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The addition of probiotic bacteria to microbial floc: Water quality, growth, non-
specific immune response and disease resistance of Cyprinus carpio in mid-
Himalayan altitude

Article  in  Aquaculture · June 2018

DOI: 10.1016/j.aquaculture.2018.06.056

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Accepted Manuscript

The addition of probiotic bacteria to microbial floc: Water quality,

growth, non-specific immune response and disease resistance of
Cyprinus carpio in mid-Himalayan altitude

Pragyan Dash, R.S. Tandel, Raja Aadil H. Bhat, Sumanta Mallik,

A.K. Singh, Debajit Sarma

PII: S0044-8486(17)32437-7
DOI: doi:10.1016/j.aquaculture.2018.06.056
Reference: AQUA 633341
To appear in: aquaculture
Received date: 8 December 2017
Revised date: 19 June 2018
Accepted date: 20 June 2018

Please cite this article as: Pragyan Dash, R.S. Tandel, Raja Aadil H. Bhat, Sumanta Mallik,
A.K. Singh, Debajit Sarma , The addition of probiotic bacteria to microbial floc: Water
quality, growth, non-specific immune response and disease resistance of Cyprinus carpio
in mid-Himalayan altitude. Aqua (2018), doi:10.1016/j.aquaculture.2018.06.056

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 ACCEPTED MANUSCRIPT

The addition of probiotic bacteria to microbial floc: Water quality, growth, non-specific
immune response and disease resistance of Cyprinus carpio in mid-Himalayan
altitude

Pragyan Dash1,*, R. S. Tandel1, Raja Aadil H. Bhat1, Sumanta Mallik1, A. K. Singh1,

Debajit Sarma1
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CAR- Directorate of Coldwater Fisheries Research, Bhimtal, Uttarakhand, India
*
Correspondence: Pragyan Dash
Email : pragyan5dash@gmail.com

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Tel : 05942- 247280 Fax : 05942- 247693

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1. Introduction

In the evolving context of current social, economic and environmental

constraints, aquaculture productions have to drive towards more environmentally

friendly systems. Illustrating their high level of water dependence, fish production

systems are mainly concerned with regards to the global context of diminishing water

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resources and the need to control waste release in recipient ecosystems

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(d’Orbcastel et al., 2009). Biofloc technology, a microbial-based technique, since its

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inception has gradually gained acceptance for healthy fish and shrimp production

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and in recent years appeared as a useful technique in the intensive culture system.

The microbial manipulation approach promises a healthy, high-density rearing

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system and has proven to maintain optimum water quality, efficient assimilation of
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feed nutrients (Hari et al., 2004 and Crab et al., 2009) by aiding in digestion with

exogenous digestive enzymes (Xu et al., 2013) and providing extra

essential nutrients for fish growth (Ekasari et al., 2010; Xu et al., 2012). Numerous
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studies have revealed the micro-habitat of gut colonizing microbes in biofloc system
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and their positive role on growth, reproduction and immune response in fish and

shrimp (Crab et al., 2012; Kim et al., 2014; Mansour et al., 2017). The natural
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probiotic effect of biofloc comprising enhancement of the non-specific defense and

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providing protection against disease infection is a well-explored useful feature of

biofloc technology (Kim et al., 2014 and Xu et al., 2013). As a whole, biofloc system

aids the host animal in acquiring a symbiotic gut microbiome that influences its

overall immunological and physiological process.

Over the last two decades, the application of probiotic bacteria through feed

or water is considered to confer the health benefit on the host animal. Several

studies reveal the use of probiotics for improvement of water quality by balancing
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bacterial populations in culture tanks and reduction of pathogenic bacteria loads

(Moriarty, 1998; Gomez-Gil et al., 2000). Bacillus spp. and Lactobacillus spp. have

been found to be effective in promoting growth, survival and immune status of the

host (Isolauri et al., 2001; Dash et al., 2017). Bacillus probiotics are known to

produce nutrient digesting enzymes that enable to improve the digestion activity of

the host (Zhou et al., 2009), enhance the immune response and confer disease

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resistance against the pathogen (Aly et al., 2008a; Zhao et al., 2012). For example,

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dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A,

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and Bacillus pumilus 47B influenced efficiently the growth, innate immunity

(phagocytic activity, respiratory bursts, and lysozyme activity) and stress responses
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in striped catfish, Pangasianodon hypophthalmus (Thy et al., 2017). Exogenous

addition of such probiotic bacteria to biofloc promises improvement in growth

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performance of host animal besides the water quality maintenance (Yuniasari, 2010;

Yusuf et al., 2015). Sartika et al. (2012) reported that the addition of Bacillus sp. with
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molasses had increased water quality and growth of common carp (Cyprinus carpio).

