Shi, Q.F., Wang, F., 1992. Suction volume and filtering effi-ciency of silver carp (Hypophthalmichthys molitrix Val.) and bighead carp (Aristichthysnobilis Rich.). J. Fish Biol. 41, 833–840.

Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichiometryof photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen inaquaculture systems. Aquaculture 257, 346–358.

Fukushima,M., Takamura, N., Sun, L., Nakagawa,M.,Matsushige, K., Xies, P., 1999. Changesin the plankton community following introduction of filter-feeding planktivorousfish. Freshw. Biol. 42, 719–735.

Gross, A., Boyd, C.E., Wood, C.W., 2000. Nitrogen transformations and balance in channelcatfish ponds. Aquac. Eng. 24, 1–14.

Hargreaves, J.A., 2006. Photosynthetic suspended-growth systems in aquaculture. Aquac.Eng. 34, 344–363.

Hari, B., Kurup, B.M., Varghese, J.T., Schrama, J.W., Verdegem, M.C.J., 2006. The effect ofcarbohydrate addition on water quality and the nitrogen budget in extensive shrimpculture systems. Aquaculture 252, 248–263.

Izquierdo, M., Forster, I., Divakaran, S., Conquest, L., Decamp, O., 2006. Effect of green andclear water and lipid source on survival, growth and biochemical composition ofPacific white shrimp Litopenaeus vannamei. Aquacult. Nutr. 12, 192–202.

Ke, Z.X., Xie, P., Guo, L.G., Liu, Y.Q., Yang, H., 2007. In situ study on the control of toxicMicrocystis blooms using phytoplanktivorous fish in the subtropical Lake Taihu ofChina: A large fish pen experiment. Aquaculture 265, 127–138.

Li, D.S., Yang, H.S., Wang, J.Q., Lu, J.R., Tian, X.L., Liu, G.C., 1998. A device of land-basedexperimental enclosure used in ponds. J. Ocean Univ. Qingdao 28, 199–204 (in Chinesewith English abstract).

Liu, H.L., Haung, Z.G., 2008. Aquaculture Science of China. Science Press, Beijing.Liu, L.P., Hu, Z.X., Dai, X.L., Avnimelech, Y., 2014. Effects of addition of maize starch on the

yield, water quality and formation of bioflocs in an integrated shrimp culture system.Aquaculture 418–419, 79–86.

McIntosh, P.R., 2000. Changing paradigms in shrimp farming: IV. Low protein feeds andfeeding strategies. Glob. Aquacult. Advocate 3, 44–50.

Rahman, M.M., Nagelkerke, L.A.J., Verdegem, M.C.J., Wahab, M.A., Verreth, J.A.J., 2008.Relationships among water quality, food resources, fish diet and fish growth inpolyculture ponds: a multivariate approach. Aquaculture 275, 108–115.

Velasco, M., Lawrence, A.L., Neill, W.H., 1998. Development of a static-water ecoassaywith microcosm tanks for postlarval Penaeus vannamei. Aquaculture 161, 79–87.

Wang, J.Q., Li, D.S., Dong, S.L., Wang, K., Tian, X.L., 1998. Experimental studies onpolyculture in closed shrimp ponds: I. Intensive polyculture of Chinese shrimp(Penaeus chinensis) with tilapia hybrids. Aquaculture 163, 11–27.

Wilen, B.M., Balmer, P., 1999. The effect of dissolved oxygen concentration on the struc-ture, size and size distribution of activated sludge flocs. Water Res. 33 (2), 391–400.

Xu, W.J., Pan, L.Q., 2012. Effects of bioflocs on growth performance, digestive enzymeactivity and body composition of juvenile Litopenaeus vannamei in zero-waterexchange tanks manipulating C/N ratio in feed. Aquaculture 356–357, 147–152.

Xu, W.J., Pan, L.Q., 2013. Enhancement of immune response and antioxidant status ofLitopenaeus vannamei juvenile in biofloc-based culture tanks manipulating high C/Nratio of feed input. Aquaculture 412–413, 117–124.

Yan, L.L., Zhang, G.F., Liu, Q.G., Li, J.L., 2009. Optimization of culturing the freshwater pearlmussels, Hyriopsis cumingiiwith filter feeding Chinese carps (bighead carp and silvercarp) by orthogonal array design. Aquaculture 292, 60–66.

Yang, S.F., Li, X.Y., 2009. Influences of extracellular polymeric substances (EPS) on thecharacteristics of activated sludge under non-steady-state conditions. ProcessBiochem. 44, 91–96.

Zhao, Z.G., Dong, S.L., Wang, F., Tian, X.L., Gao, Q.F., 2011. The measurements of filteringparameters under breathing and feeding offilter-feeding silver carp (Hypophthalmichthysmolitrix Val.). Aquaculture 319, 178–183.

Zhao, Z.G., Xu, Q.Y., Luo, L., Yin, J.S., Wang, C.A., 2013. Effect of adding carbon source ongrowth of fish and water quality in Songpu mirror Carp (Cyprinus specularis Songpu)pond. J. Northeast Agric. Univ. 44 (9), 105–112 (in Chinese with English abstract).

Zhao, Z.G., Dong, S.L., Wang, F., Tian, X.L., Gao, Q.F., 2014. Effect of algae density onbreathing and feeding of filter-feeding silver carp (Hypophthalmichthys molitrix Val.).Aquaculture 433, 133–136.