Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus.Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administeringAPV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.

Bioprocess Technology for Antagonistic Pseudomonas MCCB 102, 103,Micrococcus MCCB 104, and Evaluation of Synechocystis MCCB 114, 115 as Probiotics for the Management of Vibrio in Shrimp Culture Systems

National Centre for Aquatic Animal Health, School of Environmental Studies, Cochin University of Science and Technology

Development of nitrifying and photosynthetic sulfur bacteria based bioaugmentation systems for the bioremediation of ammonia and hydrogen sulphide in shrimp culture

This thesis entitled Development of nitrifying ans photosynthetic sulfur bacteria based bioaugmentation systems for the bioremediation of ammonia and hydregen sulphide in shrimp culture. the thesis is to propose a sustainable, low cost option for the mitigation of toxic ammonia and hydrogen sulphide in shrimp culture systems. Use of ‘bioaugmentors’ as pond additives is an emerging field in aquaculture. Understanding the role of organisms involved in the ‘bioaugmentor’ will obviously help to optimize conditions for their activity.The thesis describes the use of wood powder immobilization of nitrifying consortia.Shrimp grow out systems are specialized and highly dynamic aquaculture production units which when operated under zero exchange mode require bioremediation of ammonia, nitrite nitrogen and hydrogen sulphide to protect the crop. The research conducted here is to develop an economically viable and user friendly technology for addressing the above problem. The nitrifying bacterial consortia (NBC) generated earlier (Achuthan et al., 2006) were used for developing the technology.Clear demonstration of better quality of immobilized nitrifiers generated in this study for field application.

Bioremediation in Shrimp Culture Systems

Budidaya menghasilkan sejumlah besar limbah, terdiri dari metabolisme oleh-produk, makanan sisa, materi feses dan residu dari masukan profilaksis dan terapeutik, mengarah pada penurunan kualitas air dan wabah penyakit. Bioremediasi, aplikasi mikroba / enzim untuk tambak, adalah metode yang sedang digunakan untuk meningkatkan kualitas air dan menjaga kesehatan dan stabilitas sistem akuakultur. Bioremediasi melibatkan mineralisasi bahan organik menjadi karbon dioksida, memaksimalkan produktivitas primer yang merangsang produksi udang, nitrifikasi dan denitrifikasi untuk (1) menghilangkan kelebihan nitrogen dari tambak dan (2) mempertahankan beragam dan stabil masyarakat kolam dimana patogen dikeluarkan dari sistem dan spesies diinginkan mendapatkan didirikan. Selain dari bahan organik merendahkan (detritivorous) bakteri heterotrofik, nitrifikasi, bakteri denitrifikasi dan fotosintesis umumnya digunakan dalam bioremediasi.

Carbon/Nitrogen Ratio Optimization and Periphyton Development on the Production and Sustainability of Penaeus Monodon(Fabricius)in Extensive Culture System

Controlling the inorganic nitrogen by manipulating carbon / nitrogen ratio is a method gaining importance in aquaculture systems. Nitrogen control is induced by feeding bacteria with carbohydrates and through the subsequent uptake of nitrogen from the water for the synthesis of microbial proteins. The relationship between addition of carbohydrates, reduction of ammonium and the production of microbial protein depends on the microbial conversion coefficient. The carbon / nitrogen ratio in the microbial biomass is related to the carbon contents of the added material. The addition of carbonaceous substrate was found to reduce inorganic nitrogen in shrimp culture ponds and the resultant microbial proteins are taken up by shrimps. Thus, part of the feed protein is replaced and feeding costs are reduced in culture systems.The use of various locally available substrates for periphyton based aquaculture practices increases production and profitability .However, these techniques for extensive shrimp farming have not so far been evaluated. Moreover, an evaluation of artificial substrates together with carbohydrate source based farming system in reducing inorganic nitrogen production in culture systems has not yet been carried-out. Furthermore, variations in water and soil quality, periphyton production and shrimp production of the whole system have also not been determined so-far.This thesis starts with a general introduction , a brief review of the most relevant literature, results of various experiments and concludes with a summary (Chapter — 9). The chapters are organised conforming to the objectives of the present study. The major objectives of this thesis are, to improve the sustainability of shrimp farming by carbohydrate addition and periphyton substrate based shrimp production and to improve the nutrient utilisation in aquaculture systems.

