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Vol. 9(7), pp. 404-413, 18 February, 2015 DOI: 10.5897/AJMR2014.7249 Article Number: 562D89A50630 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR Full Length Research Paper Bacterial community in two subtropical fishponds in São Paulo, Brazil Aylan Kener Meneghine1, Daniele Belarmino da Silva1, Rodrigo Ney Millan2, Lucia Helena Sipaúba-Tavares3, Eliana Gertrudes de Macedo Lemos1 and Lúcia Maria Carareto Alves1* 1Faculdade de Ciências Agrárias e Veterinárias. UNESP – Univ Estadual Paulista, Campus de Jaboticabal, Departamento de Tecnologia. Programa de Pós-graduação em Microbiologia Agropecuária, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Brazil. 2Universidade do Estado de Minas Gerais. Av. Prof. Mario Palmerio, 1001, Centro, 38200000 - Frutal, MG – Brazil. 3Faculdade de Ciências Agrárias e Veterinárias, UNESP – Univ Estadual Paulista. Campus de Jaboticabal, Aquaculture Center. Via de Acesso Prof. Paulo Donato Castellane, s/n, 14884-900, Brazil. Received 4 November, 2014; Accepted 2 February, 2015 The knowledge concerning fishpond microorganisms is important considering the aquatic microbial ecology and water quality. In this study, we compared the bacterial communities present in freshwaters of two fish farm ponds from Brazil, one used as water reservoir (P1) and the other with conditions of high nutrient load (P4). The aim of this study was to identify and compare the bacterial population in two fishponds during dry and rainy seasons. The determination of the bacterial population was conducted through sequencing of the 16S rRNA gene. The results show that the bacterial population is different in the ponds and it changes according to the climatic period. Moreover, the diversity of microorganisms in P1 was greater than P4. Proteobacteria predominated in the rainy season in both ponds varied in proportion and classes composition. In the P4 during dry season, Cyanobacteria were predominant due to favorable conditions of environment. This study provides the first local investigation that demonstrates that the bacterial community is differ according to the trophic state of aquatic systems and between rainy and dry seasons, the latter of wich exerted more influence in bacterial communities. It can be suggested that the management in P4 promotes favorable environment for the high density of Cyanobacteria, demonstrating the need for improvements in management. Key words: 16S rDNA, biodiversity, freshwater, metagenome. INTRODUCTION Aquaculture is an important social and economic activity, that affect society as a whole. In this respect, the currently accounting for 40% of world production of fish, deterioration of water is a major socio-environmental which corresponds to approximately 150 million tonnes effect that occurs in aquaculture ponds in the absence of (FAO, 2012). This activity causes environmental impacts good management practices in aquaculture ponds *Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Meneghine et al. 405 (Calijuri et al., 2008). tions in the food chain: they are directly involved with the In aquaculture ponds, there is a constant inflow and process denoted microbial loop, which was first described outflow of water that has pronounced effect on the distri- by Azam et al. (1983). bution of organisms, and the outflow effects on the According to these researchers, an important conse- residence time of water have become essential to the quence of the microbial loop is the ability of bacteria to understanding of the internal processes of artificial soak up the nutrients in the water column, which results systems (Pereira et al., 2004). Rainfall directly affects the in a rapid recycling of these nutrients. Fenchel (2008) dynamics in these environments by promoting the reported that the principal effect of the microbial loop on transport of nutrients and allochthonous materials, visual the nutrient cycle in the water column is the acceleration changes, and changes in the physical and chemical of the mineralization and the regeneration of production characteristics of the water (Sipaúba-Tavares et al., in nutrient-limited systems. 2007). Furthermore, the expansion of aquaculture leads to increase in nutrients in the aquatic environment which MATERIALS AND METHODS can cause algae accelerated growth (Macedo and Sipaúba-Tavares 2010; Chislock et al., 2013) and Study area alteration in diversity of microrganisms (Leff, 2009). Microbial activity is important in the removal and The study was carried out in two fishponds of the University of São transformation of organic matter in fish farms (Forster et Paulo State (UNESP at Jaboticabal (São Paulo, Brazil) 21º15'S and 48º18'W) during the rainy (January and February/2012) and dry al., 2003). In microbial ecology, one of the most critical (July and August/2012) seasons (Figure 1). The water samples determinants of an aquatic ecosystem it is trophic state were collected in the water outlet with two fishponds