In L. Vannamei Fed On Biofloc Grown With Different Carbon Sources (PDFDrive) PDF

IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI
FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES
BY
DASARI PAMANNA , B.F.Sc.
THESIS SUBMITTED TO
SRI VENKATESWARA VETERINARY UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
MASTER OF FISHERY SCIENCE
IN THE FACULTY OF FISHERY SCIENCE
(AQUACULTURE)
DEPARTMENT OF AQUACULTURE
COLLEGE OF FISHERY SCIENCE, MUTHUKUR - 524 344
SRI VENKATESWARA VETERINARY UNIVERSITY
TIRUPATI - 517 502, Andhra Pradesh
AUGUST, 2015
CONTENTS
Chapter
TITLE Page No.
No.
1 INTRODUCTION 1-11
2 REVIEW OF LITERATURE 13-33
3 MATERIALS AND METHODS 34-46
4 RESULTS 47-82
5 DISCUSSION 83-88
6 SUMMARY 89-91
7 REFERENCES 92-119
LIST OF TABLES
Table
Title Page No.
No.
Proximate composition of experimental feed & different carbon
1 40
sources
Weekly variation of Dissolved Oxygen (mg/l) in the tanks treated
2 49
with various carbohydrate sources as biofloculating agents
Weekly variation of Temperature (0C) in the tanks treated with
3 51
various carbohydrate sources as biofloculating agents
Weekly variation of pH in the tanks treated with various
4 53
carbohydrate sources as biofloculating agents
Weekly variation of Total Alkalinity (mg/l) in the tanks treated
5 55
Weekly variation of TAN (mg/l) in the tanks treated with various
6 57
Growth performance of L. vannamei in the tanks treated with
7 61
8 Statistical analysis for Growth Performance 62
Weight gain in L. vannamei fed on biofloc grown with different

9 64
carbon sources
10 Statistical analysis for weight gain 65
The percentage of survival of L.vannamei fed on biofloc grown

11 68
with different carbon sources
12 Statistical analysis for Survival 69
Specific Growth Rates (%) of L.vannamei fed on biofloc grown

13 72
Feed Conversion Ratio of L.vannamei fed on biofloc grown with
14 76
different carbon sources
15 Statistical analysis for FCR 77

LIST OF ILLUSTRATIONS
Figure
Title Page No.
No.
Weekly variation of Dissolved Oxygen (mg/l) in the tanks treated
1 50
Weekly variation of Temperature (0C) in the tanks treated with
2 52
Weekly variation of pH in the tanks treated with various
3 54
Weekly variation of Total Alkalinity (mg/l) in the tanks treated
4 56
Weekly variation of TAN (mg/l) in the tanks treated with various
5 58
Growth performance (g) of L. vannamei fed on biofloc grown with
6 63
Weight gain in L. vannamei fed on biofloc grown with different
7 66
carbon sources
The percentage of survival of L. vannamei fed on biofloc grown
8 70
Specific Growth Rates (%) of L. vannamei fed on biofloc grown
9 73
Feed Conversion Ratio of L. vannamei fed on biofloc grown with
10 78
LIST OF PLATES
Plate
Title Page No.
No.
1 Litopenaeus vannamei 12
Experimental carbon sources molasses, wheat flour and tapioca

2 36
flour used as biofloc agent
3 Experimental set-up in the wet-laboratory 37
3.1.Experimental animals fed with wheat flour as biofloc agent 37
3.2. Experimental animals fed with tapioca flour as biofloc agent 38
3.3. Experimental animals fed with molasses as biofloc agent 38
4 L. vannamei length measurement 45
5 L. vannamei weight measurement 45
6 Biofloc in different carbon source treatment tanks 46
7 Biofloc composition 80
8 Rotifer- Brachionus placatilis observed in biofloc 80
9 Ciliophora protozaon - Stenter roeseli observed in biofloc 81
10 Globigirina sps observed in biofloc 81
11 Verticella sps observed in biofloc 82
12 Parametium sps observed in biofloc 82

ABBREVATIONS
T Treatment
0
C Degree Centigrade
Cm Centimeter
PL Postlarvae
mg/l Milligram per liter
Ltr Litre
ANOVA Analysis Of Variance
% Percentage
Fig. Figure
G Grams
Kg Kilograms
ppm parts per million
Temp. Temperature
FCR Feed Conversion Ratio
ppt parts per thousand
DO Dissolved Oxygen
TAN Total Ammonia Nitrogen
SGR Specific Growth Rate
ADG Average Daily Weight Gain
SPF Specific Pathogen Free
SCP Single Cell Protein
ProPO Pro Phenol Oxidase

CERTIFICATE
This is to certify that the thesis entitled “IMPACT ON GROWTH AND

SURVIVAL OF LITOPENAEUS VANNAMEI FED ON BIOFLOC GROWN WITH
DIFFERENT CARBON SOURCES” Submitted in partial fulfillment of the
requirements for the award of the degree of MASTER OF FISHERY SCIENCE in
AQUACULTURE in the Faculty of Fishery Science of Sri Venkateswara Veterinary
University, Tirupati is a record of the bona fide research work carried out by
Mr. DASARI PAMANNA, ID. No. MFM/2013-002 under our guidance and
supervision. The subject of the thesis has been approved by the Student’s Advisory
Committee.
No part of the thesis has been submitted for the award of any other degree
or diploma. All assistance and help received during the course of investigations
have been duly acknowledged by the author of the thesis.
( A.CHANDRASEKHARA RAO)
Chairperson of the Advisory Committee
Thesis approved by the Student’s Advisory Committee
Chairperson: Dr. A. CHANDRASEKHARA RAO

Assistant professor
Dept. of Aquaculture
College of Fishery Science
Muthukur – 524 344
Member : Dr. D. RAVINDRA KUMAR REDDY

Professor & Head
Dept. of Aquaculture
Member : Dr. N. MADHAVAN

Associate professor & Head
Dept. of Fishery Engineering
CERTIFICATE
This is to certify that Mr. DASARI PAMANNA, I.D. No. MFM/2013-002
has satisfactorily persecuted the course of research and that the thesis entitled
“IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI
FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES”
submitted is the result of his original research work and so sufficiency high in
standard enabling its presentation to the evaluation. I also certify that the thesis
or part thereof has not been previously submitted by him for a degree or
diploma of any University.
Date : Dr. A.CHANDRASEKHARA RAO
(Major Advisor)
DECLARATION
I Mr. DASARI PAMANNA, hereby declare that the thesis entitled
“IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI
FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES”
submitted to Sri Venkateswara Veterinary University, Tirupati for the degree of MASTER
OF FISHERY SCIENCE majoring in AQUACULTURE is the result of original research
work done by me. I also declare that the materials contained in this thesis have not been
published earlier.
Date: DASARI PAMANNA)

ACKNOWLEDGEMENTS
I solicitly convey my gratitude to the authorities of Sri Vekateswara
Veterinary University, Tirupati for granting me the permission to pursue postgraduate
studies and providing me all the necessary facilities at College of Fishery Science,
Muthukur. I wish to record my sincere thanks to Dr. Manmohan Singh, I.A.S, Hon’ble
Vice Chancellor and Dr. P. Sudhakar Reddy, Registrar, of Sri Venkateswara Veterinary
University, Tirupati for encouragement without which I would not be in a position to
complete this programme.
I am grateful to Dr. T.V.Ramana, Dean, Faculty of Fishery Science, SVVU,

Tirupati for his kind co-operation and providing necessary facilities to carry out this
research.
My sincere thanks and gratitude to Dr. K.S. Krishna Prasad, Associate Dean,
College of Fishery Science, Muthukur, for providing me all the necessary facilities to
complete my research work.
I profess my heartful gratefulness and sincere regard to my esteemed guide

Dr. A. Chandrasekhara Rao, Assistant Professor, Department of Aquaculture, College of
Fishery Science, Muthukur, for his indefatigable effort, constructive criticism, erudite
counseling, instinctive attention, constant encouragement, inspiring suggestions, benign
attitude and sagacity coupled with patience in guiding me at every stage of this
investigation and preparation of this thesis.
I express my sincere gratitude to Dr. D. Ravindra Kumar Reddy, Professor

and Head, Department of Aquaculture, College of Fishery Science, Muthukur, for his
positive support, untiring effort and good wishes extended to me during my research
work.
I owe a deep sense of reverence to Dr. N. Madhavan, Associate Professor &

Head, Department of Fishery Engineering, for his valuable advice and unconditional
support, which enforced me to specially thank him forever.
My alma mater of College of fishery science, Muthukur deserves special
appreciation for the way in which it has educated, developed and gave me opportunities
to understand and practice fisheries science Dr. P. Hari Babu, Professor and University
Head, Department of Aquatic Animal Health Management Dr. K. Dhanapal, Associate
Professor, Department of Fish Processing Technology, Dr. D. Ramalingaiah, Associate
Professor, Department of Fisheries Resource Management, , Dr.O. Sudhakar, Professor
and Univ.Head, Department of Fisheries Engineering, Head of Instructional Freshwater
Fish Farm, Dr. T.Neeraja, Assistant Professor, Department of Aquatic Animal Health
Management, Mrs. Madhavi, Assistant Professor and Mrs. R.R. Anupama, Assistant
Professor, Department of Aquatic Environment Management, have been a constant source
of inspiration to me.
I mostly thankful to Dr.V.Venkateshan, senior scientist,CMFRI cochin, for

their valuable support and moral suggestions.
My sincere thanks to BMR hatchery, nellore who helped me in providing
quality shrimp seed.
I owe my obligations to the faculty member of Mrs. Jesinta, Assistant

Professor, Department of Fisheries Resource Management, Mrs. Deepthi, Department of
Fishries Extension, Miss. Sravani, Department of Fish Processing Technology, Mrs.
Triveni, Department of Fishery Statistics and Mr. Shyam Prasad, for their cordial co-
operation and constant encouragement given at various stages for the improvement of this
study.
I offer my gratitude to Mr. Sri. K. Jayaramaiah, Department of Fishery
Statistics, Mr. Obulesu, Mr. Ravi and Mr. Rajesh Teaching Assistants, for
their valuable suggestions, moral support.
Valuable assistance and co-operation provided by Dr. B.Vijaya Kumar, Asst
Professor in Library Science, Mr. Rama Raju, Shelf Assistant and Mr. P.Lakshmi
narayana, Office subordinate CFSC is highly acknowledged.
I set forth my sincere thanks to Mr. Narasa Raju, Mr. Nageswararao, Mr.
Praveen, Mr.Pavan for their outstanding help in every aspects of my Research work
despite their busy schedule.
I express my thanks to Mr. Murali (Ele.), Mr. Krishnamohan, Mohan and
Ravi for their help during the period of my research.
I am ever indebted to my friends Nehru, Venky, Adnan, Aditya, Jagan,
Santosh, Goutham, Ramesh, Joshna, Jhansi, Anusha, Pravalika, for their moral support.
I am at loss of words to thank my loving juniors Raveendra, Suresh, Bobby,
Tandekar, Bhavitha, Ranjith, Pragna, Kumuda, Anusha, Phani, Dharmakar, Anil,
Swaroop, Srinivasulu, Panga Vishnuvardhanraj, Vivek, Bolt, Mohan Krishna, Mohith,
Hementh, Lokesh, Rajachandra reddy, Sandeep, Shashi, Harika, Gowri, Chinnari,
Lavanya, stephen for their affection, inspiration, support, words of encouragement during
my Research work and for a memorable hostel stay.
I fell short of words to express my wholehearted gratitude to my parents
D.Abanna and Vimalamma, my Brother Nagarathnam and Sujatha, Suresh and Lakshmi,
Prabhu and Anitha and my sister Prabhavathamma whose selfless, unfeigned love,
empathy, sky high inspirations and for being the guiding force of my life propelling me
towards brighter horizons of happiness.
I express my heartfelt devotion and foremost indebtness to my wife C.
Madhukumari whose encouragement, inspiration, sacrifice and love made me what I am
today.
I sincerely acknowledge Sri Venkateswara Veterinary University, Tirupati for
providing me fellowship, without which I would not be in a position to complete this
programme.
Lastly, I bow my head and bend my knees before the Almighty, for blessings, grace
and compassion showered on me.
Date:
Place: (DASARI PAMANNA )
Dedicated to my Parents
&
my A 'lma M 'ater
Abstract
Author : D.PAMANNA
Title of the thesis : “Impact on growth and survival of Litopenaeus vannamei
fed on biofloc grown with different carbon

