Comparative Efficacy of Biofloc and (PDFDrive) PDF

COMPARATIVE EFFICACY OF BIOFLOC AND FEED BASED
COMMON CARP (CYPRINUS CARPIO, L.) PRODUCTION SYSTEM

WITH SPECIAL REFERENCE TO ENVIRONMENTAL HEALTH
A Thesis
Submitted to the
West Bengal University of Animal and Fishery Sciences,
In partial fulfilment of the requirements for the Degree of
Master of Fishery Sciences
In
AQUACULTURE
By
Madhurima Sarker ~'{
B. F. Sc \,.\'o~
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Department of Aquaculture
Faculty of Fishery Sciences
WEST BENGAL UNIVERSITY OF ANIMAL AND FISHERY SCIENCES
Chakgaria Campus, Kolkata - 700 094, West Bengal, India
2015
(J)etficatetf to
fM.y (J)itfun antffM.aa43a6a
West Bengal University of Animal and Fishery Sciences

Department of Aquaculture
5, Bhuderhat Road, Chakgaria;
P .0. Panchasayar, Kolkata- 700 094
Dr. S. K. Das Ref. No. -------------------

Associate Professor Date
Certificate
This is to certify that the research work embodied in the thesis entitled "Comparative
efficacy of biofloc and feed based common carp (Cyprinus carpio, L.) production
system with special reference to environmental health" Submitted by Miss.
Madhurima Sarker in partial fulfillment of requirement for the degree of Master of
Fishery Science (Aquaculture) in the Faculty of Fishery Sciences, West Bengal
University of Animal And Fishery Sciences is the faithful and bonafide research work
carried out under my direct supervision and guidance. The results of investigation
reported in this thesis have not so far been submitted for any other degree or diploma.
The assistance and help received during the course of investigation have been duly
acknowledged.
Date: .......... .
Place: Chakgaria
Kolkata- 700094
WEST BENGAL UNIVERSITY OF ANIMAL AND FISHERY SCIENCES

5-Budherhat Road, P.0.-Panchasaya r
Chakgaria, Kol-94
APPROVAL SHEET
APPROVAL OF EXAMINERS FOR THE AWARD OF THE

DEGREE OF MASTER OF FISHERY SCIENCE
(Aquaculture)
We, the undersigned, have been satisfied with the perfonnance of

Miss Madhurima Sarker, in the Viva-Voce Examination, conducted today, the
....S":tk ...... October, 2015, recommended that the thesis be accepted for the
award of the Degree of Master of Fishery Science in Aquaculture.
Signature
Name
1. Dr. S. K. Das Chairman,

Advisory Committee
2.~~1:.~.. f ~ .... External Examiner
3. Dr. T. K. Ghosh Member,

Advisory Committee
4. Dr. B.K. Das Member,

Advisory Committee
5. Prof. S. S. Dana Member,

Advisory Committee
It is my proudprivilege to convey my deepest sense ofgratitude andfatfwmfess regartfs to my
reverend" guufe ([)r. Sfii6 'l(in~r (])as, )f.ssociate <Professor, ©epartment of )f.quacuCture, 'Facu[ty of
Pisfiery Sciences, West (Benga[ Vniversity of )f.nima[ and 'Fisfiery Sciences, and Cfiairman of tfie
)f.dvisory Committee for fiis 6enevofent guidance, constructive criticism, eruaite suggestions, untiring
dose supervision, constant inspiration, /isen interest in pfanning and conducting tfie present
dissertation andpreparation of tfiis tfiesis.
I am intfe6ted to (J)r. P. (J3iswas, 'Vice Cfiance[fur, W.(J3. V.)f.. P.S.; <Prof S. (J3iswas, Contro[(er
of 'E.:{amination, W.(}3. V.)f.. 'F.S and <Prof (J3. 1(, (])as, (J)ean, 'Facu[ty of Pisnery Sciences as weff as
fionoured mem6er of tfie )f.dvisory Committee, for proviaing necessary infrastructure and otfier
facifities.
I fee( de[igfited to e:wress my deep sense of gratitude to <Prof :N. CR., Cfiattopadfiyay, <£.)(:
<Professor, (J)r. rt'K., qfiosfi, Senior ~ader, (J)r. (:Mrs.) Sangfiamitra (J3fiattacfiaryya, quest Lecturer,
©epartment of )f.quacuCture for tfieir precious assistance and constant inspiration during tfie entire
period of researcfi wort
I am o6[iged to <Prof S.S. (J)ana, fionoured mem6ers of tfie )f.dvisory Committee for va(ued
support and academic advisement on affoccasions.
I wisfi to offer my tfian~ :Manoj (])a and (J'apai (])a and tfie otfier mem6ers of tfie li6rary of
tfiis facuCty for supp(ying 600~, journalr and references and arrangement of interna[ internet facifity
at aff time.
I wouU fi/is to e:wress my specia[ tfian~ to, J{irma(ya tfa, Suftumar da, (J'aresfi da, '}(flusfiift
da, <Papri tfi for tfieir assistance during tfie period ofdissertation wort
I wisfi to e)(Jent my intfe6tedness and fieartiest tfian~ to my seniors, )f.mit (])a, (J3anasree tfi,
'lara di. and a[{ my otfier seniors for tfieir R,jntf fie{p during tfie neetffu[ time ofdissertation wort
I e)(Jentf my intfe6tness antf fieartiest tfian~ to my year mates Sudfiesna, 'lanusree, Suprit~
Sayantan~ )f.ntara, <Rjya, Sourav, )f.jit, )f.rfta, Susovon, Sofiom, <Pritam, J{antfan, (J'ufafi.,, <Puspendu,
(J3aijnatfi and otfiers for tfieir co-operation rendered to me for time tfirougfiout tfie period ofstutfy.
I wi.sfi to offer my tfian~ to my juniors lpsfiita, <fufiR.9, Sanjufa, Saurav, Sni[Jdfia and otfiers
for tfieir co-operation for compfetion of tfie wor{
Once again, I wouU filzy to e:q,ress my sincere and specia{ tfian~ to ;4.mit da andSfianRg.r da
for tfieir e:{Jreme co-operation at eacfi and every step of my researcfi wor{
I wouU filzy to e:q,ress my deep sense of gratituae and Cove to my 6ewved grandparents for
tfieir 6fessitl{J upon me.
Last, 6ut not tfie feast, I wi{{fai{from my duty, if I do not express my deep jeefitl{Js, to my
6ewved parents, sister <Banhi and a{{ tfie fami{y mem6ers and pisi, pison, mama, mami, masi, masi,
meso, jfietfiu, RJZ{u for tlieir CotlfJ pat'ience, support, sacrifice, encouragement, guidance, constant
inspiration and remark,g.6fe co-operation tfirough a{{ means.
Pina{(y, I ascri6e a{{ gCory to tfie gracious (jod from whom a{{ good thitl{Js come, who has
sfiowered a{{ his fjnd 6fessitl{JS and 6enevofent graces upon me in {ife, for 6estowing me witfi tfie
6fessitl{JS to see tfiis day.
Jno.dhluu~ ~
Place: Chakgaria Campus (Madhurima Sarker)
Date: tito,\o.~1'5
Avg. Average
BFT Biofloc Technology.
CR Community Respiration
DAP Di-ammonium Phosphate
DDT Dichlorodiphenyltrichloroethane
EDTA Ethylene Diamine Tetraacetic Acid
etc. Etcetera
FAO Food and Agriculture Organization
FCE Food Conversion Efficiency
FCR Food Conversion Ratio
g Gram
GVA Gross Yalu Added
GDP Gross Domestic Production
Govt. Government
GPP Gross Primary Product
ha Hectare
ICAR Indian Council of Agricultural Research
Nos. Numbers
NPP Net Primary Product
Rs. Rupees
SGR Specific Growth Rate
SI. No. Serial Number
sp. Species
Wt. Weight
Yr. Year
CHAPTER PARTICULARS PAGE NO.
NO.
1. Introduction 1-4
2. Review of literatures 5-26
3. Materials and methods 27-32
4. Experimental Protocol 33
4. Results and Discussion 34-53
5. Summary and Conclusion 54-55
6. Future scope of Research 56
7. Bibliography I-XVIII
LIST OF FIGURES
SI.
Figure Page
No. Title of the Figures
No. No.
1. 3.1. Experimental protocol followed in the present study. 33
2. 4.1.2. Temporal changes of temperature of water in 34

different treatments employed.
3. 4.1.3. Temporal changes of pH of water in different 35

treatments employed.
4. 4.1.4. Temporal changes of dissolved oxygen of water 35

in different treatments employed.
5. 4.1.5. Temporal changes of hardness of water in different 36

6. 4.1.6. Temporal changes of total alkalinity of water in 36

7. 4.1.7. Temporal changes in the ortho-phosphate 37

concentration of water in different treatments
employed.
8. 4.1.8. Temporal changes of ammonia-nitrogen 37

concentration of water in different treatments
employed.
9. 4.1.9. Temporal changes of nitrate-nitrogen concentration 38

of water in different treatments employed.
SI. Figure Page
Title of the Figures
No. No. No.
Temporal changes of nitrite-nitrogen concentration of

10. 4.1.10. 38
water in different treatments employed.
11. 4.1.11. Temporal changes of biological oxygen demand of 39

water in different treatments employed.
12. 4.1.12. Temporal changes of gross primary productivity in 39

13. 4.1.13. Temporal changes of net primary productivity in 40

14. 4.1.14. Temporal changes of community respiration in 40

15. 4.1.15. Temporal trend in total phytoplankton in different 41

16. 4.1.16. Percent(%) distribution of phytoplankton in different 42

17. 4.1.17. Temporal trend in zooplankton in different treatments 43

employed.
18. 4.1.18. Percent(%) distribution of zooplankton in different 44

19. 4.1.19. Temporal trend in total plankton in different 45

SI. Figure Page
No. No. No.
Temporal changes of pH of soil in different treatmes

20. 4.1.20. 45
employed.
21 4.1.21. Temporal changes in the organic carbon of soil in 46

Temporal changes in the available phosphorus of

22 4.1.22. 46
soil in different treatments employed.
23 4.1.23. Temporal trend in body weight of common carp in 47

24 4.1.24. Temporal trend in body weight gain(%) of common 47

carp in different treatments employed.
25 4.1.25. Temporal trend in specific growth rate(%) of 48

common carp in different treatments employed.
26 4.1.26. Temporal trend in absolute growth of common carp 48

in different treatments employed.
27 4.2.27. Modular trend of absolute weight gain in different 49

28 4.2.28. Absolute weight gain(%) of fish in different 49

29 4.2.29. Modular trend of mortality rate of fish in different 50

SI. Figure Page
No. No. No.
30
Modular trend of FCR in different treatments
4.2.30. 51
employed.
31 4.2.31. Modular trend of FCE in different treatments

51
employed.
32 4.2.32. Relationship between BOD1 and NPP in different

biofloc treatments. 52
Relationship between total plankton and NPP in

33 4.2.33. 52
different biofloc treatments.
ABSTRACT
Biofloc Technology (BFT) is considered as an environmental friendly aquaculture system
in
which nutrients could be continuously recycled and reused. This technique is also referred
as the built in bioreactors and is successfully employed both in shellfish and finfish culture
systems. The basic principle is the production of microbial floes that attach to each other.
These microbial-floes either float or are suspended at the bottom and become a nutritiou
s
food for fish. In these systems, a co-culture of heterotrophic bacteria and algae is grown
in
floes under controlled conditions within the culture pond. The carbon sources play a pivotal
role in the biofloc formation, composition and its nutritive values. With the above
background, the present study on comparative efficacy of biofloc and feed based common
carp (Cyprinus carpio, L.) production system with special reference to environmental
healthwas undertaken and conducted in the outdoor experimental facilities of
the
Department of Aquaculture, Faculty of Fishery Sciences, W. B. U. A. F. S. for a period
of
three months from March to May, 2015. The findings of the study indicated that biofloc
alone was not sufficient enough for the comparable growth of the test fish under
supplementary feeding regime. Biofloc alone resulted only in 2. 79 times absolute weight
gain against 9.27 times with only feed@ 6% body weight/day however, mortality rate of
the
test fish was highest (33.33%) in biofloc only treatment. It was clear that biofloc improve
d
the performance of the supplementary feed both in terms FCR and FCE. Presence ofbioflo
c
improved the FCR from 1. 75-2.09 to2.31 in control. In spite of less absolute weight gain
with feeding @ 3% body weight/day because of the most favourable FCR and less nutrient
loading, the system was proved to the economically and ecologically more sustainable
in
presence of bioflocs. Biofloc not only supported the test fish nutritionally but supported
the
phytoplanktonic productivity as well.
Moreover, it also favoured phosphate mineralization in the soil phase. The
abundance myxophyceae and rotifer community in the biofloc treatments with feed
supplementation @ 6% body weight/day indicated organic loading and therefore,
was
inferior in terms of ecological health.
9ntro ducti on
1. Introduction
Aquaculture has always played a significant role in the economy by its contribution to
income and wealth through the supply of nutritious food (Pillai and Kathia, 2004).
Constituting about 5.68% of the global fish production, India today is the second largest
fish producing nation in the world. India is also a major producer of fish through
aquaculture and ranks second in the world after China. Fish production has increased from
41.57 lakh tonnes (24.47 lakh tonnes for marine and 17.10 lakh tonnes for inland fisheries)
in 1991-92 to 95.79 lakh tonnes (34.43 lakh tonnes for marine and 61.36 lakh tonnes for
inland fisheries) in 2013-14 (DAHDF, 2014). During the first two quarters of 2014-15,
the fish production is estimated at 4.37 million tonnes. The sector contributes about 0.92%
to the overall Gross Value Added (GVA) and 5.58% of the agricultural GVA at current
prices for the year 2013-14 (DAHDF, 2014). Fish and Fish Products export was 9,83,756
tonnes worth Rs. 30,213.26 crores during 2013-14 (DAHDF, 2014). The aquaculture
industry is growing fast at a rate of 9% per year since 1970's (FAO, 2008) but is going
through unsustainability in the meaning of environmentally and economically, haunted by
number of environmental and social issues (Boyd, 1990). Average growth in fish
production during 2013-14 was 5.9% (DAHDF, 2014). Fishery sector share 0.9% of total
GDP and 17.5% of total agricultural sector GDP (DAHDF, 2014).
Moreover, this sector plays a major role in the socio-economic development of the
country. It has been recognized as a powerful income and employment generator. It is a
source of cheap and nutritious food and a foreign exchange earner. Development of fishery
sector can ensure the food security as well as tackle the problem of unemployment.
Aquaculture continues to be the fastest growing animal food-producing sector and to
outpace population growth. Aquaculture contributed 48% of aquatic animal food for
human consumption in 2007 (FAO, 2012).
All forms of food production like any other human activity affect environment in one
way or another (Pillai, 1992). It is true in the case of aquaculture also. Land-based fish
farms produce effluents that may have, if not properly handled, a negative impact on the
quality of water courses, rivers and soil (Acierno and Zonno, 2010). Excessive
accumulation of toxic inorganic nitrogen from feed, faEces and other external factors in
the aquaculture ponds is always posing major threat to pond ecology, thus not only
deteriorating pond environment but also the environment of the surrounding aquatic
ecosystem (Kurup, 201 Ob). Over usage of antibiotics, disease and exotic fish transfer to
1
?nfrotfucfion
wild and omitting of heavy load nutrients are under consideration. In aquaculture, the
accumulated waste must be removed continuously to maintain optimal growth conditions
and the health of the cultured organism. The commercial success of fish farms and the
quality of aquaculture product will depend increasingly on their success in either
minimizing the production of waste or utilizing waste as inputs to other production
processes. Therefore, the management of solid wastes and dissolved substances is one of
the most important aspects in the aquaculture industry today. Over dependence of fish
meal and privatization of public common leads to physical conflicts among fishermen and
depletion of natural resources. All these factors searches opportunities for economic and
environmental developments permeate in to many facets of the industry (Kuhn et al.,
2010). The expansion of aquaculture is restricted due to land costs and by its strong
dependence on fishmeal and fish oil (Browdy et al., 2001; De Schryver et al., 2008). Such
ingredients are one of the prime constituents of feed for commercial aquaculture (Naylor
et al., 2000). Of the total production expenses 50% represents for feed cost, which is
predominantly due to the cost of protein component in commercial diet (Bender et al.,
2004). For the production of 1 kg live weight fish needs 1-3 kg dry weight feed (FCR is
about 1-3, Naylor et al., 2000). Of the total nitrogen input in culture ponds only 20-30%
retain in biomass and the rest becomes a polluting agent of the water by producing high
level toxicants such as ammonia and nitrite leads to eutrophication in the surrounding
water of the aquaculture farms (Briggs, and Funge-Smith, 1994; Jackson et al., 2003;
Thakur et al., 2003). Approximately 36% of feed excreted as in the form of organic waste.
Thus tool to make up industry more sustainable pond management should be geared
towards improving nutrient retention (Hari et al., 2006). In order to make aquaculture
industry more successful, it is imperative to develop technology that will increase
economic and environmental sustainability (Kuhn et al., 2010).
The rapid growth of aquaculture aimed at continued expansion is necessary to meet
future protein demand. This will depend upon: increasing productivity without
overburdening land and water resources, applying sustainable technologies which
minimize environmental effects and developing cost-effective production systems which
support economic and social sustainability. In order for aquaculture to be completely
successful, the industry will need to develop 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 as feed
Sarker, M., M. "F. Sc. "Thesis, 20(3, Oefl of7'.ttuacullure 2

