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Physico Shrimp Essay

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Sample collection and physico-chemical analyses
Sediment samples were taken from three bioaugmented ZWE shrimp ponds having having 4500 m2 area and 1m depth located in Kerala, India: Kodungallur-Pullut farm (TCR-S; 10˚15′16.47˚N 76˚12′35.33˚E), Kodungallur-Karuppadam farm (TCR-A; 10˚16′2.68˚N 76˚11′47.18˚E) and Ernakulam-Pizhala farm, Kerala, (EKM-B; 10˚02′49.02˚N 76˚15′38.23˚E) during different phases of culturing. Based on the location and day of culture, samples were coded as TCR-S-0, TCR-S-45, TCR-S-75, TCR-S-120 for TCR-S site, TCR-A-0, TCR-A-30, TCR-A-45, TCR-A-60, TCR-A-75, TCR-A-90 for TCR-A site and EKM-B-0, EKM-B-30, EKM-B-45, EKM-B-60 for EKM-B site, where the numbers indicated days of culture. These ponds were maintained under
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1982) using pH meter. Similarly, sediment ammonia, nitrite, nitrate was analysed. Total organic carbon (Walkley, A.; Black 1934) of dried sediment sample was determined.
Molecular analysis
Sediment DNA was extracted using the Powersoil DNA extraction kit ((MoBio Laboratories Inc., USA) following manufacturer's protocol. Recovered DNA was quantified using QuBit meter using QuBit dsDNA HS kit (Life Technologies, Portugal). Different primer pair specific to hyydrazine odi…… gene (HZO gene) was used to amplify DNA of anaerobic ammonia oxidizing bacteir (anammox). Due to low annealing temperature, the amplified product was non-specific. Later with primer (ref…) with slight modification HZO gene of anammox bacteria was amplified in a PCR reaction containing 1X EmeraldAmp® GT PCR Master Mix (Takara Bio Inc. Japan), 1mM primer each and 20-30 ng of soil DNA using the programme as follows: initial denaturation of 95°C for 5 min followed by 42 cycles of denaturation at 95°C for 60 s, 60 °C of annealing for 60 s, extension of 75°C for 2 m and a final extension of 75°C for 5 min. The pooled amplified product was ligated to pGEM-T easy vector (Promega, USA) and transformed into DH5α E. coli competent cells. The clone library was prepared from the positive transformants, and the clones were preserved using 60% glycerol at -80°C. Ninety-six positive clones were selected for plasmid isolation using alkaline lysis with resuspension
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