sources’’
Submitted for the
award of degree : Master of Fishery Science in
Aquaculture Faculty : Faculty of Fishery Science
Department : Department of Aquaculture
Major advisor : Dr. A. CHANDRASEKHARA RAO
University : Sri Venkateswara Veterinary University
Year of submission : 2015
The present study : “Impact of growth and survival of Litopenaeus vannamei
fed on biofloc grown with different carbon sources’’ was conducted in the Wet
Laboratory of the Department of Aquaculture, College of Fishery Science, Sri
Venkateswara Veterinary University, Muthukur
Impact of L. vannamei rearing with biofloc by using different carbohydrate
materials ( wheat flour, tapioca flour and molasses) as a carbon source to boost the
production by improving the conversion of nutrients into harvestable products while
maintaining good water quality. The carbohydrate sources for this study were
selected based on easy availability and economic viability. In the present study it has
been evaluated to identify the efficient carbon source to develop the quality biofloc
which play significant role in growth and survival of L. vannamei. Experiments were
conducted with three biofloc treatments and one control in triplicate in 1000 ltr
capacity indoor tanks and water level filled up to 600 ltr. Different carbohydrate
sources addition for biofloc rearing played significant impact on water quality of
shrimp rearing tanks. Water quality parameters were in the acceptable level for L.
vannamei cultured in all the biofloc treated tanks. Enhanced shrimp growth was
noticed in biofloc treatment tanks. There was a significant difference in the final
average body weight of (15.92±0.07g) in the wheat flour treatment than those
treatments and control group of shrimp. The FCR differs significantly between
biofloc treatment group and control (P<0.05). FCR lowest (0.5±0.07) was recorded in
wheat flour as carbohydrate source biofloc treatment. Highest SGR (4.59) was
observed in the wheat flour treatment than those treatments and control. Wheat flour
utilization as carbohydrate source to biofloc development for rearing of L. vannamei
was proved to be the best option among all treatments. The addition of carbohydrate
for biofloc development affected the survival of L. vannamei. The highest survival of
(73.36%) was recorded for wheat flour used as carbohydrate source in biofloc
treatments. All the carbohydrate sources ( wheat flour, tapioca flour and molasses)
utilized for biofloc treatments indicated highest growth and survival than control
treatment.
1
1. Introduction
World Aquaculture is growing with an annual rate of 8.9–9.1% since the 1970s.
This high growth rate is needed to solve the problem of shortage in protein food supplies,
which is particularly situated in the developing countries (Gutierrez- Wing and Malone,
2006; Matos et al., 2006 and Subasinghe, 2005). The global shrimp market has expanded
from less than $1 billion to $12 billion (US) from 2000 to 2013 (FAO, 2014). To meet this
growing demand, the shrimp industry is shifting from extensive rearing systems to more
intensive rearing systems.
Aquaculture is a critical industry for supporting the world's demand of seafood protein and
will play an even more important role as the global population continues to increase
(Jackson, 2007). In order for aquaculture to be completely successful, the industry need to
develop technology that will increase the economic and environmental sustainability.
In conventional and semi-intensive ponds, natural food can supply up to 70% of the
nutritional requirements of shrimp, benthic organisms and zooplankton constituting the
essential components of this food source (Martinez-Cordova et al., 2003). In biofloc
culture systems, the “natural food” consists of diatoms, macroalgae, food and faecal
remnants, exoskeletons, bacteria and invertebrates. These food items form an aggregate of
living and dead organic matter suspended in water (Avnimelech, 1999, 2006; Burford et
al., 2003; Hargreaves, 2006; McIntosh, 2000a; Michaud et al., 2006; Taw, 2010;
Wasielesky et al., 2006). Hence, the availability of this natural food allows the amount of
protein in the diet and its cost can be reduced.
2
The prime goal of aquaculture expansion in to produce more aquaculture products without
significantly increasing the usage of the basic natural resources of water and land
(Avnimelech, 2009). The second goal is to develop sustainable aquaculture systems that
will not damage the environment (Naylor et al., 2000). The third goal is to build up
systems that provides an equitable cost/benefit ratio to support economic and social
sustainability (Avnimelech, 2009). All these three prerequisites for sustainable aquaculture
development can be met by using biofloc technology.
The environmental friendly aquaculture system called “Biofloc Technology (BFT)” is
considered as an efficient alternative to the present super intensive aquaculture system
since nutrients could be continuously recycled and reused. The sustainable approach of
biofloc technology system is based on growth of beneficial microorganism in the culture
medium, benefited by the minimum or zero water exchange. These microorganisms
(biofloc) has two major roles: (i) maintenance of water quality, by the uptake of nitrogen
compounds generating “in situ” microbial protein; and (ii) nutrition, increasing culture
feasibility by reducing feed conversion ratio and a decrease of feed costs.
Increasing concerns in recent years about the environmental impact of marine
shrimp farms, associated with the incidence of diseases, has led to the development of
production systems with little or no water exchange (Hopkins et al., 1995). Biofloc
technology is a new concept in aquaculture, where manipulation of the microbial
community is carried out under controlled conditions within the culture system with the
raised animals (De Schryver et al., 2008). The biofloc system facilitates the production of
aquatic animals at high stocking densities in a sustainable and bio-secure fashion
(McAbee et al., 2003; McNeil, 2000; Vinatea et
3
al., 2009). In some cases the protein content of feed can be reduced due to partial protein
supplementation by the microbial community (Burford et al., 2004; Wasielesky et al.,
2006). One of the advantage of operating a bacterial-driven system versus a conventional
phytoplankton dominated pond is that microbial production is limited by the availability of
organic matter or substrate rather than light, giving rise to the potential for this system in
indoor conditions (Azim et al., 2008).
Biofloc technology is based upon the production of shrimp with zero or minimal
water exchange, resulting in the accumulation of organic substrates and subsequent
development of dense microbial population, mostly aggregated in bioflocs (Avnimelech,
2012). These microorganisms not only remove excess nutrients, but also have been
implicated in nutritional provision for the cultured species, including shrimp and tilapia
(Burford et al., 2003; Hari et al., 2004; Azim and Little, 2008). Several researchers
suggested that the biofloc provide sources of lipids, minerals and vitamins to cultured
animal (Moss et al., 2001; Thompson et al., 2002; Moss et al., 2006; Arnold et al., 2009).
Using the N15 isotope tagging method, Avnimelech and Kochba (2009) showed that
bacterial protein in bioflocs is taken by the cultured animals. All of these results suggest
that animals cultured in BFT can consume microorganisms and other components of
bioflocs and use it for their nutrition or other purposes. It is well reported that the cell wall
of the microorganisms such as bacteria and fungi consists of lipopolysaccharides (LPS),
peptidoglycans (PG) and β -1, 3-glucans (BG), activating the nonspecific immune system
in fish and crustaceans (Johansson and Sӧderhäll, 1985, 1989; Sӧderhäll and Cerenius,
1998) and enhancing the resistance against bacterial and viral infections in penaeid shrimp
(Itami et al., 1994; Song et al., 1997; Chang et al., 1999).
4
The application of biofloc technology (BFT) in shrimp aquaculture has gained great
attention in recent years because it provides a practical solution for effective control of
water quality with negligible water exchange and improves shrimp growth performance,
thus achieving efficient and healthy culture of shrimp (Avnimelech, 2012; Crab et al.,
2012; De Schryver et al., 2008; Stokstad, 2010; Xu and Pan, 2013). In heterotrophic
biofloc-based shrimp culture systems, the driving force is dense populations of active
heterotrophic bacteria which can be promoted by increasing the C/N ratio of feed input and
assimilate the waste nitrogen from culture water resulting in the production of new
microbial biomass (cellular proteins) (Avnimelech, 2006; Crab et al., 2007; Ebeling et al.,
2006). As the microbial communities develop, bioflocs are formed from heterogeneous
aggregates of microorganisms and organic particles (De Schryver et al., 2008; Hargreaves,
2006). As a supplemental food source available for cultured shrimp, the biofloc can be
consumed and provide a significant fraction of protein demand (Ballester et al., 2010;
Burford et al., 2004; Crab et al., 2010a; Wasielesky et al., 2006; Xu et al. 2012).
Biofloc technology is not only an adequate approach in maintaining water quality in the
aquaculture system but it also generates biomass that can contribute as a protein source for
the cultured organisms in situ (Avnimelech, 2009; Crab et al., 2010a) or can be harvested
for use as a feed ingredient (Kuhn et al., 2009, 2010). Hence, the use of biofloc as a food
source implies a decrease in the requirement of formulated feed protein and also improve
nitrogen utilization efficiency by the cultured animals (Xu et al., 2012; Avnimelech, 2006).
In order to evaluate the use of biofloc as a food source, general criteria of aquaculture
feed can therefore be
5
applied, i.e., the size of particles, attractiveness, palatability, digestibility and nutritional
content (Tacon, 1987b).
Opportunities for economic and environmental improvements permeate into many facets of
the industry. For example, if the industry successfully implements cheaper alternative
ingredients to fishmeal they could effectively reduce the costs of feed while reducing their
impact on natural fisheries. Feed costs can account for 50% of operational expense (Van
Wyk et al., 1999). Meanwhile, wild fisheries are overexploited and current trends are not
sustainable (Tacon et al., 2006; Naylor et al., 2009). A success story illustrating this
concept is the complete replacement of fishmeal with soy meal for numerous species in
aquaculture production. Over the period from January 2008 to May 2009, the cost of
fishmeal was nearly 2.5 times greater than soy meal (FAO, 2009). Thus it is important to
determine if alternative ingredients derived from biologically treating fish waste, bioflocs
(microbial flocs), could be a suitable replacement ingredient in marine shrimp diets. If
implemented successfully, this option would offer a sustainable option to replace fishmeal.
In fact, this option could potentially offer a sustainable alternative to soy meal, because
producing this ingredient would have additional benefits of treating a waste stream.
In order for aquaculture to be completely successful, the industry will need to develop a
technology that will increase economic and environmental sustainability (Kuhn et al.,
2010). This technology implements cheaper alternative ingredient to fishmeal and this will
effectively reduce the costs of feed. Thus, it is important to determine if alternative
ingredients derived from biologically treating fish waste, bioflocs (microbial flocs), could
be a suitable replacement ingredient in marine shrimp diets. If implemented successfully,
this option would offer a sustainable option to fishmeal.
6
A number of nutritional studies have been conducted on the use of various
alternative ingredients in shrimp farming, particularly with the aim of reducing or replacing
the use of fishmeal in biofloc culture systems (Amaya et al., 2007; Bauer et al., 2012;
Browdy et al., 2006; Hart et al., 2004; Kuhn et al., 2010; Ray et al., 2010; Scopel et al.,
2011). However, only a few of these studies have evaluated the effect of dietary protein
levels on L. vannamei in biofloc systems (Cuzon et al., 2004; McIntosh et al., 2001).
The availability of natural food will be influenced by the production system, which
may interfere with the dependence on and availability of nutrients, such as protein, from
inert diets.
According to Kuhn et al. (2010), initial cost estimates for biofloc production is
approximately $400 to $1000 per ton of dry ingredients which is projected to be less than
the ingredients such as fishmeal and within the range for soybean meal.
The biofloc technology system (BFT) is characterized by rich microbial communities that
form flocs in the water column. Growing shrimp under these conditions has been found to
have several advantages compared with conventional aquaculture practices. These systems
are characterized by high yields, small footprints, and reduced environmental impacts
(Browdy et al., 2001; Neal et al., 2010). The use of limited water exchange minimizes the
introduction of pathogens with the incoming water. Moreover, increasing awareness of
biosecurity reduces crop losses from disease outbreaks.
By adding a carbon source to the culture medium in limited-discharge systems
(i.e., by changing the C : N ratio), it is possible to obtain a significant enhancement of
bacterial growth and of the fixation of toxic nitrogen metabolite species (Chamberlain et
al., 2001; Ebeling et al., 2006; Hari et al., 2006;
7
Avnimelech, 2009; Crab et al., 2010a). In addition to the improvement of water quality,
increase in bacterial biomass, providing supplemental feed, improved shrimp survival,
growth and to reduce the releases of nutrient rich water into receiving streams (Timmons et
al., 2002; Wasielesky et al., 2006; De Schryver et al., 2008; Avnimelech, 2009;
Krummenauer et al., 2011).
Biofloc technology has primordial advantage of minimizing the release of water into
rivers, lakes and estuaries containing escaped animals, nutrients, organic matter and
pathogens and also, surrounding areas are benefitted by the “vertically growth” in terms of
productivity, preventing coastal or inland area destruction, induced eutrophication and
natural resources losses. Drained water from ponds and tanks often contains relatively high
concentrations of nitrogen and phosphorous, limiting nutrients that induce algae growth,
which may cause severe eutrophication and further anaerobic conditions in natural water
bodies. In BFT, minimum water discharge and reuse of water prevent environment
degradation and convert such system in a real “environmentally friendly system” with a
“green” approach. Minimum water exchange maintain the heat and fluctuation of
temperature is prevented (Crab et al., 2009), allowing growth of tropical species in cold
areas.
However, these in situ based techniques need additional oxygen demands for microbial
respiration, in addition to the oxygen demand of shrimp (Burford et al., 2003; Tacon et al.,
2002). This addition of oxygen requires additional aerators, which increases the aeration
expenses in shrimp farms compared to conventional shrimp culture systems (Tacon et al.,
2002). Moreover, increased production of microbial floc particle is also a matter of
challenge if production exceeds consumption by shrimp.
8
Aquaculture produces large quantities of wastes that contain solids (e.g. feces and uneaten
feed) and nutrients (e.g. nitrogen and phosphorus) which can be detrimental to the
environment, if managed improperly. These solids and nutrients originate from uneaten
feed, feces, and animal urea/ammonia (Maillard et al., 2005 and Sharrer et al., 2007), if
released directly to the environment, these solids and nutrients can be pollutants resulting
in environmental issues such as eutrophication (Wetzel, 2001) or could be directly toxic to
aquatic fauna (Timmons et al., 2002 and Boardman et al., 2004). The most common
method for dealing with this pollution has been the use of continuous replacement of the
pond water with new clean water from the water source (Gutierrez-Wing and Malone,
2006). For instance, Penaeid shrimp require about 20 m3 fresh water per kg shrimp
produced (Wang, 2003). For an average farm with a production of 1000 kg shrimp ha −1
yr−1 and total pond surface of 5 ha, this corresponds with a water use of ca. 270 m3 day−1. A
relatively new alternative to previous approaches is the bioflocs technology (BFT)
aquaculture (Avnimelech, 2006). In these systems, a co-culture of heterotrophic bacteria
and algae is grown in flocs under controlled conditions within the culture pond. The system
is based on the knowledge of conventional domestic wastewater treatment systems and is
applied in aquaculture environments. Microbial biomass is grown on fish excreta resulting
in a removal of these unwanted components from the water. The major driving force is the
intensive growth of heterotrophic bacteria. They consume organic carbon; 1.0 g of
carbohydrate-C yields about 0.4 g of bacterial cell dry weight-C; and depending on the
bacterial C/N-ratio thereby immobilize mineral nitrogen. As such, Avnimelech (1999),
calculated a carbohydrate need of 20 g to immobilize 1.0 g of N, based on a microbial C/N-
ratio of 4 and a 50% C in dry carbohydrate.
9
The fast development of shrimp culture increasingly requires strategies to improve
production systems, enhance bio-security and reduce environmental impacts (Avella et al.,
2010; Qi et al., 2009). The rapid expansion of penaeid shrimp culture is threatened by
diseases caused by bacteria of the genus Vibrio, which affect shrimp survival and growth
(Aguirre-Guzmań et al., 2001). These opportunistic microorganisms are part of the flora
of penaeid shrimp and may cause illnesses under un favourable environmental conditions.
Specific effects such as mortality, tissue damage or necrosis and growth retardation are
reported. Vibriosis has also been implicated as the cause of high mortalities in juvenile
penaeid shrimp worldwide (Lightner and Redman 1998; Castex Lemaire et al., 2010). The
abuse of antimicrobial drugs, pesticides and disinfectants in aquaculture has caused the
evolution of resistant strains of bacteria and brought concern to the society (Esiobu et al.,
2002). Thus, defining alternative strategies to support aquaculture productivity are
extremely necessary (Avella et al., 2010).
Pacific white shrimp, L. vannamei, is one of the most important farmed species in the
world. However, farming activities of this species have been largely affected by diseases,
mostly diseases such as the White Spot Syndrome Virus (WSSV) and Early Mortality
Syndrome (EMS). Producers and researchers are constantly looking for methods to reduce
massive shrimp losses due to disease outbreaks. Growing shrimp using biofloc technology
(BFT) was proposed as a tool to reduce water exchange and minimize the introduction of
viral pathogen through incoming water. In addition, observations on the effects of BFT on
reducing viral disease outbreaks were reported (Avnimelech, 2012).
Disease remains a limiting factor for the aquaculture industry. With respect to the shrimp
culture industry, disease outbreaks have been the primary cause of
10
production loss during the last two decades (FAO, 2012). Disease outbreaks not only result
from the mere presence of a pathogen in the system, but a compromised health status of the
cultured animals in combination with suboptimal environmental conditions are also factors
facilitating disease outbreaks (Liu and Chen, 2004; De Schryver et al., 2012). Therefore,
disease prevention and control should not only focus on implementing biosecurity
measures, but also must be performed as an integral approach involving, adequate
nutrition, enhancing the immunity of the cultured animals and maintaining a good water
quality.
Physiological functions such as immune and antioxidant systems are essential for shrimp in
maintaining their health and guaranteeing satisfactory growth performance, especially
under the condition of environmental stresses (e.g. pollutions and pathogens) (Bachère,
2000; Castex et al., 2010) and thereby ensure healthy culture of shrimp. Otherwise,
decrease in immune and antioxidant defense functions of cultured shrimp could easily lead
to the outbreak of diseases and cause a great economic loss (Bachère, 2000). The supply of
feed nutrition, mainly protein, is required for the normal physiological metabolism and
growth of cultured shrimp (Kureshy and Davis, 2002). This implies that the shrimp should
not only be fed with adequate dietary protein for superior growth but also be maintained in
a healthy nutritional and physiological state. The ingested proteins are not only involved in
the synthesis of hormones and enzymes but some are components of the immune and
antioxidant systems.
A wide range of microorganisms and their cell components or metabolic products
have been studied and applied as probiotics and/or immunostimulants to improve innate
immunity and/or antioxidant capacity of shrimp, thereby enhancing their resistance against
pathogens (Ninawe and Selvin, 2009; Smith et al., 2003;
11
Vazquez et al., 2009). Therefore, to improve rearing techniques for L. vannamei, this study
is undertaken to evaluate the effect of biofloc on growth and survival of shrimp.
The objectives of present study are:
1. To collect the selective carbon sources like wheat flour, tapioca flour and
molasses which will be utilized for growing biofloc.
2. To study the effect of biofloc on growth and survival of L. vannamei.
3. To find out the effect on growth and survival of L. vannamei fed on biofloc
grown with different carbon sources.
13
Plate 1:
Litopenaeus vannamei
14
2. Review of Literature
Biofloc technology is a technique of enhancing water quality through the addition of
extra carbon to the aquaculture system, through an external carbon source or
elevated carbon content of the feed (Hargreaves, 2006).
Use of Carbohydrate Sources as a Management
tool for the control of ammonia in Biofloc System:
The carbon sources applied in BFT are often by-products derived from human
and/or animal food industry, preferentially local available. Cheap sources of
carbohydrates such as molasses, glycerol and plant meals (i.e. wheat, corn, tapioca,
etc) will be applied before fry/post-larvae stocking and during grow-out phase,
aiming to maintain a high C:N ratio (~15-20:1) and to control N compounds peaks.
Organic carbon sources such as wheat flour(Mahanand et al.,2012), tapioca flour
(Hari et al., 2004; Rajkumar et al., 2015), corn flour (Asaduzzaman et al., 2010),
molasses (Samocha et el., 2007). Also, a mix of plant meals can be pelletized (“green-
pellet”) and applied into ponds (Taw, 2010) or low protein diets containing high C:N
ratio can also be carried out (Avnimelech, 2012; Azim and Little, 2008). There are
many considerations for its selection such as costs, local availability, biodegradability
and efficiency of bacteria assimilation.
Organic matter that breaks down easily and quickly is the best. Heterotrophic bacteria
in biofloc systems can act on simple organic matter rapidly, within minutes to hours.
Simple carbohydrates such as sugar (sucrose or dextrose) or starch will have the
quickest effect. The best carbon source to add during system start-up, when the most
rapid response is needed, is simple sugar.
15
To promote exclusive control of ammonia concentration by the heterotrophic
pathway, carbohydrate additions must be made in accordance with feeding rate.
More carbohydrate is needed at the higher protein level. It is clear that relatively
large quantities of carbohydrate must be added to control ammonia concentration
this way. Less carbohydrate can be added if other ammonia removal pathways are
operating simultaneously in a biofloc system (Hargreaves, 2013).
Biofloc role in the enhancement of growth of L. vannamei :
Recently, manipulation of carbon nitrogen ratio (C:N ratio) for development
of biofloc has shown promise in aquaculture (Anand et al., 2013a; Avnimelech, 1999).
The C:N ratio is manipulated by supplementation of external carbon source or
elevated carbon level in the feed (Ballester et al., 2010; Crab et al., 2012; McIntosh,
2000b). At high C:N ratio, heterotrophic bacteria immobilize the ammonium ion for
production of microbial protein and maintain inorganic nitrogen level within the
limit (Avnimelech, 1999). Biofloc enhances the growth performance of P. monodon
(Anand et al., 2013a; Arnold et al., 2009; Hari et al., 2006), L. vannamei (Wasielesky
et al., 2006; Xu and Pan, 2012), F. paulensis (Ballester et al., 2010) and
M. japonicus (Zhao et al., 2012).
Apart from being a source of quality proteins, bioflocs are rich source of growth
promoters and bioactive compounds (Ju et al., 2008a) which enhance digestive
enzymes (Xu and Pan, 2012) and health status of the cultured shrimps (Singh et al.,
2005). In general, most research makes use of in situ developed microbial floc for
growth performance of shrimp (Hari et al., 2006; Xu and Pan, 2012). It has been
demonstrated that dietary inclusion of Biofloc enhanced the growth performance of L.
vannamei (Bauer et al., 2012; Ju et al., 2008b; Kuhn et al., 2010).
16
Beneficial role of in situ based biofloc system in penaeid shrimp culture are
well documented (Hari et al., 2006; Xu and Pan, 2012). It has been reported that use
of biofloc as a dietary ingredient in shrimp diet enhances the growth rate of L.
vannamei (Kuhn et al., 2009, 2010).
Wheat flour was used as carbohydrate source for its easy availability and
production of good quality floc (Azim and Little, 2008; Ballester et al., 2010).
The major microbial community developed in the biofloc belonged to
Vibrionaceae, Bacillus sp., and Lactobacillus sp. with majority as gram negative
bacteria. Aggregates of particulate organic matter, zooplankton communities like
ciliates, rotifers and a small amount of autotrophic microalgae were also noticed.
Similarly, Ballester et al. (2010) reported that biofloc is composed of attached
heterotrophic bacteria, filamentous cyanobacteria, dinoflagellates, ciliates, flagellates
and rotifers. Various factors like salinity, light and type of culture system affects the
microbial composition of floc.
Nutritional composition of biofloc varies with type of carbohydrate source,
microbial community structure, culture condition etc. Inclusion of biofloc as a dietary
ingredient in shrimp diet found to improve the growth performance of L. vannamei (Ju
et al., 2008b; Kuhn et al., 2009, 2010). Dietary supplementation of biofloc at 4 and 8%
levels significantly enhanced the growth, PER and reduced the FCR in tiger shrimp. It
has been documented that bioflocs are the rich source of many bioactive compounds
such as carotenoids, chlorophylls, phytosterols, bromophenols, amino sugars (Ju et al.,
2008a) and anti-bacterial compounds (Crab et al., 2010a). This suggests that microbial
components, unknown growth factors or probiotic microorganisms like Bacillus,
Lactobacillus present in the biofloc might
17
have resulted in significantly higher growth rate and better FCR in shrimp fed with
biofloc incorporated diet.
In biofloc based system (Xu and Pan, 2012) reported significantly higher digestive
enzyme activities and better growth performance in L. vannamei. Similarly, enhanced
level of digestive enzymes activity has been reported in fish and shrimp fed with
probiotics, microalgae and periphyton supplemented diets (Lara-Flores et al., 2003;
Ziaei-Nejad et al., 2006; Anand et al., 2013b).
The ammonia nitrogen was found as the preferred source of inorganic nitrogen
for phytoplankton in intensive biofloc shrimp culture systems as evidenced by Holl et
al. (2011).
The use of carbon sources in intensive systems promotes succession and dominance of
bacteria over microalgae (González-Félix et al., 2007; Ju et al., 2008a).
It has been suggested that natural productivity in zero-exchange shrimp
production systems provide supplemental food resources, reducing feed costs and
improving shrimp growth rate (Otoshi et al., 2011; Wasielesky et al., 2006). The ability
of Pacific white shrimp to utilize natural productivity and its effect for enhancing
shrimp growth is well documented (Burford et al., 2004; Decamp et al., 2003; Otoshi et
al., 2011, 2001; Wasielesky et al., 2006). Ju et al. (2009) suggest that microalgae in the
microbial floc may play a key role in improving shrimp growth rates.
One of the recent study (Ekasari et al., 2014) showed that according to the
essential amino acid composition, biofloc can be considered a good quality protein
source. Xu et al. (2012) suggested that the improvement of protein assimilation by
animals reared in BFT systems is also related to the increase in digestive proteinase
18
activity in the intestinal tract as a result of the contribution of both exogenous
digestive enzymes by the microbes in the biofloc and the endogenous digestive
enzymes production as stimulated by the biofloc. The enhancement of protein and
lipid assimilation in the biofloc treatments clearly showed a positive contribution of
biofloc biomass generated from nutrient waste as a food source for the cultivated
animal. This in turn can result in a lower feed conversion ratio in the biofloc systems
(Zhao et al., 2012; Xu et al., 2012; Gao et al., 2012; Megahed et al., 2010).
Biofloc technology could be combined with polyculture ponds, further
enhancing the water quality, natural food availability, dietary preference, growth and
production in an intensive set-up (Rahman et al., 2008).
The differences in growth when the biofloc was included were evident. Survival rates
did not vary (P>0.05) among dietary treatments; however, shrimp growth was
significantly improved (P<0.05) for shrimp fed microbial flocs. Even though
numerous studies have reported enhanced survival, health, and growth rates of
shrimp raised in ponds with high activity of algae, microbial flocs, and other natural
biota (Avnimelech, 1999; Moss et al., 2000; Moss et al., 2001; Tacon et al., 2002;
Burford et al., 2004; Cuzon et al., 2004; Izquierdo et al., 2006 and Wasielesky et al.,
2006).
Growth of shrimp as well as other aquatic organisms is mostly affected by
water quality, culture systems (Williams et al., 1996 and Tacon et al., 2002), nutrition
(Chen et al., 2006) and health condition (Rengpipat et al., 1998; Rodriguez and Le
Moullac, 2000 and Argue et al., 2002).
It is not known exactly how microbial flocs enhance growth, Izquierdo et al.
(2006) suggested lipid contributions of microbial flocs are important. It is also
19
speculated that microbial flocs are probiotics (Bairagi et al., 2002, 2004 and Kesarcodi-
Watson et al., 2008).
Biofloc role in the Water quality Management of L.

