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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10172011-154227 |
| Date | 17 October 2011 |
| Creators | Meyer, Wilmari |
| Contributors | Prof E van Heerden, Dr LA Piater, Prof J Albertyn |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-10172011-154227/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0024 seconds