'Jnfroducfion
costs can account for 50% of operational expense (Van Wyk et al., 1999) while reducing
their impact on overexploited natural fisheries (Tacon et al., 2006 and Naylor et al., 2009).
The prime goal of aquaculture expansion must be 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 providing an equitable cost/benefit ratio to support economic and social
sustainability (A vnimelech, 2009). All these three prerequisites for sustainable
aquaculture development can be met by biofloc technology. The environmental friendly
aquaculture system called "Biofloc Technology (BFT)" is considered as an efficient
alternative system since nutrients could be continuously recycled and reused. The
principles of growing fish or shrimp in limited water exchange intensive ponds were
developed simultaneously for shrimp in the Waddel Mariculture Centre in the USA and
for fish, mostly tilapia, in Israel (Avnimelech et al., 1989; A vnimelech et al., 1994;
Hopkins et al., 1993; Chamberlain and Hopkins, 1994) and practiced in the USA (Serfling,
2000), in the beginning of the 1990's. Bio floe technology is for increasing the production
and reducing the harmful chemicals from the aquaculture systems (Avnimelech, 2003;
2010). This technique is also referred as the built in bioreactors (Kurup, 2010b). BFT is
successfully employed both in shellfish and finfish culture systems (Milstein et al., 2001;
Burford et al., 2003, 2004; Avnimelech, 2005; Wasielsky et al., 2006; Serfling, 2006). 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. Currently, BFT has
received alternate appellation such as ZEAH or Zero Exchange. The basic principle is the
production of microbial floes that attach to each other (Burford et al., 2004). These
microbial-floes either float or are suspended at the bottom and become a nutritious food
for fish. In these systems, a co-culture of heterotrophic bacteria and algae is grown in floes
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. The carbon sources play a pivotal role in the
biofloc formation, composition and its nutritive values (Hollender et al., 2002; Oehmen et
.Sf.lrker, M., M. 'F. Sc. 'Thesis, 20f7, Oepl. of7lttuf.lcufture 3
'Jnfrolucfion
al., 2004). The bioflocs production depends on the quality of added substrate and its C:N
ratio (A vnimelech, 2007). So, the organic carbon can be supplied either as additional
organic carbon source like glucose, acetate, glycerol etc. or by changing the feed
composition by increasing its organic carbon content (Avnimelech, 1999). Carbohydrates
and lipids are cheaper energy sources compared to proteins. Optimal level of protein and
the protein-sparing effect of non-protein nutrients such as lipids or carbohydrates may be
effective in reducing feed costs (Gumus and Ikiz, 2009). Added advantages of this system
are that the protein content of the feed provided can be reduced without resulting in a high
FCR, and the feed waste can be minimized.
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. Over the period
of January 2008 to May 2009, the global fishmeal market varied from a low mean of about
$900 to a high mean of $1250 per metric ton. During the same time frame, soybean meal
varied approximately from a low mean of $375 to a high mean of $550 (FAO, 2009).
Aquaculture production of an important tilapia species, Oreochromis niloticus, more than
doubled between 2001 and 2007 from 1,030,888 tonnes to 2,121,009 tonnes (FAO, 2010).
Production of the most commonly cultured marine shrimp, Litopenaeus vannamei, more
than doubled from 9,82,663 tonnes in 2003 to 2,296,630 tonnes in 2007 (FAO, 2010).
Thus the use of biofloc represents a viable and more sustainable feed option due to cost,
the manner in which it is generated, and the potential that it can ease the pressure on wild
fisheries by reducing at least some of the demand for fishmeal.
With this background, the proposed research work is designed to investigate the
potential of biofloc as substitute of supplementary feed in common carp (Cyprinus carpio,
L.) culture and to assess the qualitative change in environmental health of the culture
system under biofloc treatment over conventional feed applied culture system. Therefore,
the present study has been designed with the following objectives:
i) To investigate the potential of biofloc as substitute of supplementary feed
in Common carp (Cyprinus carpio, L.) culture.
ii) To assess the qualitative change in environmental health of the culture
syatem under biofloc treatment over conventional feed applied culture
system.
Sarker, M., M. 'F. Sc. 7heJiJ, 2.0fj, 1Jeff. of:AtJuacufture 4

9l ev ie w of
L it er at ur es
2. Review of Literatures
The human population has grown from 1.5 to 6.4 billion from 1900
till now and
malnourishment is one of the biggest challenges with an estimated 840 million
being in a state
of malnourishment (UNWFP, 2005). So it is essential to ensure the health
of the world
population by providing nutritionally balanced, especially protein-rich food.
Animal
husbandry and fisheries are the two sources of animal protein for the world popula
tion while
fish and fishery products are considered to be among the safest food of
animal origin
(MPEDA, 1992) containing all essential amino acids, and is an excellent source
of essential
highly unsaturated fatty acids (de Deckere et al., 1998; Horrrocks and Yeo,
1999; Connor,
2000; Ruxtone et al., 2005). Therefore, the global demand for fish will intensi
fy in future and
world will need an extra 40-60xl06 tonne of food fish by 2020 (De Silva and Davy,
2010).
For many years aquaculture played a relatively minor role in global fish produc
tion, but
its significance has increased dramatically over the past 20 years. This is
a fast growing
industry with an average growth rate of about 12% during the past few decade
s. However,
intensification in aquaculture coincides with pollution of the culture water by
an excess of
organic materials and nutrients that are likely to cause acute toxic effects
and long term
environmental risks (Piedrahita, 2003). This increased pressure on pond
ecosystem and
increased demand for environment protection has driven aquaculture from
extensive
cultivation towards closed water intensive cultivation (Crab et al., 2007). For
long, the most
common method for dealing with this pollution has been the use of continuous
replacement
of the pond water with external fresh water (Gutierrez-Wing and Malone, 2006).
2.1. Status of aquaculture
2.1.1. World aquaculture
Global production of fish, mussels and crab in 2010 was almost 60 million
tonnes.
Aquaculture production is now about three quarters of that from ocean fish and
seafood caught
in the wild. With per capita supply from aquaculture increasing from 0.7 kg in
1970 to 19.2
kg in 2012 (PAO, 2014), an average annual growth rate of6.6% was recorded.
Fish accounted
for 16.7% of the world population's intake of animal protein and 6.5% of all protein
consumed
(PAO, 2014). According to FAO (2012), among total fish production of 154
million tonne,
5
'Rftview ofl:.iferafure
aquaculture production comprise 41 % ( 63 .6 million tonne) and capture fisheries production

comprise 90.4 million tonne. By 2015, aquaculture will pass capture fisheries as the leading
source of food fish for the human population and the proportion contributed by aquaculture
will continue to increase each year thereafter (Lowther, 2007). In 2009, Asia accounted for
89% of world aquaculture production by quantity and 79% by value (FAO, 2010). China
alone produces more than 62% of the world's aquaculture volume and 51 % by value (F AO
2010).
When considered in terms of total weight, in 2005 mariculture accounted for
approximately 51 % of production while freshwater accounted for 43% (FAO, 2007).
Freshwater becomes more important, accounting for 60% of production by quantity and 48%
by value, compared to 32% and 31 %, respectively for mariculture and 8% and 13%,
respectively for brackish water (F AO, 2009). Worldwide in 2008, freshwater fishes were the
dominant group in terms of productions (71.9%, 24.2 million tonnes in 2010) and most of this
is composed of different species of carps (FAO, 2010).
2.1.2. Indian aquaculture: Its growth and development
In India fish culture is practiced in less than 30% of the total areas available. This sector
has a potential to create huge market, provided fish cultivation is done on a scientific basis.
India occupies the 2nd position in fish production next to China, which produced 90.62 lakh
tonne in 2012-2013. It also occupies the 2nd position in aquaculture production and
contributes about 5.43% of global fish production. In India, fishery sector is the source of
livelihood for 14.9 million people and contributed about more than Rs. 30,213.26 crores
through export of marine products (MPEDA, 2014).
2.2. Feed in Aquaculture
Food is the most importan t and vital factor for the growth and survival of the living beings
on the face of earth (Royce et al., 1972; Joadder et al., 2006). In recent years, the main
objectives in fish farming are improvement of the foods used and the reduction of nutrients
excreted in the water (Fournier et al., 2003). Various feed ingredient of both plant and animal
origin, with different crude protein contents e.g. maize, gluten, cotton seed meal, soybean
6
'f<lview ofl.i{era{ure
meal, wheat bran, rice bran, blood and bone meal and fish meal etc. are being used in fish feed
formulation. Feeds are used in aquaculture to increase aquatic animal production above that
possible with fertilizers and manures (Boyd and Tucker, 1998). Aquafeed, being considered
the most vital input cost, occupy almost 50-70% of the budget allocation for production
(Anon, 1983; Veerina et al., 1993).
In addition to the growing demand for seafood for human consumption, the demand for
aquatic products used by the industrial sector for conversion into fishmeal and fish oil products
also increases (Peron et al., 2010). About 5-6 million tonne oflow-valu e/trash fish are used
as direct feed in aquaculture worldwide either provided without processing or as part of farm-
made feeds (FAO, 2009) and about 1.9 kg of wild fish is required for every kilogram of fish
produced (Naylor et al., 2000). Therefore, research in recent times has focused on the
development of feed substitution strategies with a minimal supply of fishmeal and fish oil,
which are then replaced by alternative and cheaper sources of protein such as plant proteins.
This inspired researchers to develop the biofloc technology, which is also applicable to
intensive and semi-intensive systems.
2.3. Biofloc technology
Biofloc technology (BFT) is a new technology that is basically the retention of waste and
its conversion to biofloc as a natural food within the culture system. Biofloc technology
promotes suspended growth in ponds, consisting of phytoplankton, bacteria, and aggregates
of living and dead particulate organic matter and grazers of bacteria (Hargreaves, 2006) in
order to recycle feed residues and raise feed efficiencies. This is based upon the basic
principle of the activated suspension technique and the C/N ratio maintenance. If carbon and
nitrogen are well balanced in water, the nitrogenous waste generated by the cultivated
organisms especially ammonia will be consumed into bacterial biomass and increase in C/N
ratio stimulates heterotrophic bacterial production (Schneider et al., 2005). Through the
decomposition of organic matter by microorganisms leads to the production of new bacterial
cell amounting 40-60% of the metabolized organic matter (A vnimelech, 1999). This aerobic
decomposition can be higher in high C/N ratio (through the addition carbohydrate source
S'arkflr, M., M. 'F. Sc. 'lhesis, 2.0f'j, Ve('l of7itjuacullure 7

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should be economically feasible) with high oxygenic conditions. By adding carbohydrates to

the pond, heterotrophic bacterial growth is stimulated and nitrogen uptake through the
production of microbial proteins takes place (Avnimelech, 1999).
BFT is a green approach in aquaculture knowing in different names such as zero
exchange autotrophic-heterotrophic system (Burford et al., 2003, Burford et al., 2004;
Wasielesky et al., 2006) active sludge or suspended bacterial based system (Rakocy et al.,
2004) single cell protein production system (Avnimelech et al., 1989) microbial floe system
etc. Now biofloc has been focused internally as an alternative protein for fish meal, mainly
produced in the form of microbial meal (Kuhn et al., 2009). 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). Immobilization of ammonium by heterotrophic bacteria
occurs much more rapidly because the growth rate and microbial biomass yield per unit
substrate of heterotrophs are a factor 10 higher than that of nitrifying bacteria (Hargreaves,
2006). The microbial biomass yield per unit substrate of heterotrophic bacteria is about 0.5 g
biomass C/g substrate C used (Erling et al., 2006). Suspended growth in ponds consists of
phytoplankton, bacteria, aggregates of living and dead particulate organic matter, and grazers
of the bacteria (Hargreaves, 2006). Typical floes are irregular by shape, have a broad
distribution of particle size, are fine, easily compressible, and highly porous (up to more than
99% porosity) and are permeable to fluids (Chu and Lee, 2004).
The success of this technology mainly depends on the selection of species, because the
cultured animals should have the ability to harvest the bacterial floccules developed in the
system, and should have the ability to digest and utilize the microbial protein. Experimental
trials done by different researchers showed that microbial floes of different sizes can be taken
up by fish or shrimp as feed (Avnimelech et al., 1989; Beveridge et al., 1989; Rahmathulla
and Beveridge, 1993; Tacon et al., 2002; Burford et al., 2004) which will help to reduce the
8
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cost of production by reducing the protein content of the artificial feed and improving the
overall economics (Mcintosh, 1999; Moss, 2002).
2.3.1. The role of microorganisms
In BFT, microorganisms play key role in nutrition of cultured animals as this macro-
aggregate is rich in protein and lipid available in situ all the time (Hari et al., 2006). Bioflocs
also provide the substrate required by most bacteria and can supply some refuge from
predators (De Schryver et al., 2008). In the water column occurs a complex interaction
between organic matter, physical substrate and large range of microorganisms such as
phytoplankton, free and attached bacteria, aggregates of particulate organic matter and
grazers, such as rotifers, ciliates and flagellates protozoa and copepods (Ray et al., 2010).
Regarding to maintenance of water quality, control of bacterial community over autotrophic
microorganisms is achieved using a high carbon to nitrogen ratio (C:N) (Avnimelech et al.,
1994) which nitrogenous by-products can be easily taken up by heterotrophic bacteria
(A vnimelech, 1999). High carbon to nitrogen ratio is required to guarantee optimum
heterotrophic bacteria growth (Avnimelech, 2007; Emerenciano et al., 2012), using this
energy for maintenance (respiration, feeding, movement, digestion, etc), but also for growth
and to produce new cells. High carbon concentration in water could supersede the carbon
assimilatory capacity of algae, contributing to bacteria growth. Aerobic microorganisms are
efficient in converting feed to new cell material (40-60% of conversion efficiency), rather than
higher organisms that spend about 10-15% to rise in weight (Avnimelech, 2012).
Bacteria and other microorganisms act as very efficient "biochemical systems" to degrade
and metabolize organic residues (A vnimelech, 1999). In other words, they recycle very
efficiently nutrients in a form of organic and inorganic matter (un-consumed and non-digested
feed, metabolic residues and carbon sources applied as fertilizers) into new microbial cells.
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, com, rice, tapioca, etc) will be applied before
fry/post-larvae stocking and during grow-out phase, aiming to maintain a high C:N ratio (-15-
9
'l<lview ofl.iferafure
20: 1) and to control N compounds peaks. 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). The carbon source serves
as a substrate for operating BFT systems and production of microbial protein cells
(Avnimelech, 1999). There are many considerations for its selection such as costs, local
availability, biodegradability and efficiency of bacteria assimilation.
2.3.2. Mechanisms of binding microbial cells into floes
The flocculation of microbial communities is a complex process. Within the floe's
matrix, a combination of physical, chemical and biological phenomena is operating. The exact
mechanisms and the methods to engineer microbiological floes remain largely unknown. The
main constituents that can be found within the floe matrix are the extracellular polymeric
substances. These structures form a matrix that encapsulates the microbial cells, and play a
major role in binding the floe components together. The Factors influencing floe formation
in bio-flocs technology presence of these structures in activated sludge systems can be
substantial, up to 80% of the total mass (Hantula and Bamford, 1991; Liu and Fang, 2003).
They are typically made up of polysaccharides, proteins, humic compounds, nucleic acids and
lipids (Zita and Hermansson, 1994). They are produced as slime or capsule layers under
various nutritional conditions but particularly in case of limitation by nutrients such as e.g.
nitrogen (Steiner et al., 1976).
2.3.3. Basic types of biofloc systems
2.3.3.1. In-Situ Bioflocs
In-situ bioflocs are formed in culture ponds or tanks by manipulating the carbon: nitrogen
ratio (C:N) to values of 8: 1 or higher. This can be accomplished by supplementing a carbon
source such as sucrose, molasses, and glycerin or calcium acetate. Alternatively, it has been
shown that low-protein feeds can also result in a higher C:N ratio in the culture water.
Providing the appropriate C:N ratio promotes the growth of heterotrophic bacteria while
assimilating ammonia directly from the water column into bacteria biomass. Under high C:N
Sark,pr, M., M. 'F. Sc. 1"hesis, 20(5, 'Dept. of7!.ttuacufture 10