Similarly, improved growth performance and reduced feed conversion ratio were
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observed in African catfish reared in biofloc system inoculated with Bacillus cereus
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(Hapsari, 2016). In fact, the addition of Bacillus sp. and Lactobacillus sp. to the

biofloc system is also shown to enhance the health status of culture animal. For
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example, Fennerpenaeus chinensis reared in the biofloc system with added Bacillus

sp. and Lactobacillus sp. showed increased pro-phenol oxidase, lysozyme and

serum peptidase activities. Considering these results, biofloc development with the

addition of single or combination of known probiotics is regarded as a promisable

approach for improving the physiological status of animal.

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Coldwater aquaculture in the Indian Himalayas is closely associated with the

farming of exotic trouts and chinese carps; while trout farming is commercially

practiced in higher altitude region, common carp till now is the most likely farmed

species in mid hill and foot hill Himalaya. Slow growth of fishes and unavailability of

quality seed are the primary bottleneck for aquaculture development and successful

commercialization of the local or endemic species, i.e., snow trout and mahseer. The

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ICAR-Directorate of Coldwater Fisheries Research has developed the breeding and

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culture protocol of indigenous fishes such as Labeo dyocheilus, Bangana dero,

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Labeo pangusia which could provide potential opportunity to strengthen the

aquaculture production in the mid-Himalaya sector (Singh, 2016). Nevertheless,

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water scarcity, lack of infrastructure and limited seed supply restricts the productivity

and economic activity of these potential candidates. A promising solution would be

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the scaling up of culture technique with high-density fish rearing having a small water

footprint. For which a feasible alternative is the use of zero or minimal water
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exchange technology such as biofloc technology with intensive rearing.

The present study investigated the possibility of biofloc technology application

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for the rearing of Cyprinus carpio in mid-Himalayan altitude. The study in detail was
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to examine the effect of two C/N ratios and probiotic bacteria addition on biofloc

development, water quality, growth and non-specific immune response of C. carpio.

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2. Material and Method

2.1. Experimental design

A 60 days trial was conducted in the month of May and June under the outdoor

experimental facility at the Directorate of Coldwater Fisheries research at Bhimtal,

Uttarakhand. Total 225 no. of C. carpio fingerlings of average weight 7.69±0.03 to

8.56±0.06 gram were obtained from DCFR fish farm and distributed in 15 no. of
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tanks. Three replicate tanks were randomly assigned to each of the four treatments

and control viz., C (clear water), B-15 (C/N 15 without probiotic bacteria addition), B-

15P (C/N 15 with probiotic bacteria addition), B-20 (C/N 20 without probiotic bacteria

addition), B-20P (C/N 20 with probiotic bacteria addition).

2.2. Microbial inoculum preparation

For biofloc development initially, a bacterial inoculum was prepared following the

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method of Avnimelech, (1999). Briefly, twenty gram of pond bottom soil was aerated

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in 1000 mL of water for 24 hours enriched with 10 mg L-1 (NH4)2 SO4 and 200 mg L-1

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molasses. 1000 mL of the inoculum was added to each of the biofloc treatment

tanks.
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2.3. Carbon and probiotic bacteria addition

Molasses (41.2% carbon) was used as organic carbon source to maintain the
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required carbon ratio of C/N 15 and C/N 20. The amount of molasses as carbon

source was calculated based upon the protein percentage of feed, assuming that
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protein contains 16% nitrogen and 50% of protein nitrogen will be excreted by fish

(Avnimelech, 1999). Fishes were fed with commercial feed with 28% protein content
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close to C/N ratio 10 twice a day at 2 % body weight. In order to maintain the C/N
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ratio 15:1 and 20:1, 0.56 g and 1.27 g molasses was applied per each g of feed.

The carbon content of molasses was measured as per formula by Hart et al.,
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2007.

Carbon = 0.8 × Lipid + 0.53 × Protein + 0.42×Carbohydrate +

0.42×Fibre

Probiotic bacteria, Bacillus pumilus and Lactobacillus delbrueckii used for the

experiment were procured from bacteriology laboratory of Directorate of Coldwater

Fisheries Research. Both probiotics were individually inoculated into the conical flask
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containing Brain heart infusion broth and incubated for 24 h at 30°C in a shaker

incubator. The culture was centrifuged and washed with phosphate buffer saline

(PBS; pH 7.2) and quantified in macfullen units. 5 mL of B. pumilus culture at

12.5×108 cells mL-1 and 5 mL of L. delbrueckii at 13.5 ×108 cells mL-1 suspended in

PBS was added to each of the probiotic treatment tank in every four days interval.