vannamei:
Biofloc technology (BFT) has been studied at several occasions and contributes
to the maintenance of good water quality in the system and to the nutrition of the
cultured animals (Avnimelech, 2009). The basic principle of the biofloc system is to
recycle waste nutrients, in particular nitrogen, into microbial biomass that can be
used in situ by the cultured animals or be harvested and processed into feed
ingredients (De Schryver et al., 2008; Avnimelech, 2012; Crab et al., 2010a; Kuhn et
al., 2009; Hari et al., 2004). Heterotrophic microbial aggregates are stimulated to
grow by steering the C/N ratio in the water through the modification of the
carbohydrate content in the feed or by the addition of an external carbon source
(Avnimelech, 2009) so that the bacteria can assimilate the waste ammonia for new
biomass production. Biofloc systems have been shown not only to maintain ammonia
below toxic levels and to improve the feed nutrient utilization efficiency of the
cultured animals (Avnimelech, 2009; Hari et al., 2004; Zhao et al., 2012) but also to
provide extra nutrients ( Xu et al., 2012) and exogenous digestive enzymes (Xu and
Pan, 2012). Biofloc application can also lead to increased growth, survival and
reproductive performance of the cultured animals (Ekasari et al., 2013; Emerenciano
et al., 2012).
It has been demonstrated that the formation and development of bioflocs in
the culture water are most likely linked with the direct assimilation of dissolved
organic and inorganic matters from feed residues and shrimp excretions by
heterotrophic bacteria (Avnimelech, 1999; Schneider et al., 2005; Ebeling et al., 2006),
and thereby levels of TAN and NO2 - N can be controlled within