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ratio conditions, heterotrophic bacteria are the primary components of bioflocs. Shrimp and
fish can graze on these bioflocs for nutrition (Kuhn et al., 2010).
2.3.3.2. Ex-Situ Bioflocs
Ex-situ bioflocs are formed in suspended-growth biological reactors. Biological reactors
can be employed to remove accumulated solids and nitrate from aquaculture production
effluent waters. Carbon supplementation can also be used to promote biological activity.
Bioflocs produced in the biological reactors can be used as a feed supplement for shrimp or
fish. The cleaned effluent water can then be reused for aquaculture production or discharged
from the production facility (Kuhn and Lawrence, 2012).
2.4. Factors influencing floe formation and floe structure in bioflocs technology
2.4.1. Mixing intensity
The mixing intensity imposed by a chosen aeration device at a certain power input will
determine the steady-state floe size, this is the equilibrium between the rate of aggregation
and the rate of breakage, and the floe size distribution (Chaignon et al., 2002; Spicer and
Pratsinis, 1996). In aquaculture, energy dissipation in general is in the range of 1-10 W/m3
(Boyd, 1998). However, in highly intensive systems, more realistic values can reach up to
100 W/m3. At higher mixing intensities and thus higher shear rates, the average floe size
decreases due to increased floe breakage. Biggs and Lant (2000) showed in case of activated
sludge that for a mean shear rate or G-value of 19.4/s (0.4 W/m3), the stable floe-size was ca.
130 µm, whereas, this was decreased to ca. 20 µm for a velocity gradient of346/s (120 W/m3).
The relationship between floe size and mixing intensity has been represented by Parker et al.
(1972) with the power law relationship d = C.G-x, where d is the maximum stable floe size,
G is the average velocity gradient, C is the floe strength component and x is the stable floe
size component. For BFT, the steady-state floe size is an important feature as it has already
been shown that the quality of food for different aquaculture species is also dependent on the
food size (Garatun-Tjeldsto et al., 2006; Knights, 1983). In order to represent a nutrition
source, the food particle size in case of e.g. cod larvae and Macrobrachium rosenbergii larvae
should be within the range of250-1200 µm (de Barros and Valenti, 2003).
Sark,.er, 7vf., 7vf. "F. Sc. "Thesis, 2.0{5, '[}e('{, of1'.tjuacullure 11

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2.4.2. Dissolved oxygen

The DO level is not only essential for the metabolic activity of cells within aerobic floes
but it is also thought to influence floe structure. A trend towards larger and more compact
floes at higher DO concentrations was noted by Wilen and Balmer (1999), although no clear
relation could be found with average floe diameter. Poorer settling properties, a sludge
volume index (SVI) of on average 250 mg/1, occurred at low DO values (0.5 - 2.0 ml/g)
compared to settling at higher DO values (2.0 - 5.0 ml/g) where the SVI was ca. 100 ml/g.
This can be ascribed to the presence of a higher amount of filamentous bacteria compared to
the zoogloeal bacteria at DO-levels of less than or equal to 1.1 mg 02/l as was observed by
Martins et al. (2003). As filaments have a higher affinity towards oxygen, they are able to
outcompete their zoogloeal counterparts at periods of oxygen limitation and thus dominate the
microbial floes (Martins et al., 2003). The bioflocs with a higher floe volume index or FVI
(ml/g) are produced at lower DO-levels in the bioflocs ponds. The FVI should be higher than
200 ml/g to avoid the floes from sedimenting too fast in regions of lower turbulence. This
gives the aquaculture organisms enough opportunity to filter the floes from suspension before
they sediment to the bottom of the ponds and are lost as food. Negative impacts of a higher
FVI however, like e.g. possible clogging of fish gills, have to be taken into account as well.
2.4.3. Organic carbon source
The dosing of an organic carbon source to the culture water in bioflocs ponds induces a
decrease in dissolved oxygen levels due to aerobic microbial metabolism. This may induce
sub-lethal effects on sensitive culture species (Landman et al., 2005). In such cases, it can be
advised to grow the heterotrophic biomass in external biofloc reactors rather than within the
culture unit itself. The externally grown floes can be redirected to the pond as food but without
inducing stress through varying DO levels. The organic carbon can be supplied either as
additional organic carbon source (e.g. glucose, acetate, glycerol etc.) or by changing the feed
composition thus increasing its organic carbon content (A vnimelech, 1999). The organic
carbon source of choice will to a large degree determine the composition of the floes produced,
this mainly regarding the type and amount of storage polymers (Hollender et al., 2002;
.Sarker, M., M. "F. Sc. 1'he1i1, 20f!j, 'Dept. of:lf.ttuacullure 12

'Rlview ofl.iferafure
Oehmen et al., 2004). It was observed e.g. that the dosing of acetate in a sequence batch
reactor (SBR) resulted mainly in poly-B- hydroxybutyrate (PHB) as storage polymer while
these were 3- hydroxy-2-methylvalerate and polyhydroxyvalerate in case of propionate dosing
(Yagci et al., 2007). Also, the costs of the different organic carbon sources will be a
determining choice factor (Salehizadeh and Van Loosdrecht, 2004).
2.4.4. Organic loading rate
The organic loading rate at which the organic carbon source is dosed in the water is a
major process technical factor. Filamentous bacteria have an advantage over non- filamentous
bacteria at low substrate levels due to their higher surface-to-volume ratio. Moreover, the
filaments can penetrate outside the floes and thus are exposed to higher substrate
concentrations than the non-filamentous bacteria that mainly grow within the floes (Martins
et al., 2003). The organic carbon feeding strategy can also be important for BFT. The organic
carbon can be added in small amounts and thus almost continuous mode or be added in larger
doses but at regular time intervals (e.g. 1/d). The second type of application is also known as
a feast and famine regime (Salehizadeh and Van Loosdrecht, 2004) and results in transient
conditions of substrate availability. The microbial biomass stores cellular reserves like PHB
under conditions of excess nutrient availability with which the microorganisms can bridge the
periods of nutrient shortage. The storage products may be of high importance to the added
value that bioflocs bring to aquaculture. As such, it may not be advisable to apply the organic
carbon sources in continuous mode if the goal is to produce reserve materials.
2.4.S. Temperature and pH
Changes in pH determine the stability of the bioflocs present in the ponds (Mikkelsen et
al., 1996). The influence of temperature on biofloc is complex. Wilen et al. (2000) found that
deflocculation of the floes occurred at lower temperature (4 °C) compared to higher
temperatures (18-20°C), probably due to a decrease of the microbial activity within the floes.
Krishna and Van Loosdrecht (1999) observed that higher temperatures (30-35°C) resulted in
bulking of the sludge (SVI % 500 ml/g) due to the excessive production of extracellular
polysaccharides. Therefore, it can be expected that an intermediate water temperature of 20-
.Sark.er, M., M. 'F. Sc. 'Thesis, 2.0{5, Oe('l ofJf.t;uacufture 13

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25°C would be best to obtain stable floes with an intermediate floe volume index of about 200
ml/g. The temperature is of major importance for the microbial metabolism, also concerning
the previously mentioned storage polymers that may be important for aquaculture. It was
shown that higher temperatures (35°C) can result in up to 75% less PHB formation compared
to lower temperatures (l 5°C) (Krishna and Van Loosdrecht, 1999).
2.5. Composition and nutritional value of bioflocs
Bioflocs are aggregates (floes) of algae, bacteria, protozoans, and other kinds of
particulate organic matter such as feces and uneaten feed. Each floe is held together in a loose
matrix of mucus that is secreted by bacteria, bound by filamentous microorganisms, or held
by electrostatic attraction. Diatoms are found both in and outside ofbiofloc particles and have
been implicated in improving shrimp growth (Ju et al., 2009). The biofloc community also
includes animals that are grazers of floes, such as some zooplankton and nematodes. Large
bioflocs can be seen with the naked eye, but most are microscopic. Floes in a typical green
water biofloc system are rather large, around 50 to 200 microns, and will settle easily in calm
water.
The nutritional quality of biofloc to cultured animals is good but rather variable. The dry-
weight protein content of biofloc ranges from 25-50%. Biofloc communities can have fatty
acid profiles distinctly unique from the feed administered to culture systems (Johnson et al.,
2008). Fat content ranges from 0.5-15% and 15-20% carbohydrate. There are conflicting
reports about the adequacy of bioflocs to provide the often limiting amino acids methionine
and lysine. Diatoms in and around biofloc contain relatively high levels of the essential fatty
acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Volkman et al., 1989),
providing a potential nutritional advantage for culture animals. Bioflocs are good sources of
vitamins and minerals, especially phosphorus. Dried bioflocs have been proposed as an
ingredient to replace fishmeal or soybean meal in aquafeeds. The most important factors
determining the feasibility of biofloc as a feed in aquaculture is the high protein,
polyunsaturated fatty acids (PUFA) and lipid content (De Schryver, 2010). Floe enhanced
animal growth from its nutrients and micronutrients (Moss and Pruder, 1995) and improved
Sark.er, M., M. 'F. Sc. 'Thesir, 2.0{5, Ve(1f. of1'.tjuacufture 14

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animal product quality, such as color and sensory properties from pigments such as
astaxanthin and flour materials (e.g. bromophenols) (Whitefield et al., 1997; Breithaupt,
2004). Ju et al. (2008) reported that on the basis of EAA and EAAI protein fractions of the
floe, if it is algae dominated or bacteria dominated were comparable to those of fish or soybean
meal. Due to this comparability biofloc is now used as a replacement ingredient for meal and
it was applied in nursery, grow out and in latest the breeding system of shrimp and fish.
2.6. Present status of biofloc aquaculture in World and India
The number of shrimp farms currently using biofloc technology is not known, but some
prominent examples are Belize Aquaculture Ltd., in Belize and P. T. Central Pertiwi Bahari
in Indonesia. The success or failure of the technology is mainly due to the degree of
understanding of basic concepts of the technology in commercial application. Belize
Aquaculture was the first commercial farm to use biofloc technology successfully. Its
production of 13.S MT shrimp/ha was quite an achievement at the time. Biofloc technology
has become a popular technology in the farming of tilapia, Penaeus monodon, Litopenaeus
vannamei and M rosenbergii. It has been applied with success in shrimp farming in Indonesia
and Australia (Taw, 2010). BFT farms are successfully run in Maryland, USA and
successfully expanded in Latin and Central America, South Korea, Brazil, China, Italy,
Indonesia, Australia (Taw, 2010) and India. Research institutes of many countries now
concentrate to more application of Biofloc technology such as energy kinetics, bacterial
identification and economics, low cost processing etc.
In Belize 13.5 mt shrimp/ha was an achievement at the time. The same model is applied
m Indonesia. The technology combined with partial harvest was repeated in northern
Sumatra, Medan, Indonesia with better results during 2008 and 2009. Now biofloc technology
is common in Bali and Java. In Indonesia intensification of shrimp aquaculture is doing in
HDPE lined and plastic sheet covered ponds by using this technology. Malaysia is currently
initiating a 1000 ha integrated shrimp farming project at Seitiu, Terengganu by Blue
Archipelagio. The technology combined with partial harvest was repeated in Medan,
Indonesia, with better results. During 2008 and 2009, biofloc technology was used in Java
15
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and Bali successfully. In Indonesia, biosecurity protocols were incorporated within .the
technology. Most Indonesian shrimp farmers are interested in biofloc technology, but with
some reservations, as a number of projects have failed due to incomplete understanding of the
technology. For example, the correct number and position of paddle wheel aerators used in
ponds are essential (Taw, 2010). Malaysia is currently initiating a 1,000-ha integrated
intensive shrimp-farming project at Setiu, Terengganu by Blue Archipelago.
In India this technology is not yet popular. This technology was first applied in the
extensive culture system of P. monodon, in Kerala, by School of Industrial Fisheries, Cochin
University of Science and Technology (Hari et al., 2004, 2006). It was proved that its
application is possible in the larval culture of Macrobrachium rosenbergii by biofloc system
(Saritha, 2009; Saritha and Kurup, 2011). Nursery rearing of the same species with the BFT
gave an opportunity for improving the larval stocking density and animal health welfare
compared to normal conventional systems (Prajith and Kurup, 2011). This technology also
applied in the hatchery phase of P. monodon by Devi and Kurup (2011 ).
2.7. Biofloc systems and water quality in ponds
Bioflocs technology has become a sustainable technique used in aquaculture to maintain
good in-situ water quality. This biological phenomenon happens through the development
and control of dense heterotrophic microbial bioflocs by adding carbohydrate to the water
(A vnimelech et al., 1989; Avnimelech, 1999; Crab et al., 2007; Crab et al., 2009). However,
research has shown that the capacity of the BFT to control the water quality in the culture
system and the nutritional properties of the floes are influenced by the type of carbon source
used to produce the floes (Crab, 2010). According to Avnimelech and Ritvo (2003), only
about 25% of the feed nutrients are converted into harvestable products hence contributing to
high nitrogen residues in aquaculture water, especially total ammonia nitrogen. The main
nitrogen pathways that naturally remove ammonia nitrogen in aquaculture systems include
photoautotrophic removal by algae, autotrophic bacterial conversion of ammonia-nitrogen to
nitrate-nitrogen, and heterotrophic bacterial conversion of ammonia-nitrogen directly to
microbial biomass (Ebeling et al., 2006). Therefore, development of dense heterotrophic
16
'R§view of£.iferafure
microbial floes in ponds can accelerate the biological 'cleaning' of organic and inorganic
wastes in ponds (Avnimelech, 2005; Azim et al., 2003a). The periphyton community also
play significant role in water quality control in aquatic systems. They consist of attached
aquatic biota on submerged substrates and harbours algae, bacteria, fungi, protozoa,
zooplankton and other invertebrates (Azim and Wahab, 2005). Given adequate light ofup to
about 0.5 meter depth in the water, periphytonne can perform high rates of photosynthesis and
autotrophic production (Craggs et al. 1996; Vermaat, 2005). In this process, periphytonne
entraps organic detritus, removes nutrients from the water column and helps controlling the
dissolved oxygen concentration, suspended solids and the pH of the surrounding water (Azim
et al., 2002; Dodds, 2003). Periphyton has an average C/N ratio of 10 (Azim and Asaeda,
2005) and nitrogen assimilation capacity of about 0.2 gNm-2day- 1• From this it is clear that
one needs a large surface, which allows periphytonne growth, to treat intensive aquaculture
wastewater.
2.8. The "natural probiotic" effect of biofloc
Biofloc can be a novel strategy for disease management in contrast to conventional
approaches such as antibiotic, antifungal, probiotic and prebiotic application. The "natural
probiotic" effect in BFT could act internally or externally against, Vibrio sp. and ectoparasites,
respectively. This effect is promoted by large groups of microorganisms, but mainly bacteria.
Internally, bacteria and its synthesized compounds could act similar to organic acids and might
be effective biocontrol agents, also given beneficial host's microbial balance in the gut (Sinha
et al., 2008). The regular addition of carbon in the water is known to select for
polyhydroxyalkanoates (PHA) accumulating bacteria and other groups of bacteria that
synthesize PHA granules. The microbial storage product poly-B-hydroxybutyrate (PHB), a
biodegradable polymer belonging to the polyesters class, is only one compound of a whole
family of polyhydroxyalkanoates. PHB is produced by a widely variety of microorganisms
such as Bacillus sp., Alcaligenes sp., Pseudomonas sp. from soluble organic carbon and is
also involved in bacterial carbon metabolism and energy storage (Sinha et al., 2008). This
polymer could comprise -80% of the bacteria's cell dry matter and up to 16% on biofloc dry
Sark.er, M., M. 'F. Sc. 'ThesiJ, 2.0<5, 1Je('f. of7'.ttuacufture 17

