Chapter I INTRODUCTION and REVIEW of LITERATURE

Chapter I

INTRODUCTION AND REVIEW OF LITERATURE Chapter I INTRODUCTION AND REVIEW OF LITERATURE

1.1 Cyanides, thiocyanates and nitrites

Cyanide is a compound, which played a principle role in the evolution of life on Earth (Oro and Araujo, 1981). It is not only a notorious poison but also an indispensable industrial chemical. Cyanide is a singly-charged anion containing unimolar amounts of carbon and nitrogen atoms triply-bonded (-C=N) together. It is a strong ligand, capable of complexing at low concentrations with virtually any heavy metal. The same complexing capability makes it useful in different industries such as metal extraction (particularly gold), metal finishing, manufacturing of herbicides and pesticides, paint industries, production of organic chemicals such as nitrile, nylon, synthetic rubber and acrylic plastics. (Akcil et al, 2003).

Considering the toxicity there are four major categories of cyanide compounds viz. free cyanides, strong acid dissociable cyanides (SAD), weak acid dissociable cyanides (WAD)'and cyanide related compounds. Table 1.1 enlists cyanides, thiocyanates and organic cyanides based on these categories. Free cyanides are highly volatile and most toxic forms of cyanides and include hydrogen cyanide (HCN) and CN' ions. WAD cyanides refer to cyanide complexes with metals such as cadmium, copper, nickel and zinc. SAD cyanides are cyanide- complexes with metals such as cobalt, gold, iron and silver. Cyanide related compounds include inorganic as well as organic forms of cyanides. The inorganic forms of cyanide include different thiocyanates (SCN"). Thiocyanate although a WAD cyanide, is often considered as a separate category. The organic cyanides include different aliphatic and aromatic nitriles. Nitriles are cyanide-substituted, carboxylic acids of the general structure R-CN. These different forms of cyanides are present in the environment as natural or industrial products. These cyanides are converted from one form to other depending on the environmental conditions. Table 1.1 Major categories of cyanides based on toxicity

Toxicity to Types of Example Groups humans cyanides Highly toxic Free cyanide HCN, CN- Cadmium cyanide, Weak acid copper cyanide, nickel Toxic dissociable cyanide and zinc cyanides (WAD) cyanide Cyanides

Strong acid Cobalt cyanide, gold Toxic dissociable cyanide, iron cyanide cyanides (SAD) and silver cyanide

Potassium thiocyanate, sodium thiocyanate, Thiocyanates Less toxic ammonium (SCN-) Cyanide related thiocyanate, mercuric compounds thiocyanate

Aliphatic Nitriles Organic Less toxic Aromatic nitriles cyanides

1.2 Uses of cyanides, thiocyanates and nitriles

Although a deadly poison, cyanide is one of the most indispensable industrial chemicals. Due to the highly complexing nature of cyanide, it has been used to extract precious metals from crushed rock for more than 100 years. Modern recovery methods that utilize cyanide in water-based solution can recover nearly 100% of the contained precious metals, making it profitable for mining companies to process low-grade ores. Because of properties similar to cyanide, sodium and potassium thiocyanate as well as nitrile are also widely used in metal leaching. In the electroplating industry, alkali cyanides and thiocyanates are used to a great extent. Noble metals such as silver and gold can be satisfactorily deposited from solutions containing cyanides. Thiocyanate is used as the electrolyte in the production of black nickel. In cases where the highest plating quality is required, cyanide pretreatment and degreasing baths are in use even today. Both cyanide and thiocyanates play an important role in hardening of steel. Cyanides and nitriles are used in various organic syntheses (acetophenone, alpha-naphthalene acetic acid, thiamine, acetamidine). The introduction of a CN- group into an organic substance is the first step in developing a wide variety of well-known materials that find application in pharmaceuticals as well as polymer chemistry. For example acetonitrile and malononitrile are used as the starting material for the synthesis of organic compounds such as acetophenone, alpha- naphthalene acetic acid, thiamine and acetamide. Nitriles are also used in the synthesis of polyacrylonitrile plastics. In laboratories, acetonitrile is widely used in high-performance liquid chromatographic (HPLC) analysis and as a solvent for DNA synthesis and peptide sequencing.

Other important uses of thiocyanates and nitriles are in the synthesis of different pesticides and herbicides. Ammonium thiocyanate and copper thiocyanate are used as pesticides whereas sodium thiocyanate is a raw material in the synthesis of number of pesticides and fungicides, for example methylene bis thiocyanate. Copper thiocyanate is used in anti-foulant paints for application to ships and boats for the control of aquatic organisms. Bromoxil is a contact herbicide containing benzonitrile. Nitriles and thiocyanates are also used in the synthesis of acrylic fibers and different pharmaceutical products such as vitamin Bi2 (Beekhuis, 1975).

1.3 Sources of environmental exposure to cyanides, thiocyanates and nitriles

Cyanides, because of the industrial uses, are widely present in the environment. Apart from its anthropogenic origin, cyanides are also produced naturally.

1.3.1 Natural origin

Cyanide and chemically related compounds are formed, excreted and degraded in nature by hundreds of species of bacteria, algae, fungi, plants and insects (Knowles, 1976). 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. 3 At least 1,000 species of plants from 90 families have been shown to contain one or more cyanide compounds (Seigler, 1976). 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 (Eyjolfsson, 1970). Cyanogenic glycosides such as linamarin and amygdalin, present in several plants, can be converted to cyanide under certain conditions (Legras ef a/, 1990). Cyanide poisoning of livestock by forage sorghums and other cyanogenic plants is well documented (Mudder, 1997).

Vegetables in the family Brassicaceae contain high levels of thiocyanate with concentrations ranging upto 660 pg/g, whereas other commonly consumed vegetables (e.g., spinach, radish, celery) generally contain thiocyanates at concentrations <2 pg/g. The reported thiocyanate concentrations in milk and dairy products as well as in meat are <1.0-9.0 and 0.5-0.7 pg/g, respectively (Thurkow e^ a/, 1982). In crushed plant tissues, cellular glucosinolates (thioglucosides) are hydrolyzed by glucosidase to produce thiocyanate (Wood, 1975). Glucosinolates are widely distributed in plant families such as Cruciferae (Kelly ef a/, 1993).

Nitrile compounds are numerous and fairly widespread in the natural environment mainly in the form of cyanogenic glycosides (Legras et al, 1990). Toxicity of different plants, such as cassava, white clover bamboo, peach etc. is due to the presence of different cyanogenic glycosides.

