Bacterial degradation of cyanide and its metal complexes under alkaline conditions

A bacterial strain able to use cyanide as the sole nitrogen source under alkaline conditions has been isolated. The bacterium was classified as Pseudomonas pseudoalcaligenes by comparison of its 16S RNA gene sequence to those of existing strains and deposited in the Coleccion Espanola de Cultivos Tipo (Spanish Type Culture Collection) as strain CECT5344. Cyanide consumption is an assimilative process, since (i) bacterial growth was concomitant and proportional to cyanide degradation and (ii) the bacterium stoichiometrically converted cyanide into ammonium in the presence of l-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. The bacterium was able to grow in alkaline media, up to an initial pH of 11.5, and tolerated free cyanide in concentrations of up to 30 mM, which makes it a good candidate for the biological treatment of cyanide-contaminated residues. Both acetate and d,l-malate were suitable carbon sources for cyanotrophic growth, but no growth was detected in media with cyanide as the sole carbon source. In addition to cyanide, P. pseudoalcaligenes CECT5344 used other nitrogen sources, namely ammonium, nitrate, cyanate, cyanoacetamide, nitroferricyanide (nitroprusside), and a variety of cyanide-metal complexes. Cyanide and ammonium were assimilated simultaneously, whereas cyanide strongly inhibited nitrate and nitrite assimilation. Cyanase activity was induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source. This result suggests that cyanate could be an intermediate in the cyanide degradation pathway, but alternative routes cannot be excluded.     

The cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT5344 responds to cyanide by defence mechanisms against iron deprivation, oxidative damage and nitrogen stress

Two-dimensional (2-D) electrophoresis approach has been used to test protein expression changes in response to cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344. This is a cyanide-assimilating strain which also grows in media containing cyanide-enriched effluent from the jewellery industry. The bacterium efficiently uses this residue as the sole nitrogen source for aerobic growth under alkaline pH with negligible nitrogen losses as HCN. Cell-free extracts isolated from P. pseudoalcaligenes grown with a jewellery residue, free cyanide or ammonium chloride as nitrogen source were subjected to 2-D electrophoresis and the spot patterns were examined to determine differential protein expression. Electrophoretic plates exhibiting an average of 1000 spots showed significant differences in the expression of about 44 proteins depending on the nitrogen source. Some of these protein spots were analysed by Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Characterization of five of these proteins reveals that cyanide shock induces proteins related to iron acquisition, regulation of nitrogen assimilation pathways and oxidative stress repairing and protection.     

Bacterial cyanide degradation is under review: Pseudomonas pseudoalcaligenes CECT5344, a case of an alkaliphilic cyanotroph

There are thousands of areas in the U.S.A. and Europe contaminated with cyanide-containing wastes as a consequence of a large number of industrial activities such as gold mining, steel and aluminium manufacturing, electroplating and nitrile pesticides used in agriculture. Chemical treatments to remove cyanide are expensive and generate other toxic products. By contrast, cyanide biodegradation constitutes an appropriate alternative treatment. In the present review we provide an overview of how cells deal in the presence of the poison cyanide that irreversible binds to metals causing, among other things, iron-deprivation conditions outside the cell and metalloenzymes inhibition inside the cell. In this sense, several systems must be present in a cyanotrophic organism, including a siderophore-based acquisition mechanism, a cyanide-insensitive respiratory system and a cyanide degradation/assimilation pathway. The alkaliphilic autochthonous bacterium Pseudomonas pseudocaligenes CECT5344 presents all these requirements with the production of siderophores, a cyanide-insensitive bd-related cytochrome [Cio (cyanide-insensitive oxidase)] and a cyanide assimilation pathway that generates ammonium, which is further incorporated into organic nitrogen.     