2.4. Physicochemical parameters of water

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Selected water quality parameters like pH, temperature, and dissolved oxygen were

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estimated on a daily basis using multi-parameter instrument (Eutech instrument,

Oakton). Total ammonia nitrogen (TAN), nitrate-N (NO3- -N) and phosphate (PO43-)

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were estimated by test kits and quantified by a spectrophotometer (spectroquant
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multii, Merck). Quantitative evaluation of biofloc was weekly determined by

measuring settling floc biomass (SS), total suspended solids (TSS) and total
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dissolved solids (TDS). Settling floc biomass (SS) was measured in 1 L Imhoff cone.

Total suspended solids (TSS) and total dissolved solids (TDS) were estimated
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following APHA methods 2540 F and 2540 D. There was no water exchange up to

the sixth week after which 10% water exchange was carried out. Floc development
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was quantified up to the sixth week of experimental trial, and the nutrient level was
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estimated till the end of experiment.

2.5. Estimation of floc microbial community

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Floc was collected from the imhoff cone, and the floc morphostructure and

composition was observed by a light microscope (Leica Microsystems). Total

heterotrophic bacteria (THB) count in water samples were recorded in 10 days

interval up to 40 days using standard plate count agar (Qualigen).

2.6. Response variables

2.6.1. Growth
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The fish from different experimental tanks were weighed for growth estimation after

60 days experimental trial. Specific growth rate (SGR), percentage of body weight

gain (WG %), daily weight gain (WGD), feed conversion ratio (FCR) were calculated

as follows: SGR= [ln (final weight) - ln (initial weight)] ×100/(days of experiment), WG

(%)=(final weight –initial weight) ×100/ initial weight, WGD = (final weight –initial

weight)/ days of experiment, FCR = (feed consumed, dry)/ (live weight gain, wet).

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2.6.2. Immunological assays

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Blood was collected from the caudal vein of anesthetized fish (50 mg clove oil

L-1) using 2 mL sterile syringe and 24 gauge needles. Blood samples were taken

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from two fish per replicate after 8 weeks and pooled. Then, pooled blood from each
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replicate was allowed to clot at room temperature for 30 mins and kept at 4oC for 3

hs. After that blood was centrifuged at 6000g for 5 mins to collect the serum and
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stored at -20 oC until further use (Kheti et al., 2017). An aliquot of blood was also

collected with EDTA and kept for other immunological assay.

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2.6.2.1. Lysozyme activity

Lysozyme activity in serum was determined by Salazar et al. (2016). The

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protocol determines serum lysozyme activity based on the lysis of the lysozyme
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sensitive Micrococcus lysodeikticus by correlating with decreased optical density. M.

lysodeikticus (0.25 mg mL-1, Sigma) was suspended in potassium phosphate buffer

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(0.004M, pH, 6.5). The hen egg white lysozyme (Sigma) was taken as the standard

in the same buffer. This along with the undiluted serum sample (10µl) was placed

into the 96-well plate in quadruplicate. 70 µl of M. lysodeikticus suspension was

added to each well. Data were immediately collected in a microplate reader (BioTek,

USA) over five minutes at an absorbance of 450 nm in 1 minute interval. Control

group used was 10 µl of PBS in the 70 µl M. lysodeikticus suspension. The initial

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rate of reaction was used to calculate the activity, with 1 U of activity being defined

as the amount of enzyme that catalyzed a decreased absorbance (450 nm) of 0.001

min-1.

2.6.2.2. Myeloperoxidase activity

Myeloperoxidase activity in serum was determined following modified method

of Sahoo et al. (2005).15 µl of diluted serum with 135 µl of Hank’s Balanced Salt

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Solution (Sigma) was plated in 96 well plate. To which, 25 µl of 3,3̍-5,5̍-tetramethyl

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benzidine hydrochloride (20 mM, Sigma) and 25 µl of H2O2 (5 mM, Qualigens) were

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added. 50 µl of sulphuric acid (4 M, Qualigen) was added to stop the reaction. The

plate was centrifuged for 10 min, and 150 µl of the supernatants from each well,
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were transferred to new 96 well plates. The OD was recorded at 450 nm in a micro

plate reader (BioTek, USA).