Cyanide is one of the virulence factors in fungi and bacteria. For example, Pseudomonas aeruginosa PA01 kills Caenoiiiabditis elegans by cyanide poisoning (Blumer and Haas, 2000, Larry and Colin, 2001). In the pathogenic fungus, Basidiomycete W2, cyanide is the main factor involved in etiology (Ward etal, 1971, Lebeau etal, 1959).

In addition to plants and microorganisms, insects have been shown to produce cyanide and cyanogenic glycosides. Species of centipedes, millipedes, beetles. moths and butterflies synthesize and excrete cyanide for defensive purposes (Duffey, 1981).

1.3.2 Anthropogenic origin

Cyanides, thiocyanates and nitriles are produced industrially in large amounts for use in metal, pesticide, herbicide, organic chemical and polymer industries. The major sources of cyanide in water are discharges from some metal mining processes, organic chemical industries, iron and steel works, and electroplating industries. The release of different cyanides in the environment by these industries is estimated to be >14million kg/year (Agency for toxic substance and disease registry (ATSDR): Toxicological Profile for Cyanide). The concentration of total cyanide released varies from industry to industry. Apart from water streams, soil surrounding gas work sites are also contaminated with cyanide and thiocyanate ions (Meehan, 1999). The iron-complexed cyanides are often found as a Prussian blue (ferri ferrocyanide Fe(lll)4(Fe(ll)(CN)6)3) coloration in the soil (Kjeldsen, 1999). Other cyanide sources include vehicle exhaust, exhaust releases from certain chemical industries, municipal waste burning and use of cyanide-containing pesticides. Hydrogen cyanide is also contained in vehicle exhaust and in tobacco smoke. The smoke of burning plastics contains hydrogen cyanide so also house fires often result in cyanide poisonings.

Another important source of cyanide in the water bodies is cyanide fishing, which was first practiced in Philippines and has spread to other places including Indonesia, Cambodia, the Maldives, Thailand and Vietnam (Barber and Pratt, 1997). Cyanides are used to capture live fish near coral reefs for the aquarium and seafood market. In this method, a diver uses a large, needle-less syringe to squirt a cyanide solution into areas where the fish are hiding, stunning them so that they can be easily gathered (Rubec, 1988). Since the 1960s, it is estimated that over 1 million kg of cyanide has been used in Philippine reefs (Mak et al, 2005). The high concentration of cyanide is extremely dangerous since it can kill both targeted fish as well as non-targeted organisms such as coral and invertebrates along with their eggs and thus destroy coral reef habitats (Johannes and Riepen, 1995). Thiocyanates are present in water primarily because of discharges from coal processing as well as gold and silver mining industries (Kelly and Baker, 1990; Laemmli, 1970; Wood, 1975; Hung and Pavlostathis, 1997). Thiocyanate is a by-product in the gold mines, where the cyanide used for the cyanidation reacts with the free sulfur to form thiocyanate (McGill et al, 1984). Thiocyanate concentrations in coal plant wastewaters (Tuan and Jensen, 1993) and mining wastewaters (Boucabeille et al, 1994b) have been found to be 100-1,500 and 300-450 mg/L, respectively. In some industries the cyanide in the effluents is converted to thiocyanate by the addition of sulfur at high temperature (Marsden and House, 1992). Furthermore, thiocyanate is used for manufacturing some insecticides and herbicides, and for chemical synthesis, which increases its release to the environment (Sorokin et al, 2004).

Nitriles arise into the environment as toxic industrial by-products as well as pesticides (Sorokin et al, 2004). Industries concerned with the production and use of cyanide and nitrile such as paint and polymer manufacturing industry, chemical and pharmaceutical industry, produce wastes and waste waters with a high content of cyanide and nitriles. Although acetonitrile has a variety of industrial uses as a solvent or starting material of various organic syntheses, it is generally released into the environment by direct discharges to air or by leaching into underground sites. It may be released from the thermal combustion of polyurethane foams or from municipal waste treatment plant discharges or spills.

1.4 Discharge of effluents containing cyanides, thiocyanates and nitriles

Due to its unique nature, cyanides, thiocyanates and nitriles are largely used in different industries. It has been found that the substitution of these compounds is very difficult on practical scale and often leads to cost escalation. Therefore, the use of cyanides in the industries might increase in the years to come. Under these conditions possible measures for the discharge of cyanides, thiocyanates and nitriles in the environment attain paramount importance. Table1.2 Permissible limits prescribed by IS! Permissible limits for discharge of cyanide and Waste discharged into Parameter Inland surface Public sewers, metal containing effluents waters, mg/L mg/L prescribed by the Indian Total cyanide 0.2 2.0 Thiocyanate NA NA Standard Institution are Nitriles NA NA given in Table 1.2. Copper 3.0 3.0 Nickel 3.0 2.0 Thiocyanate is not Zinc 5.0 15.0 regulated or monitored in Silver NA NA Gold NA NA most of the industries Iron 0.1 0.1 and no regulation is Lead 0.1 1.0 mandatory for nitriles. Cadmium 1.0 NA Chromium 0.1 2.0 Considering the toxic NA - Not Available effect and environmental impact of these compounds they have to be detoxified before releasing into the environment. Even though different environmental regulations are in force in countries where cyanide is primarily used in mining, cyanide will remain an indispensable chemical in these processes until there is a commercial, economical and environmentally safe alternative. Therefore, there is a need for developing effective methods to treat cyanides, thiocyanates and nitriles.

1.5 Toxicity of cyanides, thiocyanates and nitriles

Cyanide is notorious for its poisonous characteristics, affecting all classes of living cells (Fuller, 1984). All forms of cyanides including thiocyanates and nitriles are toxic to a number of microorganisms inhibiting growth, respiration and cellular metabolism (Knowles, 1976; Collins and Knowles, 1983). However, its toxicity is critically dependent on speciation and quantity. Acute toxicity to life forms is generally restricted to the free hydrogen cyanides in the solution.

In humans cyanides are readily absorbed through nasal, oral and dermal routes of exposure. The central nervous system (CNS) is the primary target organ for cyanide toxicity. Neurotoxicity has been observed in humans and animals following ingestion and inhalation of cyanides. Cardiac and respiratory effects. possibly thiocyanate mediated, have also been reported. Short-term exposure to high concentrations of cyanide elicits almost immediate collapse, respiratory arrest and death (Hartung, 1982). Symptoms resulting from occupational exposure to lower concentrations include breathing difficulties, nervousness, vertigo, headache, nausea, vomiting, pericardial pain and electrocardiogram abnormalities (Muir, 1977; Sandberg, 1967; Wuthrich, 1954). Thyroid toxicity has been observed in humans and animals following oral and nasal exposure to cyanides (Philbrick et al, 1979; EPA, 1984). In animal studies, cyanides have produced fetotoxicity and teratogenic effects, including exencephaly, encephalocele, and rib abnormalities (Doherty et al, 1982; Frakes et al, 1986; Tewe and Maner, 1981b; Willhite, 1982).