Biological cyanide destruction mediated by microorganisms

Many microorganisms have an inherent capacity to degrade the toxic organic compounds that enter the environment as a result of pollution and natural activities. Significant degradation of these compounds may take many years and it is frequently necessary to consider methods that can accelerate this process. There have been several demonstrations of enhanced biological degradation of toxic wastes, both in the laboratory and under field conditions. The prospects for enhanced biological cyanide degradation are reviewed. Compared with bench-scale processes, there are very few reports of field-scale processes for cyanide bioremediation. The implementation of such field-scale degradation requires inputs from biology, hydrology, geology, chemistry and civil engineering. A conceptual framework is emerging that can be adapted to develop new processes for bioremediation of toxic organic wastes. In terms of cyanide biodegradation, this framework incorporates identification of microbes, determination of the optimal conditions for degradation, establishment of the metabolic pathways involved in cyanide degradation, identification and localization of the genes involved, identification of suitable microbial strains for practical application and development of practical engineering processes. The present review addresses the progress that has been made in each of these aspects of cyanide biodegradation. It also examines the existing field applications of biological cyanide degradation and makes recommendations for future research.Dr S.K. Dubey is and Dr D.S. Holmes was with the Department of Biology, Clarkson University, Potsdam, NY 13699, USA. Dr D.S. Holmes is now affiliated with Centro de Estudios Cientifigos de Santiago, Av. Presedente Errazuriz 3132, Casilla 16443, Santiago 9, Chile.     

Biodegradation of cyanide by Rhodococcus UKMP-5M

A new bacterial strain, Rhodococcus UKMP-5M isolated from petroleum-contaminated soils demonstrated promising potential to biodegrade cyanide to non-toxic end-products. Ammonia and formate were found as final products during growth of the isolate with KCN as the sole nitrogen source. Formamide was not detected as one of the end-products suggesting that the biodegradation of cyanide by Rhodococcus UKMP-5M may have proceeded via a hydrolytic pathway involving the bacterial enzyme cyanidase. No growth of the bacterium was observed when KCN was supplied as the sole source of carbon and nitrogen even though marginal reduction in the concentration of cyanide was recorded, indicating the toxic effect of cyanide even in cyanide-degrading microorganisms. The cyanide biodegradation ability of Rhodococcus UKMP-5M was greatly affected by the presence of organic nutrients in the medium. Medium containing glucose and yeast extract promoted the highest growth rate of the bacterium which simultaneously assisted complete biodegradation of 0.1 mM KCN within 24 hours of incubation. It was found that growth and cyanide biodegradation occurred optimally at 30°C and pH 6.3 with glucose as the preferred carbon source. Acetonitrile was used as an inducer to enhance cyanide biodegradation since the enzymes nitrile hydratase and/or nitrilase have similarity at both the amino acid and structural levels to that of cyanidase. The findings from this study should be of great interest from an environmental and health point of views since the optimum conditions discovered in the present study bear a close resemblance to the actual scenario of cyanide wastewater treatment facilities.     

Cyanide Degradation under Alkaline Conditions by a Strain of Fusarium solani Isolated from Contaminated Soils

Several cyanide-tolerant microorganisms have been selected from alkaline wastes and soils contaminated with cyanide. Among them, a fungus identified as Fusarium solani IHEM 8026 shows a good potential for cyanide biodegradation under alkaline conditions (pH 9.2 to 10.7). Results of K(sup14)CN biodegradation studies show that fungal metabolism seems to proceed by a two-step hydrolytic mechanism: (i) the first reaction involves the conversion of cyanide to formamide by a cyanide-hydrolyzing enzyme, cyanide hydratase (EC 4.2.1.66); and (ii) the second reaction consists of the conversion of formamide to formate, which is associated with fungal growth. No growth occurred during the first step of cyanide degradation, suggesting that cyanide is toxic to some degree even in cyanide-degrading microorganisms, such as F. solani. The presence of organic nutrients in the medium has a major influence on the occurrence of the second step. Addition of small amounts of yeast extract led to fungal growth, whereas no growth was observed in media containing cyanide as the sole source of carbon and nitrogen. The simple hydrolytic detoxification pathway identified in the present study could be used for the treatment of many industrial alkaline effluents and wastes containing free cyanide without a prior acidification step, thus limiting the risk of cyanhydric acid volatilization; this should be of great interest from an environmental and health point of view.     

