Ten bacteria capable of growth on the metal-cyano complex, tetracyanonickelate (II) {K2 [Ni(CN)J } (TCN), supplied as the sole nitrogen source, were isolated. Seven isolates were identified as pseudomonads while the remaining three were classified as Klebsiella species. In addition to TCN, all isolates were able to utilize KCN although it was significantly more toxic. The degradation of TCN was most complete when supplied at growth-limiting concentrations, did not occur when ammonia was present, and resulted in the formation of nickel cyanide [Ni(CN)2] as a degradation product.

Pseudomonas fluorescens NCIMB 11764 was capable of utilizing cyanide as a sole nitrogen source for growth. Cyanate (OCN") and S-cyanoalanine could also serve as nitrogenous substrates, but do not appear to play a role as intermediates in cyanide metabolism. Growth of this strain on cyanate as the sole nitrogen source led to the induction of an enzyme characterized as a cyanase (EC 3.5.5.3) based on its stoichiometric conversion of cyanate to ammonia, and dependence on bicarbonate for maximal activity. However, since cyanase activity was not elevated in cyanide-grown cells it was concluded that it serves no role in cyanide metabolism. Related studies aimed at examining a possible role for S-cyanoalanine as a cyanide-assimilation intermediate showed that while this compound also serves as a nitrogen source, it also is not important in cyanide metabolism. Studies focused on the utilization of free cyanide as a growth substrate led to the development of a fed-batch cultivation procedure greatly facilitating further experimentation aimed at the identification of cyanide metabolites. In addition to CO_2 and NH_3 as described earlier, two additional metabolites including formamide and formate were detected by using nC-NMR, HPLC, radioisotrapping methods and other analytical means. The formation of metabolites was shown to be induced after growth on cyanide with the relative product yields dependent on the availability of oxygen. These findings support earlier work in which an oxygen-dependent mechanism was proposed for the formation of C02 and NH3. However, at least two additional oxygen-independent pathways of cyanide conversion can be elaborated by this organism. One of these involves conversion to formate and ammonia while the other leads to the formation of formamide, which is not further degraded. Thus, growth on cyanide appears to occur by several mechanisms of chemical transformation presumably serving both detoxification and nutritional roles. Since two of these mechanisms generate ammonia, which is readily assimilated, growth is presumed to proceed via ammonia as a provisionary nitrogenous substrate.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Based on the review of different research studies, we could assess that, due to their unique biological features, microbes, specifically bacteria, could be used to repair damaged soils with heavy metal and toxic compound contents. Furthermore, these microorganisms are metabolically capable to oxidize cyanide and its by-products to generate less-toxic compounds at the end of the process. This research proposal seeks to improve conventional mine closure designs, thus counteracting their negative short-term, medium-term, and long-term after-effects to the environment. The proposed technique as a solution, therefore, is microbial remediation, using pseudomonas fluorescens bacteria to oxidize this compound to non-toxic components. It will ensure operational continuity for the deposits and, in turn, the sustainability of the entire mining industry.

One of the challenges in biology is placing a function on the myriad of gene sequences having become available from rapid advances in genome sequencing. One such example is a gene cluster (Nit1C) found in bacteria that is tied to the unusual ability of certain bacteria to grow when supplied cyanide as the sole nitrogen source. The term cyanotrophs has been applied to such bacteria, for which a genetic linkage between cyanotrophy and Nit1C was demonstrated for 10 separate bacteria. In addition to growth, cyanide induced the expression of Nit1C genes in all organisms tested, and in one case, deletion of one of the Nit1C genes (nitC) caused a loss of growth. Of the ten bacteria able to grow cyanotrophically, all gave evidence of harboring Nit1C on their genome except for two (Pseudomonas fluorescens Pf11764 and P. monteilii BCN3), which were sequenced and the presence of Nit1C was also confirmed. A broader search of bacteria identified 270 separate strains with the cluster, all limited to bacteria spanning the phyla Firmicutes, Actinobacteria, Proteobacteria and Cyanobacteria. Remarkably, many examples of a single representative of a given taxon contained Nit1C, most poignantly displayed by Pf11764 and PmBCN3; the interpretation being the cluster was likely acquired by horizontal gene transfer in response to cyanide as an environmental cue. Consistent with its absence in Archaea is the time line for the emergence of cyanide producing organisms (cyanogens) on earth dating back only 400-500 million years.