In humans and animals cyanide blocks the oxidative energy metabolism by binding with the heme iron in the oxygen-binding site of the mitochondrial oxidase. Thus cyanide inhibits the ATP production. Brain cells are highly susceptible to cyanide. When ingested simple cyanide readily get converted to free cyanide ion. The free cyanide ion can bind to hydrogen ion to form HCN and can be absorbed through gastrointestinal tract.

Thiocyanates are much less toxic than free cyanide, human fatal dosage ranges from 50-80 mg/kg body weight. Chronic effects are seen with a daily dose of approximately 2-12 mg/kg body weight. It is reported to be toxic to fish at concentrations between 90 and 200 mg/L (Ingles and Scott, 1987). Although thiocyanate is less toxic than cyanide compounds, chronic absorption of thiocyanate can cause dizziness, skin eruption, running nose, vomiting, and nausea. Thiocyanate is toxic to many higher organisms at relatively low concentrations (1-2 mM) because it has a strong tendency to bind to proteins and acts as a noncompetitive inhibitor (Wood, 1975). The toxic effects of thiocyanate include inhibition of halide transport to the thyroid gland, stomach, cornea and gills as well as the inhibition of a variety of enzymes. Thiocyanate- induced effects on the central nervous system of humans include irritability, nervousness, hallucination, psychosis, mania, delirium and convulsions (Lewis, 1992). Nitriles are readily absorbed in to the gastrointestinal tract, through the skin and the lungs. Nitriles induce toxic effects similar to those observed in acute cyanide poisoning, although the onset of symptoms is somewhat delayed compared to inorganic cyanides or other saturated nitriles. The oral LD50 in the rat varies from 1.7 to 8.5 g/kg. The levels nitriles causing toxicity in man are unknown but are probably in excess of 840 mg/m^ (500 ppm) in air. Symptoms and signs of acute acetonitrile intoxication include chest pain, tightness in the chest, nausea, emesis, tachycardia, hypotension, short and shallow respiration, headache, restlessness, semiconsciousness and seizures. Other non-specific symptoms may be due to the irritant effects of the compound. Acetonitrile can cause severe eye burns when exposed to the vapor.

Some microorganisms such as the green alga, Scenedesmus quadricauda are highly sensitive to cyanide and have toxicity thresholds as low as 0.16 mg/L (Becker and Thatcher, 1973). Acetonitrile has low toxicity to microorganisms (bacteria, cyanobacteria, green algae and protozoans) with thresholds at 500 mg/L or more.

1.6 Environmental impact of cyanides, thiocyanates and nitriles

Cyanides can be present in environmental matrices and waste streams as simple cyanides, metal cyanide complexes, thiocyanates and nitriles (Ebbs, 2004). Cyanide containing wastewaters are characterized by very high toxicity and low degradability. Cyanides cause severe environmental problems when produced in high amounts by anthropogenic activities, such as mining and electroplating. There are several reports on the loss of wild life due to cyanide discharge in the environment. An event showing the potential risk of cyanide occurred on 30 January 2000, when almost 100,000 m^ of wastewater with a high concentration of cyanide was discharged into the Tisza River, in Europe causing a massive poisoning of the ecosystem and killing large number of fishes and other organisms (Cunningham, 2005). Since the mid- 1980s, cyanide in heap leach solutions and mill tailing ponds at gold mines in Nevada, USA has killed a large number of wildlife (Henny et al, 1994). Again on the 21st of March 2000 in Papua New Guinea, while flying from the capital Port Moresby to the Tolukuma mine, a Dome helicopter accidentally dropped a crate containing one ton of sodium cyanide into the rainforest. In Mainland China, the State Environmental Protection Administration reported a release of cyanide into watersheds. For example, 11 tons of sodium cyanide solution leaked into a tributary of the Luohe River in the Henan Province in October 2001 after a traffic accident. Livestock were poisoned and at least one person became sick from this contamination. Earlier, after a truck accident in September 2000 in the Shaanxi Province, 5.2 tons of liquid sodium cyanide was spilled into the Wugan River, a tributary of the Hanjian River. Dead fish were found downstream from the accident site, while no human casualty was reported (Yu et al, 2004)

Over 200,000 residents of Bhopal, India, were exposed to a cloud of methyl isocyanate (MIC) that leaked from the Union Carbide plant in December 1984. The accident has now claimed more than 6,000 lives, and the number of survivors with chronic health effects has been estimated at upwards of 50,000 (Dhara, 2002). The MIC when mixed with large volume of water along with iron and sodium converted to cyanide by an exothermic reaction and became the reason of the accident (Dhara, 2002).

These accidents have caused significant environmental damage such as —--^

• Chronic poisoning in humans and animal species

• Cyanide and heavy metal contamination in catchment areas and loss of potable water

• Extinction of some fish species in the neighborhood of the contaminated rivers

• Death of water birds and carnivorous animals after consumption of contaminated fish

10 • Contamination of surface and groundwater resources of communities along rivers with heavy metals

• Socio-economic problems for communities along rivers due to temporary or long-term loss of livelihood.

Even though there is no such environmental impact data available for thiocyanate, Heming and Blumhagen (1989) reported about sudden death syndrome in trout due to thiocyanate accumulation. Lanno and Dixon (1994) reported that juvenile fathead minnows showed numerous negative effects after chronic (124 days) exposure to thiocyanate: thyroid tissue changes started as low as 1.1 mg/L; reproduction effects were noted at 7.3 mg/L and above; overt goiter was noted at as low as 7.3 mg/L concentration. Many of these effects are believed to be controlled by the antithyroid activity of thiocyanate.

Very little information is available on the ecological impact of organic nitriles and their derivatives. It is highly conceivable that the direct discharge of wastewater containing some of these nitrile compounds could cause severe health hazards, since most of them are highly toxic and some are mutagenic and carcinogenic. Moreover nitriles can be converted to cyanide by different microbial activities. Biodegradation of acrylonitrile by Bacillus subtilis produced increasing levels of cyanide in the media, which in turn initiated the autolysis of the bacteria (Reyes et al, 2000).

These reports show that since cyanides are highly toxic, they have to be handled carefully and have to be detoxified before releasing into the environment. Several major accidental incidences of release of cyanide affecting large population have received attention. Fewer small incidences are probably unnoticed. This indicates that such incidents of cyanide contamination may occur in the future. Since no chemical was explored to eliminate cyanide, different strategies have been developed to remove cyanide and related compounds from the wastewaters.

