Tetracyanonickelate (K2(Ni(CN)4), TCN) is one of the most toxic organics to living organisms. In this study, Klebsiella oxytoca (K. oxytoca) SYSU-011 (a cyanide- degrading bacterium), which was isolated from the wastewater of a metal-plating plant, was shown to be able to biodegrade TCN under anaerobic conditions. Two different media (Burk and NFG) were used to grow K. oxytoca. Results indicate that higher TCN biodegradation rate was observed when Burk medium was used as the growth media for K. oxytoca. In the nitrogen source addition experiment, TCN degradation was inhibited by the addition of nitrite. In the carbon source addition experiment, TCN degradation was enhanced by the addition of glucose and fructose. These findings would be helpful in designing a practical in situ or on-site treatment system inoculated with K. oxytoca for the treatment of TCN-containing wastewater.

Klebsiella oxytoca SYSU-011 isolated from wastewater of a metal-plating plant in southern Taiwan had been proven to be able to degrade cyanide. In this study, we performed proteomic analyses to understand the mechanism of tetracyanonickelate (TCN) resistance in K. oxytoca by using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and MALDI-TOF-MS techniques. There were 91 protein spots had been induced or overexpressed (≥2 fold) by TCN. Among them, 44 protein spots were successfully identified by MALDI-TOF-MS. The expressed proteins that had been escalated including chaperone, glutathione S-transferase (GST) and alkyl hydroperoxide reductase (AHR) were involved in the TCN detoxification process. Fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase and 6-phosphogluconolactonase were involved in energy-producing process; nitrogenase and glutamine synthetase (GS) were required to regulate nitrogen assimilation. We also analyzed the K. oxytoca membrane proteins. Twenty six proteins spots had been successfully identified by MALDI-TOF-MS (out of 41 protein) that were induced ≥2 fold by TCN. These proteins induced RND-drive efflux proteins (RND family, MFP subunit, outer membrane factor A and outer membrane factor TolC) and cation efflux system. These efflux pumps could transport nickel ion out of the cells. The induced ATP-binding cassette (ABC) proteins may also play a role in transportation of metal-cyano complexes TCN and nutrition. By this study, we had a better understanding on the defense mechanism of K. oxytoca after exposure to TCN.

The cyanide-degrading bacterium Klebsiella oxytoca SYSU-011 was isolated from the waste water of a metal-plating plant. In this study, we found out that K. oxytoca was capable of utilizing tetracyanonickelate {K2[Ni(CN)4]}(TCN) as its sole nitrogen source. This organism could degrade TCN both aerobically (D.O.¡×100¢H) and anaerobically (D.O.¡×0¢H).The addition of ammonia (5 mM) in the growth medium would inhibit TCN-degrading. The TCN-degrading by-product, a greenish precipitate, was found in the spent medium and was identified as nickel cyanide [Ni(CN)2] by FT-IR spectroscopic studies. Ammonia was demonstrated as a product of the TCN-degrading process by K. oxytoca resting cells. The addition of glucose could greatly enhance the TCN-degradation. Nitrogenase was found to be the cyanide degrading enzyme in this organism. The activity of nitrogenase was inhibited by ammonia but could be induced by the addition of TCN or KCN.

In this study, Azotobacter vinelandii ATCC13705 (A. vinelandii), which is a free-living, nitrogen-fixing, gram-negative, and aerobic rod bacterium, was need to evaluate its ability to biodegrade tetracyanonickelate (TCN) under different conditions. Results show that A. vinelandii was able to biodegrade various concentrations of TCN (1, 10, and 20 mM) under aerobic conditions. Oxygen consumption and nitrogenase activity were investigated at 1 mM of TCN. Results indicate that the production of ammonia and methane was observed when TCN was consumed. Results suggest that nitrogenase was possibly involved in the enzymatic degradation of TCN. Moreover, higher degradation rate of TCN, higher nitrogenase activity, higher oxygen consumption, and higher specific growth rates were also observed at log growth period. Results suggest that the hypothesis of respiratory protection of nitrogenase is supported. Moreover, the addition of ammonia (1, 5, and 10 mM) would cause the decrease of TCN degradation rate (28%) during a 24-hr incubation period. Inhibition of TCN degradation (degradation rate¡G16% for 24 hrs) was observed when nitrite (5 and 10 mM) was added into the growth medium. Furthermore, the addition of 8% of glucose would significantly enhance the TCN degradation by the resting cells (degradation rate¡G43% for 8 hrs) . Results from this study provide us insight into the characteristics and mechanisms of TCN conversion by A. vinelandii.