Removal of metal cyanides from aqueous solutions by suspended and immobilized cells of Rhizopus oryzae (MTCC 2541)

This paper presents a study on biodegradation and simultaneous adsorption and biodegradation (SAB) of zinc and iron cyanides by Rhizopus oryzae (MTCC 2541), with a brief process review. Granular activated carbon was used for the immobilization of Rhizopus oryzae (MTCC 2541) for the SAB study. pH and temperature were optimized at an initial cyanide concentration of 100 mg/L for biodegradation and SAB. The microbes adapted to grow at maximum cyanide concentration were harvested and their ability to degrade cyanide was measured in both biodegradation and SAB. The removal efficiency of the SAB process was found to be better as compared to the biodegradation process. In the case of biodegradation, removal was found up to a maximum cyanide concentration of 250 mg CN^?/L for zinc cyanide and 200 mg CN^?/L for iron cyanide, whereas in the case of SAB, about 50% removal of cyanide at 400 mg CN^?/L zinc cyanide and 300 mg CN^–/L iron cyanide was possible. It was found that the SAB process is more effective than biodegradation.     

Natural attenuation potential of cyanide via microbial activity in mine tailings

Biological removal by indigenous microflora of cyanide, contained in old (6-9 years) and fresh tailings (3 months), was studied in order to assess its natural attenuation potential via biodegradation. To investigate the presence of indigenous microflora in tailings, total heterotrophic and cyanide resistant bacteria were counted using the spread-plate method. The free cyanide mineralization potential was estimated using K14CN in the presence of various unlabeled cyanide concentrations (0, 5, and 10 mg CN/kg). The biodegradation of cyanide contained initially in the samples was also investigated by monitoring formate, formamide, ammonia and total cyanide (CNT) concentrations over 111 days. The enumeration of total heterotrophic and cyanide-resistant bacteria in old tailings showed an average population of 105 cfu/g. However, no growth was detected in fresh tailings. Nevertheless, cyanide mineralization tests indicated the presence, in both old and fresh tailings, of a cyanide-degrading microflora. In old tailings, maximum mineralization percentages of free cyanide ranging from 85% to 100% were obtained after 65 days at all concentrations tested. A mineralization percentage of 83% after 170 days was also observed in fresh tailings. No decrease of total cyanide concentration in old tailings was observed when the biodegradation of endogenous cyanide was tested whereas a significant decrease was recorded in fresh tailings after 96 days. The presence of strong metal-cyanide complexes resistant to biodegradation could explain the absence of biodegradation in old tailings. This study demonstrated the presence of an indigenous free cyanide-degrading microflora in both old and fresh tailings, and suggests that natural attenuation of cyanide in gold mine tailings is likely to occur via microbial activity.     

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.     

Cyanide detoxification by the cobalamin precursor cobinamide

Cyanide is a highly toxic agent that inhibits mitochondrial cytochrome-c oxidase, thereby depleting cellular ATP. It contributes to smoke inhalation deaths in fires and could be used as a weapon of mass destruction. Cobalamin (vitamin B12) binds cyanide with a relatively high affinity and is used in Europe to treat smoke inhalation victims. Cobinamide, the penultimate compound in cobalamin biosynthesis, binds cyanide with about 10(10) greater affinity than cobalamin, and we found it was several-fold more effective than cobalamin in (i) reversing cyanide inhibition of oxidative phosphorylation in mammalian cells; (ii) rescuing mammalian cells and Drosophila melanogaster from cyanide toxicity; and (iii) reducing cyanide inhibition of Drosophila Malpighian tubule secretion. Cobinamide could be delivered by oral ingestion, inhalation, or injection to Drosophila, and it was as effective when administered up to 5 mins post-cyanide exposure as when given pre-exposure. We conclude that cobinamide is an effective cyanide detoxifying agent that has potential use as a cyanide antidote, both in smoke inhalation victims and in persons exposed to cyanide used as a weapon of mass destruction.     

Biological degradation of cyanide compounds

Cyanide compounds are produced as waste products of a number of industrial processes and several routes for their removal from the environment are under investigation, including the use of biodegradation. The most recent developments in this area have come from studies of the hydrolytic and oxidative pathways for biodegradation and the conditions that affect their activity. The biodegradation of cyanide under anaerobic conditions has also recently demonstrated the feasibility for concomitant biogas generation, a possible economic benefit of the process. Significant advances have been reported in the use of plants for the phytoremediation of cyanide compounds and evidence for the biodegradation of thiocyanate and metal-cyanide complexes has become available. Despite these advances, however, physical and economic factors still limit the application of cyanide biodegradation, as do competing technologies.     