Studies on the bacteria associated with Penaeus indicus in a culture system

Ocean persists as a rich and renewable source of cheap protein for the whole world. Among the prawns/shrimps landed from the Indian Ocean and her backwaters, more than 90% are exported to affluent countries. The Indian white prawn Penaeus indicus, constitutes the major portion of the frozen shrimps exported from India every year. The present study is aimed at gathering information on the total heterotrophic bacteria (THB) associated with B. indicus, with special reference to eggs, nauplii, zoeae, mysis, and post larvae in hatchery, and juveniles and adults in culture pond. Simultaneously, IHB associated with E. indicus in its natural habitat also is studied for comparison. It is envisaged that this information will be highly useful for modifying the existing hatchery and pond management-practices.

Studies on The Ecology And Production of Algae in Prawn Culture Systems

Existing method of culture were largely based on empirical knowledge. Lacking a scientific basis as such methods did, they were often wasteful and suffered severe limitation. Modern methods of fish and prawn culture based on scientific research, have revolutioned the industry in recent years and not only extended its scope to cover the whole country but led to increased fish and prawn production. An understanding of the biological capability of the water in the perennial and seasonal culture ponds, and the nature and extent of the influence of the abiotic factors on the production of organisms in the primary level of food chain would contribute to effectively implement management measures in the stocking strategies and in the evaluation of economics of production of prawns. It is against this background that the present topic of investigation "Studies on the ecology and production of algae in prawn culture systems” was selected.

Studies on the induction of variation through in vitro culture in Jatropha curcas L

Given the economic importance of Jatropha curcas, and its limited availability in the wild, it would be desirable to establish plantations ofthe tree so as to obtain assured supply of raw material for extraction of phytochemicals, and seeds for production of biodiesel. However both seed propagation as well as propagation by cuttings is unsatisfactory in this tree species. Seeds have poor viability and are genetically heterozygous leading to genetic variability in terms of growth, biomass, seed yield, and oil content. Stern cuttings have poor roots and the trees are easily uprooted. Tissue culture techniques could possibly be gainfully employed in the propagation of elite plants ofJaIropha. When plant tissue is passaged through in vitro culture, there is possibility of induction of variations. An estimation of somaclonal variability is useful in a determination of culture protocols. Molecular markers could be employed to estimate the amount of variations induced in callus and regenerants by different honnonal combinations used in culture. In this context the present study aims to develop an in vitro propagation protocol for the production of plantlets and to evaluate the variation induced in callus and regenerants in comparison with mother plant by the use of molecular markers and by studying phytochemicals and bio active compounds present in callus and regenerated plants

Immobilization of nitrifying bacterial consortia on wood particles for bioaugmenting nitrification in shrimp culture systems

Shrimp grow out systems under zero water exchange mode demand constant remediation of total ammonia nitrogen (TAN) andNO2 −–Nto protect the crop. To address this issue, aninexpensive and user-friendly technology using immobilized nitrifying bacterial consortia (NBC) as bioaugmentors has been developed and proposed for adoption in shrimp culture systems. Indigenous NBC stored at 4 °C were activated at room temperature (28 °C) and cultured in a 2 L bench top fermentor. The consortia, after enumeration by epifluorescence microscopy,were immobilized on delignifiedwood particles of a soft wood tree Ailantus altissima (300–1500 μm) having a surface area of 1.87m2 g−1. Selection of wood particle as substratumwas based on adsorption of NBC on to the particles, biofilm formation, and their subsequent nitrification potential. The immobilization could be achievedwithin 72 h with an initial cell density of 1×105 cells mL−1. On experimenting with the lowest dosage of 0.2 g (wet weight) immobilized NBC in 20 L seawater, a TAN removal rate of 2.4 mg L−1 within three days was observed. An NBC immobilization device could be developed for on site generation of the bioaugmentor preparation as per requirement. The product of immobilization never exhibited lag phase when transferred to fresh medium. The extent of nitrification in a simulated systemwas two times the rate observed in the control systems suggesting the efficacy in real life situations. The products of nitrification in all experiments were undetectable due to denitrifying potency, whichmade the NBC an ideal option for biological nitrogen removal. The immobilized NBC thus generated has been named TANOX (Total Ammonia Nitrogen Oxidizer)