'/<tview ofl.iferafure
weight (De Schryver et al., 2012). Different carbon sources or structures of carbon substrate
will result in varying types of PHA (De Schryver et al., 2012). Such granules are synthesized
under conditions of physiological and nutrient stress, i.e., when an essential nutrient like
nitrogen is limited in the presence of an excess carbon source (Sinha et al., 2008). When these
polymers are degraded in the gut, they could have antibacterial activity similar to short chain
fatty acids (SCF As) or organic acids. The breakdown of PHA inside the gastrointestinal tract
can be carried out via chemical and enzymatic hydrolysis (Yu et al., 2005). Enzyme
hydrolysis is carried out by extracellular depolymerases activities of bacteria and fungi, acting
as a preventive or curative protector against Vibrio sp. infections and stimulates growth and
survival of shrimp and fish larvae (De Schryver et al., 2012). The working mechanism of
PHAs with respect to their antibacterial activity is not well understood (Sinha et al., 2008).
As they could act similarly to SCFA, some studies speculated the working mechanism by (i)
reduction of pH, in which antibacterial activity increases with decreasing pH value (Ricke,
2003); (ii) inhibiting the growth of pathogenic bacteria by interference on cell membrane
structure and membrane permeability, as well as instability of internal proton balance,
lowering ATP and depletion of cellular energy (Russel, 1992); and (iii) down-regulate
virulence factor expression and positively influence the gut health of animals (Teitelbaum and
Walker, 2002). Further research is need to maximizing PHA content in bioflocs applied, i.e.,
for fish/shrimp feed, characterizing and analyzing their bio-control efficacy in different host-
microbe systems (Sinha et al., 2008). Externally, the working mechanism of biofloc
microorganisms against pathogens seems to be by competition of space, substrate and
nutrients. Some essentials nutrients such as nitrogen are required by both groups (i.e.
heterotrophic bacteria vs. Vibrio sp.) limiting their growth. Inhibiting compounds excreted by
BFT microorganisms, light intensity and type of carbon source also could reduce pathogens
growth. Unfortunately, limited information is available on this field. In a study with fish
fingerlings (Emerenciano et al., 2009) was reported that tilapia (initial weight 0.98 ± O.lg)
reared under BFT limited water-exchange condition (FLOC) presented less ectoparasites in
18
'Rflview of f:iferafure
gills and ectoderm's mucous as compared to conventional water-exchange system (CW) after
60 days.
2.9. Bioflocs as biosecurity in aquaculture systems
Green water technology in aquaculture development has been received with varied
perceptions. With the bad publicity that aquaculture has received in regards to mishandling
of antibiotics, scientists have continued to explore safe ways of arresting pathogenic infections
in aquaculture facilities without using antibiotics (Naylor et al., 2000). According to Defoirdt
et al. (2011), most antibiotics are no longer effective in treating bacterial disease because of
resistance developed to them by bacteria. Crab et al. (2010) reported that biofloc technology
could be a possible alternative to fight pathogenic bacteria in aquaculture facilities. Some
groups of bacteria in bioflocs have been found to conduct quorum quenching, which is the
disruption of bacterial cell-to-cell communication (quarumsensing) with small signal
molecules (Defoirdt et al., 2008). In fact, Defoirdt et al. (2004) reported that disruption of
bacterial cell-to-cell communication mechanism prevents expression of virulence factors thus
creating a disease free environment. Studies of Lezama-Cervantes and Michel (2010) on the
role of biofloc in shrimp production revealed that primary production and promotion of in situ
microbial populations, as is the case in biofloc technology, are beneficial for shrimp. Indeed
this can be extended to tilapia culture as well. Recently, scientists have hypothesized
possibilities of immunostimulatory features of the bioflocs leading to enhancement of the
immunity of fish to provide broad-based resistance towards many infections (Crab et al.,
2012). According to Wang et al. (2008), existing immuno-stimulants are group of live and
synthetic compounds including bacteria and bacterial products, complex carbohydrates,
nutritional factors, animal extracts, cytokines, lectins and plant extracts. Therefore bioflocs
might also contain immunostimulatory compounds since biofloc technology deals with
bacteria and bacterial products.
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2.10. Applications of bioflocs in aquaculture

2.10.1. Bioflocs as feed
With biofloc technology, where nitrogenous waste generated by the cultivated organisms
is converted into bacterial biomass (containing protein), in situ feed production is stimulated
through the addition of an external carbon source (Schneider et al., 2005). Although bioflocs
show an adequate protein, lipid, carbohydrate and ash content for use as an aquaculture feed
(Crab et al., 2010a), more research is needed on their amino acid and fatty acid composition.
Several studies were performed on the use of bioflocs as an in situ produced feed and
they indicate that bioflocs can be taken up by aquaculture species and uptake depends on the
species and feeding traits, animal size, floe size and floe density (Avnimelech, 2009; Crab,
201 O; Crab et al., 2009, 201 Oa). Giant freshwater prawn (Macrobrachium rosenbergii), white
leg shrimp (Litopenaeus vannamei) and tilapia ( Oreochromis niloticus, Oreochromis aureus)
were all able to take up bioflocs and profit from this additional protein source. This indicated
that biofloc technology is applicable to both freshwater and seawater systems, both to control
water quality and to produce as an additional feed source in situ. The potential feed gain of
the application ofbiofloc technology is estimated to be in the order of 10-20% (De Schryver
et al., 2008). With this, production costs will decline considerably since food represents 40-
50% of the total production costs (Craig and Helfrich, 2002).
Although bioflocs meet nutritional standards to serve as an 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 floes are influenced by the type of carbon source
used to produce the floes (Crab, 2010; Crab et al., 2010a). Balancing the carbon content of
the feed fed to the culture organism could be an alternative to elevating the organic carbon to
nitrogen ratio through addition of an external organic carbon source (Crab et al., 2009).
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, 2010). Feeding experiments revealed that
besides these characteristics, the type of carbon source also influenced the availability,
Sark.er, M., M. "F. Sc. 'Thesis, 2.015, 1Jepf. of11.r.juacufture 20

'/qview ofLiterature
palatability and digestibility for the cultured organisms (Crab, 201 O; Crab et al., 2010a).
Overall, bioflocs produced on glycerol gave the best results (Crab, 2010) and tend to have
higher n-6 fatty acids content when compared to bioflocs grown on acetate or glucose (Crab
et al., 2010a).
In addition, not only the carbon source, but also the indigenous microbiota present in the
pond water will put forth a characteristic effect that needs to be considered. An important
factor here is to determine the role of algae and their interaction with the bacteria in the
bioflocs. Crab (2010) showed that with L. vannamei, bioflocs grown on glucose lacked
accessibility and palatability for good survival and growth. A worthy carbon source to look
at in this regard is molasses obtained during sugar processing of sugar beet (Beta vulgaris L.
v. altissima), which contains glycine betaine, a known attractants used in aquaculture (Felix
and Sudharsan, 2004; Makela et al., 1998).
The choice of cultivated species with biofloc technology should take into account their
capability of dealing with high suspended solid concentrations, since this negatively affects
certain fish species. Another important factor that is essential for the growth and survival of
aquaculture species are vitamins. Vitamin C concentrations in bioflocs ranging from Oto 54
µgig dry matter (Crab, 2010). These values are below the required concentration for fish and
shrimp. Besides vitamin C, other vitamins such as thiamine, riboflavin, pyridoxine,
pantothenic acid, nicotinic acid, biotin, folic acid, vitamin B 12, inositol, choline, vitamin A,
vitamin 03, vitamin E and vitamin K, are usually not sufficiently synthesized by the cultured
organism either and need to be supplied through the feed (Crab et al., 201 Oa). Hence, it needs
to be established to what extent bioflocs can contribute to the supply of these essential
nutrients.
2.10.2. Nursery and grow-out
Nursery phase is defined as an intermediate step between hatchery-reared early postlarvae
and grow-out phase (Mishra et al., 2008). Such phase presents several benefits such as
optimization of farm land, increase in survival and enhanced growth performance in grow-out
ponds (Apud et al., 1983; Sandifer et al., 1991; Arnold et al., 2009). BFT has been applied
21
'R.eview off.iferafure
successfully in nursery phase in different shrimp species such as L. vannamei (Mishra et al.,
2008; Samocha et al., 2007), P. monodon (Arnold et al., 2009), F. paulensis (Ballester et al.,
2010; Asaduzzaman et al., 2008), F. brasiliensis (Emerenciano et al., 2012; Souza et al.,
2011) and F. setiferus (Emerenciano et al., 2009). The primary advantage observed is related
to a better nutrition by continuous consumption of biofloc, which might positively influence
grow-out performance (AQUACOP, Le Moullac and Damez, 1991; Emerenciano et al.,
2007). In addition, optimization of farm facilities provided by the high stocking densities in
BFT nursery phase seems to be an important advantage to achieve profitability in small farms,
mainly in cold regions or when farmers are operating indoor facilities. It (Emerenciano et al.,
2011 b) was observed that presence of bioflocs resulted in increases of 50% in weight and
almost 80% in final biomass in F. paulensis early postlarval stage when compared to
conventional clear-water system. This trend was observed even when postlarvae were not fed
with a commercial feed (biofloc without commercial feed). In L. vannamei nursery in BFT
conditions, references (Mishra et al., 2008) and (Cohen et al., 2005) reported survival rates
ranging from 55.9% to 100% and 97% and 100%, respectively. Growth and survival was not
affected by stocking density (2500 vs. 5000 PUm2 ), therefore greater production outputs were
achieved at the higher density (Arnold et al., 2009). Furthermore, in (Emerenciano et al.,
2012) was found that F. brasiliensis postlarvae grow similarly with or without pelletized feed
in biofloc conditions during 30 day of nursery phase, which was 40% more than conventional
clear-water continuous exchange system.
In grow-out, BFT has been also shown nutritional and zootechnical benefits. It (Burford
et al., 2004) was estimated that more than 29% of the daily food intake of L. vannamei
consisted of microbial floes, decreasing FCR and reducing costs in feed. The reference
(Wasielesky et al., 2006) showed that juveniles of L. vannamei fed with 35% CP pelletized
feed grew significantly better in biofloc conditions as compared to clear-water conditions. It
(Ray et al., 2010) was showed that controlling the concentration of particles in super-intensive
shrimp culture systems can significantly improve shrimp production and water quality. It
(Krummenauer et al., 2011) was evaluated the stocking density in a 120 day of L. vannamei
22
"R$view of fJferalure
BFT culture, reporting consistent survival of 92, 81 and 75% with 150, 300 and 450
shrimp/m2, respectively. Moreover, the study (Samocha et al., 2004) performed in a
heterotrophic-based condition detected no significant difference in FCR when feeding L.
vannamei 30% and 45% CP diets and 39% and 43% CP diets, respectively. It well known
that protein, peptides and amino acids participate fully in synthesis of new membranes,
somatic growth and immune function and biofloc can potentially provide such ingredients.
For fish and other species, BFT also has been demonstrated encouraged results. Intensive
BFT Oreochromis niloticus tilapia culture could produce an equivalent of 155 tonne/ha/crop
(Rakocy et al., 2004). Besides high yields, decrease of FCR and decreased of protein content
in diets have also been observed. It (Avnimelech et al., 1994) was estimated that feed
utilization by tilapia is higher in BFT with a ration 20% less than conventional water exchange
system. Studying the effect of BFT in juvenile tilapia, the reference (Azim and Little, 2008)
showed no difference in fish growth/production between 35% and 24% CP fed tanks under
BFT, but both were higher than clear-water control without biofloc with 35% CP. Moreover,
it Crab et al. (2009) was investigated the effectiveness of BFT for maintaining good water
quality in over-wintering ponds for tilapia. The BFT emerge as an alternative to overcome
over-wintering problems, particularly mass mortality of fish due to low temperatures. In the
study (A vnimelech, 2007) was observed that biofloc consumed by fish (tilapia) may represent
a very significant feed source, constituting about 50% of the regular feed ration of fish
(assuming daily feeding of2% body weight). Using glucose or a combination of glycerol plus
Bacillus as a carbon source in bioreactors led to higher biofloc protein content, higher n-6
fatty acids, which resulted in improved survival rates.
2.10.3. Bioflocs on reproduction
Emerenciano et al. (2013) described the possibility of BFT in brood stock rearing tanks
considering its in-situ nutritive capability i.e., 24 hours a day. Bioflocs could be helped to
first stages of gonad formation and ovary development. It avoids the unstability of water
quality parameters, related to the conventional brooders rearing system. This opinion
coincides with the study of (Emerenciano et al., 2011 ). BFT is applied in the breeding ponds
.Sar~er, M, M 'F. Sc. 'Thesis, 2.0f:}, 1Je/'f. of11.tjuacufture 23