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2.6.2.3. Respiratory burst activity

The respiratory burst activity was measured by nitroblue tetrazolium (NBT)

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assay according to the modified method by Stasiack and Baumann (1996). 50 µl of

blood was incubated at 37 °C for 1h in a 96 well U bottomed microtitre plate to

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facilitate adhesion of cells. The cells were washed with phosphate-buffered saline
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(Sigma) and 50 µl of 0.2 % NBT (Himedia) was added. Following 1h of incubation,

the cells were fixed with 100% methanol (Himedia) and washed thrice with 70%
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methanol. The plate was air dried and 60 µl of potassium hydroxide (2N, Sigma) and

70 μl of dimethyl sulphoxide (Sigma) were added to dissolve the formazan blue

precipitate formed. The OD was recorded in a micro plate reader (BioTek, USA) at

620 nm.
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2.7. Challenge study

The fishes from the biofloc and control groups were challenged with Aeromonas

hydrophila on the final day of the experiment. Prior to the challenge test LD 50 was

determined and 100 µl of 8×108 cells mL-1 of A. hydrophila strain, ATCC 7966 was

injected intramuscularly to 27 fishes from each treatment except in B-20P where 23

fishes were challenged. Daily mortality was recorded up to 7 days.

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2.8. Statistical analysis

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The statistical analysis was performed using IBM SPSS statistics 20.0 software.

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Data were analyzed by one-way ANOVA and Duncan test was used to determine the

significant difference (P < 0.05) among the treatments.

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3. RESULTS

3.1. Biofloc community and density

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After one week of the trial initiation, the colour of the biofloc changed to light brown in

B-15, and dark brown in B-15P, B-20 and B-20P; and later on the colour of all the
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treatments remained dark brown throughout the experiment. The biofloc community

comprised of zooplankton and protozoa such as rotifers (Branchionus, Rotaria),

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copepods, and ciliates (Vorticella, Coleps, Euplotes) irrespective of the treatments.

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Different classes of phytoplankton; Protococcus, Scenedesmus, filamentous algae,

diatoms were identified from all the treatments (Fig. 1). The floc water from all the
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treatments was dominated with pinnate diatom colony stuck upon with gelatinous

matrix. Overall, carbohydrate addition in the biofloc system significantly increased (P

<0.05) the total bacterial load compared to control (Fig. 2). Among biofloc groups,

both C/N ratio with probiotic bacteria addition significantly recorded higher level of

total bacteria count in the entire sampling interval. Furthermore, the highest bacteria

population (log 8.23 CFU mL-1) was observed on the 20th day in B-20P. There was a
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declined trend observed in total bacterial population in the floc water from the 20th

day onwards during this trial. The possible reason may be due to the increasing rate

of bacterial accumulation in the floc formation. The flocculated microbes were not

separated from the floc water during the sample processing.

Weekly estimation of settleable solids (SS), suspended solid (TSS) and

dissolved solids (TDS) is depicted in Fig. 3. The biofloc volume (SS), as well as TDS

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and TSS, increased gradually over the experimental trial. The higher concentration

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of suspended solid (TSS) was quantified from the B-20 P treatment after one week

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of trial initiation; nevertheless it was statistically similar with B-20. From the second

week onwards the suspended solid load in B-20 P started to increase significantly
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from the other groups (p<0.05), followed by B-15 P and this trend was continued

throughout the experiment. Also, B-15 P showed a significantly higher suspended

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solid level (p< 0.05) than B-15 throughout the experiment. Similar trend was

observed in the dissolved solids concentration (TDS) which was significantly higher
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in B-20 P from 2nd week onwards followed by B-15 P. The biofloc biomass

development in terms of floc volume (SS) was quantified through the settleable
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solids measurement, depicted in Fig. 3C. Higher concentration of biofloc volume

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(SS) was observed in B-20 P group throughout the experimental trial, though it was

not statistically different with B-20.

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3.2. Physicochemical parameters of water

The water quality parameters such as temperature, pH and dissolved oxygen in

biofloc and control are depicted in Table 1. Temperature and oxygen did not vary

among the treatments. pH decreased gradually in all the biofloc treatments as the

trial progressed. The weekly changes in TAN, NO3- -N and PO43- are presented in

Fig. 4. TAN concentration fluctuated significantly in all the treatments and reached its
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peak on 6th week with maximum concentration of 1.15 in B-15 (Fig. 4A). From the 2nd

week onwards, the higher concentration of TAN was observed in the B-20 P

treatment, which was followed by the B-15. A significantly lower concentration of

TAN was observed in the B-15 P group, compared to B-20 P. A sudden decline in

TAN concentration was observed in all the treatments after the 7 th week onwards

following 20% water exchange.

