Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 1(7), 15-21, Oct. (2011) Res.J.Chem.Sci. International Science Congress Association 15 Biological Treatment of Cyanide Containing WastewaterDwivedi Naveen*1, Majumder C.B., Mondal P.3 and Dwivedi Shubha4 Uttarakhand Technical University, Dehradun-248007, U.K., INDIA 1,4Department of Biotechnology, S.D. College of Engineering and Technology, Muzaffarnagar-251001, UP, INDIA 2,3Department of Chemical Engineering, Indian Institute of Technology Roorkee, Roorkee-247667, UK, INDIA Available online at: www.isca.in (Received 23rd June 2011, revised 18th July, accepted 19th August 2011)Abstract Cyanide compounds are released in to the water stream from a number of industrial effluent and several routes including biodegradation for their removal from the waste water are under investigation. The present review describes the mechanism and advances in the use of biological treatment for the removal of cyanide compounds and its advantages over other treatment processes. It also includes various microbial pathways for their removal. The feasibility of the concomitant biogas generation during anaerobic biodegradation of cyanide has also been described. A comparative study has been done on the efficiency of aerobic and anaerobic biological degradation of cyanide. The limitations of this process have also been discussed. Key words:Biodegradation, cyanide, algae, simultaneous adsorption and biodegradation, waste water.Introduction“Cyanide is a triple-bonded molecule with a single negative charge consisting of one atom of carbon in the +2 oxidation state and one atom of nitrogen in the -3 oxidation state”. Immensity of cyanide amount in environment is mainly due to metal finishing and mining industries. Although cyanide can be removed and recovered by several processes, it is still usually discussed and examined due to its potential toxicity and environmental impact. The chemical speciation of cyanides depends on their sources and also in response to a variety of environmental factors. Different forms of cyanide include: free cyanide, cyanide ion, cyanide salt, metallocyanide complexes and synthetic organocyanides, also known as nitriles and total cyanide. Cyanide occurs in water as hydrocyanic acid (HCN), cyanide ion (CN), simple cyanides, metallocyanide complexes and as simple chain and complex ring compounds. Simple cyanides include water-soluble salts of alkaline earth, alkali and heavy metals. Typical simple cyanides are NaCN, KCN, Ca(CN), Hg(CN), Zn(CN), Cd(CN), Ni(CN) and AgCN. The simple cyanides such as potassium cyanide and sodium cyanide ionize in water to release a cation and cyanide ion. Metallocyanide complexes have a wide range of stabilities. Zinc [Zn(CN)-2], cadmium [Cd(CN)-2] and Cd(CN)-2] complexes dissociate rapidly and nearly completely in dilute solutions. Nitriles are organic compounds (R-CN) containing the cyanide group (CN). Cyanide bound to carbon as nitriles (other than as cyanogenic glycosides) is comparatively innocuous in the environment, low in chemical reactivity and is biodegradable. Cyanide and chemically related compounds are formed, excreted and degraded in nature by hundreds of species of bacteria, algae, fungi, plants and insects. As a result, low levels of cyanide can appear in naturally occurring surface or groundwater samples which normally would not be expected to contain it. At least 1,000 species of plants and micro-organisms from 90 families have been shown to contain one or more of nearly twenty compounds capable of producing cyanide. About 800 species of higher plants from 70 to 80 families, including agriculturally important species such as the cassava, flax, sorghum, alfalfa, bamboo, peach, pear, cherry, plum, corn, potato, cotton, almond and beans are cyanogenic. Cyanide is fast-acting broad spectrum toxin and it affects all living organisms. Cyanide ion exerts an inhibitory action on certain metabolic enzyme systems, most notably cytochrome oxidase, the enzyme involved in the ultimate transfer of electrons to molecular oxygen. In presence of even weak acids, HCN gas is liberated from cyanide salts. Cyanide poisoning can occur through inhalation, ingestion and skin or eye contact. One teaspoon of a 2% solution can kill a person. In general, fish and other aquatic life are killed by cyanide concentrations in the microgram per liter range (part per billion), whereas bird and mammal deaths result from cyanide concentrations in