Conversely, media inoculated with protozoan isolates showed the highest removal of only Ni (12%) and Zn (18%) for only dead Peranema sp. Statistical evidence revealed no significant difference (p > 0.05) between the heavy metal removal in the media inoculated with both dead-bacterial and dead-protozoan

isolates. None of the dead-test isolates was able to remove more than 25% of the heavy metal in the culture media, with Aspidisca sp. indicating the highest of all (Ti-23%). This could have been due to the presence of several metals and high concentrations. However, when comparing the removal efficiency of both dead and living test isolates, statistical evidence revealed significant differences (p < 0.05). Figure 3 The percentage removal of Talazoparib manufacturer heavy metals from the industrial wastewater samples by heat-killed microbial isolates (n = 3). To evaluate the click here resistance ability of the microbial isolates and whether the heavy-metal removal ability of test isolates is active, the genomic DNA was amplified with specific genes such as copA, copB and copC (Cu-resistance), nccA (Ni, Co, Cd-resistance), cnrA3 and cnrC2 (Ni and Co-resistance), chrB (Cr-resistance) and czcD (Co, Zn,

Cd-resistance) using the conventional PCR (Figure  4). Of all the genes targeted in the gDNA of microbial isolates, nccA, cnrA3, chrB and copC were the only genes to show positive amplification. For bacterial isolates (Pseudomonas putida, Bacillus licheniformis and Brevibacillus laterosporus), amplified products of approximately 400 bp, 450 bp, 1141 bp Histidine ammonia-lyase and 1447 bp revealing the presence of copC (Cu sequestration

and transport), chrB, nccA and cnrA3 genes were reproductively detected, whereas, the metal-resistant genes such as copA, copB, cnrC2 and czcD were not found. However, for protozoan isolates (Peranema sp., Trachelophyllum sp. and Aspidisca sp.), amplified products of approximately 400 bp, 450 bp and 1447 bp revealing the presence of copC, chrB and cnrA3 genes were found. Peranema sp. was the only protozoan isolate with the gene cnrA3 (RND (Efflux)). None of the protozoan isolates revealed the presence of copA, copB, cnrC2, czcD and nccA. Figure 4 Agarose gel electrophoresis of PCR products of total genomic DNAs with primer pair nccA -fwd and nccA -rev, primer pair copC- fwd and copC -rev, primer pair copB- fwd and copB -rev, primer pair czcD -fwd and czcD -rev, primer pair cnrA3 -fwd and cnrA3 -rev and primer pair chrB- fwd and chrB -rev. Lanes: M: DNA ladder (Marker), N: Negative (No template DNA), 1 to 6, amplified PCR product of: Pseudomonas putida (1), Bacillus licheniformis (2), Brevibacillus laterosporus (3), Trachelophyllum sp. (4), Peranema sp. (5) and Aspidisca sp. (6).