'R§view of f.iferafure
of Litopenaeus stylirostris and Farfantepenaeus duorarum respectively and obtained high

number of eggs per spawn and higher spawning activity. Emerenciano et al., (2013) also
suggested that in breeding system should control Total suspended solids (biofloc) such as 15
ml/I. Biofloc contained high amount of ARA {Azim and Little 2008), fatty acid crucial for
reproduction and as hormone precursor, is suggestive for its application in breeding.
2.11. BFf in Aquaponics
Aquaponics is a sustainable production system combines traditional aquaculture with
hydroponics. Now a day's aquaponics is common in western countries. The waste excreted
from aquaculture species and uneaten feed is converted in to beneficiary nutrients in water.
These aquaculture waste water is directly pumped to raceways of hydroponics vegetables.
Typical plants raised in aquaponics include lettuce, chard, tomato and fruits such as passion
fruit, strawberry, watermelon etc. Now a day BFT has been successfully applied in
Aquaponics. The presence of rich microbiota and variety of micro and macronutrients
especially Nitrate (In BFT systems have high amount of nitrate compared to other systems).
A well-known BFT and aquaponics interaction was developed by United Virgin Islands
(UVI). But this BFT aquaponics system is questionable and needs more investigation. It
require solid management and excess adhesion of microbes in the roots of plants is
problematic because of reduced oxygen and growth (Ray et al., 2010).
2.12. Advantages of biofloc technology
Freshwater scarcity is for sure becoming global concern due to high human population
growth. Use of biofloc technology encourages water conservation. Significant reduction of
inorganic nitrogen accumulation; increased utilization of protein feed and reduction of feed
expenditure in biofloc mediated systems have been reported in biofloc systems (Avnimelech,
1999, 2009; Kuhn et al., 2009; Crab et al., 2010, 2012). The BFT approach can decrease fish
oil and fishmeal utilization in aquaculture by providing high quality cheap alternative protein
source to the cultured organism. The biofloc technology provides opportunity to produce high
fish yield all year-round, and the flexibility to locate production facilities (tanks) near large
markets to deliver a fresher, safer product and lower transport cost (Schneider et al ., 2005).
Sar(er, M., M. 'F. .Sc. 1"~eri!, 2.CJf5, Vc{1f. of11.tjuacuffure 24

'/qview ofl.iferalure
The fact that there are no cases of fish escape to the environment, biological pollution is thus
prevented. Being more independent from the external environment due to increased levels of
control, BFT systems improved hygiene, disease risk management, lower feed quantity
supplied and thus overall cost of production (Summerfelt, 2006). However, the entire biofloc
process is completely dependent on the availability of sunlight (Azim and Asaeda, 2005) and
therefore maximum nitrogen uptake is limited to light sufficiency.
2.13. Disadvantages of biofloc technology:
The task of harvesting the periphyton 1s laborious making the application of the
periphyton treatment technique in the intensive aquaculture sector expensive. Nevertheless,
the technique may be significant in smaller extensive aquaculture systems in developing
countries. The biofloc technology is not yet fully predictable and can therefore be risky to
implement at farm level in developing countries (Crab et al., 2012). It is not easy to convince
farmers to implement the technique, since the concept of biofloc technology goes in against
common wisdom that water in the pond has to be clear (Avnimelech, 2009). Release of
polluted effluents from bioflocs also pause challenges as environmentalists are keen to prevent
such happenings. Some microbial community in the bioflocs may tum to cause diseases to
the cultured fish. The most challenging issue is the experience and technical knowledge
regarding management ofbiofloc technology and the economic benefits that goes with it.
2.14. Perspectives of BFT
Biosecurity is a priority in aquaculture industry. For example, in shrimp farming,
considerable impact of disease outbreaks during the past two decades greatly affected the
operational management of shrimp farms worldwide (Wasielesky et al., 2006). Infected PLs
and incoming water seem to be the main pathway for pathogen introduction. This scenario
forced farmers to look for more biosecure culture practices to minimize the risk associated
with exposure to pathogens (Browdy et al., 2001). Biofloc technology brings an obvious
advantage of minimizing consumption and release of water, recycling in situ nutrients and
organic matter. Furthermore, pathogens introduction is reduced, improving the farm
biosecurity. Biofloc technology will enable aquaculture grow towards an environmental
.Sark,.er, "M., "M. "F. Sr:. 1hcsis, 2.0f:j, fJepl of11.tjuacufture 25

'f<p.Jiew off.iferafure
friendly approach. Consumption of microorganisms in BFT reduces FCR and consequently

costs in feed. Also, microbial community is able to rapidly utilize dissolved nitrogen leached
from shrimp faeces and uneaten food and convert it into microbial protein. These qualities
make minimal-exchange BFT system an alternative to extensive aquaculture.
Microorganisms in biofloc might partially replace protein content in diets or decrease its
dependence of fishmeal. Moreover, generated from a process that cleans aquaculture effluents
(Kuhn et al., 2009; Crab et al., 2010) biofloc meal production avoids discharge of waste water
and excessive damage to natural habitats (Naylor et al., 2000). This ingredient seems to be
free of deleterious levels of mycotoxins, antinutritional factors and other constituents that limit
its use in aquafeeds (Kuhn and Lawrence, 2012). Large-scale production ofbiofloc meal for
use in aquaculture could result in environmental benefits to marine and coastal ecosystems,
as the need for wild fish as an aquafeed ingredient is reduced (Kuhn and Lawrence, 2012;
Bauer et al., 2012). Sensorial quality of BFT products is also an important issue. BFT may
bring higher profit if fresh non-frozen shrimp/fish is sold to near-by market, mainly at inland
locations. These advantages certainly should be more explored and niche markets achieved,
contributing to social sustainability.
An interesting topic for further research could be the identification of micro-organisms
(bacteria and micro-algae) that are able to produce bioflocs with the desired nutritional
properties and a good ability to control the water quality. Such microorganisms could be used
as an inoculum for the start-up of aquaculture systems with biofloc technology. All these
findings and possible modus operandi emphasize the need for further study of biofloc
composition in order to achieve a desired nutritional outcome, since different research groups
have obtained different results in respect to biofloc nutritional composition (Avnimelech,
2009).
26
Materials and
Methods
3. Materials and methods
The present study on Comparative efficacy of biojloc and feed based Common carp
(Cyprinus carpio, L) production system with special reference to environmental health
production has been conducted in the Department of Aquaculture, Faculty of Fishery
Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria,
Panchasayar, Kolkata-94, 22°28'46"N 88°24'4"E. The duration of the study lasted for a-
period of about three months from March to May, 2015.
3.1. Preparations of cistern
Ten outdoor experimental cylindrical cement tanks (180 L) were selected for the
present investigation. After thorough washing and sun drying the tanks were provided
with agriculture soil base of 15 cm. and then filled with ground water (pH-7.5). All the
tanks were manured with cowdung @ I 0,000 kg/ha as practiced in traditional pond
preparation for fish farming in the locality. The requisite amount of cow dung were mixed
with water in a bucket and dispensed in the form of slurry into the tanks. They were then
randomly grouped into two batches in triplicate for the three systems designed. All the
tanks were applied with lime @ 200 kg/ha after seven days of manure application and kept
undisturbed for another seven days. The water colour turned to bluish green and became
ready for stocking of fish.
3.2. Preparation of biofloc
Boiled rice (150 L) water mixed with molasses (3 kg) in a tub. After that, yeast
powder (250 g) and filtered freshwater (50 L) were added to the mixture and vigorously
mixed together to make a solution and also kept for 72 hours in open air for aerobic
fermentation. When the colour of solution tum to deep yellow the solution was transferred
to another tub. Finally the solution was ready to use as a medium of biofloc growth
enhancer.
3.3. Feeding
Fish in first and second batches of treatment tanks were fed with crumble feed (crude
protein: 35%) once daily between 9.00 a.m. to IO a.m. @6% (Tl), 3% (T2) of body weight
respectively along with biofloc medium @ 15 ml/tank. Fish in the third batch of tanks
(T3) were applied only with biofloc medium @ 15 ml/tank daily without the above
supplementary feed. Fish in the fourth batch of tanks (CI) were supplied only with the
above diet@ 6% of body weight but were not applied with biofloc medium. The last batch
.Sar(er, M., M. '1. .Sc. 'Thesis, 20{3, 'Depl of:11.tjuacufture 27

Maferiafs anl'Metfwl
of treatment tanks without fish were applied only with biofloc medium@ 15 ml/tank daily
acted as second control (C2). The required amount of feed was broadcasted over the water
surface in respective treatments (Tl, T2 and CI).
The biofloc medium was also broadcasted by using a measuring cylinder over the
water surface of respective treatments (Tl, T2, T3 and C2). The biofloc medium was
applied to the treatments once daily at early morning between 5.30 a.m. to 6 a.m. or late
afternoon between 5 p.m. to 5.30 p.m. up to 45 days of the investigation period.
3.4. Setting of aerator
To aerate all the treatments two electromagnetic air pump (35 Watt and 220 Volt each)
were used. Aeration was provided by using two pumice stone in each treatment. Peumice
stone was set above the 40 cm. of bottom surface. Aeration was continued for 12-14 hours
per day depending upon the daily weather condition.
3.5. Stocking of fish
Healthy fry of Common carp (Cyprinus carpio L.) (Wt. 0.841 g) were collected from
faculty pond. Stocking of fish was done in the tanks @ 18 nos./tank. Two weeks after
application of cowdung when the colour of the water changes to greenish blue indicating
development of planktonic organisms. They were reared for 90 days.
3.6. Water replenishment
A fixed level of water was maintained in the experimental tanks by periodic addition
of ground water to compensate the losses due to evaporation and sampling.
3.7. Collection of water samples
Water samples were collected at 7 days interval from each of the tanks at a fixed hour
of the day (9.00 a.m.) by completely dipping the collection bottle at 15 cm depth for
physic-chemical analyses. During collection of water samples, cautions were taken so as
to prevent air bubbling, which might influence water parameters such as dissolved oxygen.
3.8. Collection of soil samples
Soil samples from each of the cisterns were collected from at 15 days interval from
the soil bed using own hand. They were then mixes, air dried, pulverized with pestle and
mortar and sieved through 150 µm mesh sieve and stored in labeled polythene packets for
analyses.
3.9. Collection of plankton
A conical plankton net made up of bolting silk cloth (no. 21 with 77 meshes per square
centimeter) was used to collect the plankton sample. About 2 L of water from each of the
.Sar~er, 'M., 'M. '1. Sc. 1"~esir, 2017, 1Jepl. of11.quacufture 28

Maferiafs anJ MetfioJ
cistern was collected from randomly selected locations with the help of a 500 ml beaker
and pooled together for filtering through plankton in 4% formalin solution and stored in
labeled vials for subsequent quantitative and qualitative analysis. Plankton samples are
collected at 20 days intervals.
3.10. Analysis of samples
3.10.1. Water quality
3.10.1.1. Temperature
The water temperature was measured using a centigrade thermometer on spot and
expressed as 0c.
3.10.1.2. pH
pH of water samples was estimated by a digital pH meter (Systronics-VI) on spot.
3.10.1.3. Dissolved oxygen
For estimation of dissolved oxygen content of water, the samples were collected with
all necessary precautions. Winkler's method was followed for the same (APHA, 1995).
3.10.1.4. Total Alkalinity
Estimation of total alkalinity of water samples were done immediately after
collection. Carbonate alkalinity of water samples were analyzed by titrating the samples
against N/50 H2S04 using phenolphthalein as indicator. Bicarbonate alkalinity was
determined against N/50 H2S04 using methyl orange indicator (APHA, 1995).
3.10.1.5. Total hardness
Total hardness of water samples was measured on the sampling day by titrating the
samples against EDT A (Ethylene Di-amine Tetra Acetic acid) after adding ammonia
buffer and Eriochrome Black T (APHA, 1995) as indicator.
3.10.1.6. Ammonia-nitrog en (NH3-N)
After proper filtration of the sample, phenol solution, sodium nitropruside solution
and oxidizing solution were added to the sample. The samples were then wrapped with
paper and kept at room temperature (22-27°C) in subdued light for at least 1 hour. A blue
colour appeared which was stable for 24 hrs. The ammonia concentration of the samples
was directly estimated through a double beam UV-vis-Spectrophotometer (CECIL CE-
4002) 640 run wavelengths (Wetzel and Likens, 1991).
3.10.1.7. Nitrate-nitrogen (N03-N)
After proper filtration of the samples, 1(N) hydrochloric acid was added to each of
the sample and after 10 minutes nitrate nitrogen concentration was directly estimated
.Sarker, M., M. '1. Si:. 'Thesis, 2.(Jf7, 1Jeff, of1l!juacullure 29

'Maleriafs anl 'Methol
through a double beam UV-vis-Spectrophotometer (CECIL CE-4002) at 220 and 275 run
wavelengths (APHA, 1995).
3.10.1.7. Nitrite-nitrogen (N02-N)

The concentration of nitrite was measured through a double beam UV-vis-
Spectrophotometer (CECIL CE-4002) at 543 run wavelengths using a-napthylamine and
sulphalinic acid (Wetzel and Likens, 1991 ).
3.10.1.8. Orthophosphate
The orthophosphate level of water was determined colorimetrically through a double
beam UV-vis-Spectrophotometer (CECIL CE-4002) at 690 nm wavelengths following the
stannous chloride method (APHA, 1995).
3.10.1.9. Primary productivity
The dark and light bottle technique by Winberg (1963) was followed for the
measurement of primary productivity of phytoplankton. Water samples were collected in
125 ml Borosil glass bottle in triplicate from each cistern taking all necessary precautions
during filling to prevent air bubble from remaining in the bottle. All the bottles were then
exposed at the surface layer of water under normal light conditions for 3-4 hours of day
light depending upon the photo period. The oxygen content of all the dark and light bottles
was modified Winkler's method. The calculation described by Vollenweider (1974) was
used to measure the rate of primary production. The results of primary productivity in
terms of oxygen were converted into mg carbon by multiplying with a factor 0.375
(Natarajan and Pathak, 1983).
3.10.2. Soil quality
3.10.2.1. pH
The pH was determined with a digital pH meter (Systronics-Vl) using 1:2 suspensions
of soil and water (APHA, 1995).
3.10.2.2. Organic carbon
For estimation of organic carbon, air-dried powdered sediment sample (1 g) was
digested with 1 N K2Cr201 (IO ml) and concentrated H2S04 (20 ml) and kept for 30
minutes at dark. The digested sample was then diluted with 200 ml distilled water and 10
ml ortho-phosphoric acid and 1 ml diphenyl amine indicator was added. It was the titrated
against 1 N ferrous ammonium sulphate (Mohr's salt) until brilliant green colour appeared
(Walkey and Black, 1934).
S'ar(er, M., M. 'F. .Sc. 'Thesi1, 2.013, 'fJefl of1t.tjuacu«ure 30

Maferia& and Me!hol
3.10.2.3. Available phosphorus

Available phosphorus was determined using 1:20 soil to Olsen's extractant (0.5
NaHC03 adjusted to pH 8.5) (Olsen et al., 1954) followed by Dickman and Bray's (1940)
Chlorostannous reduced molybdophosphoric acid blue colour method in hypochloric acid
system as described by Jackson (1967).
3.10.3. Analysis of plankton
Five litres of water sample was pooled by collecting one litre sub-samples from
different spots of each treatment tanks and passed through a plankton net made of bolting
silk cloth (no. 42). A filtrate of 20 ml was collected and preserved with 4% formaldehyde
solution. The samples were analyzed by Drop Count Method (APHA, 1995) under a
binocular microscope.
3.10.4. Fish growth
Fish growth was recorded at 15 days intervals from each cistern. Half of the stocked
fish were caught randomly with a hand net and their weight (g) increments were recorded
for estimation of absolute weight gain, body weight gain (%),feed conversion ratio (FCR),
specific growth rate (SGR) (%), feed conversion efficiency (FCE) (%), mortality rate(%),
absolute weight gain(%). They were then released again as quickly as possible for further
growth. The following estimates were done as:
Absolute weight gain= (Final wet body weight- Initial wet body weight)
Body weight gain(%) = (Final body weight - Initial body weight) x 100
Initial body weight
Feed Conversion Ratio (FCR) = {Total dry feed fed in gm I Fish weight gain in gm.)
(De silva and Anderson, 1995).