11 1.7 Treatment of waste waters containing cyanides, tliiocyanates and nitriles

Due to their toxic effects, cyanide, thiocyanate and nitrile containing effluents cannot be discharged without detoxification into the environment. The US Environmental Protection Agency has proposed a drinking water standard of 0.2 mg/L for cyanide. In order to minimize the exposure of public and aquatic ecosystems, the cyanides, thiocyanate and nitriles in the effluents require proper treatment. Conventionally cyanide containing effluents are treated by different chemical or physical methods.

1.7.1 Chemical treatment methods

1.7.1.1 Alkaline chlorination

Alkaline chlorination has been practiced ever since cyanide leaching of gold was commercially developed in 1889 and, consequently, has been the most commonly applied technique for cyanide destruction (Dobson, 1947, Staunton et al. 1988, Ingles and Scott, 1981) Alkaline chlorination removes toxic WAD and free cyanide species completely, quickly and economically.

CN~+Cl2(g) -^ CNCI(aq) + cr

CNCI(aq) + 20H~ ^ 0CN~+H2 0 + Cr

Chlorine gas also reacts with thiocyanate and metal-complexed cyanides to produce cyanate.

SCN~+4CI(g)+10OH~ -^ OCN + SOf + 8Cr+5H2O

M(CN)x^-"+XCl2(g) + (2x+y)OH" -> xOCN~+2xCr+M(OH) y+xHjO

When in excess, chlorine gas can also react with cyanate to produce nitrogen and carbon dioxide.

20CN~+ 3CI2 (g) + 40H~ ^ N, (g) + 2CO2 (g) + 6Cr+ 2H2 O

12 In such a case, chlorine and hydroxide consumptions become excessive, only increasing the difficulty in maintaining alkalinity above pH 10 to avoid the volatilization of hydrocyanic and chlorocyanic gases.

1.7.1.2 Hypochlorite treatment

Calcium, sodium and magnesium hypochlorites are used in presence of chlorine gas. The reactions are similar to that of chlorine gas. The hypochlorite reacts with cyanide to produce chlorocyanogen, which in turn hydrolyses to form cyanante. Cyanate is also formed by the reaction of hypochlorite with thiocyanate and other WAD. SAD is inert with hypochlorite. Excess of hypochlorite can also react with cyanate to produce nitrogen and carbon dioxide.

1.7.1.3 Ozonation

Ozonation for cyanide destruction has been examined extensively because it is a superior to oxygen as an oxidant (Garrison et al, 1975, Rowley and Otto, 1980, Novak and Sukes, 1981) Ozone reacts with cyanide to produce cyanate. Continued addition of ozone will convert cyanate to carbonate and nitrogen gas

CN~+03(aq) -> 0CN~+02(aq)

20CN"+303(aq) + H2 0 -> 2HCO3-+N2(g) + SOjCaq)

Furthermore, ozonation is highly destructive towards thiocyanate and WADs but is not destructive towards SADs. This method is used against nitriles also. Acetonitrile can be decomposed at 35°C by ozonation.

1.7.1.4 tiydrogen peroxide treatment

Hydrogen peroxide is an oxidant stronger than oxygen but weaker than ozone. It is often considered better because it is relatively cheaper, water soluble, and easy to handle and store. Hydrogen peroxide reacts with cyanide to produce cyanate and, when added in excess, nitrite and carbonate and, eventually, nitrate. The hydrogen peroxide method is successful at oxidizing most WADs but

13 has little or no effect on thiocyanate and SADs. Even so, it has been incorporated into numerous mining operations in Canada and the U.S. as a primary cyanide destruction process as well as a stand-by process for emergency situations.

CN~ +H2O2 -^ OCN'+HJO

OCN~+3H2 02 ^ NO' +COf+ 2H2O + 2H"

NOj" +H2O2 -^ NO," +H,0

1.7.1.5 Acidification/volatilization

In this method cyanides are volatilized to toxic HCN gas at low pH below 2 by the addition of sulfuric acid. This is an age old method and can be used to recover cyanide. Acidification is very effective against WAD. Thiocyanate and SADs also release hydrocyanic acid but the pH has to be reduced to below 2. After acidification the solution is predominantly free of cyanide but must be reneutralized before releasing into the environment. This reneutralization causes the metals, which were released, to be precipitated as hydroxide.

1.7.1.6 Sulfur addition

This method of cyanide destruction involves mixing sulfur dioxide and air with free and metal-complexed cyanide as well as thiocyanate to yield cyanate.

CN~+S02(g) + H20 + O2(g) -> OCN~+H2S04(aq)

SCN~+4 S02(g) + 5H2 0 +402(g) -^ OCN~+5H2S04(aq)

However, SADs do not react. Since the reactions generate sulfuric acid and are most efficient near pH 9, lime is added for pH control. This generates sludge due to metal hydroxide and gypsum precipitation, which, can be difficult to clarify. This process has been incorporated into numerous mining operations in Canada and the U.S. since the technology was commissioned in 1983.

14 1.7.1.7 Polysulfide

Solid polysulfide reacts with cyanide to form thiocyanate. This reaction is very slow at room temperature and therefore needs to be carried out near boiling temperature. This method, although practiced has several disadvantages such as production of thiocyanate and inability to degrade SAD cyanides.

1.7.2 Physical treatment methods

1.7.2.1 Dilution

Dilution is the only treatment method, which does not separate or destroy cyanide (Young and Jordan, 1995). This method involves combining a toxic cyanide waste with an effluent that is low in or free of cyanide to yield a wastewater below discharge limits. Consequently, dilution is simple and cheap and is often used as a stand-alone or back-up method to ensure that discharge limits are satisfied. Dilution is usually considered unacceptable since the total amount of cyanide discharge is not altered and since naturally occurring processes such as adsorption and precipitation can attenuate and thereby concentrate the cyanide in ground and surface waters.

1.7.2.2 Membranes

Free and metal complexed cyanide can be separated from water using membranes with either electrodialysis or reverse osmosis (Hinden and Bennet, 1979, Young and Jordan). In electrodialysis, a potential is applied across two electrodes separated by a membrane permeable to cyanide. The cyanide solution requiring purification is placed in the half-cell containing the cathode or negative electrode. Cyanide is negatively charged, hence diffuses through the membrane and gets concentrated in the half-cell containing the anode or positive electrode. In reverse osmosis, pressure is applied to a cyanide solution needing treatment but, in this case, water is forced through a membrane impermeable to cyanide.

