Characterization of the assembly of nitrifying bacteria by molecular biology method (Fluorescent In Situ Hybridization - FISH) in the biofilm and water of Farfantepenaeus paulensis cultivation systems

Author: Simone Soares Oliveira (Currículo Lattes)
Supervisor: Dr Paulo Cesar Oliveira Vergne de Abreu
Co-supervisor: Dr Wilson Francisco Britto Wasielesky Junior


One of the main concerns in farming systems is the accumulation of nitrogen products, especially ammonia (NH4). The exchange of water from tanks, as well as the addition of phytoplankton, are some of the techniques used to reduce the level of ammonia in the cultivation systems. Another possibility would be the addition of nitrifying bacteria like probiotics. However, it is necessary that these microorganisms are obtained from the microbiota present in the tanks so that there is no problem due to the addition of an exotic organism. Due to technical limitations, the identification and isolation of bacteria in cultivation environments, as in nature, was always done on a small scale and in specialized laboratories. With the advancement of molecular biology techniques it is possible to identify phylogenetic groups,determine the spatial and temporal variation of microorganism species and elucidate the function and activity of communities collected from the environment. In order to establish aspects of bacterial succession in culture tanks using molecular biology techniques and to relate the presence of nitrifying bacteria in biofilm and water, the FISH method (Fluorescent in situ hybridization) was used in this study. This method is based on hybridization with fluorescent markers of 16S rRNA segments that demarcate specific groups of bacteria. The probes -, - and -proteobacteria were used, which includes the majority of nitrifying bacteria. The experiments were carried out at the Marine Aquaculture Station - EMA/FURG, from November 2002 to February 2003, in the larviculture and nursery phases of the pink shrimp Farfantepenaeus paulensis. Artificial substrate for biofilm formation was added to the tanks, which were collected daily to analyze the composition of the bacteria and chlorophyll a. Water samples were collected for ammonia, nitrite and nitrate analysis, in addition to bacteria abundance data. In the larviculture experiment, the highest concentration of ammonia in water was observed, reaching 413.98 μM in time 2, decreasing to 73.92 μM in the next time. The groups - and -proteobacteria were more related to the variation of ammonia in the tanks and, through the mass balance of nitrogen in the larviculture, it was possible to verify that the greater nitrogen absorption from the tanks occurred due to nitrification (22.3% ).In the second experiment (nursery) it was possible to evaluate the impact of shrimp predation on the biofilm, observing a positive effect on microalgae and on biofilm bacteria, that is, the greatest abundance of bacteria was obtained on substrates subject to predation. of shrimp, as well as the highest concentration of chlorophyll a. The concentration of nitrogenous compounds influenced the composition of the bacteria assembly in the biofilm and in the water, mainly with the predominance of bacteria from the - and -proteobacteria groups, related to the variation of ammonia, whereas bacteria from the -proteobacteria group were related with the nitrite variation.