Cyanide is a known toxic chemical, the production of plastics, electroplating, tanning, chemical syntheses, etc. At short-term exposure, cyanide causes rapid breathing, tremors, and long-term exposure to cyanide cause weight loss, thyroid effects, nerve damage and death. Although chemical and physical processes can be employed to degrade cyanide and its related compounds, they are often expensive and complex to operate. A proven alternative to these processes is biological treatment, which typically relies upon the acclimation and enhancement of indigenous microorganisms. Biological degradation of cyanide has often been offered as a potentially inexpensive and environmentally friendly alternative to conventional processes. The aims of first part of study were to evaluate the biodegradability of tetracyanonickelate (TCN) by Klebsiella oxytoca under anaerobic conditions. Results reveal that TCN can be biotransformed to methane by resting cells of K. oxytoca. Results also show that TCN biodegradation was inhibited by the addition of nitrate, nitrite, or ammonia at higher concentrations (5 and 10 mM). Moreover, it was found that the optimum pH for TCN conversion by K. oxytoca was about 7.1. Results from the fermenter experiment show that TCN can be completely degraded within 14 days. K. oxytoca is capable of using TCN as the nitrogen source under anaerobic conditions. TCN could be biotransformed to non-toxic end product (methane) by resting cells of K. oxytoca. Those studies provide us insight into the characteristics of TCN conversion by K. oxytoca under anaerobic conditions. In second part of this study, the technology of immobilized cells can be applied in biological treatment to enhance the efficiency and effectiveness of biodegradation. In this study, potassium cyanide (KCN) was used as the target compound and both alginate (AL) and cellulose triacetate (CT) gels were applied for the preparation of immobilized cells. The free suspension systems reveal that the cell viability was highly affected by initial KCN concentration and pH. Results show that immobilized cell systems could tolerate a higher level of KCN concentration and wider ranges of pH. In the batch experiments, the maximum KCN removal rates using alginate and cellulose triacetate immobilized beads were 0.108 and 0.101 mM h-1 at pH 7, respectively. Results also indicate that immobilized system can support a higher biomass concentration. Complete KCN degradation was observed after the operation of four consecutive degradation experiments with the same batch of immobilized cells. This suggests that the activity of immobilized cells can be maintained and KCN can be used as the nitrogen source throughout KCN degradation experiments. The maximum KCN removal rates using AL and CT immobilized beads in continuous-column system were 0.224 and 0.192 mM h-1 with initial KCN concentration of 3 mM, respectively. In third part of this study, a microbial process for the degradation of propionitrile by K. oxytoca was studied. The free and immobilized cells of K. oxytoca were then examined for their capabilities on degrading propionitrile under various conditions. The efficiency and produced metabolic intermediates and end-products of propionitrile degradation were monitored in bath and continuous bioreactor experiments. Results reveal that up to 100 mM and 150 mM of propionitrile could be removed completely by the free and immobilized cell systems, respectively. Furthermore, AL and CT immobilized cell systems show higher removal efficiencies in wider ranges of temperature (20-40¢XC) and pH (6-8) compared with the free cell system. Results also indicate that immobilized cell system could support a higher cell density to enhance the removal efficiency of propionitrile. Immobilized cells were reused in five consecutive degradation experiments, and up to 99% of propionitrile degradation was observed in each batch test. This suggests that the activity of immobilized cells can be maintained and reused throughout different propionitrile degradation processes. A two-step pathway was observed for the biodegradation of propionitrile. Propionamide was first produced followed by propionic acid and ammonia. Results suggest that nitrile hydratase and amidase were involved in the degradation pathways of K. oxytoca. In the continuous bioreactor, both immobilized cells were capable of removing 150 mM of propionitriles completely within 16 h, and the maximum propionitriles removal rates using AL and CT immobilized beads were 5.04 and 4.98 mM h-1, respectively. Comparing the removal rates obtained from batch experiments with immobilized cells (AL and CT were 1.57 and 2.18 mM h-1 at 150 mM of propionitrile, respectively), the continuous-flow bioreactor show higher potential for practical application. These findings would be helpful in designing a practical system inoculated with K. oxytoca for the treatment of cyanide-containing wastewater.

The square-planar tetracyanonickelate(II) anion was intercalated into 2:1 and 3:1 Mg-Al layered double hydroxide systems (LDHs). In the 2:1 material, the anion holds itself at an angle of about 30° to the layers, whereas in the 3:1 material it lies more or less parallel to the layers. This is confirmed by orientation effects in the infrared spectra of the intercalated materials and by X-ray diffraction (XRD) data. The measured basal spacings for the intercalated LDH hosts are approximately 11 Å for the 2:1 and approximately 8 Å for the 3:1. The IR of the 2:1 material shows a slight splitting in the ν(CN) peak, which is suppressed in that compound's oriented IR spectrum, indicating that at least some of the intercalated anion's polarization is along the z-axis. This effect is not seen in the 3:1 material. A comparison between chloride LDHs and nitrate LDHs was made with respect to intercalation of tetracyanonickelate(II) anions. Both XRD data and atomic absorption spectroscopy (AAS) data of the LDH tetracyanonickelates confirms that there are no significant differences between the products from the two types of starting materials. The presence of a weak ν(NO) peak in the IR spectra of those samples made from nitrate parents indicates the presence of small amounts of residual [NO3]- in those systems. Small amounts of Cl- present in the chloride-derived samples, while perhaps detectable using AAS, would not be detectable in this manner. An attempted synthesis of Mg-Al LDH carbonates starting from reduced Mg and Al was unsuccessful due to pH constraints on hydroxide solubility in the solvent system used (water). The pH required to precipitate Al(OH)3 in the system was too high to allow precipitation of Mg(OH)2. Consequently, we found it impossible to have both of the required metal hydroxides present simultaneously in the system. An additional synthesis using a halogen as an oxidizing agent also failed to produce material of any characterizable quality.