15 1.7.2.3 Electro winning

Electro winning is a method in whicli complex cyanides are converted to free cyanide by applying a potential between two electrodes immersed in a solution containing metal cyanides. This method cannot be used for thiocyanate. Even though the recovery is less this method is used for recovery of metals from metal cyanides, predominantly in gold processing.

1.7.2.4 Hydrolysis/Distillation

Free cyanides naturally hydrolyze in water to produce aqueous hydrogen cyanide. The aqueous hydrogen cyanide can then volatilize as hydrocyanic gas. Because hydrocyanic gas has a vapor pressure of 100 kPa at 26°C, which is above that of water (34 kPa at 26°C), and a boiling point of 79°C, which is below that of water (100°C), cyanide separation can be enhanced at elevated temperatures and/or reduced pressures (Longe and DeVries, 1988). Increasing the agitation rate, the air/solution ratio, and the surface area at the air/solution interface, can also increase distillation rates. Hydrocyanic gas can be captured and concentrated for recycling in conventional absorption- scrubbing towers. It can also be vented to the open atmosphere and has been noted to occur naturally in tailings ponds, especially in warm and arid environments (Grosse, 1991). In such cases, it is paramount that environmental regulations be satisfied. Thiocyanate, WADs and SADs are not affected by this method.

1.7.3 Disadvantages of chemical and physical methods of treatment.

A large number of processes for removal of cyanides, thiocyanate and nitriles from wastewaters have been discussed so far. However, many of these methods need strict process control, such as adjusting the pH, temperature etc. Most of these methods are relatively expensive because of high operational costs. Moreover, metal-cyanides cannot be removed completely from the wastewaters by chemical and physical methods. Most commonly used methods such as alkaline chlorination and hypochlorite treatment produce large amount of sludge.

16 Therefore, the effluents generated need further treatment. The disadvantages of physical and chemical treatments can be summarized as follows:

1. Detoxification or removal of cyanides may not be complete 2. The chemical or physical methods are expensive 3. There is sludge formation during most chemical treatments 4. Most of the chemical and physical methods need strict process control 5. All the methods require further treatment of effluents

These disadvantages have led many researchers to look for alternative methods for removal of cyanide and related compounds. Biological methods are an important alternative to chemical and physical methods. They are not only "green technology" but also cost effective.

1.7.4 Biological degradation of cyanides, thiocyanate and nitriles

Various species of bacteria, fungi, algae, yeasts and plants, along with their associated enzymes and amino acids, are known to oxidize cyanides and cyanide related compounds such as thiocyanate and nitriles. Due to the diverse enzyme mechanisms different biological agents can utilize cyanide and related compounds as source of nitrogen or carbon or both. Following are some examples of cyanide utilizing organisms.

1.7.4.1 Plants

Vascular plants possess the enzymes |3-cyanoalanine synthase and |3- cyanoalanine hydrolase that convert cyanides into the amino acid asparagine and thus detoxify it (Gastric et al, 1972). Because of these properties, vascular plants such as Chinese elder and snow pine tree are already being used to treat cyanide-polluted soil at a former gas workstation in Denmark (Trapp and Christiansen, 2003). In a study, using detached leaves of 28 species of 28 families of Chinese vegetation, most of the plant leaves were found degrading potassium cyanide (Yu, 2004). The fastest cyanide removal was by Chinese elder, Sambucus chinensis, with a removal capacity of 8.8 mg CN/kg/h. In 17 another study Larsen et al (2004) showed that the leaves of a woody plant, basket willow hybrids {Salix viminalis x Salix schwerinii) could degrade potassium cyanide at a rate of 9.3 nng CNVkg fresh weight/h and proposed that the these trees can be used for phytoremediation in gold mines. Aquatic plants were also considered for cyanide removal by several researchers. Water hyacinth {Eichomia crassipes) was able to remove 3-300 mg of cyanide in batch level (Granto, 1995).

1.7.4.2 Microorganisms

Microorganisms exhibit a wide range of metabolic functions and are capable of degrading a wide range of chemicals. Microorganisms utilize a combination of extracellular and intracellular, inducible as well as constitutive enzymes in the degradation of cyano-compounds. Various organic and inorganic cyano- compounds are broken down and assimilated for the purposes of energy production and cell synthesis by the microorganisms (Akcil, 2003).

For over three decades many researchers have investigated bacterial degradation, of cyanides, thiocyanates and nitriles. Various microorganisms are reported for cyanide degradation. Table 1.3 enlists different microorganisms capable of degrading cyanide compounds. Ware and Painter (1955) first reported bacterial degradation of cyanide. They isolated an Actinomycete sp. from sewage treating percolating filters, which could grow in the presence of 1.5 mM of cyanide. Several microorganisms were reported for degradation of different cyano-compounds in heterotrophic as well as chemolithotrophic conditions (Nawaz, 1989; Katayama et al, 1993; Akcil et al, 2003; Harris and Knowles, 1983).

18 Table 1.3 Microorganisms degrading different cyanides

Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Gold nnine CN nitriles pH 6.5 Finnegan, Acinetobacter sp. effluent (Nitrogen) 30°C etal, 1991 Sewage Ware and KCN (Nitrogen Actinomycetes sp. treatment 28°C Painter, and carbon) plant 1995 Sfiivaraman Alcaligenes and NA NaCN (NA) NA faecalis Parfiad, 1985 Garcia et al, Alcaligenes sp. NA NaCN (NA) Anaerobic 1995 Alcaligenes xylosoxidans Ingvorsen, Soil CN- (NA) NA subsp. 1991 Denitrificans Tropical Azotobacter and Nickel cyanide pH 7.0, Kao et al, vinelandii temperate (Nitrogen) 30°C 2005 region NaCN and Meyers et Bacillus pumilus NA KCN (Nitrogen Neutral al, 1991 and carbon) Garden Soil Bacillus Cereus percolated KCN pH 7.0- 8.0 Kisfiore, Var Mycoides witfi (Nitrogen) 30°C 1999 cyanide Fargo clay Skowronski KCN pH 7.5 Bacillus pumilus planted in and Strobel, (Nitrogen) 40°C flax 1969 Gastric and Bacillus Fargo clay KCN (Carbon) Strobel, megaterium soil 35°C 1969 NaCN Bacillus Atkinson, NA (Nitrogen and Neutral stearothemophilus 1975 carbon) Burkholdeha Adjei, and NA CN- (Nitrogen) Alkaline cepacia Ohta, 2000 Cryptococcus Activated Nickel cyanide pH 7.5, Hyouk, humicolus sludge (Nitrogen) 25°C 2002

Continued.