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The mean concentration of nitrate-nitrogen (NO3--N) fluctuated in all the

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treatment groups between 3.2 to 31.2 mg L-1 and started to differ from control on the

3rd week (Fig. 4B). A significantly higher concentration of nitrate-nitrogen (NO3--N)

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was observed in B-15 P group on the 3 rd week, similar with the B-15 group,
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nevertheless B-20 P showed the lower concentration of nitrate among the treatment

groups. The fluctuation of nitrate was concurrent on the 7th week showing peak in B-
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15 P treatment and lower concentration in B-20 P. The nitrate concentration in B-20

group reached above 20 mg L-1 in 5th (p<0.05), 6th and 8th week while in B-15 the
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nitrate fluctuation was comparatively steady. The mean phosphate concentration in

the treatment groups increased significantly (p <0.05) from the control after one
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week of trial initiation and a significantly high phosphate was obtained in the B-20
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group in 5th and 6th week (Fig. 4C). With the increase in carbon supplementation

PO43- concentration in the cultured water increased but showed the comparably
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lower level in both of the probiotics added treatments.

3.3. Growth performance

At the end of 60 days trial, the growth of the common carp fingerlings improved

significantly in all the biofloc treatments compared to the control (Table 2). No

significant difference was found among the biofloc treatments regarding growth rate,

FCR, and SGR. The average daily growth rate of reared common carp was within
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0.05 to 0.09, highest observed in B-20 treatment. B-20 showed highest weight gain

(%) of 67.749±2.2 compared to 46.175±2.3 obtained under control group.

3.4. Non-specific immune response

The production of reactive oxygen species (ROS) as detected by NBT reduction (OD

at 650 nm) was significantly influenced by the carbon addition in culture water. The

higher amount of NBT reduction value (1.12±0.05) was observed in B-15P; however,

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it was not significantly different from the B-15 and B-20 group (Fig. 5A). Similarly, an

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increasing trend of lysozyme activity (U mL -1) and myeloperoxidase activity (OD at

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450 nm) was observed in all of the biofloc based treatments. A significantly higher

level of myeloperoxidase activity (1.10±0.08) and lysozyme activity (1666.66±176)

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was found in the B-15P group (Fig. 5B and 5C).

3.5. Survival (%)

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Survival (%) of C. carpio fingerlings in different groups after challenge with A.

hydrophila is presented in Fig. 6. Among the treatment groups, the highest survival
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(92.5%) was recorded in the B-20 group followed by B-15P and B-15, with least

survival percent recorded in the B -20P group (79.36%). Fishes reared in B-20 and
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B-15 P showed significantly higher survival (p < 0. 05) compared to the control, but
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the survival rate was not significantly higher than the B15 group.

4. Discussion
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The rationale for choosing the combination of these two probiotic bacteria B.

pumilus and L. delbreuckii were manifold. B. pumilus and L. delbreuckii have strong

gut colonizing ability, and use of these bacteria as probiotic supplements is

recommended for improving growth and well-being of fish. Also, B. pumilus is known

for its nitrogen removal capability, the bacterium along with periphytic algae could

efficiently reduce TAN and NO2 in an aquaculture system (Banerjee et al., 2010).
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Recently, many studies have conferred that multispecies or strain of probiotics are

more efficient than single species and may imply synergistic probiotic properties

(Hauville et al., 2016). For this reason, it is apparent to develop a consortium of

bacteria in the microbial floc system using the exogenous addition of mixed

probiotics. More importantly, the reason behind choosing these two probiotics also

lies in their isolation and culture environment. It was plausible to expect the

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proliferation of both Bacillus pumilus and L. delbrueckii in the culture system as

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these were previously isolated from the same climatic condition where the

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experiment was conducted.

The microscopic composition of the biofloc from the present study comprised of
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extensive diversity of microorganisms such as autotrophic algae, heterotrophic bacteria, and

protozoans. Pennate diatoms were the main autotrophic organisms dominated in all the
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biofloc groups, probably originated from the pond water used as inoculum and apparently

developed in the nutrient-rich medium. The diatoms have a strong interdependency with the
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heterotrophic bacteria due to their cross feeding habit, and their dominance is prevalent in a

heterotrophic biofloc system (Llario et al., 2018). Heterotrophic bacteria dominated in the
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biofloc tank use organic matter for their energy metabolism and food for their proliferation.

Furthermore, protozoa such as ciliates, rotifers also absorb organic matter for the energy
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source, and in this way, 40% of the nutrients are utilized for protozoa biomass production

(Lal 2006; Manan et al., 2017). As a whole, the diverse microbial community in the system
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together implements a symbiotic process and trigger the bioremediation process

characterized by the removal of nitrogenous compounds.