Haematology of Fenneropenaeus indicus in response to environmental alterations and microbial infections

Aquaculture has developed to become one of the fastest growing food producing sectors in the world.Today India is one among the major shrimp producing countries in the world.There are extensive and intensive shrimp culture practices. In extensive shrimp culture, shrimps are stocked at low densities (< 25 PLs m'2)in large ponds or tidal enclosures in which little or no management is exercised or possible. Farmers depend almost entirely on natural conditions in extensive cultures. Intensive shrimp culture is carried out in high densities (>200 PLs m'2). Much of the world shrimp production still comes from extensive culture.There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which some times has been accompanied by ecological impacts and economic loss due to diseases. The expansion of shrimp culture always accompanies local environmental degradation and occurrence of diseases.Disease out breaks is recognised as a significant constraint to aquaculture production. Environmental factors, water quality, pollution due to effluent discharge and pathogenic invasion due to vertical and horizontal transmission are the main causes of shrimp disease out breaks. Nutritional imbalance, toxicant and other pollutants also account for the onset of diseases. pathogens include viruses, bacteria, fungi and parasites.Viruses are the most economically significant pathogens of the cultured shrimps world wide. Disease control in shrimp aquaculture should focus first on preventive measures for eliminating disease promoting factors.ln order to design prophylactic and proactive measures against shrimp diseases, it is mandatory to understand the immune make up of the cultivable species, its optimum culture conditions and the physico chemical parameters of the rearing environment. It has been proven beyond doubt that disease is an end result of complex interaction of environment, pathogen and the host animal. The aquatic environment is abounded with infectious microbes.The transmission of disease in this environment is extremely easy, especially under dense, culture conditions. Therefore, a better understanding of the immune responses of the cultured animal in relation to its environmental alterations and microbial invasions is essential indevising strategic measures against aquaculture loss due to diseases. This study accentuate the importance of proper and regular health monitoring in shrimps employing the most appropriate haematological biomarkers for application of suitable prophylactic measures in order to avoid serious health hazards in shrimp culture systems.

Apptication of Biofloc Technology (BFI) in Ihe nunery rearing and farming of giant freshwater prawn, Macrobrachillm rosenbergii (de Man)

This thesis Entitled Application of Biofloc technology (BFT) In the Nursery Rearing and Farming of Giant Freshwater Prawn,Macrobrachium Rosenbergii(De Man). Aquaculture, rearing plants and animals under controlled conditions is growing with an annual growth rate of 8.3% in the period 1970-2008 (FAO, 2010). This trend of growth is essential for the supply of protein-rich food for ever increasing world population. But growth and development of aquaculture should be in sustainable manner, preferably without jeopardizing the aquatic environment.In the present study, the application of BFT in the nursery rearing and farming ofgiant freshwater prawn, M. rosenbergii, is attempted. The result of the study is organised into eight chapters. In the first chapter, the subject is adequately introduced. Various types of aquaculture practices followed, development and status of Indian aquaculture, present status of freshwater pravm culture, BF T and its use for the sustainable aquaculture systems, theory of BFT based aquaculture practices, hypothesis, objective and outline of the thesis are described. An extensive review of literature on studies carried out so far on biofloc based aquaculture are given in chapter 2. The third chapter deals with the application of BFT in the primary nursery phase of freshwater prawn. Several workers suggested the need for an intermediate nursery phase in the culture system of freshwater prawn for the successful production. Thirty day experiment was conducted to study the effect of BFT on the water quality, and animal welfare under the various stocking densities. The study concluded that stocking finfishes in biofloc-based monoculture system of freshwater prawns has the potential of increasing total yield. Prawns having a higher commercial value than finfishes besides ensuring economic sustainability. Results showed that prawn yield and survival was better in catla dominated tanks. Based on the results of the study, it is recommended to incorporate 25% rohu and 75% catla in the biofloc-based culture system of giant freshwater prawns. The results of the present study also recommend to stock relatively larger catla for biofloc-based culture system. Fish production was also higher in the 100% catla tank. When catla was added in higher percentages it should ensured that the hiding objects in the culture ponds shall be used in order to reduce the chance of cannibalism among prawns. rohu and catla equally have the ability to harvest the biofloc, catla consumes the planktonic contributes in the floc whereas rohu grazed on the bacterial consortium suspended in the water column. In Chapter 8, recommendations and future research perspectives in the field of biofloc based aquaculture is presented.