the milligram per liter range (part per million). The release of cyanide from industries worldwide has been estimated to be more than 14 million kg/yr. Industries concerned with the production and use of cyanide compounds generate wastes which contain large amounts of cyanide. Especially paint manufacture and use, polymer production, chemical and pharmaceutical industry; steel industry, mining operations and coal manufacturing all produce wastes and waste water with high cyanide content. Electroplating industrial wastes contain 0.5% to 20% cyanide. Complex cyanide, Prussian blue is used for dyeing Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(7), 1-21, Oct. (2011) Res.J.Chem.SciInternational Science Congress Association 16 jeans. Since, cyanide is a toxic compound well-known as a metabolic inhibitor, cyanide-containing effluents cannot be discharged without being subjected to treatment to reduce their cyanide contents to very low levels (�0.1 mg of CN per liter). US-health service cites 0.01mg/L as guideline and 0.2 mg/L as permissible limit for cyanide in effluent. Indian standard has set a minimal national standard (MINAS) limit for cyanide in effluent as 0.2 mg/L. Material and MethodsCyanide and its related compounds can be removed from industrial effluent through various physical, chemical and biological/biodegradation treatment methods. Physical methods for cyanide treatment can be accomplished using dilution, adsorption, membranes, electrowinning and hydrolysis/distillation. Adsorption is commonly used in physical treatment methods for the removal of cyanide. In chemical methods, some of the more common processes used to degrade cyanide are the hydrogen peroxide process, alkaline chlorination, sulphur dioxide-air oxidation, electrolytic oxidation etc. These methods are used to purify effluents for discharge by concentrating and recovering the cyanide for recycling. On the other hand, oxidation processes are used to destroy the cyanide and include various biological, catalytic, electrolytic, chemical and photolytic methods10. These aforesaid methods are really effective removal methods but have a few drawbacks over biodegradation method. These are much expensive, produce secondary pollution also and suitable for low concentration of cyanide, some time pretreatment is also required. Biological Degradation of Cyanide: Mechanism of biological degradation: Biological degradation is the process of breaking and transforming hazardous materials into simple nontoxic substances by a biological treatment. Cyanide can be broken into simpler substances by microorganisms in the wastewater, piles and in the soil. Some bacteria, fungus, algae and plants can decompose cyanide and cyanide compounds. The bacterial detoxification would be safer, quicker and cheaper than the chemical treatment to handle cyanide wastes. The process that demands oxygen is an aerobic biodegradation and the process that occurs in the absence of oxygen is an anaerobic biodegradation. Cyanide-oxidizing bacteria normally break it down into harmless compound. Under aerobic conditions, biological process may consume hydrogen cyanide and generate hydrogen cyanate as shown in the equation 1. The hydrogen cyanate is in turn hydrolyzed into ammonia and carbon dioxide as shown in equation 2. 2HCN + O2 + Enzyme 2HCNO (1) HCNO + HO NH + CO (2) Anaerobic biodegradation of cyanide and hydrogen cyanide is restricted to the moderately to strongly reduced portions of the heap environment and can only occur if HS2 or HS are present. The Sulphur species present depends on pH. At a pH value greater than 7, HS is the dominant species. At a lower pH, HS present predominantly. CN + H S HCNS + H2 (3) HCN + HS HCNS + H2 (4) The HCNS then hydrolyze, to form NH, HS and CO. Anaerobic biodegradation is much slower than aerobic biodegradation of cyanide, and anaerobic bacteria have a cyanide toxicity threshold of only 2 mg/l compared to 200 mg/l for aerobic bacteria. Consequently, anaerobic biodegradation is a much less effective cyanide removal mechanism12. In the waste streams and environmental matrices, cyanide can be present as a form of simple cyanides (HCN, CN, NaCN), metal cyanide complexes, cyanates and nitriles. Degradation pathways are susceptible to the concentration and form of the cyanide compounds, the physicochemical conditions of the media and the presence of interfering and inhibitory compounds. The development of biodegradation strategies for