微生物降解3,5,6-三氯-2-吡啶醇的研究进展

随着高毒性有机磷杀虫剂的限制和禁止使用,近年来以毒死蜱为代表的低毒性有机磷杀虫剂的市场份额有所增加。然而,毒死蜱的使用也导致了环境中3,5,6-三氯-2-吡啶醇(TCP)的产生,因为TCP是毒死蜱和甲基毒死蜱在环境中降解的主要中间代谢产物。它具有较高的水溶性和迁移性,容易进入深层土壤及水体环境,从而引起广泛的污染。释放到环境中的TCP不仅可以抑制TCP及其母体化合物毒死蜱和甲基毒死蜱的生物降解,而且也能抑制其他有机污染物的生物降解,从而进一步加重环境中TCP以及其他有机污染物的累积残留,影响环境系统的自我修复功能。本文概述了TCP及其母体化合物的结构、TCP的生态毒性、TCP降解菌的多样性及其微生物降解的最新研究进展,为毒死蜱和TCP污染地区进行经济可行的生物修复提供参考。     

Effect of HRT on the biological pre-denitrification process for the simultaneous removal of toxic pollutants from cokes wastewater

A lab-scale serial anoxic-aerobic reactor for the pre-denitrification process was continuously operated to efficiently and economically treat actual cokes wastewater containing various pollutants, such as phenol, ammonia, thiocyanate and cyanide compounds. The biodegradation efficiencies of the pollutants were examined by changing hydraulic retention time (HRT) as a main operating variable. The long-term operation of the pre-denitrification process reactor showed that approximately 100% phenol, approximately 100% free cyanide, approximately 100% SCN(-), 97% ammonia, 85% COD, 84% TOC (total organic carbon) and 83% TN (total nitrogen) were removed at HRT above 11.9h. Removal efficiency of total cyanides significantly decreased with a decrease in the HRT. Free cyanide and some of total cyanides were removed in anoxic reactor, whereas thiocyanate was removed in aerobic reactor. Phenol was completely removed under successive anoxic and aerobic conditions. Although actual cokes wastewater contained high concentrations of various toxic pollutants, the pre-denitrification process showed stable and successful performances in both nitrification and denitrification reactions.     

Enzymatic detoxification of cyanide: clues from Pseudomonas aeruginosa Rhodanese

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.     

Degradation of ferrous(II) cyanide complex ions by Pseudomonas fluorescens

Degradation of ferrous(II) cyanide complex (ferrocyanide) ions by free cells of P. fluorescens in the presence of glucose and dissolved oxygen was investigated as a function of initial pH, initial ferrocyanide and glucose concentrations and aeration rate in a batch fermenter. The microorganism used the ferrocyanide ions as the sole source of nitrogen. The ferrocyanide biodegradation rate was 30.7 mg g⁻¹ h⁻¹ under the conditions of initial pH: 5, stirring rate: 150 rpm, aeration rate: 0.15 vvm, initial ferrous(II) cyanide complex ion and glucose concentrations: 100 mg l⁻¹ and 0.465 g l⁻¹, respectively. The culture utilized glucose as the main substrate following the non-competitive toxic component inhibition model in the presence of 100 mg l⁻¹ initial ferrous(II) cyanide complex ion concentration. The inhibition of ferrous(II) cyanide complex ions as a secondary substrate began at very low concentrations. A mathematical model, based on non-competitive substrate inhibition was used to describe the inhibitory effect of ferrous(II) cyanide complex ions on the growth of microorganism and the best fitted model parameters were determined by non-linear regression techniques.     

Biodegradation of toxic and environmental pollutants.

Organic chemicals that are toxic to humans and to the environment can be transformed and metabolized by a variety of microorganisms. Such chemicals include trichloroethylene, chloroform, carbon tetrachloride, toluene, phenols, chlorinated phenols, polychlorinated biphenyls and polyaromatic hydrocarbons. This review focuses on some of the most important recent developments in the biodegradation of these toxic chemicals. Depending on the compound and the organism, the extent of our understanding ranges from the molecular level to the conceptual.