20
recommended ranges for shrimp culture (Samocha et al., 2007; Arnold et al., 2009;
Ballester et al., 2010; Xu et al., 2012a). In other words, organic residues and excreted
nitrogen are continually converted into microbial biomass rather than accumulating as
toxic ammonia and nitrite in the system. Meanwhile, the moderate accumulation of
NO3 -N concentration, which occurred in all the four treatments, indicates that
nitrifying bacteria were also present in the bioflocs (Ebeling et al., 2006; Xu et al.,
2012a). In fact, both heterotrophic assimilation and autotrophic nitrification processes
occurred in the tank systems and were responsible for inorganic nitrogen control. In
general, whether using high or low protein feed, the addition of appropriate amounts
of sucrose can be effective in promoting the development of bioflocs and
simultaneously controlling inorganic nitrogen.
The biofloc technology has been widely used in shrimp culture for reducing the
accumulation of toxic metabolites like ammonia by its conversion to heterotrophic
bacterial biomass, especially in static systems. The modified static green water system
of seed production of Macrobrachium rosenbergii faces the problem of elevated levels
of metabolites like ammonia and nitrite. Saritha, 2009; Kurup and Saritha, 2010;
Saritha and Kurup (2011) made an attempt to evaluate the effectiveness of biofloc
technology in larval rearing of the giant freshwater prawn where in the quantity of
carbohydrate addition has been optimised to assimilate the toxic metabolites generated
and consequently converting them into bacterial floccules.
Avnimelech (2009) identifies four routes for controlling inorganic nitrogen
accumulation: water exchange, the control of algae, nitrification, and nitrogen control
using a bacterial biofloc. Several authors (Ebeling et al., 2006; Samocha et al., 2007;
Avnimelech, 2009) documented the successful removal of ammonia in
21
limited-exchange culture systems via adjustment of the carbon : nitrogen ratio to 15:1
to favour the assimilation of the ammonia and the production of microbial biomass.
Cohen et al. (2005) reported a gradual increase in the ammonia level, followed by a
similar increase in nitrite, in a limited water-exchange system. These authors stated
that the nitrifying bacteria required 5–7 week to develop in the culture medium before
a significant reduction in the ammonia levels occurred. The level of TAN in the control
treatment (0%) followed the normal route usually observed in BFT systems.
The biofloc forms in pond water as the flocculent aggregates organic material,
nitrogen fixing bacteria, and algae in suspension, which serve as food for cultivated
fish or crustaceans and promotes direct use of toxic metabolites by degrading the
activity of the bacteria and algae (Avnimelech, 2007). The biofloc feed results from
adding carbon sources to regulate the ratio of C:N that naturally varies between 15:1
and 20:1 (Asaduzzaman et al., 2008). The presence of bacteria in bioflocs has the
advantage of decreasing concentrations of ammonium in the water, which, in turn,
significantly improves the quality of the water for cultivation (Avnimelech and
Lacher, 1999; Crab et al., 2007; Schryver et al., 2008). Biofloc technology has been
successfully tested for shrimp (Burford et al., 2004) and to a lesser extent tilapia
(Crab et al., 2009). Many aquaculture species can be grown efficiently in biofloc,
including prawns, which are a detritivorous, opportunistic species that feeds on
bacteria, fungi, and decomposing material (Milstein et al., 2001; Serfling, 2006).
In general, in tanks where the biofloc technology is applied, benefits will be
higher than in traditional production; however, it is important to maintain control of
the concentration of biofloc (algae, bacteria, and zooplankton within the floc)
22
because it can lead to anoxia. Wasielesky et al. (2006) show that biofloc culture
increases turbidity, as did Beveridge et al. (1991), Brune et al. (2003), Hargreaves
(2006) and Rakocy (1989) describes a simple method to maintain the concentration of
biofloccules and eliminate solids that cause turbidity in the culture by using conical-
shaped settling tanks in the recirculation systems.
To control sediment and suspended matter, maintaining the concentration of
biofloc requires temperature limits (25.9 ± 2.19 °C). Lowering temperature regulates
the metabolism and growth of microalgae and the composition of their biomass. The
optimal temperature range for the growth of most algae is 18–22 °C (FAO, 2008).
Avnimelech (2012) pointed out different effects of simple versus more complex
carbohydrates applied as the carbon source in biofloc-based ponds. Simple sugars,
such as sucrose, result in a faster ammonia removal, while more complex
carbohydrates require more time for decomposition into simple sugars, thereby
resulting in slower ammonia removal.
The application of biofloc technology (BFT) in zero-exchange shrimp culture
systems has gained popularity because it offers a practical solution to maintain water
quality and recycle feed nutrients simultaneously (Avnimelech, 2006, 2008; Crab et
al., 2007; De Schryver et al., 2008). In such systems, inorganic nitrogen control is
based on the promotion of dense populations of active heterotrophic microorganisms
that can assimilate the nitrogen from the water column resulting in the production of
new microbial biomass (cellular proteins) (Avnimelech, 2006; Crab et al., 2007). As
these microbial communities develop, bioflocs (microbial flocs) are formed from
heterogeneous aggregates of microorganisms and organic particles (Hargreaves
2006; De Schryver et al. 2008). These bioflocs can be consumed as a supplemental
food source by the shrimp, creating a nutrient recycling process within the systems
23
and subsequently increasing feed utilization efficiency (Burford et al., 2004; Schneider
et al., 2005; Hargreaves, 2006; Wasielesky et al., 2006; Xu and Pan, 2012; Xu et al.,
2012). More importantly, several studies noted that the application of BFT could
improve growth performance of cultured shrimp, primarily attributed to the
promotion of bioflocs (Wasielesky et al., 2006; Arnold et al., 2009; Megahed, 2010; Xu
and Pan, 2012; Xu et al., 2012). It was thought that the biofloc, apart from providing a
portion of nutritional demand, can also exert a positive effect on digestive enzyme
activity of shrimp and produce some microbial extracellular enzymes, both of which
could facilitate feed digestion and utilization (Xu and Pan, 2012).
Biofloc technology is a technique of enhancing water quality through the addition of
extra carbon to the aquaculture system, through an external carbon source or
elevated carbon content of the feed. This promoted nitrogen uptake by bacterial
growth decreases the ammonium concentration more rapidly than nitrification
(Hargreaves, 2006).
In addition to biofloc technology on its own, several researchers are looking at
combinations of this technology with other innovative techniques to control water
quality in aquaculture and its effluents. Researchers are now investigating the
combination of periphyton with carbon to nitrogen ratio control (Asaduzzaman et al.,
2008, 2010). Lezama-Cervantes and Paniagua-Michel (2010) investigated microbial
mats that are able to adapt to large fluctuations in dissolved oxygen and pH and were
able to remove and stabilize different organic and inorganic substrates partly due to
the mixed autotrophic and heterotrophic communities that co-exist in the substrate
matrix.
24
In addition, there are several studies reporting the development of intensive
shrimp culture systems without water exchange, as a way to improve biosecurity and
reduce environmental impacts (Burford et al., 2003; Wasielesky et al., 2006; Ballester
et al., 2010).
A potential alternative to intensive shrimp production are zero water exchange
systems with microbial flocs (BFT), which has the benefits of bacterial uptake of
nitrogen, including ammonia (Burford et al., 2003) and conversion of ammonia into
cellular protein, which also provides a supplemental source of nutrition (McIntosh,
2000a; McIntosh, 2000b; Burford et al., 2004b; Wasielesky et al., 2006).
The microbial community formed in this system is able to rapidly utilize
dissolved nitrogen and convert it into microbial protein (McIntosh, 2000a; Burford
and Williams, 2001). This system offers the possibility to simultaneously maintain a
good water quality within aquaculture systems and produce additional food for the
aquaculture organisms (de Schryver et al., 2008). Juvenile L. vannamei grown in a
microbial floc based system have demonstrated higher growth rates and survival
compared with juveniles grown in clear water systems (Wasielesky et al., 2006).
Biofloc role in the Feed Management of L. vannamei:
Avnimelech et al. (1994) estimated that feed utilization is higher in bioflocs
technology ponds, while tilapia in such ponds is fed a ration 20% less than
conventional one. The feed requirement of shrimp growing in bioflocs technology
ponds was studied recently by Panjaitan (2004). It was found that lowering feed
application by up to 30% of conventional feeding ration, did not lower shrimp
growth, probably due to the partial replacement of feed by the microbial flocs.
25
From proximal analysis, protein levels were higher in prawns with biofloc treatment,
which have a strong preference for natural food, probably because these are more
efficiently digested, compared to commercial diets. Hence, biofloc material
contributes to better nutrition and feeding efficiency (Ekasari et al., 2010).
Additionally, the biofloc reduces expenditure of energy to find food, which leads to
greater storage of energy in muscles and tissues (protein and lipid), which in turn,
reduced the quantity of moisture present in BFT prawns.
Proteins are the most expensive ingredients in practical feeds; and because feed
represents at least 50% of the total cost of shrimp production (Naylor et al., 1998), it
is more cost-effective to use relatively low protein feeds. Several authors have
reported that reduction in dietary protein levels had no significant effect on shrimp
growth when the shrimp were cultured in the presence of bioflocs (Wasielesky et al.,
2006; Ballester et al., 2010; Xu et al., 2012). On the other hand, as the C/N ratio of
most practical feeds (25% ~ 35% crude protein) is around 10:1, requiring the
addition of an alternative carbohydrate source to further increase the feed C/N ratio,
thereby promoting the development of bioflocs within the systems (Avnimelech, 1999;
Hari et al., 2004, 2006; Asaduzzaman et al., 2008; Asaduzzaman et al., 2010; Xu et al.,
2012). Furthermore, several studies suggest that manipulating a higher feed C/N ratio
could increase biofloc community volume without compromising the nutritional
quality of the biofloc (Azim et al., 2008; Asaduzzaman et al., 2010). Although BFT has
been applied and developed in intensive culture systems of several shrimp species,
especially L. vannamei (Crab et al., 2007; Ballester et al., 2010; Xu and Pan, 2012; Xu
et al., 2012; Zhao et al., 2012), the information concerning how biofloc can be
manipulated to maximize nutritional benefits to the cultured shrimp is limited,
and therefore the further
26
investigation into the nutritional composition and extracellular enzymes activity of
biofloc is necessary for the continued development of BFT.
The bacterial protein and new cells (single-cell protein) synthesized by the
heterotrophic bacterial population are utilized directly as a feed source by the cultured
fish and shellfish species, thus lowering the demand for supplemental feed protein
(Avnimelech, 1999). Hari et al. (2004) reported that P. monodon could effectively utilize
the additional protein derived from the increased bacterial biomass as a result of
carbohydrate addition. Burford et al. (2004) suggested that “flocculated particles” rich
in bacteria and phytoplankton could contribute substantially to the nutrition of the L.
vannamei in intensive shrimp ponds. Bacterial flocculation was observed, probably
supporting filtering out by the shrimp and thus supplying protein that was available
and suitable for shrimp nutrition.
Biofloc technology (BFT), a new type of water treatment technology, is often cited as
an approach to reduce feed costs and environmental problems associated with
discharge of waste products (Avnimelech, 1999). Biofloc technology involves
stimulating the process of inorganic nitrogen assimilation by heterotrophic bacteria.
These bacteria utilize ammonium, in addition to the organic nitrogenous wastes, to
synthesize new cells by consumption of carbohydrates, simultaneously utilizing
organic matters, microorganisms and some suspended solids to form bioflocs
(Hargreaves, 2006). This process reuses uneaten feed and excrement and reduces the
need for water exchange, thereby reducing the risk of disease infection through water
intake (Crab et al., 2009). The C/N ratio of most of the feeds used in semi-intensive
aquaculture ponds is around 10, whereas bacteria require about 20 units of carbon
per unit of nitrogen assimilated (Avnimelech, 1999). Therefore, if the C/N ratio is
increased by adding a carbohydrate source, such as tapioca starch, in addition to the
27
regular feed, the increased availability of carbon allows the heterotrophic bacterial
population to increase in abundance (Asaduzzaman et al., 2010), resulting in bioflocs.
Burford et al. (2004) evaluated the consumption of bioflocs by shrimp using N15
labeling and noted that 18–29% of the protein consumed by shrimp on a daily basis
was derived from bioflocs. Hari et al. (2006) also noted that the addition of tapioca
starch in shrimp culture systems improved water quality, shrimp growth and feed
utilization efficiency.
Previous studies have demonstrated the beneficial effects of promoted bioflocs on
shrimp nutrition (Burford et al., 2004; Kuhn et al., 2008; Xu et al., 2012). It is plausible
to speculate that the nutritional contribution of bioflocs may be related to their
nutritional contents and extracellular enzymes (Burford et al., 2004; Wasielesky et al.,
2006; Ju et al., 2008; Xu and Pan, 2012; Xu et al., 2012).
Although bioflocs meet nutritional standards to serve as a aquaculture feed in
general, research has shown that the capacity of the technique to control the water
quality in the culture system and the nutritional properties of the flocs are influenced
by the type of carbon source used to produce the flocs (Crab et al., 2010b). Different
organic carbon sources each stimulated specific bacteria, protozoa and algae, and
hence influenced the microbial composition and community organization of the
bioflocs and thereby also their nutritional properties (Crab, 2010a). Feeding
experiments revealed that besides these characteristics, the type of carbon source also
influenced the availability, palatability and digestibility for the cultured organisms
(Crab et al., 2010a; 2010b).
Addison et al. (2010) studied the affect of Biofloc replaced fish meal and
soybean meal in semi-purified shrimp diets. Ju et al. (2008) studied the enhanced
growth effect on shrimp (L. vannamei) from inclusion of whole shrimp floc or floc
28
fractions to a formulated diet. The results suggest that inclusion of the floc material
in shrimp diets could enhance the shrimp growth. They observed enhanced growth
when compared with that of the control (P<0.05); the diet preference and pellet
stability study were also positive. Shrimp preferred the experimental diets over
commercial diet and the feed stability was same as that of commercial diet. Addison et
al. (2010) reported increased growth of L. vannamei by replacing fishmeal and
soybean with biofloc. Microbial flocs produced in suspended growth bioreactors
could offer the shrimp industry a novel alternative feed. Study by Kuhn et al. (2009)
showed that bioflocs harvested from the sequencing batch reactors (SBRs) and
membrane biological reactors proved to be a suitable and often superior ingredient,
to soybean protein and fishmeal in lab-scale feeding trials with L. vannamei. High
quality control diets were compared against experimental diets in 35- day feeding
trials. Result was that experimental diets were varied greatly and notable
independent variables included complete replacement of soybean protein, two-thirds
replacement of fishmeal, and no fish oil. Biofloc inclusion always increased growth
rates and ranged from a low average increase of 4% to a high average of 67% over
the control diets; the latter percent increase was significant at P < 0.01. It seems that
Biofloc technology represents a promising option for sustainability of the
aquaculture industry. Ballester et al. (2010) compared the performance of F.
paulensis reared with partial diet with different protein levels under zero exchange
microbial floc intensive system. Forty five days experimental trial concluded that
shrimps fed with a protein percentage of 35 which are maintained in biofloc ponds
showed maximum growth parameters. It was significantly greater when compared to
the feed with 25, 30, 40 and 45% of crude protein. Kuhn et al. (2010) compared the
possibilities of incorporating two types of microbial flocs in the feed of L.vannamei
29
derived from biological treatment of fish effluent. Logan. (2010) presented the
possibilities of commercializing the biofloc feeds in recent World Aquaculture Society
conference held at California, the product is expected to be available in the market by
2012 as the trade name Obron SCP ingredient.
The floc biomass could provide a complete source of cellular nutrition as well as
various bioactive compounds (Akiyama et al., 1992; Fast and Menasveta, 2000; Tacon
et al, 2002; Truus et al., 2004; Singh et al., 2005) and may contain an, as yet,
undiscovered growth factor (Ju et al., 2008). Floc carotenoids have been reported to
provide essential nutritional and many bioactive physiological functions in animal
tissues, including stimulating animal immune systems. Ju et al. (2008) concluded that
these results suggest that floc materials that develop in low-water exchange shrimp
culture systems could be added to diets to obtain better shrimp growth in clear water
systems.
Biofloc role in the Disease Management of L. vannamei:
In biofloc systems, nitrogenous waste is reduced through microbial assimilation
aided by the addition of extra carbonaceous materials (Avnimelech, 1999). The
heterotrophic microbial biomass is suspected to have a controlling effect on pathogenic
bacteria (Michaud et al., 2006). Several studies have been conducted using biofloc
technology for culture of tilapia (Azim and Little, 2008; Asaduzzaman et al., 2009) and
shrimps (Kuhn et al., 2008, 2009, 2010; Asaduzzaman, 2010; Ray
et al., 2011).
In addition to water quality control and in situ feed production, bioflocs technology
has protected brine shrimp (A. franciscana) larvae from vibriosis. (Crab et al., 2010b).
30
Bioflocs technology have been applied and developed in high intensive
farming systems of several shrimp species, such as P. monodon, L. vannamei and M.
rosenbergii farming (Burford et al., 2003; Crab et al., 2010b; Hari et al., 2006).
However, due to the specific hiding sand behaviour of M. Japonicus by daylight, little
is known about the practicability of biofloc technology for the intensive farming of
the shrimp M. japonicus. Moreover, few investigations were shown about the
microbial diversity in bioflocs technology ponds.
The reduction in nitrogenous compound through carbon addition could lead to an
increased microbial flocs, which immobilized nitrogen for microbial synthesis
(Avnimelech, 1999; Hari et al., 2004). Microbial flocs performed in bioflocs
technology ponds are demonstrated to be an effective potential food source for tilapia
through the monitoring of floc volume (Avnimelech, 2007).
Manipulating the C/N ratios through carbon addition could result in a shift from
an autotrophic to a heterotrophic system (Avnimelech, 1999; Avnimelech et al., 1994;
Browdy et al., 2001). Further study showed heterotrophic bacteria was suspected to
have a controlling effect on pathogen (Defoirdt et al., 2007; Michaud et al., 2006).
Biofloc technology (BFT) has recently gained great attention as a sustainable
solution, which can not only effectively control water quality under zero-water
exchange but also sustain intensive and healthy culture of shrimp (Avnimelech, 2012;
Crab et al., 2012; Stokstad, 2010). The driving force of BFT culture systems is
microbial biofloc, which is a heterogeneous aggregate of suspended organic particles
and many varieties of active microorganisms associated with extracellular polymeric
substances (De Schryver et al., 2008; Ju et al., 2008b; Ray et al., 2010). The biofloc has
been reported to confer many beneficial effects on shrimp culture, including: (1)
31
improving water quality through removal of toxic nitrogen species such as ammonia
and nitrite (De Schryver et al., 2008; Ray et al., 2011; Xu et al., 2012a); (2) increasing
feed utilization and growth performance of shrimp through supplementing natural
food and stimulating digestive enzyme activities (Ballester et al., 2010; Emerenciano et
al., 2012; Xu and Pan, 2012; Xu et al., 2012); and (3) enhancing biosecurity and health
management through zero-water exchange and possible probiotic effect (Crab et al.,
2010a; Haslun et al., 2012; Moss et al., 2012; Zhao et al., 2012).
It is also interesting to note that the application of biofloc in shrimp culture results in
similar effects in terms of growth, feeding efficiency, pathogenic bacteria inhibition
and immune responses as the application of probiotics (Zokaeifar et al., 2013;
Zokaeifar et al., 2012; Zhang et al., 2011; Xia et al., 2013; Li et al., 2009; Castex et al.,
2009). For instance, Zokaeifar et al. (2012), Zokaeifar et al. (2013) reported that adding
Bacillus subtilis into water or the feed of white shrimp resulted in better growth and
survival, inhibition of Vibrio growth in the intestine, enhanced protease and amylase
activities, as well as up-regulation of immune related genes such as proPO,
peroxinectin, and serine protease. The mechanisms by which probiotic bacteria affect
shrimp performance have been reviewed by several authors (De Schryver et al., 2012;
Ninawe et al., 2009).
Crab et al. (2010b) have recently shown that biofloc technology constitutes a possible
alternative measure to fight pathogenic bacteria in aquaculture. Intensive
aquaculture of crustaceans is one of the fastest-growing sectors in aquaculture
production (Wang et al., 2008). Despite its huge success, shrimp culture is facing
severe outbreaks of infectious diseases, which have caused significant economic losses.
Due to the haphazard mishandling of antibiotics in aquaculture, pathogenic
32
bacteria are now becoming resistant to numerous antibiotics and as a result,
antibiotics are no longer effective in treating bacterial disease (Defoirdt et al., 2011).
The disruption of quorum sensing, bacterial cell to-cell communication with small
signal molecules (Defoirdt et al., 2008), has been proposed as a new strategy to control
bacterial infections in aquaculture as this cell-to-cell communication mechanism
regulates the expression of virulence factors (Defoirdt et al., 2004). Interestingly, we
recently found that bioflocs grown on glycerol were able to protect gnotobiotic brine
shrimp (A. franciscana) against pathogenic V. harveyi, and that the beneficial effect
was likely due to interference with the pathogen's quorum sensing system (Crab et al.,
2010b). Indeed, survival of challenged nauplii increased 3-fold after the addition of live
bioflocs. This complies with former research that revealed that primary production
and promotion of in situ microbial populations, as is the case in biofloc technology,
were found to be beneficial for shrimp (Lezama Cervantes and Paniagua-Michel,
2010).
Interesting feature of bioflocs to further investigate with respect to biocontrol
effects is the capability to accumulate the bacterial storage compound poly-β
hydroxybutyrate (PHB). PHB and PHB accumulating bacteria have been shown
before to protect different aquaculture animals from bacterial infections (De
Schryver et al., 2010; Defoirdt et al., 2007; Dinh et al., 2010; Halet et al., 2007). PHB-
accumulating bacteria are present in bioflocs as we have measured PHB levels in
bioflocs of between 0.5 and 18% of the dry matter (Crab, 2010a; De Schryver and
Verstraete, 2009). The latter bioflocs contain a sufficient PHB level to protect
cultured animals from infection by pathogenic bacteria (Halet et al., 2007).
In earlier studies, preliminary results showed that the water of shrimp tanks
fed bioflocs inoculated with Bacillus had an on average 5 times lower Vibrio load
33
when compared to the shrimp tanks fed an artificial feed (Crab, 2010a). These results
indicate that inoculating biofloc reactors with probiotic bacteria might have
biocontrol effect toward Vibrio spp., but the inoculation of biofloc systems with
specific desired microorganisms needs further investigation in order to confirm these
beneficial effects. Other interesting fields of research regarding this subject are
possible immunostimulatory features of the bioflocs. Enhancement of the innate
immunity of cultured organisms may provide broad-spectrum resistance to
infections. Existing immunostimulants include bacteria and bacterial products,
complex carbohydrates, nutritional factors, animal extracts, cytokines, lectins, plant
extracts and synthetic drugs such as levamisole (Wang et al., 2008). Bioflocs might
also contain immunostimulatory compounds since biofloc technology deals with
bacteria and bacterial products.
2.6 . Biofloc role in Survival of L. vannamei:
In earlier studies, it was shown that the application of BFT in general results in an
increased growth performance, FCR and survival of the cultured shrimp (Hari et al.,
2004; Zhao et al., 2012; Krummenauer et al., 2011; Ray et al., 2011).
The differences in growth when the biofloc was included were evident. Survival rates
did not vary (P>0.05) among dietary treatments; however, shrimp growth was
significantly improved (P<0.05) for shrimp fed microbial flocs. Eventhough numerous
studies have reported enhanced survival, health, and growth rates of shrimp raised in
ponds with high activity of algae, microbial flocs, and other natural biota (Avnimelech,
1999; Moss et al., 2000; Moss et al., 2001; Tacon et al., 2002; Burford et al., 2004;
Cuzon et al., 2004; Izquierdo et al., 2006 and Wasielesky et al., 2006).
34
With this above review, it can be concluded that there is limited research work over
the effect on growth and survival of L. vannamei reared with biofloc developed by
different carbohydrate sources. Hence, the present study is aimed to evaluate the
impact on growth and survival of L. vannamei fed on biofloc grown with different
carbohydrate sources.
34
3. Materials and Methods
Site of the Experiment:
The experiment was conducted in Wet Laboratory of the Department of
Aquaculture, College of Fishery Science, Sri Venkateswara Veterinary University,
Muthukur, for a period of 60 days.
Experimental animals and their acclimatization:

