Cyanide-degrading Activity Research Articles

Biodegradation of Cyanide by a New Isolated Aerococcus viridans and Optimization of Degradation Conditions by Response Surface Methodology

Microbial treatment of cyanide pollution is an effective, economical, and environmentally friendly method compared with physical or chemical approaches. A cyanide-degrading bacterium was isolated from electroplating sludge and identified as Aerococcus viridans (termed A. viridans T1) through an analysis of the biochemical reaction and 16 S rDNA gene sequence. A. viridans T1 showed a maximum resistance to 550 mg L−1 CN−. The effect of pH and temperature on cyanide degradation and bacterial growth was evaluated. The highest cyanide removal efficiency and bacterial growth occurred at pH 8 and pH7, respectively. The optimum temperature for cyanide degradation and bacterial growth was 34 ∘C. In addition, the carbon source and nitrogen source for cyanide degradation were optimized. The optimal carbon source and nitrogen source were glycerol and peptone, respectively. The cyanide degradation experiment indicated that A. viridans T1 was able to remove 84.1% of free cyanide at an initial concentration of 200 mg L−1 CN− within 72 h and 86.7% of free cyanide at an initial concentration of 150 mg L−1 CN− within 56 h. To improve the cyanide-degrading efficiency of A. viridans T1, eight process variables were further optimized using a response surface methodology. Three significant variables (soybean meal, corn flour, and L-cysteine) were identified using a Plackett–Burman design, and the variable levels were optimized using a central composite design. The optimal values of soybean meal, corn flour, and L-cysteine were 1.11%, 1.5%, and 1.2%, respectively. Under these optimal conditions, the confirmatory experiments showed that the actual degradation rate was 97.3%, which was similar to the predicted degradation rate of 98.87%. Its strong resistance to cyanide and cyanide-degrading activity may allow A. viridans T1 to be a candidate for the bioremediation of cyanide-contaminated environments.

Isolation of cyanide-degrading bacteria and molecular characterization of its cyanide-degrading nitrilase.

Hydrogen cyanide is an industrially important chemical, and its annual production is more than 1.5 million tons. Because of its toxicity, the cyanide-containing effluents from industries have caused many environmental problems. Among various methods to treat the contaminated soils or water, the biological degradation is regarded to be promising. We isolated two cyanide-degrading microorganisms, Pedobacter sp. EBE-1 and Bacillus sp. EBE-2, from soil contaminated with cyanide. Among these bacteria, Bacillus sp. EBE-2 exhibited significantly a high cyanide-degrading ability. Bacillus sp. EBE-2 might be used for the remediation of cyanide contaminated water or soil. A nitrilase gene was cloned from Bacillus sp. EBE-2. Bacillus nitrilase was expressed in Escherichia coli and purified. Bacillus nitrilase exhibited cyanide-degrading activity as a large oligomer. Since formic acid formation from cyanide was observed, Bacillus nitrilase is likely to be a cyanide hydrolase. Although there exist various homologous enzymes annotated as carbon-nitrogen family hydrolases, this is the first report on the cyanide degrading activity. The structure and catalytic site of Bacillus nitrilase were studied by homology modeling and molecular docking simulation.

Enhanced cyanide biodegradation by immobilized crude extract of Rhodococcus UKMP-5M

ABSTRACTThe capability of the crude extract of Rhodococcus UKMP-5M was enhanced by adopting the technology of immobilization. Among the matrices screened to encapsulate the crude extract, gellan gum emerged as the most suitable immobilization material, exceeding the activity of cyanide-degrading enzyme by 61% and 361% in comparison to alginate carrier and non-immobilized crude extract, respectively. Improved bead mechanical strength which supported higher biocatalyst activity by 63% was observed when concentration of gellan gum, concentration of calcium chloride, number of beads and bead size were optimized. The immobilized crude extract demonstrated higher tolerance towards broad range of pH (5–10) and temperature (30°C–40°C), superior cyanide-degrading activity over time and improved storage stability by maintaining 76% of its initial activity after 30 days at 4°C. Furthermore, repeated use of the gellan gum beads up to 20 batches without substantial loss in the catalytic activity was documented in the present study, indicating that the durability of the beads and the stability of the enzyme are both above adequate. Collectively, the findings reported here revealed that the utilization of the encapsulated crude extract of Rhodococcus UKMP-5M can be considered as a novel attempt to develop an environmentally favourable and financially viable method in cyanide biodegradation.

Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP−5M

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min−1 mg−1 protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP−5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

Purification and characterization of a cyanide-degrading nitrilase fromTrichoderma harzianum VSL291

An intracellular nitrilase (Nit1) with cyanide-degrading activity was isolated from Trichoderma harzianum VSL291, cultivated on benzonitrile as the sole carbon source. Nit1 was purified to homogeneity by ion exchange and gel filtration chromatography with a recovery of 7.15% and a fold of 22.5. The molecular weight was estimated to be 47.7 kDa and the purified enzyme was sequenced with a system of liquid chromatography and mass spectrometry (LC-MS). The enzyme consists of 436 amino acids with a predicted molecular weight of 47.088 kDa. The sequence revealed conserved domains for a nitrilase super family such as putative active and binding sites and a Glu-Lys-Cys catalytic triad. Nit1 exhibited maximum activity (19.6 U mg^{?1}) at 40 °C and a pH of 7.5. Nit1 had a strong inhibition in the presence of Al^{3}+, Cu^{2+}, Zn^{2+}, and Ag^+ ions and was able to degrade KCN completely at 0.02 mmol/L, 0.05 mmol/L, and 0.1 mmol/L in 15 min, 40 min, and 45 min, respectively. The effect on KCN (0.02 mmol/L) degradation was tested in the presence of Cu2+ and Ag+ ions (0.025 mmol/L to 1.0 mmol/L) and the enzymatic activity was not affected significantly at 0.025 mmol/L, 0.075 mmol/L, and 0.125 mmol/L concentrations. However, when both ions were combined, the activity of the enzyme decreased significantly.

Biodetoxification of cyanide-containing industrial wastewaters by Rhodococcus UKMP-5M

The utilization of cyanide in many industrial processes despite its toxicity generated voluminous effluents, which necessitated treatment prior to discharge into the environment. In the present study, Rhodococcus UKMP-5M was solely employed as biological tool to detoxify three different cyanide-containing industrial wastewaters with different characteristics since initial attempt to employ mixed culture of Rhodococcus strains was unsuccessful due to synergistic interaction which yielded only 19% biodetoxification of 12 mM cyanide. The bacterium was able to achieve 97% cyanide removal efficiency in Wastewater A (pH 8.4) after 19 days of incubation. However, the growing cells needed the supplementation of nutrients, which was uneconomical besides requiring extensive time for complete detoxification. In contrast, the employment of immobilized beads of Rhodococcus UKMP-5M, which required a relatively straightforward method, were cost-effective in detoxifying almost 100% of 15 mM cyanide in Wastewater C (pH 9.5) within a short period of 3 days of incubation. This discovery is highly significant since most wastewaters pHs originating from various industrial applications involving cyanide often range from 6 to 9.5 and this strain is therefore seemingly effective for biodetoxification of cyanide-containing wastewaters with high pH values. In addition, it is interesting to note that the presence of heavy metals in the wastewaters did not inhibit the cyanide-degrading activity of the bacterium. The findings from this study provided essential information to develop a pilot plant on a technical scale for the biotreatment of industrial effluents bearing cyanide, which should be of great interest from an environmental and economic point of views.

An effective method for the detoxification of cyanide-rich wastewater by Bacillus sp. CN-22

The biodetoxification of cyanide-rich wastewater has become increasingly popular because of its cost-effectiveness and environmental friendliness. Therefore, we have developed an effective method, optimised by response surface methodology, for detoxifying cyanide-rich wastewater using Bacillus sp. CN-22, which was newly isolated from a cyanide-contaminated electroplating sludge and could tolerate a CN⁻ concentration of 700 mg L⁻¹. The concentration of CN⁻ in the treated wastewater decreased from 200 to 6.62 mg L⁻¹ after cultivation with 2.38 % inocula for 72 h on the medium, consisting of 0.05 % KH₂PO₄, 0.15 % K₂HPO₄, 1.0 mM MgCl₂, 1.0 mM FeCl₃, 0.1 % NH₄Cl, and 0.1 % glycerol. The CN⁻ degradability of 96.69 % is similar to the predicted value of 96.82 %. The optimal cultivation conditions were controlled as follows: initial pH, 10.3; temperature, 31 °C; and rotary speed, 193 rpm. The maintenance of higher pH in the overall treatment procedures may avoid the production of volatile HCN and the risk associated with cyanide detoxification. Additionally, the bacterial strain Bacillus sp. CN-22, with its potent cyanide-degrading activity at the initial CN⁻ concentration of 200 mg L⁻¹, may be employed to effectively treat cyanide-rich wastewater, especially electroplating effluent.

