Isolation and genetic charcterization of a microbial consortium capable of cyanide degradation

[摘要] English: Cyanide is highly toxic to living organisms due to the potent inhibitory effect on the respiration system. This toxic compound can be deposited in the environment through various sources. Naturally occurring cyanide compounds can be synthesized (cyanogenesis) by various taxa including fungi, plants and bacteria. Cyanogenesis in bacteria is mostly linked to antagonistic activity against various microorganisms competing for the same nutrients in the same environment. Anthropogenic sources of cyanide include a wide variety of industries but the major contributor is the cyanidation process. This process extracts gold (silver can also be extracted) from ore and is responsible for the formation of metal-cyanide complexes in soil which can dissociate to form free cyanide under the correct conditions. Various microorganisms are capable to degrade free cyanide. The aims of this study were to identify microorganisms capable of utilizing cyanide as both a carbon and nitrogen source and to elucidate the mode of degradation. Samples were obtained from the Klipspruit Calcium Cyanide Factory site and were inoculated into minimal medium supplemented with NaCN. Eighteen isolates were identified from the samples and included organisms that could possibly be novel isolates based on the maximum identity percentage obtained when the 16S rRNA gene sequences (~1 500 bp) were used in a BLAST analysis against the NCBI database. The MIC was calculated for each of the 18 isolates and indicated that most of the organisms were capable of degrading cyanide at concentrations of above 2 M. This, in correlation with literature, is far above average. Gram stains were performed on the eighteen isolates. Five isolates were chosen for further studies based on 16S rRNA sequencing results, MIC determinations as well as information from literature that states that Bacillus and Pseudomonas species are often employed in bioremediation strategies. The five selected organisms included three gram positive (Bacillus sp.; Paenibacillus sp. and Leifsonia sp.) and two gram negative (Achromobacter sp. and Brevundimonas sp.) isolates. For comparative studies three control organisms (Bacillus pumilus, Pseudomonas fluorescens and Pseudomonas stutzeri) that are known and described in literature to be capable of cyanide degradation, were included in this study. The cyanide assay (100 mM NaCN) was performed on the five selected and three control organisms. The control organisms were unable to utilize the cyanide as the sole carbon and nitrogen source at this high concentration. In contrast, the selected organisms were capable of increasing their biomass over time indicating that these organisms can utilize the NaCN as the sole carbon and nitrogen source. To elucidate the mode of cyanide degradation primers were designed specific for the known genes involved in cyanide utilization in the three control organisms, and screening the five isolates with these primers for the presence of the these genes. The genes targeted were cyanide dihydratase (Bacillus pumilus), hydrogen cyanide synthase (Pseudomonas fluorescens) and cyanide degrading enzyme (Pseudomonas stutzeri). The specifically designed primers were used on the gDNA from the selected organisms and this led to various non-specific product formations and in many of the samples no product was obtained. With the failure to identify the presence of known cyanide degrading genes in the five selected organisms, one of these organisms, Bacillus sp. B4H3, was selected for pyrosequencing to elucidate the complete cyanide metabolism in this microorganism. The sequencing data was analyzed and it was observed that the 16S rRNA gene sequence obtained in Chapter 2, section 2.3.2.7 was not present in the genome of the isolate after pyrosequencing. The pyrosequencing data was analyzed and a total of twenty one enzymes involved in the cyanide metabolism of this isolate were identified. From the complete metabolic pathway it can be concluded that the single nitrogen can be utilized through the conversion of cyanide to formamide which in turn can be converted to ammonia. The utilization of the single carbon is based upon the hypothesis that the reaction between cyanide and glycine is reversible. This will lead to the carbon fixation metabolism which will prove that the isolate is capable of utilizing the single carbon as the sole carbon source.

[发布机构] University of the Free State