Litopenaeus vannamei (1000 numbers) were obtained from BMR Hatchery,
Nellore, who has been authorized by Coastal Aquaculture Authority (CAA), Chennai
to produce the seed. Shrimp seed were packed in double plastic bags filled with
oxygen and water in the ratio of 3:1 in each bag and the density of shrimp was
300/bag. Post larvae (PL 20) transported by road in plastic bags containing 5 ppt
saline water. PL transferred to the same salinity water in the wet lab. Acclimatization
was carried out over 2 weeks. During this time salinity was lowered from 5 ppt to
3ppt. During this period the seed were fed with crumble, sinking starter feed having a
crude protein percentage of 35 (Manamei shrimp feeds AVANTI Company). The
number of shrimp seed to be packed in oxygen inflated polythene bags was calculated
as per the following formula (Jameson et al., 1995).
N = (DO – 2) X V/CH
Where:
DO: Dissolved oxygen content of water
(mg/l) V: Volume of water used for
transport (Lt)
C: Rate of oxygen consumption of shrimp (ml/kg of
shrimp) H: Duration of transport (Hours)

35
Tank allocation:
Indoor experiments were conducted in FRP tanks with 1000 ltr capacity and
with an effective bottom area of 1.03 m2, three triplicate treatments were maintained
in the Wet Laboratory (Plate 3). Tanks were filled with bore water with a depth of 60
cm. All tanks were facilitated with 2 air stone-hoses type of diffuser system which is
fitted to 2 HP blower. Aeration was provided 24 hours throughout the experiment for
better biofloculation. Tanks were kept one week for dechlorination. Urea and super
phosphate were added as fertilizers at a dosage of 4 and 1 g/m2 during the first three
weeks (Varghese, 2007). After two week all tanks were stocked with shrimps at a rate
of 15/m2 (New, 2002). Before stocking initial weight of the organism (1.025±0.05g),
initial water parameters were recorded. Commercial pelletized sinking shrimp feed
with a dietary protein level 35% was selected as experimental feed in pellet form and
for initial feeding it was repelletized into smaller size.
Preparation of carbohydrate source and feeding:

Three easily and locally available carbohydrate sources Viz, tapioca flour
(Manihot esculaneta), wheat flour (Triticum aestivum), and molasses (Saccharum
officinarum) were selected as carbohydrate sources for biofloclation (Plate 2). Wheat
flour were purchased from the local market in powdered form which was meant for
the culinary purpose. Molasses were purchased from the local suger factory. While
tapioca were purchased from vegetable market. Raw tubers were purchased, peeled
and washed thoroughly, made into small pieces and soaked in water overnight. Next
morning water drained and the pieces were kept in oven at 600C till it dried
completely. After that slices were powdered in a mixer grinder, sieved through 35 μm
sieves and powder stored in air-tight container (Saritha, 2009). By processing 1 kg of
raw tuber, 500 g of corresponding powder was obtained.
36
Molasses
Wheat flour Tapioca flour
Plate 2: Experimental carbon sources molasses,

wheat flour and tapioca flour used as biofloc
agent
37
Plate 3: Experimental set up in the wet - laboratory
Plate 3.1: Experimental animals fed with wheat flour as biofloc agent
38
Plate 3.2: Experimental animals fed with tapioca flour as biofloc agent
Plate 3.3: Experimental animals fed with molasses as biofloc agent
39
Shrimps were fed with experimental feed at 12 % of initial body weight and
adjusted gradually to 2.5% at the end of the culture (1-60 days). The daily feeding
ration for each treatment was calculated and adjusted by estimating the weekly
sampled mean biomass. The ration was divided and distributed twice daily with
similar portions between 9:00 and 10:00 hours in the morning and between 17:00 and
18:00 hours in the evening. The C:N ratio of the treatments was calculated using the
formula of Avnimelech (2000) and it was found to be 10:1 for all the treatments. The
quantity of carbohydrate added was calculated following the theory of Avnimelech
(1999) and Hari et al. (2004, 2006). Pre weighed carbohydrate source was mixed in a
glass beaker with the water collected from the corresponding culture tanks. The
culture tanks treated with wheat flour, tapioca flour, molasses were represented as T 2,
T3, and T4, respectively (Plate 2). All the systems were maintained for 60 days
without any water exchange. Water loss due to evaporation was compensated by the
addition of dechlorinated water as per requirement.
Calculation of quantity of carbohydrate required

for biofloculation:
Wheat flour, tapioca flour and molasses was purchased from local market. The
quantity of carbohydrate added to the BFT system was calculated using the equation
of Avnimelech (1999) and assuming that the added carbohydrate contains minimum
of 50% carbon, the CH addition needed (ΔCH) to reduce the total ammonia nitrogen
concentration by 1g N/m3 is 20 g/m3.
ΔCH= ΔN/0.05 --------------------------------------------------------- (1)
It can be assumed that the ammonium flux into water, ΔNH4 +, directly by
excretion or indirectly by microbial degradation of the feed residues, is roughly
around 50% of the feed nitrogen (Avnimelech, 1999):
ΔN=quantity of feed × % N in feed × %N excretion -------------- (2)

40
The amount of carbohydrate addition needed to assimilate the ammonium flux into
microbial protein is calculated using equations (1) and (2):
ΔCH=quantity of feed × % N in feed × % N excretion/0.05------ (3)
According to equation (3), wheat flour, tapioca flour and molasses are calculated
which are required for each kg of 35% dietary protein.
Proximate composition:
Proximate analysis of the feed was estimated by the method of AOAC, 1995.
Table 1: Proximate composition of experimental
feed & different carbon sources:
Feed code Protein (%) Fat (%) Ash (%) Fiber (%) Moisture(%)
3S 35 5 4 4 11
Wheat flour 12.8 1.8 13.3 1.2 6
Tapioca flour 2.1 1.23 0.6 0.89 9.4
Molasses 5.9 0.06 9.4 0.1 29.6
Moisture:
A known weight of the feed sample was taken and dried in an oven at
1050C to constant weight and the moisture content was calculated by using the
following formula:
Weight of wet sample - Weight of dried sample
Moisture (%) = ---------------------------------------------------------------x100
Weight wet of sample
41
Crude protein:
Nitrogen content of the sample was estimated by Kjeldahl method and
the crude protein was estimated by multiplying nitrogen percentage by a
constant factor 6.25.
Crude protein (%) = Nitrogen (%) x 6.25
Ether extract:
Ether extract was estimated by soxhlet apparatus using petroleum ether
as a solvent.
Weight of initial sample – Weight after extraction
Ether extract (%) =- ----------------------------------------------------------------
x100
Weight of initial sample
Ash:
Ash content was estimated by taking a known weight of sample in silica
crucible and placing it in a muffle furnace heated at 600 0C for 6 hours.
Weight of ash
Ash (%) = ------------------------------------

x100
Weight of sample
Crude fibre:
Crude fibre was estimated by treating the moisture and fat free sample
successively with dilute acid (1.25%) and alkali.
Weight of crude fibre
Crude fibre (%) = ------------------------------------ x 100
Original weight of sample

42
Shrimps were fed at the rate of 12% body weight initial days and then reduced
to 2.5% body weight. fed twice a day (9.00 AM and 7.00 PM ).
Assessment of water quality parameter:

