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Microbial remediation of cyanides

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4  cha p t e r

Microbial Remediation of Cyanides

Tek Chand Bhalla*, Vijay Kumar and Virender Kumar
Department of Biotechnology, Himachal Pradesh University,
Summer Hill, Shimla–5, Himachal Pradesh, India

1.  Introduction
Cyanides are widely present in biotic and abiotic components of the ecosystem and
act as defense molecule in various life forms. Cyanide compounds are being used
in gold extraction, electroplating industries, synthesis of agrochemicals, etc. Rapid
industrialization has resulted in enormous increase in the amount of cyanides in
environment. According to International Cyanide Management Institute, 1.1 million
metric tons of cyanide is produced annually worldwide. Although cyanides have a
range of applications, yet these are highly toxic and need to be degraded into nontoxic
or less toxic forms. However, chemical and physical treatments detoxify cyanide at a
rapid rate, their applications are limited due to environmental variations, high cost
and operational hazards. Thus biological method of cyanide removal or precisely
bioremediation of cyanide is more attractive. A number of microorganisms (fungi,
bacteria and algae) have been reported which use cyanide as sole source of nitrogen
or carbon and degrade cyanide to amide or acid. In this chapter, sources, applications
and potential of microorganisms for bioremediation of cyanide contaminated land and
wastewater is discussed.

2.  Cyanide
Cyanides are organic molecules that contain a cyano (–C∫N) group, i.e., a triple-bonded
carbon nitrogen functional group. Inorganic cyanides are generally salts of the anion
CN−. Most of the cyanide compounds are solids and few are in gaseous or liquid form.
The toxicity and reactivity of these cyanide species is dependent on their form, which

* Corresponding Author E-mail: bhallatc@rediffmail.com

 Microbial Remediation of Cyanides  89

determines their environmental fate and transport (Luque and Almagro, 2011; Kumar
et al., 2013). Cyanide compounds or complexes that can release the cyanide ion (CN −)
are highly toxic in nature. Hydrogen cyanide (HCN), cyanogen chloride (CNCl), and
cyanogen bromide (CNBr) are the three gaseous forms of cyanide. All these gases are
highly toxic to human and animals if they are inhaled or absorbed dermally. These
gases are highly soluble in water, but can be easily hydrolyzed to form cyanate, which
subsequently degraded to ammonia (NH3) and carbon dioxide (CO2) at alkaline pH
conditions.
In IUPAC nomenclature, organic compounds that have a (–C∫N) functional group
are called as nitriles, e.g., acetonitrile (CH3CN) which is also known as methyl cyanide.
Nitriles usually do not release cyanide ions. It is the carbon-nitrogen chemical unit
which combines with many organic and inorganic compounds. Cyanide containing
waste is a potent health hazard and is a worldwide problem. It comes from both natural
and manufactured sources, and occurs as both inorganic (HCN) and organic cyanide or
nitriles (RCN). It inhibits some of the key biochemical reaction of life, i.e., the reduction
of oxygen in the cytochrome respiratory chain, electron transport chain and the activity
of enzymes like catalase and oxidase (McMahon et al., 1995). Hydrogen cyanide and
metal cyanides are potent poisons. Common symptoms of cyanide poisoning in humans
include gastric problems, vomiting, respiratory distress, convulsions, and coma. The
lethal single dose of cyanide for vertebrates has been reported to be between the range
of 35–150 μmol kg−1 body weight, although much higher amounts of HCN can be
tolerated if consumed over a long period (Zagrobelny et al., 2008). Due to high toxicity
of cyanide as strong inhibitor of cytochrome oxidase, it is inevitable to degrade cyanide
in industrial effluents or remediate contaminated soil and water to reduce its level to
permissible limit of 0.2 mg L−1 in effluents (Kumar et al., 2013; Kumar et al., 2014).
An organism can metabolize cyanide if it possesses a biodegradable pathway to
convert cyanide into an assimilable product (NH4+), cyanide resistance mechanism
and a system for taking up Fe3+ from the medium (siderophores). Although some
organisms synthesize cyanide, yet comparatively a larger number of microbes are
capable of cyanide biodegradation. The existence of pathways in these organisms has
made it possible to develop biotechnological methods to degrade cyanide compounds
in industrial waste streams (Ebbs, 2004). These degradation pathways are sensitive to
the form and concentration of the cyanide compound, the physicochemical conditions
of the media, and the presence of interfering and inhibitory compounds. Some of the
common cyanide compounds and their properties are mentioned in Table 4.1.
90  Bioremediation Current Research and Application

Table 4.1 Classification of cyanide compounds (Raybuck, 1992)