Specific Growth Rate(%) = In (Final body weight}- ln (Initial body weight) x 100
Number of days
Feed conversion efficiency (FCE) (%) = Fish weight gain x 100

Food consumed
(Goddard, 1996)
Mortality rate (%) = (Initial no. of fish - Final no. of fish) x 100
Initial number of fish
Absolute weight gain(%) = Absolute weight gain x 100

Initial body weight
.Sark,.er, M., M. 'F. Sc. 'Thesis, 2.015, fJef !. of71.tjua,:u(ture

31
Materials anrf Metfwrf
3.10.5. Statistical analysis

All the results were subjected to statistical analysis. One way analysis of variance
(ANOVA) were applied to test the significance among the treatments. Correlation
between the selective parameters were tested and subsequently the relationship was fitted
with appropriate curve.
,Sark.er, M., M. 'F. .Sc. 'ThesiJ, 2.0f5, 1Jepl. of71.ijuacuffurc 32

Maferiafr anl Mefhotfr
EXPERIMENTAL
PROTOCOL
SOIL+WATER+FISH SOIL+WATER+FISH+ SOIL+WATER+FISH+ ONLY SOIL+WATER+FISH+6% SOIL+WATER+NO

BIOFLOC + 6% BODY BIOFLOC + 3% BODY BIOFLOC, NO FEED (T3) BODY WEIGHT. FEED, NO BIOFLOC, NO FISH (C2)
WEIGHT FEED (T 1) WEIGHT FEED (T2) BIOFLOC (Cl)
ANALYSES
Fish body wt.

SOIL SAMPLES FISH GROWTH ANALYSES
Fish body wt.
gain(%) WATER SAMPLES
Specific growth
Physico-chemical analyses rate
Absolute growth Biological analyses Physico-chemical analyses
pH
Temperature
I Plankton analyses I Primary productivity

Alkalinity
Organic carbon Gross primary productivity
N02-N
Net primary productivity
Available-P Total Phytoplankton Total Zooplanl..ion population
population NO,-N
Community respiration
Orthophosphate BOD 1
Percentage distribution of Phytoplankton Percentage distribution of Zooplankton
Statistical Analyses
Fig. 1. Experimental protocol followed in the present study
,Sark.er, M., M. .-,:_ Sc. 'Thesis, 2.015, 1Je(1l of7'.tjuacuffure 33

!Results a n d
D is c u s s io n
4. Results and Discussion
The results of the study on comparative efficacy of biofloc and feed based common
carp (Cyprinus carpio, L.) production system with special reference to environmental
health with regards to physico-chemical parameters, nutritional parameters, biological
parameters of water quality, soil quality parameters and fish growth are documented
herein.
4.1. Results
4.1.1. Water quality
4.1.1.1. Physico-chemical parameters
4.1.1.1.1. Temperature
Surface water temperature did not vary much among the treatments and ranged from
31 ·c to 36"C during the period of investigation (Fig. 2).
37
36
35
.-..
~ 34
'-'
~ 33
.; 32
~
I-
~ 31
E
~
30
~
29
28
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 2. Temporal changes of temperature of water in different treatments employed.
4.1.1.1.2. pH
pH of water in any treatment declined up to week 4 followed by gradual increase up
to week 9-10 after which it declined again except in T3 and C2 (Fig. 3 ). Range of variation
in water pH was highest in T2 (7.29 to 8.2) and lowest in Tl (7.1 to 7.64). Difference
among the treatments exhibits strong significance (F = 11.57; P < 0.01) during the
investigation period.
.Sark.,er, M., M. "F. Sc. T~esis, 2015, fJef f. of7vjuacullure 34

'R§sufts antf 1Jircumon
8.2 ---Tl ~Tl

--+-Cl -C2
8
7.8
7.6
:c
Q.
7.4
7.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 3. Temporal changes of pH of water in different treatments employed.
4.1.1.1.3. Dissolved Oxygen

Dissolved oxygen of water declined sharply after the initiation of the study followed
by sharp increase up to week 4 in any case (Fig. 4). It declined again thereafter and
followed random pattern of changes. The highest mean value was encountered in C2 (8.13
mg 1- 1) and lowest in T2 (7.57 mg 1- 1). Difference among the treatments remained
insignificant (P > 0.05).
10
9.5
9
:::-' 8.5
.:..
t=.11
8
._..
=
O 7.5
Q
7
6.5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 4. Temporal changes of dissolved oxygen of water in different treatments

employed.
4.1.1.1.4. Hardness
Hardness of water declined sharply almost up to the first half of investigation in all
the systems including control after which in exhibited two minor peaks during week 7 and
11 respectively (Fig. 5). The overall mean value was highest in Tl (375.57 mg 1- 1)
followed by C2 (348.50 mg 1-1), T2 (329.29 mg 1- 1), Cl (326.86 mg J- 1) and lowest in T3
(319.43 mg J-1). Overall treatment difference exhibited significant (F = 7.85; P < 0.01) .
.Sarker, M., M. 'f. .Sc. "Thesis, 2.013, Vepl of~uacullure 35

'Rtsuf& anl Oiscussion
700
---Tl ~ T2 ---T3 -+--- Cl - C2
600
~ 500
E
'-'
"'~
c 400
"Cl
i..
~
:C 300
200.!---.---,~-r---.--.::~- r----r---,r---r----r-~~~~
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 5. Temporal changes of hardness of water in different treatments employed.
4.1.1.1.5. Total Alkalinity

Similar to the hardness, total alkalinity of water declined during the initial phase and
then recovered following liming during week four, eight and twelve (Fig. 6). The average
value of total alkalinity was highest in T3 (134.29 mg 1- 1) and lowest in T2 (123.71 mg 1-
1). Difference among the treatments exhibited marginal significance (F = 3.186; P < 0.05).
_._ Tl ~ T2 ---T3 - CI - C2
150
£' 140
~
E 130
'-'
.f'
.!: 120
]
< 110
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 6. Temporal changes of alkalinity of water in different treatments employed.
4.1.1.2. Nutritional parameters

4.1.1.2.1. Ortho-phosphate
Ortho-phosphate tended to increase up to week 3-4 in any of the systems employed.
The values then remained near stationary (Fig. 7). The overall mean value was maximal
in Tl (0.08 mg 1- 1) followed by Cl (0.075 mg 1- 1), T2 (0.073 mg i-1), T3 (0.067 mg 1- 1) and
minimal in C2 (0.062 mg 1- 1). Overall difference exhibited strong significance (F = 33.18;
P < 0.001).
36
'R,esuffJ ant!1Jiscusrion
---Tl --e-T2 -.-T3 -+-Cl --C2

0.1
r
'-'
o.09
u
~ 0.08
..c
Q.
rll
Q
,g_ 0.07
I
Q
..c
..c 0.06
t::
0
0.05 +----.----.--... --~---.--.----- -,.-....-----,--- ......,....--,
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 7. Temporal changes of ortho-phosph ate concentratio n of water in different
4.1.1.2.2. Ammonia-Ni trogen

Concentration of ammonia-N tended to increase gradually in all the systems employed
during the investigation period except in T3 and C2 where a decline was observed during
the second half (Fig. 8). The overall mean concentration of ammonia-N either in Tl or C 1
(0.27 mg. 1- 1) was higher by 1.56%, 49.44%, and 64.65% compared to that of T2, C2 and
T3 respectively. Overall treatment difference exhibited high level of significant at 0.001 %
level (F = 17.60).
0.45
- - - Tl --e-T2 ---.!r-T3 --+--- Cl - C2
0.4
0.35
-
.:....
~
0.3
E 0.25
'-'
:z..,
I 0.2
::i::
:z 0.15
0.1
0.05
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 8. Temporal changes of ammonia-nit rogen concentratio n of water in different

4.1.1.2.3. Nitrate-Nitro gen
Nitrate-nitrogen increased gradually as the period of investigation progressed except
in C2, whereas, the value remained almost stationary after 2nd week of investigation (Fig.
9). The highest overall mean value was encountered in Tl (0.24 mg. 1- 1) and lowest in C2
Sarker, M, M 'F. Sc. 'TheriJ, 2.0flj, 1le('f, of?ltjuacufture 37

'f<lsufls anlViscussion
(0.18 mg. 1- 1). Analysis of variance indicated strong significance among the treatments
during the investigation period (F = 16.64; P < 0.001).
0.35
---Tl --B-T2-6-T3-Ct - C 2
0.3
--'
~ 0.25
'-'
z
ci' 0.2
z
0.15
0.1 ---r--..-~-..--.---,-..... .---,,-~--.---.---,---,

1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 9. Temporal changes of nitrate-nitrogen concentration of water in different

4.1.1.2.4. Nitrite-Nitrogen
Nitrite-nitrogen was conspicuous either by absence or representing with very low
concentration during the period of study in any of the treatments employed (Fig. I 0). The
average value of nitrite-N did not differ much among the treatments employed (0.02-0.025
mgJ- 1). Difference among the treatments remained insignificant (P > 0.05).
0.06 ---Tl --&-T2 ..........,_ T3 - Ct - - C2
0.05
£t)II
0.04
e
'-'
0.03
z I
0"' 0.02
z
0.01
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Weeks
Fig. 10. Temporal changes of nitrite-nitrogen concentration of water in different

4.1.1.3. Biological parameters
4.1.1.3.1. Biochemical Oxygen Demand (BOD1)
Biochemical oxygen demand, in general, sharply increased after initiation of the
experiment in any of the systems employed. The values then remained more or less
.Sar(er, M., M. 'F. Sc. 7fiesis, 20fj, Vepf. of:Attuacuffure 38

'/<truffs (,jnrl'/Jirr,urrion
stationary during most part of the investigation period except in T 1 and T2 where an
increasing trend was observed during the later phase (Fig. 11 ). The overall mean value of
BOD 1 was highest in Tl (3.54 mgJ-1), followed closely either by T2 (2.97 mgJ- 1) or Cl
(2.74 mgJ- 1) and lowest in C2 (2.35 mgJ- 1). Overall difference among the treatments
exhibited strong significance (F = 15.40; P < 0.001).
s.s -e--Tt -a-T2 ---+-TJ ~Cl -C2
4.5
,-...
..i..
bf) 3.5
e
.._,
0.- 2.5
0
CCI
1.5
0.5 +--...--.---...--.---...--.-- -...--.---...--.---...--.---- ,

1 2 3 4 S 6 7 8 9 10 11 12 13 14
Weeks
Fig. 11. Temporal changes of biological oxygen demand of water in different

4.1.1.3.2. Gross Primary Productivity (GPP)

Gross primary production in general, tended to increase after 4-5 weeks of inception
of this study and continued up to week I 0-11 after which a decline was observed (Fig. 12).
The overall mean value ofGPP in Tl (150.18 g C m 3 day- 1) was 8.97 to 47.51% higher
than the rest of the treatments. Overall difference was marginally significant at 5% level
(F = 3.34).
-e--TI -a-TI ---+-T3 ~ Cl - C2

300
::;-- 250
;.,....
= 200
..."O
e
U 150
.._,
bf)
i:i..
Q.,
100
~
so
0
1 2 3 4 S 6 7 8 9 10 11 12 13 14
Weeks
Fig. 12. Temporal changes of gross primary productivity in different treatments

employed.
Sar(er, M., M. 'F. Sc. 7herir, 2.015, 1Jept. of11.iju(,lr,ullure 39

'f<tsuftr and1Jimmion
4.1.1.3.3. Net Primary Productivity (NPP)

The temporal trend ofNPP was almost identical to that of GPP. The maximal mean
value (130.78 g C m3 day · 1) was observed in Tl and minimal (80.48 g C m3 day- 1) in C2.
Difference among treatments was significant (F = 3.65; P < 0.05).
300 ---Tl -e-n -T3

--e-c1 -CZ
250
--
-~ 200
..."Cl
e 150
ul)fl
.._, 100
=--
z=-- 50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 13. Temporal changes of net primary productivity in different treatments

employed.
4.1.1.3.4. Community Respiration (CR)

The values of community respiration declined after an initial increase till week 4-6
after which it remained nearly stationary (Fig. 14). Highest overall mean (24.63 g C m 3
day -1) was observed in Cl and lowest (17.53 g C m 3 day -1) in C2. Treatment difference
remained insignificant (P > 0.05).
80
---Tl -e-n - T 3 --e-ct - C Z
70
--
1.,..
('ii
60
:;' 50
e
U 40
l)fl
.._, 30
c.::
U 20
10
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 14. Temporal changes of community respiration in different treatments

employed.
40
'R,suftr anti1Jiscurrion
4.1.1.4. Plankton Population

4.1.1.4.1. Phytoplankton
4.1.1.4.1.1. Total phytoplankton
Total phytoplankton population in any of the systems employed was represented by the
members of the family Chlorophyceae (Scenedesmus sp., Volvox sp., Chlorella sp., etc),
Myxophyceae (Anabaena sp., Merismopedia sp. ), Baccillariophyceae (Navicula sp.,
Asterionella sp., etc), Xanthophyceae (Gonochloris sp.), Euglenophyceae (Euglena sp.),
Zygnematophyceae (Spirogyra sp.)
Temporal trend indicated a gradual increase in any case, however, the magnitude of
increase was highest in Tl (52.81 %) and lowest in C2 (11.95%). The overall mean value
in Tl (6235 nos.i-1), T2 (5035 nos.I-') and T3 (4711.25 nos.I-') was 37.55 to 52.81 % higher
than C2 (Fig. 15). Treatment difference during the period of study was highly significant
(F = 46.01; P < 0.001).
8000 ---Tl -a-- T2 ---.--- T3 ---+- Cl - Cl
::--
~ 7000
c
---§ 6000
-
.:,r:
;5000
c.
,S 4000 -=.;;e=:.----
....
.c
C.. 3000
3
~2000
1000+-~~~~---.~~~~~--~~~~~
20 40 Days 60 80
Fig. 15. Temporal trend in total phytoplankton in different treatments employed.