19 Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Copper Gold mine cyanide, iron pH 7.0, Figueira et Escherichia coli solution cyanide and SOX a/, 1995 zinc cyanide Nickel cyanide Fusarium Yanase, NA (Nitrogen) NA oxysporum 2000 Contaminat pH 9.2-10.7 Dumestre, Fusarium solani KGNNA ed soil 30°C 1997 Ezzi and Fusarium sp. NA CM- (Nitrogen) pH6.5 30°C Lynch, 2005 Nickel cyanide Spent oxide and iron PH 4 and Barclay, Fusarium solani mixed with cyanide 1998 b soil 7.5 25°C (Nitrogen) Fry and Gloeocerocospora CN (Nitrogen) NA Munch, sorghi 1975 Industrial KCN Kao et al, Klebsiella oxytoca 7.5 30°C waste water (Nitrogen) 2003 Cyanide KCN and Liu et al, Klebsiella oxytoca Contaminat nickel cyanide SOX 1992 ed water (Nitrogen) Hope and Klebsiella KCN NA NA Knowles, planticola (Nitrogen) 1991 KCN Kang and Klebsiella sp. NA NA (Nitrogen) Kim,1993 Nickel cyanide, Silva- Klebsiella sp. Creek water KCN SOX Avalos et (Nitrogen) al. 1990 KCN Kang and Morexella sp NA NA (Nitrogen) Kim,1993 Nickel cyanide Penicillium and iron Barclay et NA NA miczynski cyanide a/, 1998 a (Nitrogen) Phanerochaete KCN (Carbon Shah et al, NA 25-SOX chrysosporium and Nitrogen) 1991 Historical pH 10.5 Akcil et al, Pseudomonas sp. CN- (Nitrogen) slags SOX 2003

Continued.

20 Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Shivaraman Pseudomonas NaCN pH 7.5-7.9 and Soil acidovarans (Nitrogen) SOX Parhad, 1985 Harris and Pseudomonas Activated KCN 7.5 and Knowles, fluorescens sludge (Nitrogen) SOX 1983 Pseudomonas Silver cyanide Podol'skya NA NA fluorescens NA etal, 1994 CN-, metal Whitlock Pseudomonas cyanides and NA NA paucimobilis (Nitrogen and Mudder, carbon) 1984 Pseudomonas Almagro, et Sludge CN- (Nitrogen) Ph11.5 pseudoalcaligenes al, 2005 Pseudomonas Arps et al, NA NaCN NA 7.5 pseudoalcaligenes 1993 NaCN and Pseudomonas Babu e^ al, NA metal cyanides NA putida 1993 (Nitrogen) Cyanide Pseudomonas pH 7.0 Babu et al, contaminat CN- (Nitrogen) putida 1996 ed soil S5X Nickel cyanide Scytalidium and iron Barclay et NA NA thenvophilium cyanide al, 1998 (Nitrogen) KCN Fry and Stemphylium loti NA 21-25X (Nitrogen) Millar, 1972 Nazly and KCN Stemphylium loti NA NA Knowles, (Nitrogen) 1981 Ezzi and Trichoderma KCN NA 6.5 Lynch, harzianum (Fungi) (Nitrogen) 2005 Nickel cyanide Trichoderma and iron Barclay et NA NA polysoporum cyanide al, 1998 a (Nitrogen) Ezzi and Trichoderma sp. PH 6.5 NA CN- (Nitrogen) Lynch, SOX 2005

Continued.

21 Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Boucabeill Acinetobacter sp. Sludge SCN (Energy) 28°C e et al, 1994 Acremonium Activated Hyouk. et SCN- (Energy) pH 6.5 25°C strictum sludge al, 2002 Boucabeill Arthrobacter sp NA SCN- (Energy) NA e et al, 1994 SCN- Betts, Arthrobacter sp. NA NA (Nitrogen) 1979 Paruchuri, Bacillus sp. NA SCN (Energy) NA efa/, 1990 Hung and Methanogeni Escherichia sp. NA SCN- (Energy) Pavlostathi 0 s, 1998 SCN- Pereira et Fusarium sp NA Acidic (Nitrogen) al, 1996 Soil sample SCN- (Carbon Lee et al, Klebsiella sp. around gold pH 7.0 38°C and nitrogen) 2003 mine SCN- Ahn et al, Klebsiella sp. Gold mine pH 7.0 38°C (Nitrogen) 2004 Soil from the root Methylobacten'um SCN- pH 7.7 30 Wood et balls of A. thiocyanatum (Nitrogen) al, 1975 aflatunenen -37°C se Boucabeill Pseudomonas Sludge SCN (Energy) 28°C e et al, diminuta 1994 Paracoccus Katayama NA SCN (Energy) pH 7.0-8.0 thiocyanates etal, 1995 Paruchuri, Pseudomonas NA SCN (Energy) Neutral et al, 1990. Pseudomonas SCN-, CN- Babu, et NA pH7.5 putida (Nitrogen) al, 1996 Pseudomonas SCN-, CN- Karavaiko NA pH9.0 putida (Nitrogen) et al, 2000 Pseudomonas SCN-, CN- pH 9.0 -9.2 Karavaiko NA stutzeri (Nitrogen) 20-22°C et al, 2000

Continued.

22 Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Stafford SCN- Pseudomonas Activated and (Nitrogen and pH 7.0 38°C stutzeri sludge Callely, sulfur) 1969 Thialkalivibrio Hypersaline pH 10.0 Sorokin et thiocyanodenitrifica soda lake SCN- (Energy) 38°C al, 2004 ns (PH 10) Thioalkalivibrio pH 8.0-8.5 Sorokin et Soda lakes SCN- (Energy) paradoxus 28°C al, 2001 Thioalkalivibrio pH 8.5 Sorokin et NA SCN- (Energy) thiocyanoxidans autotrophic al, 2002 Thiobacillus Katayama, NA SCN- (Energy) pH 8.5 thioparus etal, 1993 Thiobacillus Activated Katayama, SCN- (Energy) pH 7.0 30°C thioparus sludge etal, 1993 Finnegan, Gold mine Nitriles Acinetobactersp. pH6.5 30°C et al effluent (Nitrogen) 1991 Malononitrile Glutaronitrile Succinonitrile Chloroacetonitr Gavagan, Acidovorax facilis Soil pH7.2 ile 1999 Acetonitrile Benzonitrile (Nitrogen) Acrylonitrile Narayana Arthrobacter sp (Nitrogen and NA samy, et carbon) a/, 1991 Acetonitile Pereira, Bacillus sp. Soil pH7.5 (Nitrogen) 1998 Soil exposed at Acrylonitrile Takashim Bacillus smithii high pH 7.5 55°C (Nitrogen) a, 1998 temperature (40-80°C) acrylonitrile, butyronitrile, isobutyronitrile, Gold mine methacrylnitrile Linardi et Candida famata NA effluent , propionitrile, al, 1996 succinonitrile, valeronitrile, (Nitrogen)

Continued.