these varied surroundings requires a comprehensive understanding of the biological pathways. Common biochemical pathways responsible for cyanide degradation:There are four common pathways for the biodegradation of cyanide. These are; hydrolytic, oxidative, reductive and substitution/transfer, described in figure 1. Various reviews have already explained these pathways and the organisms in which they are found11-12. More than one pathway can be utilized for cyanide biodegradation in some organisms13. Hydrolytic reactions are catalyzed by cyanide hydratase, forming formamide, or cyanidase, which produces formate and ammonia figure 1. Cyanide hydratase is mainly a fungal enzyme and is highly conserved between species14. Cyanidase (cyanide dihydratase) is mainly bacterial enzyme. Cyanide hydratase and cyanidase have recently been shown to have similarity at both the amino acid and structural levels to nitrilase and nitrile hydratase enzymes15. The oxidative reactions for the biodegradation of cyanide form ammonia and carbon dioxide figure 1. Cyanide monoxygenase converts cyanide to cyanate, with cyanase then catalyzing the bicarbonate-dependent conversion of cyanate to ammonia and carbon dioxide. A second oxidative pathway utilizes cyanide dioxygenase to form directly carbon dioxide and ammonia figure 1. The biodegradation of thiocyanate (cyanide compound) has also been studied figure 1. Thiocyanate is used in a variety of chemical processes and can be produced from the reaction of cyanide with pyretic materials in waste streams. Thiocyanate is produced in vivo by the action of thiosulfate-cyanide sulfurtransferase enzymes figure 1. Biodegradation of thiocyanate can take place by two pathways16-17. According to the first pathway, Cyanate is converted in to ammonia and carbon dioxide in the presence of high concentration of Cyanase17. In a second pathway, thiocyanate is converted to ammonia and carbonyl sulfide. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(7), 1-21, Oct. (2011) Res.J.Chem.SciInternational Science Congress Association 17 Hydrolytic Reactions Cyanide hydratase HCN + HO HCONH2 Cyanidase HCN + 2H 2 O HCOOH Reductive Reactions HCN + 2H+ + 2e CH=NH + HO CH=O CH2 = NH + 2H + 2e CH3 – NH + 2H+ + 2e– CH + NH Oxidative Reactions Cyanide monoxygenase HCN + O + H + NAD(P)H HOCN + NAD(P) + HO Cyanide dioxygenase HCN + O + 2H + NAD(P)H CO + NH3 + NAD(P) Substitution/Transfer Reaction Thiosulfate:cyanide sulfurtransferase CN + S 2 O 3 2- SCN + SO 3 2- Thiocyanate biodegradation Carbonyl pathway {Thiocyanate hydrolase} SCN + 2HO COS + NH + OH- Cyanate pathway {cyanase} SCN + 3HO + 2O2 CNO + HS HS- + 2OSO2- + H+ CNO + 3H + HCO NH 4 + 2CO 2 Figure 1 Chemical reactions for the biodegradation of cyanide and thiocyanate Microorganisms responsible for cyanide degradation: The microorganisms primarily responsible for degradation of cyanide include a diverse group of anaerobic and aerobic bacteria. These single-celled microorganisms exhibit a wide range of metabolic functions and are capable of degrading a wide range of chemical structures and concentrations. Bacteria utilize a combination of extracellular and intracellular adaptive and constitutive enzymes in the biochemical breakdown and assimilation of the organic and inorganic compounds present in liquid wastes. These compounds, along with various nutrients (i.e. nitrogen, phosphorous and trace metals) are degraded and/ or assimilated for the purposes of energy production and cell synthesis. The extracellular enzymes found on the cell wall or in the capsule layer surrounding the cell wall aid in the breakdown of larger and more complex molecules, converting these compounds to smaller, more soluble by-products. The breakdown products are then transported into the bacterial cell for conversion to cellular material and energy through the use of intracellular enzymes18. Whilst bacteria and fungi have often been identified as cyanide detoxifying microorganisms, cyanide detoxification by algae has been shown in only a few studies18. Several algal cultures can effectively degrade cyanide as carbon and/or nitrogen source for their growth. Cyanide effluent degradation by Scenedesmus obliquus has been examined19. Microbial detoxification of cyanide by algae species was significantly influenced by the various factors, including initial concentration of cyanide, initial cell density and time. There are few microbes such as Klebsiella oxytoca, a bacterial strain, which can utilize these compounds as a source of nitrogen and carbon for their own growth. Several microorganisms such as Pseudomonas fluorenscens, Escherichia coli, Klebsiella oxytoca, Fusarium solani, Stemphylium loti, R. oryzae etc. are responsible for cyanide degradation. The bacterium Pseudomonas fluorescens utilizes KCN under nitrogen-limiting conditions in fed batch culture20-21. Metabolism of cyanides by strains of Escherichia coli, Acinetobacter, Alcaligenes, and Bacillus, Stemphilium loti has also been studied. Many other bacteria including P. