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Molecular-genetic analysis of the microflora composition of water of biofilter in rsa

The main difference in the growth of rainbow trout in RSA, as opposed to growing on open water, is a limited area, which at the same time should provide favorable conditions for the growth of fish and the operation of the system of treatment of reversible water.  An important role in these processes is performed by the microflora, which represents the biocenoses formed in different biotopes of the RSA (Recirculating aquaculture system). It takes an active part in the regeneration of reversible water and is an active integral part of the biological filter in which the biological film is in the attached form or in the form of active sludge.  Molecular genetic methods are considered to be the most accurate and reliable for the identification of the available microflora, which forms biocenosis and shows a nitrification function. Therefore, along with the classical routine methods, we examined the water of the biofilter reactor using molecular genetic methods, in particular by using the polymerase chain reaction (PCR) method to identify the available microflora that forms biocenose and exhibits a nitrification function.

The purpose of the work was to sequenced cDNA of the microflora of the biofilters of the closed water supply system and compared the 16S rDNA and 16S RNA clones available in the library base.

Materials and methods.  The amplification of 16S cDNA was performed using PCR (Michaud et al., 2009)]. They were performed using the ABI 9600 (Applied Biosystems) thermal cycler using the front primer 27F (5'-AGA GTT TGA TCC TGG CTC  AG-3 ') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). PCR was performed in a mixture of 50 μl (total volume) containing 1 × Q solution (Qiagen), 10 × Qiagen reaction buffer, 1 μM of each specimen, 10 μM of dNTPs (Fermentas), 50 ng of cDNA matrix and 2.0  U Taq Polymerase (Qiagen).  The PCR program included the following steps: 3 minutes.  at 95 ° C, then 30 cycles for 1 min.  at 94 ° C, 1 min at 50 ° C, 2 min.  at 72 ° C and complete prolongation for 10 min.  at a temperature of 72 ° С.

11 bacterial phytopaths were identified. It was found that most of the sequenced clones belonged to the Gammaproteobacteria phylotype - 56.7 %. 12.2 % belonged to the Alphaproteobacteria class. The share of the CFB filotype was 8.88 %, which is 6.4 times (p <0.05) less than Gammaproteobacteria and 1.5 times (p <0.05) than Alphaproteobacteria. The share of the remaining filotopes such as Nitrospirae, Betaproteobacteria, Planctomycetes, Deltaproteobacteria, Uncultured bacterium Verrucomicrobia, Firmicutes did not exceed 6 %, and the smallest number was the filo type of Actinobacteria - 0.5 %.  In our opinion, a significant predominance of representatives of the Gammaproteobacteria filotype is due to the fact that it includes several large families (Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, etc.) that are widespread in the environment - in the soil, on plants, trees, in water, and naturally form the microflora of the water of the biofilter reactor.

It has been established that 182 clones were sequenced, which belonged to 11 phylotypes.  The major part among the detected clones was bacteria of the genus Vibrio spp. - 22.5 % and Gamma proteobacterium - 14.8 %. This is due to the fact that these microorganisms form a symbiotic microflora of water and the surface of the fish, despite the fact that among the representatives of the genus Vibrio is the pathogenic form of V. anguillarum, which causes septicemia of trout. The share of the main genera and species of bacteria involved in the processes of nitrification and denitrification, in particular Nitrosomonas spp., Nitrospira spp., Pseudomonas stutzeri, was 13.7 %. Other identified types of bacteria were divided evenly between identified genus.

Consequently, the conducted molecular genetic studies made it possible to detect, found the most common and stable classes and species of bacteria that form active normomicrobiocenosis in the biofilter reactor for growing rainbow trout. In general, the microflora of the biofilter reactor should be considered as biocenosis, in which the genera and species of bacteria are not accidental together, but formed as a result of the complex of environmental and human factors during the startup and operation of the ultrasound.  We believe that microbial species should be considered as peculiar informational systems that report changes in the environment, in particular, in microbiological biofilter. In this case, the violation of microbiocenosis will negatively affect the nitrification function of the biofilter reactor.  Determination and evaluation of the role of individual groups of microorganisms in such a biotope as a biofilter reactor.  Monitoring of the composition of its microbial populations has an important ecological significance.

Key words: RSA, rainbow trout, biofilter, microflora, sequencing, polymerase chain reaction.


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