Water quality parameters, temperature (Digital pH and temperature meter),
dissolved oxygen (Titrimetric Winkler's method APHA, 1995) and pH (Digital pH
and temperature meter) were measured in-situ at 9.00 hrs on daily basis. Water
samples were collected using a horizontal water sampler from three locations of each
tank and pooled together. Water samples were transported to the laboratory after
collection and analyzed. Water samples were collected on weekly basis between
9.00 and 10.00 hrs. TAN ( analyzed spectrophotometrically APHA, 1995).
Physico-Chemical parameters of water:

Sl.No. Parameter Method
1. pH Digital pH and temperature meter (ELICO-Li 120)
2. Temperature Digital pH and temperature meter (ECOSCAN)
3. Dissolved Oxygen Titrimetric Winklers method (APHA, 1995)
4. Total Alkalinity Titrimetric method ( APHA, 1995)
5. Total Ammonia (APHA, 1995)

Nitrogen
Composition of biofloc:
Suspended growth in ponds consists of phytoplankton, bacteria, aggregates
of living and dead particulate organic matter, and grazers of the bacteria
(Hargreaves, 2006). In our study the plankton samples were collected in triplicate
and concentrated to 50 ml by filtering the water using of plankton net bolting silk
cloth (no. 25) from the treatment tanks. The collected plankton samples were
43
preserved in 5% formalin for further analyses (Pennak, 1978). Typical flocs are
irregular by shape, have a broad distribution of particle size, are fine, easily
compressible, highly porous (up to more than 99% porosity) and are permeable to
fluids (Chu and Lee, 2004). In our study the photographs of all the major planktons
were taken using a camera (NIKON COOLPIX Camera, S2600 ) attached to Magnṻs
biological microscope (Model: MLX). Floc volume taken in test tubes (Plate 6).
Shrimp yield parameters:

Yield study was done by using hand net in the culture tanks. Individual length
(Plate 4) and weight (Plate 5) were recorded. individual shrimp weight gain, specific
growth rate (SGR), feed conversion ratio (FCR), average daily weight gain (ADG)
and survival rate, were calculated as follows.
3.8.1 Growth performance:

The growth parameters of all the shrimps of each aquarium were individually
estimated by taking their total body length and weight at 7 days interval.
Weight increment:
Weight increment was obtained by subtracting initial body weight from the
final body weight.
Weight increment (gm) = Final body weight (gm) – Initial body weight (gm).
Specific growth rate:

Specific growth rate was calculated by the formula
[(Ln FBW - Ln IBW) / day] x 100
FBW -- Final body weight
IBW -- Initial body weight
Ln -- Logarithm
Day -- duration of experiment (60 days)

44
Survival Rate:
Survival of the shrimps was weekly noted down and survival rates are
calculated as below
Total number of shrimp survived
Survival (%) = ------------------------------------------- x 100
Total number of shrimp stocked
Food Conversion Ratio (FCR):

Feed Conversion Ratio was calculated by dividing feed given (dry weight) by
body weight gain (wet weight)
Feed given (dry weight)
Feed Conversion Ratio = -----------------------------------------
Body weight gain (wet weight)
3.9. Statistical Analysis:

Statistical analyses were performed using web agri stat package (WASP)
version 2.0. The data obtained on Growth, Survival and Food Conversion Ratio was
statistically analyzed by applying Randomized Block Design (RBD) of two-way
classification.
45
Plate 4: L. vannamei length measurement
Plate 5: L. vannamei weight measurement
46
Plate 6: Biofloc in different carbon source
treatment tanks
47
4. Results
WATER QUALITY PARAMETERS:
In the present study important water quality parameters such as Dissolved
oxygen, Temperature, pH, Total alkalinity, TAN were observed for every seven days
of sampling in all FRP tanks and presented in tables from 2-6 and in figures from 1-5.
The water quality parameters were similar during the experimental period and
maintained in acceptable level.
The average values recorded for the various physiochemical parameters like
dissolved oxygen, temperature, pH and total alkalinity are presented in Tables (2-6).
These parameters were well within the optimum range and were not found to be
affected by the addition of different sources of carbohydrate (New and Singholka,
1985). Water quality parameters, such as dissolved oxygen, temperature, pH and total
alkalinity, were in the range of 7.12±0.05 - 7.98±0.02 mg/l, 25.8±0.01 - 30.7±0.04 0C,
7.71±0.03-8.03±0.01 and 252±1.41 - 290±1.41 mg/l, respectively. Among the
various treatments, the water TAN is lower in control (T 1) (0.04±0.01 mg/l) and
maximum (0.27±0.02 mg/l) was in where Molasses (T4) was used as carbohydrate
source. Concentrations of TAN recorded from water showed fluctuating trends.
When comparing week-wise values, the TAN concentration significantly lower in
water was in initial days of the culture period, especially in the 1 st week where higher
values were recorded in Molasses (T4) 0.09±0.01 mg/l and lower value recorded in
control (T1) 0.04±0.01 mg/l. Similarly second week (14th day) higher values were
recorded in Molasses (T4) 0.09±0.02 mg/l and lower TAN values was in Wheat flour
(T2) 0.05±0.02 mg/l. Similar trend continued on the 21st day also. The highest and
lowest water TAN observed were 0.13±0.02 and 0.09±0.02 mg/l for Wheat flour (T 2)
48
and Control (T1) respectively. During the 28th day, the highest and lowest water TAN
observed were 0.16±0.0 mg/l and 0.05±0.01 mg/l in Molasses (T 4) and Control (T1)
respectively. Wheat flour (T2) and Tapioca flour (T3) stood in second and third
positions with water TAN of 0.11±0.02 mg/l and 0.09±0.02 mg/l respectively. On the
35th day of the experiment the highest and lowest water TAN value observed were
0.18±0.02 mg/l and 0.08±0.02 mg/l in Wheat flour (T 2) and in Control (T1)
respectively. Molasses (T4) and Tapioca flour (T3) stood in second and third positions
with TAN value of 0.15±0.02 mg/l and 0.13±0.02 mg/l respectively. On the 42 nd day
highest TAN value of 0.27±0.02 mg/l and lowest value of 0.09±0.02 mg/l were
recorded in Molasses (T4) and Control (T1) respectively. On the 49th day highest TAN
value of 0.23±0.03 mg/l and lowest value of 0.08±0.01 mg/l were recorded in
Molasses (T4) and Control (T1) respectively. On the 60th day highest increment of
0.22±0.03 mg/l and lowest value of 0.09±0.02 mg/l were recorded for the Molasses
(T4) and Control (T1) respectively. Wheat flour (T2) and Tapioca flour (T3) stood in
second and third positions with TAN value of 0.21±0.02 mg/l and 0.10±0.02 mg/l
respectively. An overall study indicated that the Molasses (T 4) recorded highest TAN
value of 0.27±0.04 mg/l in the 60 days experimental period.
49
Table 2: Weekly variation of Dissolved Oxygen
(mg/l) in the tanks treated with various
carbohydrate sources as biofloculating
agents:
Treatment
Control Wheat flour Tapioca flour Molasses
T1 T2 T3 T4
Period(Days)
0 7.98±0.02 7.62±0.02 7.63±0.04 7.19±0.01
7 7.24±0.03 7.34±0.01 7.30±0.05 7.25±0.04
14 7.21±0.01 7.30±0.03 7.39±0.01 7.41±0.03
21 7.16±0.02 7.12±0.05 7.16±0.03 7.14±0.02
28 7.27±0.01 7.23±0.02 7.27±0.02 7.26±0.03
35 7.24±0.04 7.40±0.04 7.43±0.05 7.44±0.05
42 7.31±0.03 7.46±0.01 7.34±0.04 7.39±0.01
49 7.28±0.02 7.38±0.01 7.41±0.03 7.37±0.02
60 7.33±0.05 7.34±0.03 7.38±0.01 7.35±0.04
50
Figure 1: Weekly variation of Dissolved Oxygen (mg/l) in
the tanks treated with various carbohydrate
8.2
sources as biofloculating agents:
7.8
7.6
7.4 Control
DO(mg/l)
Wheat flour
7.2
Tapioca flour
Molasses
7
6.8
6.6
0 7 14 21 28 35 42 49 60
Days
51
Table 3: Weekly variation of Temperature ( 0C) in the tanks treated with
various carbohydrate sources as biofloculating agents :
Treatment
Period(Days) T1 T2 T3 T4
0 27.6±0.02 27.6±0.03 27.6±0.03 27.6±0.01
7 25.8±0.01 25.8±0.01 25.8±0.05 25.8±0.05
14 27.4±0.04 27.4±0.03 27.4±0.01 27.4±0.02
21 28.4±0.02 28.4±0.01 28.4±0.02 28.4±0.01
28 28.0±0.03 28.0±0.03 28.0±0.03 28.0±0.03
35 28.1±0.05 28.1±0.02 28.1±0.01 28.1±0.01
42 30.6±0.02 30.6±0.01 30.6±0.04 30.6±0.02
49 30.7±0.03 30.7±0.03 30.7±0.02 30.7±0.04
60 30.5±0.02 30.5±0.02 30.5±0.03 30.5±0.03
52
Figure 2: Weekly variation of Temperature (0C) in the tanks treated with
32
31
30
29
Temperature(0C)
28 Control
Wheat flour
27
Tapioca flour
26
Molasses
25
24
23
0 7 14 21 28 35 42 49 60
Days
53
Table 4: Weekly variation of pH in the tanks treated with various carbohydrate
sources as biofloculating agents :
Treatment
T1 T2 T3 T4
Period(Days)
0 8.01±0.01 7.97±0.04 8.00±0.02 7.81±0.03
7 7.92±0.03 7.93±0.02 7.89±0.03 7.93±0.01
14 7.90±0.02 7.86±0.01 7.87±0.01 7.95±0.05
21 7.94±0.02 7.88±0.05 7.86±0.04 7.89±0.03
28 7.92±0.04 7.88±0.02 7.92±0.02 7.94±0.01
35 8.03±0.01 8.01±0.01 8.02±0.02 8.01±0.04
42 7.95±0.03 7.91±0.04 7.90±0.05 7.84±0.02
49 7.78±0.01 7.74±0.01 7.79±0.01 7.82±0.01
60 7.71±0.05 7.71±0.03 7.79±0.05 7.77±0.05
54
Figure 3: Weekly variation of pH in the tanks treated
with various carbohydrate sources as
biofloculating agents :
8.1
7.9
Control
7.8
Wheat flour
pH
Tapioca flour
7.7
Molasses
7.6
7.5
0 7 14 21 28 35 42 49 60
Days
55
Table 5: Weekly variation of Total Alkalinity (mg/l) in the tanks treated with
Treatment
T1 T2 T3 T4
Period(Days)
0 252±1.41 258±2.82 254±4.24 256±5.65
7 266±2.82 278±1.41 270±1.41 268±1.41
14 268±1.41 280±1.41 268±2.82 272±2.82
21 270±1.41 276±2.82 272±1.41 278±1.41
28 258±4.24 282±1.41 270±2.82 280±1.41
35 268±2.82 262±4.24 284±2.82 268±4.24
42 276±1.41 274±1.41 280±1.41 278±2.82
49 280±2.82 286±1.41 278±2.82 274±1.41
60 288±1.41 280±2.82 290±1.41 286±1.41
56
Figure 4: Weekly variation of Total Alkalinity (mg/l) in
the tanks treated with various carbohydrate
300
290
280
Total Alkalinity (mg/l)
270
controle
Wheat flour
260
Tapioca flour
250 Molasses
240
230
0 7 14 21 28 35 42 49 60
Days
57
Table 6: Weekly variation of TAN (mg/l) in the tanks treated with various
carbohydrate sources as biofloculating agents :
Treatment
7 0.04±0.01 0.07±0.01 0.06±0.01 0.09±0.01
14 0.06±0.02 0.05±0.02 0.08±0.01 0.09±0.02
21 0.09±0.02 0.13±0.02 0.11±0.02 0.12±0.02
28 0.05±0.01 0.11±0.02 0.09±0.02 0.16±0.03
35 0.08±0.02 0.18±0.02 0.13±0.02 0.15±0.02
42 0.09±0.02 0.23±0.02 0.12±0.02 0.27±0.02
49 0.08±0.01 0.19±0.01 0.11±0.03 0.23±0.03
60 0.09±0.02 0.17±0.02 0.10±0.02 0.21±0.03
58
Figure 5: Weekly variation of TAN (mg/l) in the
tanks treated with various carbohydrate
0.3
0.25
0.2
TAN(mg/l)
control
0.15
Wheat flour
0.1 Tapioca flour

Molasses
0.05
0
7 14 21 28 35 42 49 60
Days
59
GROWTH PARAMETERS:
Growth of L.vannamei fed on biofloc grown with different carbon sources:
Weight of shrimp in grams and weight increment data observed weekly for
different treatments were presented in tables 7, 9 and figures 6 - 7. Observations on
the growth during the first week (7thday) revealed that the weight increment varied
between 2.09±0.11 and 2.70±0.08g in Control (T1) and Wheat flour (T2). On the 14th
day highest weight increment of 4.44±0.11g and lowest weight increment of
3.21±0.08g were recorded for the Wheat flour (T 2) and Control (T1) respectively.
Similar trend continued during the 21 st day also. The highest and lowest weight
increment were observed were 4.43±0.05 and 5.75±0.11g in Control (T 1) and
Molasses (T4) respectively. During the 28th day, the highest and lowest weight
increments observed were 7.97±0.09g and 5.72±0.07g in Wheat flour (T 2) and
Control (T1) respectively. In treatments of Tapioca flour (T 3) and Molasses (T4) stood
in second and third positions in weight increment of 7.42±0.08 and 7.40±0.05g
respectively. Similar trend continued during the 35th day of the experiment also.
Highest and lowest weight growth increments observed were 9.78±0.11 and
7.08±0.02g in Wheat flour (T2) and Control (T1) respectively. Tapioca flour (T3) and
Molasses (T4) stood second and third positions with growth weight gain of 9.24±0.11
and 9.11±0.01g respectively. On the 42 nd day the highest weight increment of
11.71±0.04g and lowest increment of 8.59±0.11g were recorded in Wheat flour (T 2)
and Control (T1) respectively. On the 49 th day the highest increment of 13.65±0.12g
and lowest increment of 10.26±0.12g were recorded in Wheat flour (T 2) and Control
(T1) respectively. On the 60th day the highest increment of 15.92 ±0.07g and lowest
increment of 12.27±0.09g were recorded in Wheat flour (T2) and in Control (T1)
60
respectively. Treatments of Tapioca flour (T3) and Molasses (T4) stood in second and
third positions with growth weight gain of 15.17±0.07 and 14.82±0.04g respectively.
An overall study indicated that the Wheat flour (T 2) recorded total weight increment
of 15.92±0.07g in 60 days experimental period. This was followed by the Tapioca
flour (T3) 15.17±0.07g and Molasses (T4) 14.82±0.04g, they stood in second and third
positions respectively.
The growth data was subjected to analysis of variance (ANOVA) at 5% level
of significance and the observations are given in table 8. The statistical analysis has
shown that F- value is found to be significant among treatments. Since F- value is
found to be significant, the pair wise comparison of any two Treatments could be
done by computing RBD two way classification. The Treatment Wheat flour (T 2) is
found to be significantly superior when compare to other Treatments. Treatment
Wheat flour (T2) has shown significantly different from all other Treatments. The
second and third positions were occupied by Tapioca flour (T 3) and Molasses (T4)
respectively. There was a significant difference between the culture periods also.
61
Table 7: Growth performance (g) of L. vannamei
sources:
Treatment
T1 T2 T3 T4
Period(Days)
0 1.025±0.12 1.025±0.12 1.025±0.12 1.025±0.12
7 2.09±0.11 2.70±0.08 2.53±0.12 2.54±0.05
14 3.21±0.08 4.44±0.11 4.12±0.07 4.13±0.09
21 4.43±0.05 5.19±0.05 5.74±0.02 5.75±0.11
28 5.72±0.07 7.97±0.09 7.42±0.08 7.40±0.05
35 7.08±0.02 9.78±0.11 9.24±0.11 9.11±0.01
42 8.59±0.11 11.71±0.04 11.12±0.08 10.79±0.09
49 10.26±0.12 13.65±0.12 13.05±0.12 12.64±0.12
60 12.27±0.09 15.92±0.07 15.17±0.07 14.82±0.04
62
Table 8: Statistical analysis for Growth performance
Treatment means
S. No Average
Treatment 1 6.074
Treatment 2 8.042
Treatment 3 7.712
Treatment 4 7.577
Anova Table
Source of Degrees of Sum of Mean sum of F