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(7), 1-21, Oct. (2011) Res.J.Chem.SciInternational Science Congress Association 18 putida, Pseudomonas paucimobilis and P. fluorescens utilize moderately strong metal complexed cyanides at neutral pH22. Some microorganisms are listed to having cyanide removal capability from contaminated water shown in table 1. Table 1 Microorganisms having cyanide removal capabilitiesBacteria Enrichment Source Pseudomonas putida Sewage sludge, Soil, Creek water Pseudomonas pickettii Soil Pseudomonas paucimobilis Mining waste water Klebsiella pneumoniae Soil, Creek water Klebsiella sp. Creek water Alcaligenes xylosoxidans Soil Fungi Fusarium solani Soil (contaminated) F. oxysporum - F. lateritum - Rhizopus oryzae - Cryptococcus humicolus (Yeast) Cock-plant waste water Algae Arthrospira maxima- Scenedesmus obliquus Lake Chlorella sp seawater ‘-’ not reported Simultaneous adsorption and biodegradation (SAB) process: The application of simultaneous adsorption and biodegradation processes in the same reactor is known to be effective in the removal of biodegradable contaminants in various kinds of wastewater. The use of simultaneous adsorption and biodegradation is effective in treatment of organic toxic pollutants, present in industrial wastewaters. Recently simultaneous adsorption and biodegradation process has been found to be much effective over the individual processes by using microbes Rhizopus oryzae and Stemphylium loti with granular activated carbon (GAC) as adsorbent. Maximum removal efficiency of cyanide achieved by biodegradation alone as 83% by Rhizopus oryzae while 90% by Stemphylium loti with the initial cyanide concentration of 150 mg/L at initial pH of 5.6 and 7.2 respectively as shown figure 2. In the SAB process the efficiency of cyanide removal of Rhizopus oryzae and Stemphylium loti were 95.3% and 98.6% respectively as shown figure 3. It has been observed that SAB process is suitable methodology for the removal of toxic material with higher efficiency and effectively. The results are encouraged with SAB; therefore, it can be better treatment option when compared to adsorption or biodegradation alone. The removal percentage and specific uptake have been studied in the both cases of adsorption and simultaneous adsorption-biodegradation (SAB) and have been compared. Figure 2 Cyanide Removal efficiency through biodegradation by R. oryzae and S. loti Figure 3 Cyanide removal efficiency through SAB process by R. oryzae and S. loti immobilized on granular activated carbon (GAC) Effects of Various Physio-Chemical Parameters on Cyanide Removal Efficiency: Aerobic and anaerobic biological processes have been successfully employed in the treatment of a wide variety of industrial and hazardous liquid wastes. The optimized physical and chemical parameters like temperature, pH, flow rate, pressure, hydraulic retention time and agitation are very essential parameters for cyanide degradation from contaminated water. If any alteration is made on these parameters, the cyanide removal efficiency of microorganism can be directly affected. Table 2 shows that the efficiency of aerobic treatment of cyanide removal is much better than the anaerobic treatment method. The yeast strain Cryptococcus humicolus, which is isolated from coke- plant waste water, has been reported to have better cyanide removal efficiency in aerobic treatment, (approximately 100%) than in anaerobic treatment method removed 90%. The pH has also played a leading role in cyanide removal from waste water. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(7), 1-21, Oct. (2011) Res.J.Chem.SciInternational Science Congress Association 19 Table-2 Various physio-chemical parameters of aerobic and anaerobic biodegradation with their CN removal efficiency New Biotechnological Approach Using Algae for the Detoxification of Cyanide: The detoxification of cyanide by algae was examined by exposing cultured suspensions of Arthrospira maxima, Chlorella sp. and Scenedesmus obliquus in growth media to varying concentrations in shorttime batch tests. In each experiment, the pH was adjusted to 10.3. The effect of pH, initial concentration of algal cells, temperature and cyanide concentration on microbial detoxification were examined. Under the experimental conditions, initial microbial detoxification rates of 50 and 100 mg/L free cyanide were observed for 25 h18. A. maximadid not survive due to its sensitivity to the higher cyanide concentrations in the solutions. S. obliquus removed the cyanide to a greater extent than did Chlorella sp. S. obliquusdetoxified 99% of the cyanide, while Chlorella sp. removed about 86% in the same time period. For the increase in cyanide concentrations between 100 and 400 mg/L, S. obliquus was the only microorganism tested for 67 h. 18. It has been observed that Scenedesmus obliquus was competent algal organism in trio. It removed 99% of the cyanide from cyanide containing solution. The use of algae reported in this work offers a process for removing cyanide without pH adjustment at minimal cost as only trace amounts of nutrients are required to be added. Algae can be easily obtained and cultivated and require less nutrients than bacteria. Thus, the process has the potential of becoming an economical and an eco-friendly alternative to the conventional chemical processes.Results and DiscussionBiodegradation of cyanides has been studied with a number of microorganisms, including bacteria and fungi. Most of the microorganisms have been investigated in aerobic conditions to apply for conventional activated sludge processes. On the contrary, anaerobic treatment of cyanides has not been intensively studied except for the cases of some methanogenic mixed cultures. Anaerobic wastewater treatment could be more advantageous, the biodegradation of cyanide under anaerobic conditions has also recently demonstrated the feasibility for concomitant biogas generation, a possible economic benefit of the process due to its low energy requirement compared with aerobic treatment. Algae can be simply obtained and cultivated; it requires fewer nutrients than other organisms. Thus, the process has the potential of becoming an economical and an eco-friendly alternative to the conventional chemical processes. Simultaneous adsorption and biodegradation process is also Reference Treatment Methods / Conditions Microorganism pH Temperature (cº) HRT (hrs) Rpm Reactor /column and diameter Adsorbent material Initial conc. Of cyanide (mg/lit) Efficiency (%) R. D. Fallon et al. 1991 Anaerobic (methanogenesis) Mixed 7.5 35-40 48 - Up flow, anaerobic fixed bed Activated charcoal .02;ä•100 .02;ä•70 Alain Dumestre et al. 1997 Aerobic (alkaline) Fusarium solani 9.2-10.7 30 72 120 Rotatory shaker - �100 �80 R. Roshan Dash et al. 2006 Aerobic ( SAB) Rhizopus oryzae 5.6 25 120 140 Rotatory incubator shaker Granular activated carbon 150 83 R. Roshan Dash et al. 2006 Aerobic ((SAB) Stemphylium loti 7.2 25 120 140 Rotatory incubator shaker Granular activated carbon 150 90 C.J. Gantzer et al. 1990 Nitrogen fixing anabaena High - - - Batch reactor - 3 25 Hyouk Kee Kwona et al. Aerobic Cryptococcus humicolus7.5. - 24 - fixed-bed biofilm reactors - 50 mg CN dm-3100 Anaerobic 5.0 90 Hyouk Kee Kwona et al. Aerobic Cryptococcus humicolus 7.5. - 9 - fixed-bed biofilm reactors - 50 mg CN dm-383 61 Anaerobic 5.0 Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(7), 1-21, Oct. (2011) Res.J.Chem.SciInternational Science Congress Association 20 effective in treatment of organic toxic pollutants over individual process. Due to the delayed growth of microbes in the culture medium adsorption occurred in the first phase. It was observed that the removal efficiency of SAB process is much efficient to remove cyanide as compared to biodegradation alone. A new yeast strain, Cryptococcus humicolus, showed facultative property on TCN (tetracyanonickelate (II)) degradation, which is able to grow in both aerobic and anaerobic conditions. This facultative ability can be beneficial for practical applications since oxygen limitation in aerobic operation or the presence of oxygen in anaerobic operation is not detrimental to cell activity. ConclusionIt has been concluded that, biological treatment method is an ecosociable substitutional approach for removal of cyanide. 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