23 Degradation Isolated Microorganism of (Utilized as Conditions Reference from source of) Candida Gold mine Acetonitrile Dias et al, pH7.5 guilliermondii effluent (Nitrogen) 2000

Resin manufacturi Acrylonitrile Comamonas Wang, ng (Nitrogen and NA testosterone 2004 wastewater carbon) treatment system

Acetonitrile Rezende Cryptococcus sp. NA Propionitrile NA etal, 1999 (Nitrogen)

Acrylonitrile Klebsiella Nawaz et NA (Nitrogen and pH 8.0 SOX pneumoniae al, 1991 carbon) DiGeronim Acetonitrile Nocardia Aerobic 0 and NA Propionitrile rhodochrous neutral Antoine, (Nitrogen) 1976 Pseudomonas Acetonotrile Nawaz, Soil NA aeruginosa (Nitrogen) 1991

Pseudomonas Acrylonitrile Ignatov, NA pH 7.1 30°C pseudoalcaligenes (Nitrogen) 1996

Acrylonitrile Ignatov, Brevibacterium sp. NA pH 7.5 SOX (Nitrogen) 1996 Contaminat Acetonitrile Pseudomonas ed soil from Nawaz, (Carbon and pH 7.5 25°C putida industrial 1989 Nitrogen) sites NA - Not available

These bacteria used cyanide compounds as the source of nitrogen or carbon or both. Some of these bacteria even utilize them as the source of energy. There are very few reports on biodegradation of cyanide and thiocyanate under alkaline conditions. One of them is fungus Fusarium solani, which can utilize cyanides as nitrogen source at pH 9.2 (Dumestre, 1997). The bacterium Burf

24 ions, such as iron and copper (Adjei and Ohta, 2000). P. pseudoalcaligenes CECT5344 possesses a mechanism to assimilate cyanide at pH 9.5 (Almagro, 2005). Three kinds of bacteria able to utilize thiocyanate at pH 10 were isolated from alkaline soda lake sediments and soda soils by Sorokin et al, (2002). Two strains of bacteria that utilized thiocyanate as sole energy and nitrogen source from saline process water were reported by Stott etal, (1999).

Most of these microorganisms were isolated form contaminated soils of industrial sites or industrial wastewater (Nawaz, 1989, Wang, 2004, Dias et al, 2000, Linardi et al, 1996, Finnegan et al, 1991). There are reports on the isolation of efficient microorganisms from activated sludge (Katayama etal, 1993; Harris and Knowles, 1983; Hyouk, 2002; Ware and Painter, 1955). The advantages of such isolates are, they are already acclimatized and may possess the enzyme system for degrading cyano- compounds. Another approach is the isolation of cultures from sites containing cyanogenic glycosides. Several plants contain cyanogenic glycosides in their body. These cyanogenic glycosides can enter into the soil during decaying of the plant body. Skowronski and Strobel (1969) have isolated a Bacillus pumilus culture from the soil, which had been planted with flax for 73 continuous growing seasons. It was very well known that the flax is high in different cyanogenic glucosides such as linamarine and lotaustralin. Similarly Meyers et al (1991) had isolated bacterial cultures from soil removed from root balls of A. aflatunense. However, Patil and Paknikar (2000) by enrichment techniques isolated a bacterial consortium from garden soil, which was not contaminated by cyano-compounds. The bacterial consortium was very efficient in degrading metal cyanide complexes such as copper cyanide, nickel cyanide, zinc cyanide and silver cyanide. The report showed that cyanide-degrading bacteria could be isolated from uncontaminated soil also. This report gives an idea that bacteria from any habitat can be enriched for cyanide degradation.

1.7.4.3 Biodegradation pathways of cyanide, thiocyanate and nitriles

There are four general pathways for the biodegradation of cyano-compounds: hydrolytic, oxidative, reductive and substitution/ transfer.

25 The hydrolytic pathway can be of two types; one catalyzed by cyanide dihydratase, which produces ammonia and formate, the second one that produces formamide by cyanide hydratase. The cyanide hydratase pathway is exclusive for fungi (Fry and Millar, 1972), whereas the cyanide dihydratase pathway is found in both fungi and bacteria (Jandhyala, 2003). Cyanide hydratase and cyanidase have recently been shown to have similarity at both the amino acid and structural levels to nitrilase enzymes (O'Reilly and Turner, 2003). Nitrilase converts nitrile to corresponding acid. Nitrile-utilizing enzymes have been found in a wide variety of bacterial, fungal and plant species.

The oxidative reaction is the biodegradation of cyanide to ammonia and carbon dioxide. Cyanide monoxygenase converts cyanide to cyanate, with cyanase then catalyzing the bicarbonate-dependent conversion of cyanate to ammonia and carbon dioxide. Another enzyme reported in this type of conversion is cyanide dioxygenase, which converts cyanide to ammonia and carbon dioxide.

Thiocyanate is the end product of substitution/ transfer reaction of cyanide. The enzyme thiocyanate- cyanide sulfur transferase catalyzes the conversion of cyanide to thiocyanate in the presence of thiosulfate (Ebbs, 2004). Reductive reaction of cyanide is usually seen under anaerobic conditions and involves the action of nitrogenase enzyme.

Thiocyanate can act as both energy and nitrogen source or only as a nitrogen source for microorganisms. Based on the mode of growth mainly two pathways are reported for thiocyanate biodegradation, carbonyl pathway and cyanate pathway. Thiobacillus thioparus has been postulated for the conversion of thiocyanate to ammonia and carbon dioxide via cyanate. The liberated sulfide is utilized as electron donor and energy source. The enzyme catalyzing the first step is not yet identified. The end products of both pathways are carbon dioxide, sulfate and ammonia.

26 Hydrolytic reaction 1 Cyanide Hydratase Reductive reactions HCN+H,0 -•HCONH, HCN +2H" ^+ 2 , • CH, NH+ H, O Cyanide dihydratase HCN+2H3O •HCOOH+NH- Substitution/transfer Nitrile hydratase reactions R-CN+H,0 •R-HCONH, Thiosulfate cyanide sulfurtransferase Nitrile dihydratase — ^ CN +5,0,; ->SCN +S0;-

Thiosulfate nitrile sulfurtransferase Oxidative R-CN+S.O:- •R-SCN+SO:" Cyanide Monoxigenase HCN+0,+2H HOCN+H,0

Cyanide dioxygenase HCN + 0,+2H • CO.+NH

Thiocyanate biodegradation

Thiocyanate hydrolase (Carbonyl pathway) SCN +2H.0 • COS+NH, +0H-

Cyanase (Cyanate pathway) SCN +3H, 0+20, • CNO -•SOf+NHs + CO,

Fig.1.1 Biodegradation reactions of thiocyanates cyanides, and nitriles

In carbonyl pathway carbonyl sulfide is the first product of thiocyanate hydrolysis, which is then converted to sulfate and carbon dioxide (Katayama, 1993). The enzyme involved in this reaction, thiocyanate hydrolase, has substantial homology to nitrile hydrolase. Such homology is hardly surprising, assuming that both enzymes break triple bond between carbon and nitrogen. The overall reactions of biodegradation are given in the Figure 1.1.