In this study, the biofloc development in terms of solid load occurred with the addition

of molasses as an organic carbon source; meanwhile, the water quality was maintained

within the optimal range throughout the trial period. Water temperature range (17.7°C -

20.3°C) observed throughout the trial was favorable for the biofloc production. The

concentration of settled, suspended and dissolved solids increased as the trial proceeded
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and the highest concentration was observed in probiotics added biofloc treatment in higher

C/N ratio. The significant increase in suspended solid load in B-20 P and B-15 P from the

respective control (p < 0.05) was obvious due to the probiotics addition. The result indicates

that the increase in carbon addition with probiotic bacteria causes the faster development of

suspended biomass. This was accomplished due to a relatively high heterotrophic bacterial

load rooted in the biofloc system (p < 0.05) as depicted in Fig.1, which is also similar with the

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finding by Miao et al., (2017).

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The addition of carbon significantly reduced the pH in the biofloc groups but within

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the acceptable range. Decreased pH in biofloc treatment was due to the increase in

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heterotrophic bacteria density as has been previously reported (Avnimelech et al., 1999).

Probiotics addition to the culture system had no significant effect on the pH level. Probiotics
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are capable of reducing the total nitrogen load in pond sediment (Wang et al., 2008). In our

study, the addition of probiotics to the biofloc system with lower C/N ratio showed a lower
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level of TAN, compared to the other groups, while the opposite result was observed in higher

C/N ratio, i.e., B-20. This may be due to the higher concentration of solid observed in B20-P
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treatment. Removal of solids was not carried out in this study up to the 6th week of trial,

resulting in excessive suspended solid load in B-20 P from the 4th week onwards, exceeding
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the recommended concentration of maximum 500 mg/ L by Azim and Little, (2008). An

excessive solid load may reduce the dissolved oxygen and increase the nitrogenous
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compounds (Rakocy et al., 2006; Perez-Fuentes et al., 2016). The solid removal after the 6th
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week had a positive effect on lowering the TAN concentration in all the treatment groups.

The results also agree with the previous finding that TAN concentration gets stabilized after

a certain period, once a mature biofloc is established (Xu et al., 2016).

The increase in nitrate concentration indicated the presence of enormous nitrifying

bacteria in the system. The fluctuation of NO 3--N concentration between 3.2 to 31.2 mg L-1

would not have the possible negative effect on the C. carpio performance, as the fish is

reported to tolerate nitrate up to a concentration of 1000 mg L-1 (Tilak et al., 2007). Nitrate

concentration in C/N 20 was consistently higher from the other treatments, which is similar
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with the result of Dauda et al., (2018). It is interesting to note that a significantly high nitrate

concentration (p<0.05) was observed during the initial period of trial in C/N 15 when

probiotics were added. The addition of probiotics to biofloc might have triggered the faster

nitrification process during the trial initiation in lower C/N ratio. From the consistent result of

lower nitrate level in high C/N ratio with probiotics (B-20 P), it is predicated that TAN

assimilation by the heterotrophic bacterial load could be the major pathway for nitrogen

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removal (Ebeling et al., 2006) in this treatment.

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In a zero water exchange system like biofloc technology, increase in

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phosphate concentration is inevitable. On the other hand, the higher concentration of

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phosphate also leads to the formation of cyanobacteria bloom in culture system

(Anderson et al., 2002, Silva et al., 2013). Bacterial species individually or in the
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consortium can remove phosphate by utilizing the solubilized phosphate through
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intracellular accumulation in the form of polyphosphate. Bacillus spp., Pseudomonas

spp., the dominant microbial community in a biofloc based culture system (Zhao et
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al., 2012) are also reported to be the efficient phosphate reducer (Krishnaswamy et

al., 2009). In fact, heterotrophic bacteria have a huge role in the absorption of a large
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fraction of orthophosphate in freshwater at an estimated rate of around 60%

(Kirchman, 1994). Wang et al. (2008) observed that the probiotics application
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significantly decreased the TP (total phosphorus), and TIP (total inorganic

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phosphorus) in pond sediment. In this study, carbon addition along with the probiotic

bacteria has shown a lower level of phosphate in both the treatments; B-20P and B-

15P (Fig.4C). The possible reason may be due to the utilization of phosphate by the

bacteria consortium established in the biofloc system in which a vast diversity of

microorganisms take part (De Schryver et al., 2008) and also the establishment of

dense heterotrophic bacteria load observed throughout the culture period (Fig. 2).

Also, the phosphate concentration was higher in B-20 than B-15. This is in contrary
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to a previous study on polyculture of carps in the biofloc system, where phosphate

concentration significantly declines with the increase in C/N ratio (Zhao et al., 2014)

and similar to the finding of Xu et al. (2016).

