Bioremediation of phosphates in seawater: approach for recirculating marine aquaculture effluents

Marine Recirculating Aquaculture Systems (RAS) produce great volume of wastewater, which may be reutilized/recirculated or reutilized after undergoing different treatment/remediation methods, or partly discharged into neighbour water-bodies (DWW). Phosphates, in particular, are usually accumulated at high concentrations in DWW, both because its monitoring is not compulsory for fish production since it is not a limiting parameter, and also because there is no specific treatment so far developed to remove them, especially in what concerns saltwater effluents. As such, this work addresses two main scientific questions. One of them regards the understanding of the actual (bio)remediation methods applied to effluents produced in marine RAS, by identifying their advantages, drawbacks and gaps concerning their exploitation in saltwater effluents. The second one is the development of a new, innovative and efficient method for the treatment of saltwater effluents that potentially fulfil the gaps identified in the conventional treatments. Thereby, the aims of this thesis are: (i) to revise the conventional treatments targeting major contaminants in marine RAS effluents, with a particular focus on the bioremediation approaches already conducted for phosphates; (ii) to characterize and evaluate the potential of oyster-shell waste collected in Ria de Aveiro as a bioremediation agent of phosphates spiked into artificial saltwater, over different influencing factors (e.g., oyster-shell pre-treatment through calcination, particle size, adsorbent concentration). Despite the use of oyster-shells for phosphorous (P) removal has already been applied in freshwater, its biosorptive potential for P in saltwater was never evaluated, as far as I am aware. The results herein generated showed that NOS is mainly composed by carbonates, which are almost completely converted into lime (CaO) after calcination (COS). Such pre-treatment allowed obtaining a more reactive material for P removal, since higher removal percentages and adsorption capacity was observed for COS. Smaller particle size fractions for both NOS and COS samples also increased P removal. Kinetic models showed that NOS adsorption followed, simultaneously, Elovich and Intraparticle Difusion kinetic models, suggesting that P removal is both a diffusional and chemically rate-controlled process. The percentage of P removal by COS was not controlled by Intraparticle Diffusion and the Elovich model was the kinetic model that best fitted phosphate removal. This work demonstrated that waste oyster-shells, either NOS or COS, could be used as an effective biosorbent for P removal from seawater. Thereby, this biomaterial can sustain a cost-effective and eco-friendly bioremediation strategy with potential application in marine RAS.

Routines in a European aquaculture company and the development of a Flavobacterium psychrophilum dose response pre-model in adult Rainbow Trout

An internship in a European company dealing with aquaculture and biotechnology - AquaBioTech Group, Malta - was undertaken to complete the Master Degree of Science in Aquaculture of the School of Tourism and Maritime Technology of the Polytechnic Institute of Leiria. Biotechnology and aquaculture are two areas that have been synergistically used to contribute for the progress and improvement of fish production. The AquaBioTech Group is an example of a company able to integrate these areas to maximizing their services. Located in Mosta (Malta) the company operates in a sustainable way using Recirculation Aquaculture Systems (RAS) to maintain aquaculture species. In collaboration with several companies and institutions, the AquaBioTech Group is involved and supports the development of important international research projects. The present report focuses on two important parts of the internship performed during 6 months. Initially, it will cover the operation and constitution of the company, describing the routines and techniques acquired. Then, it will describe a pathology trial that forms the practical and scientific component of this report. Despite the limitation to describe some confidential assays, this trial consisted in the infection of Rainbow Trout (Oncorhynchus mykiss) with the bacterium Flavobacterium psychrophilum in order to evaluate the mortality rates over time. The internship served to solidify theoretical knowledge acquired during the academic training, develop professional skills and provide an understanding of jobs available on the market.

Acute toxicity of un-ionized ammonia to milkfish (Chanos chanos) fingerlings

The acute toxicity of un-ionized ammonia to milkfish (Chanos chanos) fingerlings was determined using a static bioassay system. Median lethal concentrations found show that milkfish fingerlings have a high tolerance to ammonia and it is unlikely that levels as high as those employed for the acute exposure would be found to occur under natural conditions. Although the threat of acute toxicological effects induced by ammonia are remote, such conditions might be encountered in stressed natural environments or in heavily loaded aquaculture systems.