4.1.1.4.1.2. Percentage Distribution of Phytoplankton
Percent distribution of different groups among the systems in general, exhibited
gradual decrease of both chlorophyceae and bacillariophyceae with concomitant increase
in myxophyceae or other groups in any treatment. The average contribution of
chlorophyceae, bacillariophyceae and myxophyceae was 38-51 %, 25-32% and 17-27%
respectively (Fig. 16).
S'ar(er, M., M. '1. Sc. 'Thesis, 20fj, 1Jepf. of1'.tjuacullure 41

'Rflsuffs antf'[)immion
!2 MYXO liCHLORO !fiZYGNE •BACI 9:XAN ;EUG ".J MYXO !llCHLORO ::!ZYGNE •BACI ~ XAN .: EUG
cl00% 100%
-
.s
..6 80% -=
c
.Sl
80%
-
·c:
-~
"O
.-
60%
..Q
·c:
....
-~
"Cl
,-.,
60%
-;!. 0~
-;:: 40% 40%
c
41
....c
'-
...u
:_ 20%
41
...u 20o/.
41
~
0% 0%
20 40 60 80 20 40 60 80
0 ays Days
~
OMYXO UCHLORO rl ZYGNE r:l MYXO II CHLO RO r, ZYGNE • BACJ '2i XAN :J EUG
•BACJ itilXAN ,-:: EUG
c 100% 100%
-
.i:
_s 80% -=
c
.Sl
80%
-
..Q
·c:
-
·c:
-~
:e;"'
.-
~
60%
--
"O
0~
._,
60%
-...
._,
0
40% 40%
c4,1 c
41
u ...
u
41 20%
4,1 20% ~
~
0% 0%
20 40nays 60 80 20 40 60 80
0 ays
;t MYXO Cl CHLORO :::: ZYGNE • BACI ~ XAN =EUG

100%
-=
i::
.i: 80%
-~-
.Q
·c:
60%
..-
"O
~
._, 40%
-
c
4,1
...
I.I 20%
4,1
~
0%
20 40 Days60 80
~
Fig. 16. Percent(%) distribution of phytoplankton in different treatment
employed.
42
~suffs and'[}jsr,urrion
4.1.1.4.2. Zooplankton
4.1.1.4.2.1. Total Zooplankton
Total zooplankton community was represented by the members of rotifers (Keratella
sp., Brachionus sp.), copepods (Cyclops sp.), cladocerans (Daphnia sp., Moina sp.) and
ostracods. The population increased sharply up to day 40-50 in any of the treatments
followed by a decline except in C2 (Fig. 17). The overall mean value was highest in Tl
(557.5 nos. 1- 1) followed by T2 (481.25 nos. i-1}, Cl (452.5 nos. J-1), C2 (435 nos. 1- 1) and
lowest in T3 (377.5 nos. 1-1). Difference among the treatments remained significant (F =
4.06; P < 0.05).
800 ~Tl -a-Tz -TJ -+-CI -CZ
£ 700
.,;
-=g
0
600
,:!.! 500
=~
g. 400
0
N
] 300
0
r""'
200+-~~~~--.~~~~~---~~~~~
20 40 60 80
Days
Fig. 17. Temporal trend in total zooplankton in different treatments employed.

4.1.1.4.2.2. Percentage Distribution of Zooplankton
Contribution of different groups in the total zooplankton community indicated
remarkably higher abundance of rotifer (36-57%) in any of the treatments. The next
contributor was copepods (27-44%), followed by ostracods (2.8-20%) and cladocerans (5-
8%). Moreover, rotifer population tended to increase as the period of investigation
progressed in all the treatments (Fig. 18).
Sar(er, M., M. 'F. Sc. 'Thesis, 2.0f;j, '[Jeff. of7'.ttuacufture 43

'f<.pJufts and 1Jircunion
!l ROTI • COPE =CLADO -:, OSTRA II ROTI • COPE la! CLADO : : OSTRA
100%
c 100%
-=
i:
-
.s
j 80%
.s
80%
-
·c
~ 60%
-"Cl
.-..
-
.c
·c
:a"'
.-..
60%
t'-"' 40%
-i:
~ 20%
~
-
Q
'-"'
i:
11,l
u
....
40%
20%
l 11,l
Q..
0%
20 40 60 80 20 40 60 80
Days Days
11 ROTI • COPE e CLADO . . OSTRA LJ ROTI • COPE =CLADO i . OSTRA
100%
-
.s
=
i:
-=
c
.s 80%
-
.c
·c
:a.-.."'
t'-"'
60% -
.c
·c
:a.-.."'
~
60%
- i:
11,l
~
40%
20%
-
,_. 40%
Q
c
11,l
u
.... 20%
~ 11,l
Q..
0% 0%
20 40 60 80 20 40 60 80
Days Days
~ IJROTI •COPE eCLADO iJ OSTRA @]

100%
c
0
-:g 80%
.c
·c
.i 60%
"Cl
.-..
-
c4o%
i:
11,l
~
11,l
20%
Q..
0%
20 40 60 80
Days
Fig. 18. Percent(%) distribution of zooplankton in different treatment employed.
4.1.1.4.3. Total plankton

The temporal trend in the total plankton population exhibited similar trend to that of
either total phytoplankton or total zooplankton (Fig. 19). The overall mean value was
.Sark,er, M. , M. 'F. Jc. 'TheJiJ, 2013, 11epl of7liuacufture 44

~ruftr antiOirr:union
highest in Tl (6792.5 nos. 1- 1) followed by T2 (5516.25 nos.1- 1), Cl (5393.75 nos.1- 1), T3
(5088.75 nos. 1- 1) and lowest in C2 (3376.75 nos. 1- 1). Difference among the treatments
remained significant (F = 43.29; P < 0.001).
---Tl -a-T2 -T3 ~Cl -C2

9000
£' 8000
~ 7000
'=
; 6000
~ 5000
j 4000 ~~===----
i:i.
- 3000
~
~ 2000
1000
o+-~~~~~....-~~~~~....-~~~~--,
20 40 60 80
Days
Fig. 19. Temporal trend in total plankton in different treatments employed.
4.1.2. Soil quality

4.1.2.1. Physico-chemical parameter
4.1.2.1.1. Soil pH
Soil pH remained always alkaline in all the systems and ranged from 7.27 to 8.04
during the period of study. pH of soil in any of the systems remained more or less stable
during the second half compared to the first where two peaks with corresponding falls
were observed (Fig. 20).
8.2 ----n -a-n -n ~c1 -c2
7.8
-·=
Cl.
7.6
c5
rJ) 7.4
7.2
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 20. Temporal changes of pH of soil in different treatments employed.
Sarker, M., M. 'F. Sc. 1'herfr, 2.015, 'De(1f, of1f.!tuar:uffure 45

'Rtsuftr anti1Jisr:umon
4.1.2.1.2. Soil Organic Carbon

Organic carbon in general, gradually increased in all the systems during the
investigation period, however, the rates of increase was sharp at inception (Fig. 21 ). The
maximal mean value was attained in Cl (2.04 mg g- 1) and minimum in C2 (1.78 mg g- 1).
Treatment difference exhibited strong significance (F = 27.63; P < 0.001).
..-. 2.5 - n -a-n - - u -c1 -c2
'Qi,
r
'-'
2.3
_g= 2.1
..
"u
.~ 1.9
=
"
Qi,
::; 1.7
·-r.,;s 1.5 -+---.--....-- -.---.--.----r --..---.---.,- ----.-----
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 21. Temporal changes of organic carbon of soil in different treatments employed.
4.1.2.1.3. Soil Available phosphorus

Available phosphorus of soil increased gradually over time in all the treatments during
the first half of investigation followed by a near stationary phase except in T3 and C2
where a fall was exhibited during the second half (Fig. 22). The overall mean
concentration did not differ much either among Tl, T2 and Cl (0.26 to 0.27 mg g- 1) or
between T3 and C2 (0.22 mg g- 1). Overall treatment difference remained highly significant
(F = 41.60; P < 0.001).
---- TJ -B-T2 ---+-TJ - CJ - C2

0.3
"'
=
::; 0.28
.c
c.
~ 0.26
.c :'
c. 'Qi,
.!! Qi,0.24
.c E
~'-'
·; 0.22
;;,..
=s" 0.2
r.,;
0.18 +---,--..--,- ---,--r-~-~- -.--..--~--,. ...----..--,
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Weeks
Fig. 22. Temporal changes of available phosphorous of soil in different treatments

employed.
Sarkftr, M., M. 'F. Si:. 'T~sis, 2.0f!J, 11ef1{, of:Aftuar:uffure 46

'R§suftr anJViscussion
4.1.3. Fish Growth

4.1.3.1. Fish body weight
As expected, weight offish continued to increase over time in any case and the overall
mean value was highest (4.65 g) in Tl and lowest in T3 (2.14 g).
10 ---Tl --a-n - n -c1
---=-
8
t:lfl
6
t:lfl
"ii
~ 4
.....
"C
Q
i:=
2
0
0 15 30 45 60 75 90
Days
Fig. 23. Temporal trend in growth of common carp in different treatments employed.
4.1.3.2. Body weight gain(%)
Temporal trend of weight gam (%) declined sharply as the period of rearing
progressed (Fig. 24). The average value was highest in Tl (54.47%) followed by Cl
(50.15%), T2 (43.88%) and lowest in T3 (27.73%).
140 ---Tl --a-n -T3 -Cl
120
100
=
·; 80
t:lfl
i 60
.....
"g 40
.J:J
~ 20
0
15 30 45 60 75 90
Days
Fig. 24. Temporal trend in body weight gain (%) of common carp in different
4.1.3.3. Specific growth rate (SGR)

Temporal trend in SGR (specific growth rate) of fish among the treatments was
distinctly different from one treatment to another (Fig. 25). The overall mean was highest
in Tl (9.27%) and lowest in T3 (2.59%).
47
'R,§suflr and 1JiJcusrion
20 ------- Tl --a- T2 -....- T3 - Cl
15
0 +-~~---.~~~~~~~~~~~~~~--,
15 30 45 Days 60 75 90
Fig. 25. Temporal trend in specific growth rate (%) of common carp in different
4.1.3.4. Absolute growth

Absolute growth offish in Tl and Cl was distinctly higher by 62.41 % and 29.97 %
compared to T2 and T3 respectively.
9
2
Tl T2 T3 Cl
Treatments
Fig. 26. Absolute growth of common carp in different treatments employed.
4.2. Discussion
Absolute weight gain of the test fish (Cyprinus carpio L.) decreased polynomially (y
= -0.562 x2 +0.745x+8.255; R2 = 0.991) (Fig. 27) from Tl to T3 either with decreasing
ration size from 6% to 3% or with and without biofloc supplementation. Therefore, it was
established that both the rate of feed application and biofloc exerted a definite role in
growth of the test fish .
.Sar(er, M., M. '1. Sc. 'Thesis, 2.0f;j, 1Jepf. of7'.truacuffure 48

'R§sullr anl1Jiscussion
Absolute weight gain (g)

9
y = -0.5625x 2 + 0.7455x + 8.255
,-.
8 R2 = 0.9916
~ 7
c:
·; 6
-~
.c 5
-~
~
4
~
-
0
Ill
:I
3
2
"'
.c
< 1
0
Tl Cl T2 T3
Treatments
Fig. 27. Modular trend in absolute weight gain in different treatments employed.
Moreover, such intensity of impact upon growth of the fish was extremely discernible
among the treatments where in presence of biofloc, feed application @ 6% body weight
(Tl) resulted in absolute weight gain nearly by an order of magnitude (Fig. 28) against
6.07 times in T2 (@ 3% feed). However, comparing an iso-nutrient regime in Tl and Cl
effect of biofloc in increasing the absolute weight gain was only 6.42% in Tl. Again,
biofloc alone in T3 resulted only in 2.79 times absolute weight gain against 9.27 times
with only feed@ 6% body weight. Therefore, biofloc alone was not sufficient enough for
the comparable growth of the test fish under supplementary feeding regime.
Absolute wt. gain(%)
,__ 1000
..
~
'-'
c: 800 ·
·;
-
-~
~
.c 600
~
400
-
0
"'
.c
~
Ill
:I
200
< 0
Tl Cl T2 T3
Treatments
Fig. 28. Absolute weight gain (%) of fish in different treatments employed.
This was further supported by the fact that mortality of the test fish was also highest
(33.33%) in the biofloc alone treatment (T3) and such rate declined by a modular pattern
.Sarker, M., M. 'F. Sc. 'Thesis, 2.0f!J, fJept. of71.Lruacullure 49

'R.tsullr and1Jiscussion
(y = 30.68x-0 ·60 ; R2 = 0.892) either with feeding support alone or both with feeding and
biofloc support (Fig. 29).
Mortality rate y = 30.684x-0·607

35 R2 = 0.8921
•
30
-QI
f 25
....
;<,::
-;
t: 20
0
~
15
10
T3 T2 Cl Tl
Treatments
Fig. 29. Modular trend in mortality rate of fish in different treatments employed.
However, presence of biofloc improved the FCR both in Tl (2.09) and T2 (1.75)
against C 1 (2.31 ). Therefore, in spite of decrease in absolute weight gain in T2 because of
the most favourable FCR (Fig. 30) and less nutrient loading the system was proved to the
economically and ecologically more supportive. This was further corroborated by the fact
that FCE (%) (Fig. 31) decreased polynomially (y = 2.288 x2-16.04x+70.81 R2 = 1) with
an identical reverse trend in FCR with identical fit (y = -0.057X2+0.506x+ 1.302; R2 = I).
Under biofloc supported culture system reduction of feed amount by 50% in T2 resulted in
concomitant reduction of growth of the test fish by 38.42% compared to Tl (Fig. 30). So,
it was clear that biofloc improved the performance of the supplementary feed both in terms
FCR and FCE.
Sar~er, M., M. 'F. Sc. 'Thesis, 2.0f!j, 1Je(1l of1Yfuacufture 50

'R_tsu(& anl fJisr.usfion
2.4
2.3 y = -0.057x 2 + 0.5069x + 1.3027
R2 = 1
2.2
2.1
a:
U 2
~
1.9
1.8
1.7
1.6
T2 Tl Cl
Treatments
Fig. 30. Modular trend of FCR in different treatments employed.
60
y = 2.2889x 2 - 16.044x + 70.811

R2 = 1
40
T2 Tl Cl
Treatments
Fig. 31. Modular trend of FCE in different treatments employed
Increase in the concentration of all the major nutrients like ortho-phosphate,

ammonia-nitrogen and nitrate-nitrogen in the feed supplemented treatments was because
of the uneaten feed and metabolic by-products as well. Several studies (De Silva and
Hasan, 2007; Sinha, 1979; Wee, 1991 and 1998) have indicated that supplementary feed act
as source of nitrogen and phosphorus in the culture system. Moreover, the resultant
increase in nitrogen and phosphorus was subsequently manifested in higher growth of fish
in the biofloc treated systems as nitrogen mobilization in presence of suitable carbon
reserve in bioflloc was effected in microbial protein synthesis. This in turn was operative
in higher growth in fish Xu et al. (2012), Ray et al. (2009), Burford et al. (2003) have
Sark_er, M, M 'F. ..SC. 'Thesis, 2.0f7, fJept. of~uacuffure 51

'Risuffs and1.Jiscussion
reported that in biofloc technology nitrogen is transformed into protein in presence of a

strong carbon pool.
Again, higher presence of nutrients in the treatment with biofloc with fish (T3)
compared to its counterpart without fish (C2) was because of the bioturbation effect of the
test fish. Das and Jana (1992) and Jana et al. (2001) conclusively observed that
benthivorous fish like Cyprinus carpio was effective in increasing the nutrient status of
water through bioturbation of the sediment.
The increase in BOD1 was concomitant with the NPP (y = l.7365e0·0033 x; R2 = 0.71)
(Fig. 32) as well as total planktonic load (y = -243.6x2 + 2571.7x- 64.544; R 2 = 0.64) (Fig.
33) in any treatment indicated that phytoplanktonic load and resultant primary production
was operative in increasing the BOD1 particularly in biofloc treatment and with increasing
feed load from 3% to 6% of body weight.
5 y = 1. 7365e0.0033x
R2 = 0.7101
4.5 •
4
:::--
.I..
bJ) 3.5
e •
-
'-"'
Q
0
3
= 2.5
2 •
1.5
50 100 150 200 250 300
NPP
Fig. 32. Relationship between BOD1 and NPP in different biofloc treatments.
y = -243.6x 2 + 2571.7x - 64.544