The biofloc treatment significantly influenced the performance of C. carpio

during this study. It is well known that growth performance and feed utilization are

critically influenced by the floc microbial community (Xu et al., 2016) and the nutrient

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composition. This may be due to the abundance of heterotrophic bacteria used as

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cellular protein for fish consumption and floc as a source of dietary protein, lipid,

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mineral, and vitamin (Moss et al., 2006; Mansour et al., 2017). The positive effect of

biofloc on growth performance and digestive enzyme activity of C. carpio was also
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observed by Najdegerami et al., (2016). The weight gain (%) and FCR differ

significantly between control and biofloc but not among the treatment groups. There
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is sufficient evidence of benefits associated with the use of probiotic bacteria in the

biofloc system. De Souza et al., 2012 reported the higher performance of shrimp,
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Farfantepenaeus brasiliensis in the probiotics added biofloc system. Mean final

weight, specific growth rate, total protein, and granular hemocyte count was
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significantly higher in Farfantepenaeus brasiliensis reared in the probiotic combined

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biofloc system. Similarly, the study conducted by Krummenauer et al., 2014 on the

addition of probiotics to biofloc culture system revealed the higher growth, survival
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rate and lower FCR of Litopenaeus vannamei in the probiotics-treated group.

Contrary to the above findings, in this study the addition of probiotic bacteria to

biofloc culture system shown to improve water quality but did not add additional

weight gain. Similar results were obtained by de Paiva Maia et al., 2016 using a

commercial probiotic mixture of Bacillus spp. and Lactobacillus spp. in a molasses

based zero water exchange pond culture system. Use of these probiotics with
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molasses did not influence the growth of Litopenaeus vannamei compared to the

control with only molasses. Nevertheless, we have also observed the attainment of

gonadal maturation of C. carpio at the end of the experimental trial. Gonadal maturity

might have affected the growth improvement as reproduction process alters the

energy for growth metabolism. The influence of biofloc on maturational triggering

was also pointed out in C.carpio fingerlings (Manzoor et al., 2017) and red tilapia

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rearing system (Ekasari et al., 2012).

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The stimulation effect of BFT in general results in higher immune response

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and disease resistance of animals (Ekasari et al., 2014) but the extent of stimulation

may be influenced by the probiotic bacteria addition. In our study, the biofloc clearly
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affected the non-specific immune response and disease resistance of C. carpio

fingerlings, but not in the group B20-P. The increase in respiratory activity as
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observed in the biofloc treatments is associated with the phagocytosis ability of the

leucocytes in killing the pathogens (Brieger et al. 2012). Increased respiratory burst
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activity has also been reported in rohu (Irshad et al., 2016, Kamilya et al., 2017,

Kheti et al., 2017) and genetically improved farmed tilapia (Oreochromis niloticus)
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grown in a biofloc system (Long et al., 2015). Similarly, biofloc also stimulated the
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lysozyme and myeloperoxidase activity which is considered to involve in an

enhancement of antibacterial activity. Lysozyme promotes phagocytosis and

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opsonization of both gram-positive & negative bacteria (Saurabh et al., 2008) and

myeloperoxidase participates in defense against pathogens by utilizing oxidative

radicals (Castro et al., 2008).

When considering the biofloc with probiotics-based immune stimulation a

significantly higher level of immune activity was observed in B-15 P than the B-20 P.

The increased immune response in probiotics added treatment B-15 P was

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consistent with the results of Kim et al., (2015), who used multi-probiotics in a study

with Fennerpenaeus chinesis in BFT and reported a higher level of lysozyme activity.

Also, Immunological parameters of Macrobrachium rosenbergii were significantly

improved when a mixture of Bacillus subtilis and Lactobacillus were added to the

biofloc (Miao et al., 2017). The addition of Bacillus probiotic to floc water was able to

modify the gut microbial community of Litopenaeus vannamei (Hostins et al., 2017).

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Such fact can possibly explain the positive effect of biofloc with probiotics on immune

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stimulation and disease resistance of host animal. Also, the probiotics used in this

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study also have immune stimulatory potential. Srisapoome et al. (2017) and Aly et al.

(2008b) demonstrated the elevated respiratory burst activity, and disease resistance
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in Oreochromis niloticus against Streptococcus agalactiae and A. hydrophila fed with

B. pumilus probiotics. Likewise, the enhancement of lysozyme, myeloperoxidase

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activities, and disease resistance against A. hydrophila was observed in C. carpio

fed with L. delbrueckii (Zhang et al., 2017).