A f1ourometric procedure for the analysis of free cyanide is described. The method is based on the dissociation of a ternary complex of si1ver-1, 10-phenanthro1ine-tetrabromof1uorescein by cyanide to produce a fluorescent product. The fluorescence intensity is found to be directly proportional to the cyanide concentration. The method can reliably detect 0.02 μg/mL cyanide.

The Nit1C cluster is a conserved gene cluster of seven genes that confers bacterial growth on cyanide as the sole nitrogen source. Bacteria with this ability are referred to as cyanotrophs. To date, the linkage between Nit1C and cyanotrophy has only been demonstrated for environmental isolates but the cluster also exists in certain medically related bacteria. In this study, a nosocomial isolate, Acinetobacter haemolyticus ATCC 19194, carrying Nit1C also displayed the ability to grow on cyanide. Growth on cyanide was accompanied by the induction of the cluster as was the mere exposure of cells to cyanide. Expression of the cluster was determined by measuring the activity of the nitrilase (NitC) coded for by the cluster and by transcriptional analysis (qRT-PCR). However, a disconnect between nitC message and NitC protein was observed depending on the phase of the growth cycle, the disconnect being related to proteolytic digestion of the NitC protein. Ironically, the cluster was also discovered to be upregulated in the absence of cyanide under nitrogen starvation conditions paralleling biofilm formation. The basis of the genetic linkage to cyanotrophy is not understood but taken together with results showing that nitrogen starvation and biofilm formation are also physiologically associated with Nit1C expression, points to a critical role for the cluster in stress-induced adaptation.

No description available.

No description available.