Types of Cyanide Examples Properties

Hydrogen cyanide (HCN), cyanide Equilibrium depends on pH,
Free
ion (CN ­–)­ (pKa, 9.2 at 25°C)
Sodium cyanide (NaCN), Ionizes completely in aqueous
Soluble
Potassium cyanide (KCN) solution at low temperature
Simple and mostly present as HCN
Zinc cyanide (Zn(CN)2), Silver
Insoluble below pH 8
cyanide (AgCN)
Dipotassium tetracyano zincate (II) Easily ionisable
Weak
(K2Zn(CN)4)
Complex Moderately Dipotassium tricyano Moderately stable
strong cuprate K2Cu(CN)3
Strong Potassium ferrocyanide (K2Fe(CN)6) Highly stable
Thiocyanate (SCN–), Cyanate unstable
Inorganic
Cyanate (CNO–)
Acetonitrile, Acylonitrile, Stable
Aliphatic
Organic Adiponitrile, Propionitrile
Aromatic Benzonitrile

3.  Sources of cyanide

Cyanide is believed to be the first molecule formed during evolution. The cyanide
compounds are widely distributed and are produced naturally by many organisms
including bacteria, fungi, plants and some insects (Kumar et al., 2013). These are
synthesized by plants for defense purposes against pests (Moller, 2010), secreted by
fungi and bacteria as an antimicrobial compound (Frapolli et al., 2012), and synthesized
by insects as a control over their mating behaviour (Baxter and Cummings, 2006). Small
amount of cyanides are present in certain seeds, e.g., those of apple, mango, peach
and bitter almonds (ASTDR, 2006). Some agriculturally important plants like almond,
alfalfa, bamboo, cassava, cotton, corn, cherry, flex, peach, plum, potato and sorghum
are well known for their cyanogenic nature (Kumar et al., 2014). Principal natural
sources of cyanides are from over 2,000 plant species, including fruits and vegetables
that contain cyanogenic glycosides which can release cyanide on hydrolysis when
ingested. Many organisms have pathways to naturally generate cyanides and plants
are major source of cyanide compounds because they use it as defense against pests
and pathogens. When tissue injury occurs, cyanoglycosides are hydrolyzed to a sugar,
HCN and a keto or aldehyde compound (Luque-Almagro et al., 2011). Cyanate and its
derivatives have also been widely used as herbicides and precursors in the synthesis of
polymers. Protein carbamoylation by cyanate, especially in the eye and kidney, causes
 Microbial Remediation of Cyanides  91

severe health problems in mammals (Kraus et al., 2001). In addition, cyanate may chelate
metal centres in some enzymes, such as carbonic anhydrase, superoxide dismutase, and
carboxypeptidase A (Gulloton and Karst, 1987). It is present in environmental matrices
and waste streams as simple cyanides (e.g. HCN, CN−, NaCN), metal cyanide complexes,
cyanates and nitriles (Ebbs, 2004). Soil is the most abundant natural system and proven
to be an acceptable waste receptacle and will always play an important role in waste
disposal despite trends toward recycling of waste constituents. Interactions between
soil and agricultural wastes are a complex set of relationships that are dependent on
the soil environment, microbial populations, the chemical and physical properties of
the soil and wastes materials (Ubalua, 2007). Generally waste waters containing toxic
pollutants entering the environment as a result of anthropogenic activities are usually
biodegraded to less toxic forms (Ezeronye and Ubalua, 2005). Cyanide is highly toxic
for most living organisms because it forms very stable complexes with transition metals
that are essential for protein function, i.e., iron in cytochrome oxidase.
Agricultural practices also contribute to increase the levels of cyanide in the
environment because of the use of nitrile herbicides, such as dichlobenil and ioxynil
which are used to control pests of some crops, such as rice, wheat, barley, corn, etc.
Bromoxynil and chlorothalonil are the other examples of nitrile herbicides. These are
used to control diseases of broad leaf crops. However, natural quantities of cyanogenic
compounds could not be compared to those produced by industrial processes. In
addition, these compounds are extensively being used in mining, electroplating, steel
manufacturing, polymer synthesis, pharmaceutical production, dyes and agricultural
production (Dash et al., 2009; Kumar et al., 2014). Hydrogen cyanide is produced by
the combustion or pyrolysis of certain materials under oxygen-deficient conditions, e.g.,
it can be detected in the exhaust and tobacco smoke. Certain plastics, especially those
derived from acrylonitrile, release hydrogen cyanide when heated or burnt. Although
cyanide is ubiquitous in the environment, the environmental levels are found in the
vicinity of combustion sources, i.e., automobile exhaust, fires, cigarette smoke and solid
waste incineration, in waste waters from water treatment facilities, iron and steel plants,
and organic chemicals industries, in landfills and associated ground water (ASTDR 2006).
Cyanide has been used for the extraction of gold from ore for almost a century,
since it forms tight complexes with heavy metals, such as gold, and dissolves them,
allowing for the leaching of gold from ore. As gold forms a strong complex with cyanide,
this allows the usage of relatively dilute sodium cyanide solutions for gold extraction
(Ripley et al., 1996). The effluents so generated however, are difficult to treat because of
the cyanide forming complexes with other metals. Cyanide form very stable complexes
with iron, gold, and silver which are resistant to treatment. Various sources of cyanide
have been listed in Table 4.2.
92  Bioremediation Current Research and Application