F cal
variation freedom squares squares prob
Replications 8 677.211 84.658 242.001 4.992
Treatments 3 20.606 6.868 19.637 1.193
Error 24 8.391 0.347 - -
Total 35 - - - -
Coefficient of Variation = 8.049
Treatments found Significant at 1% and 5% level of significance CD(0.01) =

0.778 CD(0.05) = 0.574
Comparison of Treatment Means with Critical Difference (0.05)
Treatment No. T2 T3 T4 T1
Treatment Average 8.042 7.712 7.577 6.074
Critical Difference (CD) Compared a a a b
63
Figure 6: Growth performance (g) of L. vannamei
sources:
18
16
14
12
Growth performance (g)
10 Control
8 Wheat flour
Tapioca flour
6
Molasses
4
0
0 7 14 21 28 35 42 49 60
Days
64
Table 9: Weight gain (g) in L. vannamei fed on biofloc grown with different
carbon sources:
Treatment
T1 T2 T3 T4
Period(Days)
7 1.07±0.08 1.68±0.05 1.54±0.11 1.52±0.09
14 1.12±0.02 1.74±0.08 1.59±0.09 1.59±0.05
21 1.22±0.05 1.75±0.02 1.62±0.05 1.62±0.02
28 1.29±0.11 1.78±0.11 1.68±0.08 1.65±0.11
35 1.36±0.09 1.81±0.09 1.82±0.02 1.71±0.01
42 1.51±0.02 1.93±0.07 1.88±0.12 1.68±0.12
49 1.67±0.07 1.94±0.12 1.93±0.09 1.85±0.08
60 2.01±0.12 2.27±0.01 2.12±0.11 2.18±0.09
65
Table 10: Statistical analysis for weight gain
Treatment means
S. No Average
Treatment 1 1.402
Treatment 2 1.865
Treatment 3 1.775
Treatment 4 1.725
Anova Table

F cal
Replications 7 1.407 0.208 37.690 1.709
Treatments 3 0.943 0.314 59.207 2.041
Error 21 0.118 0.003 - -
Total 31 - - - -
Treatments found Significant at 1% and 5% level of significance CD(0.01) =

0.103 CD(0.05) = 0.079
Critical Difference (CD) Compared a b b c
66
Figure 7: Weight gain (g) in L. vannamei fed on
biofloc grown with different carbon sources
:
2.5
1.5
Control
Weight gain (g)
Wheat flour
1 Tapioca flour
Molasses
0.5
0
7 14 21 28 35 42 49 60
Days
67
SURVIVAL RATE:
Survival (%) of L.vannamei fed on biofloc grown with different carbon sources:
Survival percentages of L.vannamei shrimp in various experimental treatments
are presented in table 11. The percentage of survival in the experimental was lowest
in Control (T1). By the final sampling (60th day) the survival percentage was highest
in Wheat flour (T2) 73.33% and lowest in Control (T1) 46.66%.
The data was subjected to analysis of variance (ANOVA) presented in the
table 12. Statistical analysis has shown that F- value is found to be significant among
treatments. Since F- value is found to be significant, the pair- wise comparison of any
two treatments could be done by computing RBD two way classifications. The
treatment Wheat flour (T2) had shown highest survival rate when compared to the
other treatments. The subsequent positions were occupied by Treatments Tapioca
flour (T3) and Molasses (T4) followed by control. Treatment Wheat flour (T 2 ) has
shown significant difference from all other treatments. There was a significant
difference in between experimental period also.
68
Table 11: The percentage of Survival of L. vannamei
sources:
Treatment Control (%) Wheat Tapioca Molasses (%)
flour (%) flour(%)
0 100 100 100 100
7 100 100 100 100
14 93.33 100 93.33 93.33
21 86.66 93.33 93.33 86.66
28 80.00 86.66 86.66 80.00
35 73.33 86.66 80.00 80.00
42 66.66 80.00 80.00 73.33
49 60.00 73.33 73.33 66.66
60 46.66 73.33 66.66 66.66
69
Table 12: Statistical analysis for Survival %
Treatment means
S. No Average
Treatment 1
78.515
Treatment 2
88.145
Treatment 3
85.923
Treatment 4
82.960
Anova Table
Source of Degrees of Mean sum of F
Sum of squares F cal
variation freedom squares prob
Replications 8 5756.734 719.596 44.541 1.517
Treatments 3 467.942 155.980 9.657 0.002
Error 24 387.703 16.152 - -
Total 35 - - - -
Treatments found Significant at 1% and 5% level of significance
CD(0.01) = 5.294 CD(0.05) = 3.916
Critical Difference (CD) Compared a ab b c
70
Figure 8: The percentage of Survival of L. vannamei fed
on biofloc grown with different carbon sources:
120
100
80
Survival (%)
control
60 Wheat flour
Tapioca flour
40
Molasses
20
0
7 14 21 28 35 42 49 60
Days
71
Specific Growth Rates:
Specific Growth Rates (%) of L. vannamei fed on biofloc grown with different
carbon sources:
Specific growth rates by the end of experimental period (60 days) were
calculated for all the treatments. Specific growth rates for L. vannamei treated with
different carbon source bioflocs were calculated and presented in table 13.
Control (T1) group has the lowest Specific Growth Rate of 4.13%. The highest
value was in Wheat flour (T2) with 4.59%. The treatments that stood second and third
positions were Tapioca flour (T3) 4.49% and Molasses (T4) 4.45% respectively.
72
Table 13: Specific Growth Rates (%) of L. vannamei
sources :
Treatment
T1 T2 T3 T4
Period(Days)
Initial 1.025 1.025 1.025 1.025
Final 12.27 15.92 15.17 14.82
SGR 4.13 4.59 4.49 4.45
73
Figure 9: Specific Growth Rates (%) of L. vannamei fed on biofloc grown with
different carbon sources:
4.7
4.6
4.5
4.4
Specific Growth Rates(%)
Control
4.3
Wheat flour
4.2 Tapioca flour

Molasses
4.1
3.9
60
Days
74
FEED CONVERSION RATIO:
Feed Conversion Ratio of L. vannamei fed on biofloc grown with different carbon
sources:
The Feed Conversion Ratio (FCR) in different experiments of L. vannamei
groups were calculated and presented in the table 14.
The range for Feed Conversion Ratio varied during the experimental period
was 0.50±0.07 (Wheat flour) – 1.80±0.05 (Control).
During the first sampling (7th day), the Feed Conversion Ratio ranged
between 0.50±0.07 and 0.80±0.08. The highest value during this period was recorded
in Control (T1) and the lowest was in Wheat flour ( T 2). On the 14th day of sampling
the highest value of (FCR) 1.39±0.02 was in Control (T 1) and the lowest value
1.08±0.05 was in Wheat flour ( T2). The highest value of 1.58±0.05 was observed in
Control (T1) on 21th day while the lower value of 1.42±0.04 was recorded in Tapioca
flour (T3). On 28th day recorded Molasses (T4) with highest Feed Conversion Ratio
value of 1.58±0.05 and lowest value was in Wheat flour (T2) the value of 1.31±0.04.
The highest value of 1.28±0.04 was observed in Control (T 1) on 35th day while the
lowest value of 1.21±0.02 was recorded in Molasses (T 4). Sampling on 42ndday
recorded highest value of 1.24±0.04 in Molasses (T4) and lowest value of 1.03±0.08
was in Tapioca flour (T3). Sampling on 49th day recorded a highest Feed Conversion
Ratio value of 1.12±0.05 in Molasses (T 4) and lowest value of 1.00±0.02 in Control
(T1). In the last sampling on 60th day recorded Control (T1) with highest Feed
Conversion Ratio value of 1.80±0.05 and lowest value of 1.59±0.07 in Molasses (T 4).
75
The Feed Conversion Ratio was subjected to analysis of variance (ANOVA)
and presented in table 15. Statistical analysis has shown that F- value is found to be
significant among treatments. Since F- value is found to be significant, the pair- wise
comparison of any two treatments could be done by computing RBD two way
classification. The Control (T1) was found to be significantly superior when compared
to the other Treatments. The Molasses (T 4), Tapioca (T3) and Wheat flour (T2)
occupied second, third and fourth positions. There was a significant difference
between the experimental periods also.
76
Table 14: Feed Conversion Ratio of L. vannamei
sources:
Treatment
T1 T2 T3 T4
Period(Days)
7 0.80±0.08 0.50±0.07 0.55±0.05 0.56±0.02
14 1.39±0.02 1.08±0.05 1.19±0.02 1.19±0.05
21 1.58±0.05 1.52±0.02 1.42±0.04 1.53±0.04
28 1.56±0.05 1.31±0.04 1.54±0.07 1.58±0.05
35 1.28±0.04 1.23±0.08 1.23±0.05 1.21±0.02
42 1.08±0.07 1.15±0.05 1.03±0.08 1.24±0.04
49 1.00±0.02 1.05±0.04 1.09±0.04 1.12±0.05
60 1.80±0.05 1.65±0.02 1.69±0.02 1.59±0.07
77
Table 15: Statistical analysis for FCR
Treatment means
S. No Average
Treatment 1 1.312
Treatment 2 1.182
Treatment 3 1.215
Treatment 4 1.255
Anova Table

F cal
Replications 7 3.144 0.449 60.742 1.632
Treatments 3 0.069 0.029 3.104 0.046
Error 21 0.155 0.004 - -
Total 31 - - - -
Treatments found Significant at 5% level of Significance CD(0.05)= 0.085
Critical Difference (CD) Compared a ab b b
78
Figure 10: Feed Conversion Ratio of L. vannamei fed on biofloc grown with
different carbon sources:
1.8
1.6
1.4
1.2
Control
1
Wheat flour
FCR
0.8 Tapioca flour

