Isolamento, cultivo e identificação de bactérias com potencial uso probiótico no cultivo de organismos aquáticos: estudo de caso com bactérias do trato gastrointestinal do linguado Paralichthys orbignyanus (Valenciennes, 1839)

Dissertação (mestrado) - Universidade Federal do Rio Grande, Programa de Pós–graduação em Aqüicultura, Instituto de Oceonografia, 2008.Por definição, organismos probióticos são aqueles que melhoram a saúde do hospedeiro, seja auxiliando na digestão do alimento e na assimilação de nutrientes, estimulando o sistema imunológico, ou atuando no controle de microrganismos patogênicos. No entanto, o uso de probióticos comerciais apresenta riscos para a saúde dos animais cultivados e para o meio ambiente que precisam ser considerados. Bactérias utilizadas em produtos comerciais podem apresentar genes de resistência a antibióticos,que podem ser transferidos para microrganismos patogênicos presentes no ambiente de cultivo. Desta forma, o isolamento de linhagens do próprio hospedeiro seria uma alternativa parar solucionar este problema. O objetivo deste trabalho foi isolar, cultivar e identificar bactérias, com potencial uso probiótico em cultivo de animais aquáticos. Neste estudo as bactérias foram obtidas a partir do trato gastrointestinal do linguado Paralichthys orbignyanus (Valenciennes, 1839) cultivado na Estação Marinha de Aquacultura da Universidade Federal do Rio Grande - FURG. Diluições de material extraído do trato gastrointestinal foram semeadas em quatro meios de cultivo (TCBS,MRS, MA e NA), incubadas e as colônias foram isoladas e purificadas. Os valores de UFC foram baixos (104 UFC ml-1) devido, provavelmente, ao longo período decorrido entre a coleta dos tratos intestinais e semeadura das bactérias, ou devido ao transporte deste material no gelo. Apesar disso, foram isoladas 94 bactérias, cujas colônias se apresentaram em oito morfotipos diferentes. O DNA genômico de 46 isolados provenientes dos meios TCBS e MRSA foi extraído, amplificado e purificado, sendo seqüenciada a região codificadora do gene 16S rRNA destes microorganismos. As bactérias foram agrupadas e identificadas pela construção de fenogramas. Os resultados mostraram a presença dos gêneros Brachybacterium, Brevibacterium, Photobacterium e Staphylococcus. Dezenove isolados não agruparam com nenhuma linhagem-tipo existente, permitindo supor que se tratem de espécies não descritas. Como perspectivas futuras propõe-se a realização de testes in vitro e in vivo para se verificar a possível atuação das bactérias isoladas como inibidoras de bactérias patogênicas, ou na melhoria da performance zootécnica de P. orbignyanus. Além disso, pretende-se obter microorganismos probióticos isolados a partir de outros organismos aquáticos cultivados.Probiotic organisms are those capable to improve the health of their hosts, by assisting in the food digestion and nutrient assimilation, stimulating their immunological system or by controlling the development of pathogens. However, the use of commercial probiotics poses a serious hazard to the health of farmed animals and to the environment that must be considered. Bacteria present in such commercial products might have resistance genes to antibiotics that can be transferred to pathogenic microrganisms in the culture environment. The screening for strains isolated from the host itself would be an alternative for this issue. The aim of this work was to isolate, culture and identify native bacteria with putative use as probiotic in the rearing of aquatic animals. In the present work bacteria were obtained from the gastrointestinal tract of a laboratory-reared flounder Paralichthys orbignyanus (Valenciennes, 1839) reared at the Marine Aquaculture Station of the Federal University of Rio Grande - FURG. Serial dilutions from the gastrointestinal tract were seeded in plates with four culture mediums (TCBS, MRSA, MA and NA), incubated and the growing colonies were isolated and purified. Low values of CFU (104) were probably due to the long time taken from sampling to analysis or due to transporting conditions. Despite of it, 94 strains, present in eight different colony morphotypes, were recovered. The genomic DNA of 46 strains isolated from the TCBS and MRSA mediums were extracted, amplified, purified and the gene coding the 16S rRNA region was sequenced. The bacteria were clustered and identified by the construction of phenograms. The results showed the presence of four genera, Brachybacterium, Brevibacterium, Photobacterium and Staphylococcus. Nineteen of the isolates clustered with none of the existing typestrain, suggesting that they might be species not previously described. Future perspectives of this work are the setting up of in vitro and in vivo tests in order to define the probiotic potential of the isolated strains by improving the growth performance of P.orbignyanus or inhibiting the development of pathogens. Beside that we intend to isolate probiotic micoorganisms from other laboratory-reared aquatic organisms

Effects of pH, Salinity, Biomass Concentration, and Algal Organic Matter on Flocculant Efficiency of Synthetic Versus Natural Polymers for Harvesting Microalgae Biomass

© 2016, Springer Science+Business Media New York. This study investigated the effects of pH, salinity, biomass concentration, and algal organic matter (AOM) on the efficiency of four commercial cationic flocculants. The tannin-based biopolymers Tanfloc SG and SL and the polyacrylamide polymers Flopam FO 4800 SH and FO 4990 SH were tested for flocculation of two microalgae models, the freshwater Chlorella vulgaris and the marine Nannochloropsis oculata. Both biomass concentration and AOM presence affected all polymers evaluated, whereas salinity and pH affected only Flopam and Tanfloc, respectively. A restabilization effect due to overdosing was only observed for Flopam polymers and increasing Tanfloc dose resulted in improved efficiency. Flopam polymers showed a significant decrease in the maximum quantum yield of photosystem II as function of polymer dose for Chlorella, which supported the need for toxicological studies to assess the potential toxicity of Flopam. In overall, Tanfloc was not affected by salinity nor presented potential toxicity therefore being recommended for the flocculation of both freshwater and marine species.status: publishe