Water budgeting studies on the hatchery and nursery rearing practices for the common carp, Cyprinus carpio (Linnaeus, 1758)

Aquaculture systems are an integral element of rural development and therefore should be environment friendly as well as socially and economically designed. From the economic standpoint, one of the major constraints for the development of sustainable aquaculture includes externalities generated by competition in access to a limited resource. This study was conducted as an investigation into the water requirement for the hatchery and nursery production phases of common carp, Cyprinus carpio (Linnaeus, 1758) at the Maharashtra State Fish Seed Farm at Khopoli in Raigad Dist. of Maharashtra during the winter months from November to February. The water budgeting study involves the quantification of water used in every stage of production in hatchery and nursery systems and aimed at becoming a foundation for the minimization of water during production without affecting the yield; thereby conserving water and upholding the theme of sustainable aquaculture. The total water used in a single operation cycle was estimated to be 11,25,040 L [sic]. Out of the total water consumed, 4.74% water was used in the pre-operational management steps, 4.48% was consumed during breeding, 62.72% was consumed in the hatching phase, 21.50% was used for hatchery rearing and 6.56% was consumed during conditioning. In the nursery ponds, the water gain was primarily the regulated inflow coming through the irrigation channel. The total quantum of water used in the nursery rearing was 31,60,800 L [sic]. The initial filling and regulated inflow formed 42.60% and 57.40% respectively of water gain, while evaporation, seepage and discharge contributed 20.71%, 36.46% and 42.82% respectively to the water loss. The total water expended for the entire operation was 1,21,61,120 L [sic]. Water expense occurred to produce a single spawn in the hatchery system was calculated and found to be 0.56 L while the water expended to produce one fry was calculated as 4.86 L. The study fulfills the hydrological equation described by Winter (1981) and Boyd (1985). It also validates the water budget simulation model that can be used for forecasting water requirements for aquaculture ponds (Nath and Bolte, 1998).

Agri-aqua farming in Thailand

Integrated agriculture-aquaculture systems have been in existence in Thailand for centuries. This country has the most varied integrated farming operations in southeast Asia; pig, cattle, buffalo, chicken, duck, vegetable, aquatic plant, rice and orchard in combination with fish are practices. The systems most preferred by subsistence farmers are rice-fish, duck-fish and chicken-fish culture. A brief outline is given of these 3 systems.

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO2-) or nitrate (NO3-) as the sole nitrogen source and aerated with air or with CO2-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 mu M NaNO3 as nitrogen source. Accordingly, NO2- concentration reached 10.4 mu M after 95 h at the low CO2 condition (aerated with air); while the maximum NO2- concentration was only around 2.0 mu M at the high CO2 condition (aerated with 5% CO2 in air), furthermore, after 30 h it decreased to no more than 1.0 mu M. NO2- was almost assimilated in 80 h when C. muelleri was cultured at the high CO2 condition with 100 mu M NaNO2 as sole nitrogen source. At the high CO2 condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO2 condition. It was indicated that enriched CO2 concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO2 might be an effective way to control nitrite content in aquaculture systems.

Agar extraction from integrated multitrophic aquacultured Gracilaria vermiculophylla: Evaluation of a microwave-assisted process using response surface methodology

Microwave-assisted extraction (MAE) of agar from Gracilaria vermiculophylla, produced in an integrated multitrophic aquaculture (IMTA) system, from Ria de Aveiro (northwestern Portugal), was tested and optimized using response surface methodology. The influence of the MAE operational parameters (extraction time, temperature, solvent volume and stirring speed) on the physical and chemical properties of agar (yield, gel strength, gelling and melting temperatures, as well as, sulphate and 3,6-anhydro-Lgalactose contents) was evaluated in a 2^4 orthogonal composite design. The quality of the extracted agar compared favorably with the attained using traditional extraction (2 h at 85ºC) while reducing drastically extraction time, solvent consumption and waste disposal requirements. Agar MAE optimum results were: an yield of 14.4 ± 0.4%, a gel strength of 1331 ± 51 g/cm2, 40.7 ± 0.2 _C gelling temperature, 93.1 ± 0.5ºC melting temperature, 1.73 ± 0.13% sulfate content and 39.4 ± 0.3% 3,6-anhydro-L-galactose content. Furthermore, this study suggests the feasibility of the exploitation of G. vermiculophylla grew in IMTA systems for agar production.