Considering all the information, there is a possibility of a biogeochemical cycle of cyanide operating in nature. Microorganisms play an important role in this cycle.

27 Fig. 1.2 Possible biogeochemical cycle of cyanide in nature. R-CN' Nitrile, CN' Cyanide, SCN' Thiocyanate, CNO" Cyanate, OCS'Carbonyl sulfide, NH2O" Amide, HCOO" Carboxylic acid, S04^" Sulphate, NH3 Ammonia and CO2 Carbon dioxide

Different cyanogenic plants, fungi and bacteria produce cyanide. In plants the cyanides are released into the environment through the injuries. This cyanide is then degraded to carbon dioxide and nitrogen through different pathways as seen in the Figure 1.2. In microorganisms, the enzyme cyanide-sulfur- 28 transferase converts cyanide and nitrile to thiocyanate under autotrophic conditions in presence of pyritic minerals. The thiocyanate produced by the degradation of nitriles and cyanides can be converted into ammonia, carbon dioxide and sulfate directly or through cyanate as an intermediate. Some bacteria convert thiocyanate to ammonia and carbon dioxide, utilizing the sulfur compound as energy source. Under heterotrophic conditions the cyanides are converted to ammonia and carbon dioxide. Vascular plants convert cyanide to (3- cyanoalanine by an enzyme cyanoalanine synthase and incorporate it into the plant body.

The close observation of the cycle shows that thiocyanate is a key compound in cyanide biogeochemical cycle. In chemolithotrophic conditions, in presence of sulfur, degradation of cyanides and nitriles are directed towards thiocyanate. The thiocyanate formed can enter into heterotrophic as well as chemolithotrophic mode of degradation. In heterotrophic mode thiocyanate gets converted to cyanate followed by carbon dioxide and ammonia. Thus degradation of thiocyanate is interlinked with cyanides and nitriles in both directions, upstream and down stream. Cyanides and nitriles can be degraded by other pathway without thiocyanate as intermediate and get converted to carbon dioxide and ammonia. In addition, cyanides can be directly transformed to thiocyanate enzymatically (this is a method of cyanide detoxification). These observations indicate that there is a greater possibility for a thiocyanate degrader to degrade cyanide and nitriles.

1.7.4.4 Biological treatment plants

Several industries use microbial treatment plants for removal of cyanide and related compounds. Various commercial applications have been developed utilizing both aerobic and anaerobic processes incorporated into full-scale active, passive and in-situ treatment facilities. Homestake Mining Company was the leading mining company in the development and implementation of biological treatment systems for cyanide destruction. The first application was at the Homestake Gold Mine in Lead, South Dakota in the United States. The full-scale facility has been in continuous operation treating high volumes of tailings pond 29 solution for nearly two decades. The aerobic attached growth fixed film biological facility consisted of five stages of forty-eight rotating biological contactors (RBCs) for the removal of thiocyanate, cyanide, ammonia, and metals (Whitlock and Mudder, 1998). This combined aerobic and anaerobic, suspended growth biological treatment process has effectively removed these constituents to environmentally acceptable levels and has been operating continuously over a period of 10 years.

1.8 Definition of the problem

There are several pharmaceutical, metal furnishing, paint, chemical and polymer industries located in India. These industries release high concentration of cyanides, thiocyanates and nitriles into the environment. These wastes are treated by conventional physical-chemical methods. However, with the increasingly stringent environmental standards imposed by the statutory agencies, it is realized that these methods may not be adequate. To meet the higher standards put forth by the regulatory agencies; industries often have" to use biological treatment as a polishing step. Given the inherent high costs of physical-chemical treatments, requirement of strict process control and generation of solid wastes (sludge) that need safe disposal, etc. industries are considering biological treatment as a cost-effective, 'green' technological alternative.

Although there are several advantages of using biological (in particular, microbial) methods, there could be some impediments that are specific to the waste being treated. For example, thiocyanate and cyanide bearing wastewaters are characterized by high alkalinity, presence of different heavy metals, high salinity and combinations of different cyano-compounds. A concoction of these compounds may prove inhibitory to the growth of even potent cyanide degrading microorganisms. Since it is highly unlikely that a single microbial culture could have such a broad spectrum of degradation activity, there is a need to develop consortia of microbial cultures having such ability.

30 Considering the chemistry of cyano-compounds, microbial treatment of cyanide containing effluent should be carried out under alkaline pH to obviate the abiotic volatilization of hazardous cyanides. Moreover, the cultures used should be resistant to high concentration of heavy metals and cyanides. It is often seen that industries tend to recycle the wastewater after treatment, which leads to built up of salts in the effluents. Also, industries located in coastal and arid regions have to use saline or brackish water. Thus, cyanide degrading microbial cultures need to be active under saline conditions. Our literature search showed that reports on microbial processes for the treatment of a combination of cyanides, thiocyanate and nitriles under alkaline and saline conditions are practically non-existent.

Industries all over the world, view waste treatment as a 'cost centre' and are therefore reluctant to invest large funds for improved technologies, which adversely affects the task of environmental clean-up. This scenario can change drastically if the waste treatment becomes economically attractive. One such way could be recover products that can offset treatment costs. In our laboratory, it was shown during the previous studies (Patil, 1999) that the cost of treating metal-cyanide wastes can be brought down by recovering the metal component. As a sequel to these studies, it is felt that if precious metals such as gold and silver could be recovered as nano-sized particulates from wastes, there could be a tremendous value-addition. It is well known that gold and silver nanoparticles have uses in cutting-edge nanotechnologies encompassing all spheres of life.

In view of this background, investigations in the present work were undertaken with following aims and objectives:

• To isolate cultures capable of degrading thiocyanates under alkaline and saline conditions • To evaluate the ability of the isolates to degrade cyanides and nitriles under alkaline and saline conditions • To explore the possibility of obtaining value added products such as metal nanoparticles as a result of biodegradation of metal cyanides.