Studies on vibrio spp. in juveniles of penaeus indicus in culture systems

The present study is carried out to understand (i) the incidence and occurrence of species of Vibrio in different culture systems in and around Cochin, (ii) characteristics of vibrios isolates, their ecology including growth response to various hydrological parameters, sensitivity to about 40 antibiotics, and (iii) role of physico-chemical parameters in pathogenicity of vibrios, etc. and the results emerged from the investigations are important and encouraging for better understanding of the 'vibriosis' in the culture systems to develop remedial measures to control diseases. The Thesis begins with an “Introduction” followed by “A review of literature” on diseases of penaeid shrimps with particular reference to 'vibriosis' and “Material and methods” which details with the methods and procedures followed in the experiments and analyses of data. This Thesis consists of three chapters. Chapter Ideals with the incidence and ecology of Vibrio spp. in water, sediment and in juveniles of the Indian white prawn Penaeus indicus in the culture systems. In Chapter 2, characteristics of vibrio isolates including growth response to various levels oftemperature, salinity and pl 1, sensitivity to 40 antibiotics and minimal inhibitory concentration tests are detailed.e out-breaks. The Chapter 3 discusses the role of physico-chemical parameters in the incidence, seasonal abundance of Vibrio spp. and in 'vibriosis'. A summary of the whole work and list of references are also included at the end. This study gives a detailed information regarding the incidence and ecology of vibrios in the culture systems. their characteristics and pathogenicity

Application of Biofloc Technology (BFT) in the nursery rearing and farming of giant freshwater prawn, Macrobrachium rosenbergii (deMan)

In India a study conducted by CIFE and CIBA (1997), concluded that shrimp farming does more good than harm and it is not eco-unfriendly (Krishnan and Birthal, 2002). Upsurge in coastal aquaculture activity induced by high profitability is reported to have caused adverse impacts on coastal ecosystems and social environments (Parthasarathy and Nirmala, 2000). The crustacean farming sector has received criticism for excessive use of formulated feed containing high protein, of which around 50% gets accumulated at the pond bottom as unconsumed (Avnimelech, 1999; Hari et al., 2004, 2006). The wasted feeds undergo the process of degradation and results in the release of toxic metabolites to the culture system. Reduction of protein in the feed, manipulation and utilisation of natural food in the culture system are the remedy for the above problems. But before reducing the feed protein, it should be confirmed that the feed with reduced protein is not affecting the growth and health of the cultured animal. In the present study, biofloc technology is identified as one of the innovative technologies for ensuring the ecological and environmental Sustainability and examines the compatibility of BFT for the sustainable aquaculture of giant prawn, M. rosenbergii. This thesis starts with a general introduction (Chapter-1), a brief review of the most relevant literature (Chapter-2), results of various experiments (Chapter-3-6), summary (Chapter-7) and recommendations and future research perspectives in the field of biofloc based aquaculture (Chapter – 8). The major objectives of this thesis are, to improve the ecological and economical sustainability of prawn farming by the applicationof BFT and to improve the nutrient utilisation in aquaculture systems.

Vibrio harveyi phages: Isolation, characterization and evaluation of their potential as phage therapeutics on Vibrio harveyi in shrimp hatcheries

Aquaculture is a global industry providing food and employment thereby contributing to the economy. For the sustenance of aquaculture, disease management is a major requirement. Among the bacterial pathogens Vibrio harveyi remains to be the major one especially in shrimp culture systems. Rapid and mass mortality of shrimp larvae due to Vibrio harveyi infection is well known, and the pathogen causes serious economic losses in grow out systems as well. It suggests that a well defined management strategy has to be built up to protect the crop from Vibrio harveyi infection in aquaculture systems. Antibiotics have been the choice for quite some times which led to residues in meat and development of multidrug resistant bacteria which invited ban on their application. In this context several alternate options have been thought off such as probiotics, immunostimulants and vaccines. Phage therapy is yet another option. Phages being natural parasites of bacteria and are abundant in aquatic environments their application to control bacterial pathogens in aquaculture has commendable potential in lieu of antibiotics. For that matter the therapeutic effect of phages has been proven in several antibiotic resistant pathogens inclusive of Vibrio harveyi.