Purification and Biochemical Characteristics of Cyanide-degrading Enzyme from Alcaligenes sp.DN25

The cyanide-degrading enzyme from Alcaligenes sp.DN25 was purified through ultrafiltration,precipitation with 30 mg/mL protamine sulfate,30%~70% fractional ammonium sulphate precipitation and hydrophobic chromatography on Phenyl-Toyopearl 650M,and the pure enzyme with the specific activity of 44 U/mg was obtained.After the proper reaction conditions including enzyme concentration and reaction time were determined for the cyanide-degrading activity assay,the purified enzyme properties were then studied in order to provide a theoretical basis for the future researches on cyanide-degradation mechanism and genetic engineering of strain DN25.The results showed that the optimal pH and temperature were 8.0 and 30 ℃,respectively.Good stability of the enzyme was observed at pH 7.0~8.0 and its activity decreased quickly when pH reached up to 9.0.The activity of the purified enzyme could keep stable when preserved at 30 ℃ for 10 h while fast deactivation happened at 60 ℃.However,with glycine added,the enzyme activity still remained 19.6% after incubated at 60 ℃ for 20 min.The degradation of cyanide by the purified enzyme followed a typical Michaelis-Menten kinetics,with Km of 3.11 mmol/L and Vmax of 0.23 mmol L-1 min-1.

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10 mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25 mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.

Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase.

The cyanide-degrading bacterial strain AK61 was isolated from waste water at a metal-plating plant. The isolated strain was characterized by Gram-staining, quinone analysis, fatty acid profile and the API 20NE identification system, and identified as Pseudomonas stutzeri. Whole cells were able to degrade cyanide rapidly in a 1 mM solution containing no organic substances, and produced ammonia as a product. The induction of the cyanide-degrading activity of P. stutzeri AK61 did not depend on the presence of cyanide in the culture medium during growth. The cyanide-degrading enzyme was purified approximately 49-fold from a cell extract of P. stutzeri AK61. The enzyme had a K(m) of 1.7 mM for cyanide and a specific activity of 54.6 mumol ammonia produced min-1. The activity of the enzyme was optimal at 30 degrees C and pH 7.5. The results of SDS-PAGE, gel-filtration chromatography and NH2-terminal amino acid sequence analysis of the enzyme indicated that the functional enzyme was an aggregated protein consisting of a 38 kDa polypeptide. Like cyanidase (cyanide dihydratase), it was shown that the enzyme catalysed the hydrolysis of cyanide to ammonia and formate.

Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764.

Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalysed the degradation of cyanide into products that included CO2, formic acid, formamide and ammonia. Cyanide-degrading activity (CDA) was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM (2600 mg CN-I-1). At least two different CDAs could be distinguished by: (i) the determination of reaction product stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) plus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymic activity was identified as cyanide oxygenase as previously described [Harris, R. E. & Knowles, C. J. (1983) FEMS Microbiol Lett 20, 337-341] whereas the second activity is believed to consist of two enzymes, a cyanide nitrilase (dihydratase) and hydratase (EC 4.2.1.66). In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (EC 1.2.1.2) thereby providing a means of recycling NADH utilized by cyanide oxygenase. A mutant strain having lost the ability to grow on cyanide as a nitrogen source was isolated and shown to be defective in cyanide oxygenase, but not the cyanide nitrilase/hydratase enzymes. This finding together with results showing that the substrate affinity of cyanide oxygenase was tenfold greater than for the nitrilase/hydratase enzymes, indicates that it is this enzyme that is most important in cyanide assimilation.(ABSTRACT TRUNCATED AT 250 WORDS)

An efficient cyanide-degrading Bacillus pumilus strain

A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg l-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide l-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ l-1 and did not occur at Mn2+ concentrations below 0.002 mg l-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.