A molecular study of the copper resistant genes in the microbial population of industrial bioreactors
Ojo, Abidemi Oluranti
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Copper ions play an essential role in many biological systems but above optimum concentrations it exerts toxic effects. Some micro-organisms mediate the toxic effects of copper through resistance mechanisms the organisms possess. These vary with the resistance systems. Microbial diversity studies for 37°C and 70°C bioreactors were done to characterize the organisms present in the industrial copper bioreactors used for bioleaching of copper from its ore. After 16S rDNA amplification both reactors communities were defined. The BLASTn results obtained from the selected clones of the 37°C bioreactor revealed the presence of Sulfobacillus thermosulfidooxidans, Leptospirillum ferriphilum (these were associated) and Acidithiobacillus caldus (which was 100% similar), and Acidianus sp., Solfobales archaeon and Metallospaera sp. were present in the 70°C bioreactor. This conforms to the suggested organisms present in the bioreactor by MINTEK. Prior to determination of MIC of copper exhibited by the organisms, suitable medium was selected for the 37°C bioreactor organisms and the organisms present in this selected medium was assessed through amplification and sequencing analysis of the DGGE products. The result obtained showed the presence of the three organisms present in the 37°C reactor. Each species was isolated through selective media and the minimum inhibitory copper concentrations were determined thereafter. The single species were not able to tolerate the high copper concentrations the consortium was able to withstand. The MIC of copper of the consortium of bacteria (organisms from 37ºC bioreactor sample) were determined to be 400 mM, while isolation of each of the organisms present in the 37ºC bioreactor sample led to a drastic drop in copper MIC; Sulfobacillus isolate exhibited MIC of copper at 6 mM, Leptospirillum isolate a minimum inhibitory copper concentration at 3 mM and Acidithiobacillus sp. showed a minimum inhibitory copper concentration at 10 mM. Standard curves for copper(I) and copper(II) were set up at 390 nm to distinguish between the two valence states. Whole cell interaction with copper showed the ability of Proteus mirabilis and the consortium of bacteria to take up copper(II) and release copper copper(I) at a certain period of time. The individual isolates were subjected to copper(II) environment to assess the ability to interact with copper. The results obtained showed less uptake of the divalent state of copper in Sulfobacillus sp. and Leptospirillum sp. and no copper(I) was detected. In contrast, Acidithiobacillus sp. was able to take up copper(II) and actively reduce it to copper(I). Acidithiobacillus sp. may be dominant in the ability to reduce copper(II) and in the consortium. Efflux of copper(I) has been demonstrated by Rensing and co-workers (2000). The copper resistance mechanism of these organisms was further characterized and the whole cell reduction showed that Acidithiobacillus sp. identified as Acidithiobacillus caldus was the organism responsible for active efflux of copper(I) and was confirmed by amplification and sequencing analysis of the copA fragment. There was no PCR amplification of a copper resistance fragment in Sulfobacillus sp. and Leptospirillum sp. but amplification thereof in Acidithiobacillus sp. was obtained, while there was also amplification of the fragment in the consortium of bacteria. The BLASTn results obtained after sequencing analysis showed similarity to Acidithiobacillus ferrooxidans copA gene. As a result, full length gene specific primers were designed using the Acidithiobacillus ferrooxidans copA sequence. The efforts to amplify a full length copA fragment from Acidithiobacillus caldus were fruitless. As a result, for quality control, the previous set of designed primers was used for amplification of a copA in Acidithiobacillus ferrooxidans and there was amplification. The results confirm that Acidithiobacillus caldus possesses a copper translocating P-type ATPase which it uses to protect itself from copper toxicity and the remaining organisms in the bioreactor.