Ecological and Biochemical Studies On Cyanobacteria of Cochin Estuary and Their Application as Source of Antioxidants

The main objectives of the present investigation were to evaluate the qualitative and quantitative distribution of natural cyanobacterial population and their ecobiological properties along the Cochin estuary and their application in aquaculture systems as a nutritional supplement due to their nutrient-rich biochemical composition and antioxidant potential. This thesis presents a detailed account of the distribution of cyanobacteria in Cochin estuary, an assessment of physico-chemical parameters and the nutrients of the study site, an evaluation of the effect of physico-chemical parameters on cyanobacterial distribution and abundance, isolation, identification and culturing of cyanobacteria, the biochemical composition an productivity of cyanobacteria, and an evaluation of the potential of the selected cyanobacteria as antioxidants against ethanol induced lipid peroxidation. The pH, salinity and nutritional requirements were optimized for low-cost production of the selected cyanobacterial strains. The present study provides an insight into the distribution, abundance, diversity and ecology of cyanobacteria of Cochin estuary. From the results, it is evident that the ecological conditions of Cochin estuary support a rich cyanobacterial growth.

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.

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.

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.

Studies on finfish and shellfish diseases

In the present study, fifteen typee of diseases occurring in twenty one species of finfishes end shellfishes are documented and the causes studied. All these are being reported from India for the first time which adds considerably to our existinq knowledge on the subject.In fact, the candidate's studies embodied in this Thesis has also an applied significance aimed at the proper management_of finfish and shellfish aquaculture systems in this country.The thesis is presented in seven main chapters.

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.

Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish

Motile aeromonads isolated from the intestines of farm-raised freshwater fish such as Catla catla, Labeo rohita and Ctenopharyngodon idella have been characterized to species level. Morphological and physiological grouping revealed 61% Aeromonas hydrophila, 30% Aeromonas caviae, 7% Aeromonas sobria and 2% which remained unidentified. Hemolytic activity was detected mostly in A. hydrophila, while only half of the A. sobria and A. caviae showed this activity. Antibiotic resistance patterns of the strains revealed that they had acquired a relatively higher resistance to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B, implicating possible use of these antibiotics in the aquaculture systems.

Nitrification in a packed bed bioreactor integrated into amarine recirculating maturation system under different substrate concentrations and flow rates

BACKGROUND: A packed bed bioreactor (PBBR) activated with an indigenous nitrifying bacterial consortia was developed and commercialized for rapid establishment of nitrification in brackish water and marine hatchery systems in the tropics. The present study evaluated nitrification in PBBR integrated into a Penaeus monodon recirculating maturation system under different substrate concentrations and flow rates. RESULTS:Instantnitrificationwasobservedafter integration ofPBBRinto thematuration system.TANandNO2-Nconcentrations were always maintained below0.5 mg L−1 during operation. The TANandNO2-N removalwas significant (P < 0.001) in all the six reactor compartments of the PBBR having the substrates at initial concentrations of 2, 5 and 10 mg L−1. The average volumetric TAN removal rates increased with flow rates from 43.51 (250 L h−1) to 130.44 (2500 L h−1) gTAN m−3 day−1 (P < 0.05). FISH analysis of the biofilms after 70 days of operation gave positive results with probes NSO 190 ((β ammonia oxidizers), NsV 443 (Nitrosospira spp.) NEU (halophilic Nitrosomonas), Ntspa 712 (Phylum Nitrospira) indicating stability of the consortia. CONCLUSION: The PBBR integrated into the P. monodon maturation system exhibited significant nitrification upon operation for 70 days as well as at different substrate concentrations and flow rates. This system can easily be integrated into marine and brackish water aquaculture systems, to establish instantaneous nitrification