studied. The which is heavily influenced by the food chain and first fishpond (P1) is a water supply reservoir to the aquaculture biogeochemical cycles (Leff, 2009). The eutrophication of farm with continuous water flow surface area of 3,472 m² and water depth ranging from 1 to 1.3 m. This shallow reservoir was in the aquatic environments can be caused by man through disposal of effluent from aquaculture farm (Cao et al., past used as a fishpond, but since 1995, it is used only as a water reservoir for the aquaculture farm. The other fishpond studied is the 2007). The concentration increase of nitrogen and phos- fourth (P4) of six similary size at fishponds in a sequential line, and phorus are the principal cause of the eutrophication, which each one of them is directly or indirectly receiving water from the can affect the fast develop and excessive growth of algae previous one (Figure 1C). This fishpond studied has an area of and Cyanobacteria (Macedo and Sipaúba-Tavares, 2010). 4,268 m², a depth of 1-1.3 m, a continuous water flow with a daily The microrganisms in the water have an important exchange rate of 5% of its volume and an average fish density of 1 fish.m-2. A mix of fish species, such as Cyprinus carpio, Piaractus interaction in food chain, they are directly involved with mesopotamicus, Cichla ocellaris, Oreochromis niloticus and Brycon the process named microbial loop, first described by cephalus, were found in this pond. These fishponds are affected by Azam et al. (1983). According to these authors, in sum- continuous water flow, wind, rainfall, and the transport of nutrients mary, an important consequence of the microbial loop is and allochthonous materials. the bacterial ability to soak up the nutrients in the water The aquaculture consist of 73 small fishponds (c.a. 140 m²) with column, which leads to a fast recycle of these nutrients. a continuous water flow and six large fishponds, disposed in a sequence, ranging between 1.822 and 8,067 m². A semi intensive Fenchel (2008) relates that the principal effect of production is conducted for research. microbial loop on nutrients cycle in the water column is The climate in the region is subtropical Cwc (Peel et al., 2007), the acceleration of the mineralization and the regene- relatively dry in the winter (June to August) and rainy in the summer ration of production in systems limited of nutrients. (December to March), with mean yearly temperature of 22°C and Despite the importance of the microbial dynamic of mean yearly of rainfall 1,427 mm. population in freshwater ecosystems (Zwart et al., 2002; Taipale et al., 2009) studies in this area are scarce and Water sampling needed. In this sense, the aquatic bacteria are difficult to obtain in culture and consequently defining their biogeo- Physical and chemical parameters were sampled at 8:00 AM at 10 chemical role and understand the dynamics of popula- cm depth. Samples are collected with Van Dorn bottle and transported in refrigerated polyethethylene bottles to laboratory. tions in these systems becomes a challenge (Kemp and Temperature, pH, conductivity and dissolved oxygen were mea- Aller, 2004). sured in situ using a multi-probe Horiba U-10. Total phosphorus, The advents of molecular methods as metagenomic orthophosphate, total ammonia nitrogen, nitrite and nitrate, were strategies, especially ribosomal RNA-based techniques, quantified spectrophotometrically according to Golterman et al. can be used to explore the microbial diversity in samples (1978) and Koroleff (1976). Analyses were performed immediately after sampling or samples were duly store under a refrigerator until directly from the environment. Much of what is now analysis. All samples were carried out in triplicate. known of the diversity of aquatic bacteria is based on distinguishing among different organisms, as represented by polymerase chain reaction (PCR) of 16S RNA gene, Molecular analyses where it is unnecessary culturing them or having any Water samples for molecular analysis were stored in sterile 5 L direct knowledge of their morphology, physiology or bottles. The total DNA was extracted using UltraClean ™ Water ecology (Kemp and Aller, 2009; Petrosino et al., 2009). DNA Isolation kit (0.22 µm) (MO BIO Laboratories, Inc., Carlsbad – The microorganisms in water present important interac- CA), and then subjected to PCR amplification of the 16S rRNA 406 Afr. J. Microbiol. Res. A B C Figure 1. Cross section of aquaculture farm. A, shaded area indicates São Paulo state, in southeastern Brazil. B, Distribution of fishponds at UNESP farm. C, fishponds studied. gene using Y1 and Y2 primers (Young et al., 1991). The PCR among the seasons (dry and rainy) and fishponds and their reaction were performed using 60 ng of DNA , 1X PCR buffer [20 interactions (Sokal and Rohlf, 1981). mM Tris- HCl (pH 8.4), 50 mM KCl ] , 1.5 mM MgCl2, 0.2 mM dNTP, 10 pmols of each primer and 0.8 U of Taq DNA polymerase.

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