Thesis (PhD)--Stellenbosch University, 1992. / ENGLISH ABSTRACT: The dissolution of gold from refractory ores is a complex kinetic problem involving a number of chemical, mass transport and mineralogical factors. In most Witwatersrand ores in South Africa more than 97 % of the gold is dissolved in cyanide medium after a residence time of about 16 hours in pachuca tanks. This high percentage may be the reason why so little fundamental research has been done into the mechanism and kinetics of the leaching process. With the increasingly lower grades of ore mined, the introduction of backfill mining, and the reduction of profit margins, it has become imperative to increase the efficiency of gold dissolution. The effects of the chemistry and particle size on the dissolution of gold in each sample of ore were studied in detail. The emphasis in this study is on the effect of the leaching behaviour of various ore constituents on the rate of gold dissolution. Interferences with the leaching of gold in contact with other minerals or metals could be attributed to the galvanic interaction (electrical conductivity) between the gold and the mineral and to the formation of a surface film on the gold surface. Sulphide minerals and their oxidation products cause the largest decrease in gold dissolution rate. Galena enhances the rate of gold dissolution owing to dissolved Pb(II)-ions. Gold in contact with conductive minerals passivates as a result of the enhanced magnitude of the cathodic cu1Tent. In all experiments the rotating disc of gold passivated so that the rate of dissolution was much slower than that predicted by a mass-transport limiting model. The various films that form on the surface of the gold and associated minerals, as well as the galvanic interaction, depend largely on the pretreatment of the ore. Pre-elimination of host minerals from the gold bearing ore increases the dissolution rate of gold, and explains the kinetics of reaction on the gold surface to a large extent. The selective destruction of the various minerals with oxidative acid leaches destroys and/or decomposes certain minerals which may form films on the gold surface by precipitation. The chemical composition of these films and precipitates depends on the mineralogy of the sample. These films may be oxides, sulphides, carbonates and cyanide complexes. The complexes can be destroyed, depending on the nature of the film, by interstage dilute acid and/or cyanide washes in an agitated vessel. The destruction of the films exposes the gold surface for cyanidation. A simple distribution function similar to the King liberation model is proposed and tested to describe the dissolution step in the multi-step leaching mechanism. For the King model, good agreement is shown with experimental results. For the liberation results obtained by leaching in this study, the trend is co1Tect, but calibration is required for a close fit. A potentially important use for the liberation model by leaching is to predict the leachable or free gold in an ore from the free gold in the complete sample. This approach for studying the leaching behaviour of different gold bearing minerals has provided reasons why some ores leach better than others. / AFRIKAANSE OPSOMMING: Die loging van goud vanuit weerbarstige ertse is 'n komplekse kinetiese probleem wat verskeie faktore soos massa-oordrag, chemiese aspekte en mineralogiese ingeweefdheid insluit. Goud ekstraksies so hoog as 97 % in sianied oplossings in Pachuca reaktore na ongeveer 16 uur logingstyd word behaal in die meeste Witwatersrand ertse in Suid-Afrika. Hierdie hoë ekstraksies mag dalk die rede wees vir die min fundamentele navorsing oor die ekstraksie van goud vanuit minerale in 'n spesifieke erts. Die dalende erts grade, die verlaging van winsgrense en die terugplaas van geloogde erts in die myn noodsaak verbeterde goud ekstraksie. Verkeie faktore nl., chemie, diffusie, partikelgrootte en oplosbaarheid van goud in elke monster erts is in hierdie studie ondersoek. Die sentrale tema was om die logingsgedrag van goud vanuit verskeie minerale in 'n erts te bepaal. Galvaniese interaksie (hou verband met elektriese geleidingsvermoeë) en film vorming is die belangrikste faktore wat die loging van goud in kontak met minerale nadelig beïnvloed. Sulfied minerale en hul oksidasie produkte speel die grootste rol in die verlaging van die tempo van goudloging. Galena verhoog die tempo van goudloging as gevolg van die Pb (II)- ione in oplossing. Goud in kontak met geleidende minerale passiveer as gevolg van die verhoogde katodiese stroomdigtheid. In alle eksperimente met die roterende skyf (goudskyf) apparaat, passiveer die goudskyf in so 'n mate dat die logingstempo baie stadiger is as wat voorspel word met die massa-oordrags model. Die onderskeie films wat vorm op die goud- en geassosieerde minerale se oppervlaktes, asook die galvaniese interaksies, is 'n funksie van die voorafbehandeling van die erts. Die selektiewe eliminering van minerale vanuit 'n gouddraende erts verhoog die tempo van goudloging drasties en dit beskryf die kinetika van goudloging op die goudoppervlak in 'n groot mate. Die selektiewe eliminering van minerale deur gebruik te maak van oksiderende suurlogings, vernietig sekere van die minerale wat films op die goudoppervlakte kan veroorsaak deur middel van presipitasie. Die chemiese samestelling van hierdie films hang af van die mineralogie van die monster. Dit bestaan meestal uit oksiedes, sulfiedes, karbonate en sianiedkomplekse en hulle kan vernietig word deur middel van inter-stadia verdunde suur-en/of sianied wasse. 'n Eenvoudige distribusiefunksie, soortgelyk aan die King bevrydingsmodel word voorgestel en eksperimenteel getoets om die logingstap in die multi-stadia logingsmeganisme te beskryf. Vir bevryding deur loging, is die neiging van King se model korrek, maar kalibrasie word benodig vir goeie passing. 'n Potensiele gebruik van die aangepaste model is om vrye of loogbare goud in 'n spesi fieke partikel grootte fraksie van 'n erts te voorspel as die vry goud in die totale fraksie bekend is. Die resultate uit hierdie studie kan gebruik word om die logingsgedrag van gouddraende minerale te voorspel en te beskryf, en dus veduidelik hoekom goud uit sekere ertse beter loog as uit ander.

Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)