7000
R2 = 0.6453
6500
:::--
~ 6000
0
S 5500
c •
~ 5000
c •
..!!
Q.,
4500 .
• •
~ 4000 • •
E--o
3500
3000+-~--,~~.......,....~~~~-----.....-~~~~~~-----.
1.5 2 2.5 3 3.5 4 4.5 5
BOD 1 (mg 1-1)
52
'/qsuffs and'[);:,ccmion
Fig. 33. Relation ship between total plankton and NPP in different biofloc treatmen ts
Though water pH declined in amount of the treatments with biofloc supplemented

with feed during the first half, it remained congenial for the test fish. Jhingran (1985)
opined that pH range of 7 .5 to 8.5 is ideal for cultivation of fish. Similarly, alkalinity and
hardness also remained within the acceptable range. (Fig. 5 and 6). The decline in hardness
in biofloc treated system was observed (Bhatnagar and Devi, 2013).
Both GPP, NPP and absolute weight gain were highest in T 1 indicated that biofloc
not only directly supported the test fish as a valuable source of nutrition but supported the
phytoplanktonic community and photosynthesis as well. Therefore, the heterotrophic
components within the system in tum supported mineralization to provide more nutrients
for the phytoplankton who in return supplied the carbon as energy source to the former.
As a result a dynamic complementary process in between the autotrophs and the
heterotrophs was developed. Several studies (Wang & Priscu, 1994; Kamjunke et al.,
1997; Duvall et al., 2001; Brett et al., 1999) confirmed the importance of autotrophic
production in supporting the heterotrophic pathway in manure and feed based culture
systems.
The abundance in planktonic community in terms of relative contribution of

myxophyceae and rotifer community in the biofloc treatments with feed supplementation
particularly in feeding@ 6% body weight/day indicated organic loading, because both the
above groups are considered as indicator organisms (Dulic et al., 2006, 2009) normally
abundant in organically enriched polluted environment. Therefore, in spite of higher
growth of the test fish in Tl it was inferior in terms of ecological health compared to T2
in which the rate of feed application was reduced to half.
Though, there was high fluctuation ofpH of soil particularly in Tl, T2 and Cl during
the first half of study, it remained always alkaline. Moreover, in general, the higher
presence of organic carbon of soil in biofloc treatments (Fig. 21) might have been resulted
in comparatively higher concentration of available phosphorus in soil of the above
treatments. Therefore, biofloc not only impacted the water quality and growth of the test
fish but also favoured the phosphate mineralization in the soil phase.
Sark.Pr, M, M "F. Sc. 'Thesis, 2.0f!j, Vept. of:lufuacufture

53
Su m m ar y an d
Co nc lu sio n
5. Summary and Conclusion
From the Results and Discussion chapter of this thesis on Comparative efficacy of
biofloc and feed based Common carp (Cyprinus carpio, L.) production system with
special reference to environmenta l health, the following findings are summarized
herein:
1. It was established that both the rate of feed application and biofloc exerted a definite
role in growth of the test fish (Cyprinus carpio L.). However, biofloc alone was not
sufficient enough for the comparable growth of the test fish under supplementary
feeding regime.
2. Biofloc alone resulted only in 2.79 times absolute weight gain against 9.27 times with
only feed @ 6% body weight/day.
3. Absolute weight gain of Cyprinus carpio L. decreased polynomially (y = -0.562 x2
+0.745x+8.255; R2 = 0.991) (Fig. 26) either with decreasing ration size from 6% to
3% or with and without biofloc supplementation.
4. In presence of biofloc, feed application @ 6% body weight/day resulted in absolute
weight gain nearly by an order of magnitude against 6.07 times in feeding@3% body
weight/day.
5. Comparing an iso-nutrient regime (@ 6% body weight/day), effect of biofloc in
increasing the absolute weight gain was only 6.42%.
6. Mortality of the test fish was highest (33.33%) in the biofloc only treatment and it
declined by a modular pattern (y= 30.68x·0 ·60 ; R2= 0.892) either with feeding support
alone or both with feeding and biofloc support (Fig. 28).
7. Presence ofbiofloc improved the FCR both in Tl (2.09) and T2 (1.75) against control
(2.31 ).
8. In spite of less absolute weight gain with feeding @ 3% body weight/day compared
to feeding @ 6% body weight/day, because of the most favourable FCR and less
nutrient loading, the system was proved to the economically and ecologically more
sustainable in presence of bioflocs.
9. Under biofloc supported culture system reduction of feed amount by 50% in resulted
in concomitant reduction of growth of the test fish by 38.42%. So, it was clear that
54
Summary anlConclusion
biofloc improved the performance of the supplementary feed both in terms FCR and
FCE.
10. Increase in the concentration of all the major nutrients like ortho-phosphate, ammonia-
nitrogen and nitrate-nitrogen in the feed supplemented treatments was because of the
uneaten feed and metabolic by-products as well.
11. The increase of nitrogen and phosphorus was subsequently manifested in higher
growth of fish in the biofloc treated systems as nitrogen mobilization in presence of
suitable carbon reserve in bioflloc was effected in microbial protein synthesis.
12. Higher presence of nutrients in the treatment with biofloc with fish compared to its
counterpart without fish was because of the bioturbation effect of the test fish.
13. The increase in BOD1 was concomitant with the NPP (y = l.7365e 0 ·0033 x; R2 = 0.71)
as well as total planktonic load (y = -243.6x2 + 2571.7x- 64.544; R2 = 0.64) in any
treatment indicated that phytoplanktonic load and resultant primary production was
operative in increasing the BOD1 particularly in biofloc treatments and with increasing
feed load from 3% to 6% of body weight.
14. Biofloc not only directly supported the test fish as a valuable source of nutrition but
supported the phytoplanktonic community and photosynthesis as well.
15. The abundance in planktonic community in terms of relative contribution of
myxophyceae and rotifer community in the biofloc treatments with feed
supplementation particularly in feeding @ 6% body weight/day indicated organic
loading and therefore, was inferior in terms of ecological health compared to the
treatment in which the rate of feed application was reduced to half.
16. The higher presence of organic carbon of soil in biofloc treatments might have been
resulted in comparatively higher concentration of available phosphorus in soil of the
above treatments.
17. Therefore, biofloc not only impacted the water quality and growth of the test fish but
also favoured the phosphate mineralization in the soil phase.
Sark,r, "M., "M. 'f. Sc. 'Thesis, 20f!j, 1Je('l of7ltjuacufture

55
gu tur e Scope of
Research
6. Future scope of Research
As the present study on Comparative efficacy of hiojloc and feed based Common carp
(Cyprinus carpio, L.) production system with special reference to environmental health
was short term and time bound, it was inherent with so many limitations. Several aspects
viz. community composition of the bioflocs particularly with particular reference to the
heterotrophs, periodic qualitative and quantitative estimation of bioflocs within the culture
tanks with special reference to nutritional value, estimation of heterotrophic production and
mineralization of organic residues under biofloc treated systems etc. could not be addressed
because of the limitations of time and infrastructural facilities. Therefore, such study should
be continued further with long term basis plugging such loop holes in planning and
designing so as to get a time series data for ultimate validation of the results. Moreover,
comparative studies with variable media compositions in the light of the several media being
used in shrimp culture sectors, variable test fish and with variable feeding regimes under
different levels of crude proteins and feeding rate should be addressed by the future
researchers. This is because, as biofloc has been proved to be effective in reducing the
artificial feed of common carp (Cyprinus carpio L.) by 50% with concomitant improvement
in environmental health of the culture system through reduced organic loading, this
technology has a tremendous scope for the culture of omnivorous fin fishes.
56
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XVIII
CURRICULAM. VITAE
Full Name: Madhurima Sarker
Phone I Mobile: 9007564270
Email : madhurimasarker9@gmail.com
EDUCATIONAL BACKGROUND
Serial Education I Subjects Board I Percentage Class

No. Degree University of Marks I
10 point
2rade
1. Madhyamik Bengali, English, W.B.B.S.E 78.8 First
Pariksha Mathematics, Ph. Science,
(2007) Lf. Science, History,
Geography.
2. Higher Secondary Bengali, English, Physics, W.B.C.H.S.E 68.90 First
(2009) Chemistry, Biology,
Mathematics & Env.
Science
3. B.F.Sc (Bachelor Fishery Sciences West Bengal 72.80 First
of Fishery University of
Sciences) Animal &
(2013) Fishery
Sciences
4. M.F.Sc (Master Aquaculture W.B.U.A.F.S Appearing
of Fishery
Sciences)
(2015)
PROFESSIONAL QUALIFICATIONS:
1 One week training at Kakdwip Reaserch Centre on Brackish Water Aquaculture

(CIBA, ICAR) in 2012.
2 Training at Fish Technological Station (FTS, Junput) under State Govt. in 2012
3 Training at Freshwater Fisheries Research Training Centre (FFRTC, Kulia,

Kalyani) under State Govt. in 2012.
4 Block Survey on Status & Types of Fisheries in Nandakumar block under the Fishery
Extension Officer and Block Development Officer, Nandakumar block in 2012.
5 Training "Fisheries Work Experience Program" at North 24 Perganas Krishi Vigyan
Kendra (K.V.K), Ashokenagar, held during 17th to 23th August, 2012.
6 "On Farm Training of Various aspects of prawn breeding and hatchery

management" on 29.09.12 at Experimental Prawn Hatchery, Dept. of Fisheries, Digha,
Purba Medinipur.
7 Experimental learning on "Hands on Training" programme held in Dept. of Fish

Processing Technology F.F.Sc., W.B.U.A.F.S. Kolkata in 2013.
8 "On Farm Training on "Integrated Duck-cum Fish Farming" from 08.12.12 to

11.12.12 under the DST Project, Dept. of Fishery Extension, F.F.Sc., W.B.U.A.F.S.
Kolkata.
9 Training on different Central Institute like CIFE, Saltlake; CIFA, Rahara; CIFRI,
Barrackpore and BENFISH, Kolkata in 2012.
10 Training on "Sustainable Brackish Water Aquaculture Practices" from 21-25 July

2015 at CIBA Kakdwip Reasearch Centre.
11 Training on "Ornamental Fish Breeding and Culture" from 04-10 August 2015 at
Central Institute of Fisheries Education, Salt lake, Kolkata.
PERSONAL DETAILS
Date of birth 20.11.1990
AJ:!;e 24+
Birth place Hooglv
Gender Female
Marital Status Unmarried
Blood J:!;roup o+
Category SC
Religion Hinduism
Nationality Indian
Correspondence Address Faculty of Fishery sciences,
5,budherhatroad, Chakgaria,panchasavar,kol-94
Permanent Address Rishra, Hoo,:!;lY, 712250
Hobbies I interests Readinj:?; Books, Singing song
Languages known:
Lan2ua2es Read Speak Write

Bengali Yes Yes Yes
English Yes Yes Yes
Hindi No Yes No
I do hereby declare that the above mentioned particulars are true to the best of my
knowledge and belief.
Place: Kolkata Signature

Date:()':). \O, ~1S

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COMPARATIVE EFFICACY OF BIOFLOC AND FEED BASED

COMMON CARP (CYPRINUS CARPIO, L.) PRODUCTION SYSTEM

West Bengal University of Animal and Fishery Sciences,

In partial fulfilment of the requirements for the Degree of

Master of Fishery Sciences

. . re ...''.\Q ••~V.·j _di I, ...........

Faculty of Fishery Sciences

Dr. S. K. Das Ref. No. -------------------

Faculty of Fishery Sciences

APPROVAL OF EXAMINERS FOR THE AWARD OF THE

We, the undersigned, have been satisfied with the perfonnance of

1. Dr. S. K. Das Chairman,

2.~~1:.~.. f ~ .... External Examiner

3. Dr. T. K. Ghosh Member,

4. Dr. B.K. Das Member,

5. Prof. S. S. Dana Member,

1. 3.1. Experimental protocol followed in the present study. 33

2. 4.1.2. Temporal changes of temperature of water in 34

3. 4.1.3. Temporal changes of pH of water in different 35

4. 4.1.4. Temporal changes of dissolved oxygen of water 35

5. 4.1.5. Temporal changes of hardness of water in different 36

6. 4.1.6. Temporal changes of total alkalinity of water in 36

7. 4.1.7. Temporal changes in the ortho-phosphate 37

8. 4.1.8. Temporal changes of ammonia-nitrogen 37

9. 4.1.9. Temporal changes of nitrate-nitrogen concentration 38

Temporal changes of nitrite-nitrogen concentration of

11. 4.1.11. Temporal changes of biological oxygen demand of 39

12. 4.1.12. Temporal changes of gross primary productivity in 39

13. 4.1.13. Temporal changes of net primary productivity in 40

14. 4.1.14. Temporal changes of community respiration in 40

15. 4.1.15. Temporal trend in total phytoplankton in different 41

16. 4.1.16. Percent(%) distribution of phytoplankton in different 42

17. 4.1.17. Temporal trend in zooplankton in different treatments 43

18. 4.1.18. Percent(%) distribution of zooplankton in different 44

19. 4.1.19. Temporal trend in total plankton in different 45

Temporal changes of pH of soil in different treatmes

21 4.1.21. Temporal changes in the organic carbon of soil in 46

Temporal changes in the available phosphorus of

23 4.1.23. Temporal trend in body weight of common carp in 47

24 4.1.24. Temporal trend in body weight gain(%) of common 47

25 4.1.25. Temporal trend in specific growth rate(%) of 48

26 4.1.26. Temporal trend in absolute growth of common carp 48

27 4.2.27. Modular trend of absolute weight gain in different 49

28 4.2.28. Absolute weight gain(%) of fish in different 49

29 4.2.29. Modular trend of mortality rate of fish in different 50

31 4.2.31. Modular trend of FCE in different treatments

32 4.2.32. Relationship between BOD1 and NPP in different

Relationship between total plankton and NPP in

Sarker, M., M. "F. Sc. "Thesis, 20(3, Oefl of7'.ttuacullure 2

Sarker, M., M. 'F. Sc. 7heJiJ, 2.0fj, 1Jeff. of:AtJuacufture 4

aquaculture production comprise 41 % ( 63 .6 million tonne) and capture fisheries production

S'arkflr, M., M. 'F. Sc. 'lhesis, 2.0f'j, Ve('l of7itjuacullure 7

should be economically feasible) with high oxygenic conditions. By adding carbohydrates to

Sark,pr, M., M. 'F. Sc. 1"hesis, 20(5, 'Dept. of7!.ttuacufture 10

Sark,.er, 7vf., 7vf. "F. Sc. "Thesis, 2.0{5, '[}e('{, of1'.tjuacullure 11

2.4.2. Dissolved oxygen

.Sarker, M., M. "F. Sc. 1'he1i1, 20f!j, 'Dept. of:lf.ttuacullure 12

.Sark.er, M., M. 'F. Sc. 'Thesis, 2.0{5, Oe('l ofJf.t;uacufture 13

Sark.er, M., M. 'F. Sc. 'Thesir, 2.0{5, Ve(1f. of1'.tjuacufture 14

Sark.er, M., M. 'F. Sc. 'ThesiJ, 2.0<5, 1Je('f. of7'.ttuacufture 17

Sar(er, 'M., 'M. 'F. Sc. 'Thesis, 2.0(5, 1Je(1f. of?l.rjuacufture 19

2.10. Applications of bioflocs in aquaculture

Sark.er, M., M. "F. Sc. 'Thesis, 2.015, 1Jepf. of11.r.juacufture 20