Molasses
0.6
0.4
0.2
0
7 14 21 28 35 42 49 60
Days
4.6. Composition of Biofloc:
Composition of biofloc in the biofloc treatment group, floc was seen in
anomalous flocculation with bacteria and zooplankton especially rotifers (Plate 8).
The compositions of different floc associated planktonic organisms are given in Plate
8-12. The group of organisms was identified as Rotifer-Brachionus placatilis (Plate
8), Ciliophora protozaon - Stenter roeseli (Plate 9), Globigirina sps (Plate 10),
Verticella sps (Plate 11), Parametium sps (Plate 12). Rotifers were the most dominant
group in all the biofloc treatment tanks. The total number of organisms was
significantly high in the Wheat flour (T2) followed by Tapioca flour (T3), Molasses
79
(T4) and Control (T1). These microorganisms were found to be grazing on the flocs,
when fresh samples were observed under micro scope. In all the biofloc treatment
tanks, mostly the zooplanktons were dominant and very limited number of
phytoplankton could be visualized under the microscope.
80
Plate 7: Biofloc composition
Plate 8: Rotifer-Brachionus placatilis observed in biofloc
81
Plate 9: Ciliophora protozaon -Stenter roeseli observed in biofloc
Plate 10: Globigirina sps observed in biofloc
82
Plate 11: Verticella sps observed in biofloc
Plate 12: Parametium sps observed in biofloc
83
5. Discussion
Water quality parameters in biofloc treatments grown
with different carbon sources:
Water quality parameters did not differ between treatment and levels were within
acceptable ranges for most penaeid species (Wickins, 1976; Van Wyk and Scarpa,
1999).These findings corroborates with (Crab et al., 2009; Emerenciano et al., 2013) who
proposed that BFT as a system to control water quality fluctuations typical presented year
round in ponds.
de Souza et al. (2012) also observed high TAN biofloc treated tanks compared to
control. Mahanand et al. (2013) noticed the biofloc tanks had generally lower inorganic
nitrogen concentration than control tanks.
Correia et al. (2014) found that the only significant difference (p < 0.05) found in daily and
weekly water quality indication between with two treatments were in nitrite, N ,nitrate and
phosphates which were significantly higher in high protein (HP) 40 than in low protein
(LP) 30 treatment. The Total Ammonia Nitrogen (TAN) was peak in 3rd week in the
average of 3.46 mg TAN L-1 and 2.93 mg TAN L-1 for LP 30 and HP 40 treatment
respectively. Although TAN concentrations in the raceways increased in control on Day 26
(reaching a maximum level of 4.16 mg L -1 molasses supplementation helped keep their
under control (Samocha et al., 2007).
Megahed (2010) reported there were no significant differences (p > 0.05) in the pH, water
temperature and salinity between BFT and control green houses . The concentrations of
TAN in the control green house was significantly(p < 0.05) higher than that of other
treatments. There was a significant difference (p < 0.05) in DO
84
between control and all other biofloc treatments. The addition of carbohydrates to the
water column (p < 0.05) reduced the TAN in the Biofloc treated tanks.
The present study TAN concentration was showed fluctuating trend, initially the
accumulation of TAN was observed in biofloc treatment tanks, but from the 42 th of the
experiment TAN concentration started declining trend. As most of the ammonia in the
culture system taken up by heterotrophic bacteria, the declining trend of TAN in the
biofloc treatment tanks may be due to heterotrophic bacterial activity increased with the
progression of the experiment. Rajkumar et al. (2015), noticed similar results in biofloc
treatment tanks. They observed that a sharp decline in TAN on 75 th day. Water quality
values in the present study were found to be optimal for shrimp culture (Davis et al., 1993;
Van Wyk et al ., 1999; Cuzon et al., 2004; Fox et al., 2006; and Megahed, 2010).
Growth Performance of L.vannamei in biofloc
treatments grown with different carbon
sources:
Biofloc consist of variety of bacteria, fungi, micro algae, detritus and other suspended
organism (Hargreaves, 2006). The microbial community associated with biofloc improves
feed utilization and animal growth (Kim et al., 2014). The growth performance of shrimp
in biofloc reared with different carbon sources were significantly (p<0.05) higher than
those in control group in this study (Table 7). Many of previous studies have shown that
growing L. vannamei in biofloc system can improve shrimp survival and growth as
compared to clear water (Moss& Pruder 1995; Burford et al., 2003, 2004; Cohen et al.,
2005; Wasielesky et al., 2006; Azim & Little, 2008; Mishra et al., 2008; Kim et al., 2014).
Not only in L. vannamei biofloc technology has been demonstrated benefits for many
shrimp species in terms of growth and production such as in L. setiferus (Emerenciano et
85
al., 2009), P. monodon
(Arnold et al., 2009; Anand et al., 2014), L. stylirostris (Emerenciano et al., 2011a),
F. brasilensis (Emerenciano et al.,2012), F.paulensis (Ballaster et al., 2010), P.
semisulcatus (Megahed, 2010) and M. rosenbegii (Crab et al., 2010a; Prajith, 2011).
Growth performance was significantly higher (p<0.05) in wheat flour treated tank
than to control and other carbohydrate source treatments (Figure 6). In the present
study increasing in growth performance in biofloc with wheat flour as carbohydrate
source are in the agreement with findings of Megahed, 2010 in P. semisulcatus and
Anand et al., 2014 in P. monodon.
The growth and survival rates of shrimp in biofloc group were significantly higher
than those in control group in our study (Table 7&11). One of the reason for improved
performance is probably related to consuming biofloc by the shrimp. For example Burford
et al. (2004) reported that up to 29% of daily feed intake of this species can come from
flocculated particles in heterotrophic culture system. However, very limited information is
available for harvesting or collecting mechanisms of bioflocs by shrimp. Most bacteria are
free-living and very small, having a typical diameter of about 1µm, but in dense microbial
biomass, and they tend to congregate and create flocs, conglomerates of microbes having a
diameter in the range of 0.1 to several mm (Avnimelech, 2012). Recently, Kent et al.
(2011), based on the examination of the setae on the third maxilliped using a scanning
electron microscope speculated that juveniles (2 g in body weight) of L. vannamei were
able to potentially select and consume suspended food particles of approximately 10µm in
diameter using their net like setae arrangement. This speculation may explain the enhanced
growth performance of shrimp growing in biofloc systems.
In the present experiment tapioca flour used as carbohydrate source showed higher growth
performance than molasses and control but lower than wheat flour.
86
This may be due to ready availability of carbohydrate to microbes in tapioca flour are in
more than in molasses and lower than in wheat flour. Varghese, (2007) carried out similar
studies in extensive culture system of P. monodon with the same carbohydrates sources
and the results were similar. Increase in growth performance of
M. rosenbergii was noticed in tapioca flour as carbohydrate source treatment tanks
compare to control tanks (Hari et al., 2004; Crab et al., 2010a and Prajith, 2011). .
The different organic sources, however appeared to have an effect on the assimilation
of protein and lipid by the shrimp in the biofloc treatment was higher than that of the
control shrimp (Ekasari et al., 2014). Molasses as a carbohydrates source in the present
study showed higher growth performance than that of control shrimp. A similar
observation was obtained for the lipid and protein assimilation resulted in increased growth
that was significantly higher for the molasses and tapioca treatments (Ekasari et al., 2014).
Wheat flour as a carbohydrate source biofloc grown shrimp documented higher weight
gain (2.27±0.01) than to control and other carbohydrate source treatment grown shrimp. In
the case of biofloc treatments in the shrimp culture with different carbohydrate sources
wheat flour as carbohydrate source showed significantly higher growth performance and
weight gain in L. vannamei than those of others (Raj kumar et al., 2015).
In the present experiment biofloc grown shrimp with different carbohydrate sources
showed significant (p<0.05) reduction in FCR compared to control treatment. FCR in the
present study has been demonstrated fluctuating trend, initially from 7 th day to 21st day it
was increased and gradually followed declining trend. It may be due to fluctuating in the
density of floc in different carbohydrate sources treatments. Biofloc supplementation of
12% did not resulted in proportionate increase in growth
87
rate or improvement in FCR of P.monodon compared with control (Anand et al., 2014). On
28th day in regard to wheat flour, 42nd day for tapioca flour and 60th day for molasses
lowest FCR was noticed in different carbohydrate sources utilization for biofloc treatment.
except on 49th day all the treatments of L. vannamei fed on biofloc grown with different
carbohydrate sources performed with better FCR than control treatments. Biofloc is a
proven food source for cultured species and results in a decreased requirement for
supplemental feeding (Avnimelech, 2007; Burford et al., 2004; Kuhn et al., 2009; Li et al.,
2013). Similar results were reported by Anand et al. (2014) in P. monodon at 4% and 8%
supplementation of biofloc treatments.
In the present study the shrimp fed on biofloc grown with different carbon sources showed
better SGR than the control shrimp. Wheat flour utilized as carbohydrate source for biofloc
development to provide diet for L.vannamei was resulted higher SGR than the control. The
results reported in the present study were correlated with the finding of Rajkumar et al.
(2015) in L. vannamei and Anand et al. (2014) in P. monodon.
Avnimelech et al. (2008) also demonstrated that addition of carbohydrate can lead in
increasing in protein utilization and supply of essential lipids and vitamins for the growth
of shrimp. However Liu et al. (2014) reported that shrimp fed grow more slowly (SGR) in
the biofloc treatment tanks than in control. It might be due to the complex nature of
carbohydrates used as carbohydrate source for biofloc development.
Survival rate of L. vannamei in biofloc treatments
grown with different carbon sources:
The results in the present study demonstrated that shrimp survival was higher in wheat
flour than in control and other carbohydrate sources treatments. Significant
88
difference in survival of shrimp in carbohydrate source utilization treatments and control
(p<0.01). The survival of shrimp in biofloc group were significantly higher than those in
control (Kim et al., 2014).
The results observed in present study were correlated with findings of Anand
et al. (2014) in P. monodon, Hari et al., 2006 in extensive culture practice; Prajith,
(2011) in M. rosenbergii and Zhao et al. (2012) in Marsupenaeus japanicus.
Many of previous studies have shown that growing L. vannamei in biofloc systems can
improve shrimp survival and growth performance compared to clear water (Moss &
Pruder, 1995; Cohen et al. 2005; Azim & Little, 2008; Mishra et al. 2008). One of the
reason for improved performance is probably related to consuming biofloc by the shrimp.
Composition of biofloc:
Zooplankton was highly abundant throughout the study in all the biofloc treatment tanks.
Rotifers, ciliates and copepods were present in low abundance at the beginning of the study
and became less due to grazing by the shrimp. Rotifers and copepods were seen almost
exclusively grazing on and within the particles principally located within biofloc. Shrimp
might have consumed a portion of the zooplankton community. Previous studies have
shown that these nutritious planktons are an important food item for shrimp (Moss et al.,
2001). The similar types of plankton community were observed by Ray et al. (2010).
89
6. Summary
The present experiment was conducted in the wet lab of Department of
Aquaculture, College of Fishery Science, Muthukur, SPSR Nellore (District) to study
the “Impact on growth and survival of Litopenaeus vannamei fed on biofloc grown
with different carbon sources”. Post larvae (PL 20) of L. vannamei were brought to
the wet laboratory from BMR hatchery (SPF vannamei hatchery recognised by
CAA). Post larvae transported by road in plastic bags containing 5 ppt saline water.
PL transferred to the same salinity water in the wet lab. Acclimatization was carried
out over 2 weeks. During this time salinity was lowered from 5 ppt to 3 ppt bore well
water at an average rate of 1 ppt day-1. After acclimatization to 3 ppt, they were
transferred to experimental tanks of 1000 ltr capacity water filled up to 600 ltr and
with the stocking density of 15 PL/m2 and continued. Prior to stocking tanks were
allowed to biofloc development for 4 weeks with needful fertilization and continued
aeration. Three carbohydrate materials viz, wheat flour, tapioca flour and molasses
were selected as carbohydrate sources for biofloculation. Preweighed carbohydrate
source was mixed in a glass beaker with the water collected from corresponding
biofloc treatment tanks. All the systems were maintained for 60 days without any
water exchange. Feed supplementation as per biomass prior to carbohydrate addition
was continued throughout the experiment. Control treatment shrimps were fed
without biofloc supplementation.
 The sampling was done at weekly intervals for growth and survival of shrimps. Water
quality parameters were observed daily for all the treatments and control. Triplicates
were maintained for all the treatments and control. The results obtained were
subjected to statistical analysis.
90
 The results obtained in the present study on growth, survival, feed conversion ratio
and specific growth rate of L. vannamei are summarized.
 Important water quality parameters such as dissolved oxygen, temperature, pH, total
alkalinity and total hardness were analysed.
 The water quality parameters were recorded in the following order, dissolved oxygen
varied between 7.12 - 7.98 ppm, temperature range from 25.8 - 30.7 0C, pH values
ranged between 7.71 - 8.03, total alkalinity recorded in the range of 252 - 290 mg l-1
and TAN varied between 0.04 - 0.27 mg l-1. The water quality parameters were
observed for all the treatments and control tanks throughout the experimental period.
 Weekly sampling was done to study growth, survival, FCR, and SGR.
 In the carbohydrate source addition for biofloc treatment tanks growth performance of
15.92g and highest weight gain 2.27g was recorded in the wheat flour utilized biofloc
treatment and lowest value was recorded for the control treatment.
 All the carbohydrate added biofloc treatments were demonstrated higher growth than
to that of control.
 The analysis of variance for growth performance and survival showed significantly
difference among the treatments.
 Biofloc developed with wheat flour as carbohydrate source had showed lowest FCR
(0.5) in the initial period of experiment with the progress of experiment lowest FCR
observed in molasses treatment (1.59). All the biofloc treatments were recorded
reduced FCR levels compared to control treatment.
 The analysis of variance for FCR has shown significant difference for all the biofloc
treated tanks with the addition of different carbohydrate sources.
91
 In the biofloc treatment tanks with utilization of different carbohydrate sources
highest SGR (4.59%) was observed in wheat flour treatment than those treatments and
control.
 The highest survival of 73.33% and lowest 46.66% was recorded in wheat flour as
carbohydrate source used for biofloc treatment and control respectively .All biofloc
treatments with different carbohydrate addition demonstrated better survival than the
control.
 Regarding composition of biofloc are Ciliates, Rotifers and Copepods were identified
in all the biofloc treatment tanks.
 The higher nutritional value of the biofloc developed by wheat flour would have
increased growth and survival of L. vannamei than other treatments. Biofloc system
with different carbon sources utilization certainly play a significant role in the present
system of shrimp culture through better water quality maintenance avoiding the stress
in water exchange, decreasing feed requirement, high yield and survival to obtain
more profit in shrimp farming.
92
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IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI

FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES

DASARI PAMANNA , B.F.Sc.

2 REVIEW OF LITERATURE 13-33

3 MATERIALS AND METHODS 34-46

8 Statistical analysis for Growth Performance 62

Weight gain in L. vannamei fed on biofloc grown with different

10 Statistical analysis for weight gain 65

The percentage of survival of L.vannamei fed on biofloc grown

12 Statistical analysis for Survival 69

Specific Growth Rates (%) of L.vannamei fed on biofloc grown

15 Statistical analysis for FCR 77

Experimental carbon sources molasses, wheat flour and tapioca

3 Experimental set-up in the wet-laboratory 37

3.1.Experimental animals fed with wheat flour as biofloc agent 37

3.2. Experimental animals fed with tapioca flour as biofloc agent 38

3.3. Experimental animals fed with molasses as biofloc agent 38

4 L. vannamei length measurement 45

5 L. vannamei weight measurement 45

6 Biofloc in different carbon source treatment tanks 46

8 Rotifer- Brachionus placatilis observed in biofloc 80

9 Ciliophora protozaon - Stenter roeseli observed in biofloc 81

10 Globigirina sps observed in biofloc 81

11 Verticella sps observed in biofloc 82

12 Parametium sps observed in biofloc 82

mg/l Milligram per liter

ANOVA Analysis Of Variance

ppm parts per million

FCR Feed Conversion Ratio

ppt parts per thousand

TAN Total Ammonia Nitrogen

SGR Specific Growth Rate

ADG Average Daily Weight Gain

SPF Specific Pathogen Free

SCP Single Cell Protein

ProPO Pro Phenol Oxidase

This is to certify that the thesis entitled “IMPACT ON GROWTH AND

Thesis approved by the Student’s Advisory Committee

Chairperson: Dr. A. CHANDRASEKHARA RAO

Member : Dr. D. RAVINDRA KUMAR REDDY

Member : Dr. N. MADHAVAN

This is to certify that Mr. DASARI PAMANNA, I.D. No. MFM/2013-002

“IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI

FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES”

diploma of any University.

Date : Dr. A.CHANDRASEKHARA RAO

I Mr. DASARI PAMANNA, hereby declare that the thesis entitled

“IMPACT ON GROWTH AND SURVIVAL OF LITOPENAEUS VANNAMEI

FED ON BIOFLOC GROWN WITH DIFFERENT CARBON SOURCES”

OF FISHERY SCIENCE majoring in AQUACULTURE is the result of original research

Date: DASARI PAMANNA)

I am grateful to Dr. T.V.Ramana, Dean, Faculty of Fishery Science, SVVU,

I profess my heartful gratefulness and sincere regard to my esteemed guide

I express my sincere gratitude to Dr. D. Ravindra Kumar Reddy, Professor

I owe a deep sense of reverence to Dr. N. Madhavan, Associate Professor &

I mostly thankful to Dr.V.Venkateshan, senior scientist,CMFRI cochin, for

I owe my obligations to the faculty member of Mrs. Jesinta, Assistant

Title of the thesis : “Impact on growth and survival of Litopenaeus vannamei

fed on biofloc grown with different carbon

Submitted for the

award of degree : Master of Fishery Science in

Aquaculture Faculty : Faculty of Fishery Science

Department : Department of Aquaculture