Screening of commercial natural and synthetic cationic polymers for flocculation of freshwater and marine microalgae and effects of molecular weight and charge density

Twenty-five natural and synthetic cationic polymers of differentmolecular weightsand charge densitieswere evaluated for microalgae flocculation. Tanfloc is a naturallow molecular weight tanninpolymer whereas Zetag and Flopam are both synthetichigh!molecular weight polyacrylamide polymers. Five exponential concentrations (0.55,1.66, 5, 15 and 45 mg L-1) were tested for freshwater Chlorella vulgarisand marine Nannochloropsis oculata. All polymers were efficient (>90%at ≥ 1.66 mg L-1) for C. vulgaris. However,forN. oculata,only Tanfloc was effective. Charge density positively influenced flocculationdecreasing the required polymer dosage. Restabilisationwas observed only for synthetic polymers when overdosed. Natural polymers performed similarly for both species.In overall, Tanfloc SL and FlopamFO 4990 SH were the most efficientpolymers for microalgae flocculationthough Tanfloc is amore economic option (US$37 ton-1of biomass) and environmentally friendly than Flopam (US$ 171 ton-1of biomass)

Comparison of open-air and semi-enclosed cultivation system for massive microalgae production in sub-tropical and temperate latitudes

This study compared open-air and semi-enclosed production system of the marine microalgae Nannochloropsis oculata in a sub-tropical region (32 S; 52 W) under uncontrolled environmental conditions. The semi-enclosed system was composed of 1.2 m3 circular tanks installed inside of a greenhouse. Water temperature was 4 C higher in the indoor treatment than in the outdoor, mainly in winter although no difference was observed in warmer seasons. Moreover, variation in salinity was observed in the outdoor treatment due to rainfall (winter) and evaporation (spring), whereas indoor treatment experienced an increase (up to 100 PSU) due to evaporation only in warmer seasons. Light transmission was approximately 20% lower in the indoor treatment although cell densities and biomass yields were higher indoor during winter. As the temperature increased (spring) no differences were observed among treatments. In summary, partial control of temperature and salinity in the semi-enclosed system, especially during the colder and rainy season, allowed higher microalgae biomass production. Further experiments must be conducted with CO2 addition, larger pH range and salinity control

IMMOBILIZING MICROALGAE IN HYBRID MATRICES AS A GREEN TECHNOLOGY FOR NATURAL GAS SWEETENING USING NOVEL STATIC SYSTEM

Photosynthetic microorganisms have been widely studied as an alternative technology for CO2 capture. Aiming to overcome some operational challenges in the application of these microorganisms for gas treatment on a large scale, the immobilization of microalgae in solid matrices emerges as an alternative to facilitate effective management of microalgae culture during harvesting process. In this work, different matrices for microalgae immobilization composed of silica/alginate were obtained varying silica precursors. The synthesized materials were characterized in terms of their specific surface area, cell viability, transparency and physical-chemical properties. Additionally, a new methodology was developed to evaluate the CO2 capture by microalgae using a pressurized system with natural gas mixture. Tests were carried out exploring the influence of some variables, such as headspace volume, cell concentration, stirring and pressure. Once the optimized parameters were established, the amount of CO2 captured by immobilized microalgae was investigated for 7 days by determining the CO2 relative concentrations using gas chromatography. The results of immobilized microalgae showed levels of CO2 removal of 41.4%. This work proved the potential application of the studied biomaterial for natural gas processing, making even more feasible the adoption of this technology for selective capture of CO2 on an industrial scale

Harvesting of the marine microalga conticribra weissflogii (bacillariophyceae) by cationic polymeric flocculants

The harvesting of microalgae is currently one of the bottlenecks hindering the commercial production of microalgae-based biofuels and products. The objective of this study was to determine the best flocculant and its optimum concentration in order to harvest the marine microalga Conticribra weissflogii (previously Thalassiosira weissflogii) for further use in the production of biofuels or bioelements. Experiments were conducted with cultures in the logarithmic and stationary growth phases. The low-charge FLOPAM® FO 4240 SH was the most effective at concentrations of 2 and 4 mg m 3 in the LOG phase cultures, with flocculation efficiencies >90%. Smaller flocculation efficiencies were observed for cells in the stationary growth phase, most likely due to the production of dissolved organic carbon by the microalga. The highest microalgae density generated higher flocculation rates, whereas the pH and salinity negatively impacted flocculant efficiency

Application of 1H HR-MAS NMR-Based Metabolite Fingerprinting of Marine Microalgae

Natural products from the marine environment as well as microalgae, have been known for the complexity of the metabolites they produce due to their adaptability to different environmental conditions, which has been an inexhaustible source of several bioactive properties, such as antioxidant, anti-tumor, and antimicrobial. This study aims to characterize the main metabolites of three species of microalgae (Nannochloropsis oceanica, Chaetoceros muelleri, and Conticribra weissflogii), which have important applications in the biofuel and nutrition industries, by 1H High-resolution magic angle spinning nuclear magnetic resonance (1H HR-MAS NMR), a method which is non-destructive, is highly reproducible, and requires minimal sample preparation. Even though the three species were found in the same ecosystem and a superior production of lipid compounds was observed, important differences were identified in relation to the production of specialized metabolites. These distinct properties favor the use of these compounds as leaders in the development of new bioactive compounds, especially against environmental, human, and animal pathogens (One Health), and demonstrate their potential in the development of alternatives for aquaculture