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Enhancement of fish disease resistance is associated with the increased

activity of non-specific immune parameters. A bacterial challenge test was performed

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for evaluating the wellbeing of the animal. The significantly higher survival of C.
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carpio against A. hydrophila in biofloc groups B-20 and B-15 P compared to control

was due to the increased immune activity and better disease resistance; consistent
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to the previous finding by Irshad et al., (2016) and Kim et al., (2015). The stimulatory

effect is obvious due to the consumption of floc which eventually increased the host-

microbial interaction. The continuous exposure to microbes leads to the activation of

the non-specific immune response, as the cellular component of microbes such as β-

glucan, lipopolysaccharide are known to be the stimulator of lysozyme and

phagocytic activity (Paulsen et al., 2001). The lower immune response and disease
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resistance in B-20 P as compared to B-15 P observed in this experiment may be

related to the possible reasons. First, probiotic addition to higher C/N ratio also

contributed to excessive solid load and increased in the nitrogenous compound as

discussed earlier. Secondly, the prevalence of gonadal maturation was observed in

most of the fishes in this group (unpublished data) which may be also likely to affect

the non-specific immune responses as previously reported by Huo et al., (1999);

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Kortet et al., (2003) and Swain and Nayak, (2009).

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This study has demonstrated that biofloc technology application could be a

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promising approach for C. carpio farming in mid-Himalayan altitude. Moreover, the

performance of C. carpio regarding growth, non-specific immune response and

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disease resistance was enhanced in the biofloc based system. Certainly, probiotics

addition to biofloc could significantly influence the faster development of floc.

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Nevertheless, the prevalence of high solid load also requires more frequent solids

management to obtain better water quality. The growth performance showed no

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significant difference within the biofloc groups; whereas there was an enhanced non-

specific immune response observed in C/N 15 with probiotics addition. The study
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suggests the possibility to maintain a well-developed biofloc system in a reduced

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C/N ratio of 15:1 in the mid-Himalayan region. Further, the probiotics bacteria

addition to the B-15 treatment would elicit the faster development of floc, better water
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quality, and enhanced immune response. Further investigations are needed to

optimize the periodicity of probiotics addition and solid management in a biofloc

system.

Acknowledgements

The authors are thankful to Director, ICAR-DCFR for providing necessary facilities to

carry out this work.

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Table 1 Mean (± SE) and range of physiochemical parameters of water during a 60

days biofloc trial under two different C/N inputs with/ without addition of probiotic
bacteria
Treatment Temperature (°c) pH DO (mg/L)
Control Mean 20.3±0.28 7.94±0.01 7.1±0.13
min, max 17.7, 22.6 7.9,8.0 6.9, 7.2
CN-20P Mean 20.3±0.28 7.74±0.02 5.4±0.01
min, max 17.8, 22.6 7.6, 8.0 5.0, 5.6
CN-20 Mean 20.2±0.27 7.7±0.02 5.6±1.03
min, max 17.8, 22.4 7.56, 7.9 5.2, 5.9
CN-15P Mean 20.2±0.26 7.66±0.02 5.5±0.47

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min, max 17.8, 22.5 7.5, 7.9 5.2, 5.7

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CN-15 Mean 20.1±0.27 7.68±0.03 5.8±0.28
min, max 17.8, 22.5 7.0, 7.9 5.4, 6.3

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Table 2 Growth performance of C. carpio fingerlings in a 60 days biofloc trial under

SC
two different C/N inputs with/ without addition of probiotic bacteria
Treatment
Varia Contr B-20P B-20 B-15P B-15
NU
ble ol
a
IW (g) 7.693±0.035 7.764±0.32
a
8.088±0.2
ab
8.566±0.0 7.73±0.01
b a
1 6 6
b
FW 11.244±0.14 12.838±0.3 13.56±0.1 13.946±0. 12.636±0.
MA

b c c a
(g) 4 4 18 21
a
WG 46.175±2.3 65.549±2.4 67.749±2. 62.796±0. 63.385±3.
b b b b
(%) 2 45 1
a
WGD 0.059±0.002 0.084±0.00 0.091±0.0 0.089±0.0 0.081±0.0
ED

bc c c b
(g/d) 06 009 01 03
b
FCR 2.545±0.11 1.872±0.05 1.645±0.1 1.673±0.0 1.844±0.0
a a a a
7 2 8
a b
SGR 0.632±0.02 0.83±0.02 0.861±0.0 0.812±0.0 0.817±0.0
PT

b b b
(%/da 2 04 3
y)
Different letters in the same row represent significant differences at p < 0.05
CE

IW = Initial weight, FW = Final weight, WG = weight gain, WGD = weight gained per
day, FCR = Feed conversion rate, SGR = specific growth rate
AC
 ACCEPTED MANUSCRIPT

Highlights

 Biofloc technology could be a feasible alternative for high density fish rearing in mid-
Himalyan altitude.
 Exogenous probiotics addition causes faster development of suspended biomass in
biofloc system.
 Higher immune response and disease resistance of Cyprinus carpio in C/N ratio of 15:1
along with probiotic bacteria addition.

P T
RI
SC
NU
MA
ED
PT
CE
AC
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