Table 4.2 Natural and anthropogenic sources of cyanide

Natural Sources Anthropogenic Sources

Cyanogenic algae Electroplating industry
Chlorella vulgaris Mineral processing
Plectonema boryanum Processing of cyanogenic crops
Anacystis nidulans Pharmaceutical industry
Cyanogenic bacteria Paint-manufacturing industry
Pseudomonas aeruginosa Coal-coking plants
Pseudomonas fluorescens Coal-gasification plants
Chromobacterium violaceum Synthetic fibre producing plants
Pseudomonas aureofaciens Manufactured gas plants
Pseudomonas chlororaphis Photo-finishing industry
Cyanogenic plants Plastics industry
Manihot esculenta (cassava) Agricultural sector
Sorghum bicolor (Sorghum)
Linum usitatissimum (flax)
Alocasia macrorrhizos (Giant Taro)
Bambuseae (bamboo)

Soil as major repository of microorganisms, is probably the most complex and diverse
on the planet. Soil has different layers down from surface which may act as membrane
and can be a source or sink for most gases. A further source of complexity in biological
activity of soil is the existence of extracellular enzymes, presumably derived from past
populations of organisms, but stabilized by sorption on mineral surfaces and retaining at
least a part of their activity. Soil is also used for waste disposal, so its detoxification and
filtration roles are very important. A vast range of organic wastes released into the soil
including sewage sludge, composted municipal waste and effluents from bio industries,
such as the processing of oil palm and cassava which contain cyanide and cyanide
degrading microbes (Powlson et al., 2001). The applications of cyanide compounds in
various sectors are illustrated in Figure 4.1.
Hydrogen cyanide is ubiquitous in nature. The variation in concentrations of
cyanogenic glycosides is due to genetic and environmental factors, location, season,
and soil type. Known cyanogenic glycosides in plants include amygdalin, linamarin,
prunasin, dhurrin, lotaustralin and taxiphyllin. Transports of cyanide in soils are mostly
influenced by volatilization and distribution. Accordingly, high volatility of cyanide
and the action of soil microbes ensure that high levels of cyanide do not persist or
accumulate in soil under natural conditions (Fuller, 1984). Although cyanides may be
absorbed by several materials, including clays and biological solids (Chatwin, 1987), yet
existing data indicate that the rate of hydrogen and metal cyanide adsorption in soils is
not significant when compared with rates of volatilization and biodegradation (ATSDR,
 Microbial Remediation of Cyanides  93

2006). However, small amounts of cyanide in soil may be oxidized to cyanate (HCNO)
(Chatwin and Trepanowski, 1987).

GOLD
EXTRACTION
HCN, NaCN
MEDICINE
FISHING (Ferrocyanide &
(HCN, NaCN) ferricvanide)

AGRICULTURE SYNTHETIC FIBRES

(HCN, Ferrocyanide &
(HCN, Metal CYANIDE ferricyanide)
cyanide)

FOOD CHEMICAL INDUSTRY

(HCN, NaCN) (HCN, KCN & Thiocyanate)

Figure 4.1  Applications of cyanide compounds in various sectors

The industrial effluents contain between 0.01 and 10 mg L−1 of cyanide. However,
some cyanide wastes from individual operations at electroplating and metal finishing
plants can be stored for periods of years, after which the effluent may contain from
1 percent to 3 percent (10,000–30,000 mg L−1) of cyanide (Wild et al., 1994). In fact,
some industrial effluents from electroplating plants have been reported to contain even
higher cyanide levels of 100,000 mg L−1 (Wild et al., 1994). The cyanide concentrations
in effluents of these industries are found to be very high as compared to cyanide levels
(0.001–0.05 mg L−1) found in unpolluted stream and lake water (Wild et al., 1994). The
relationship between simple and complex cyanide species in industrial effluents is not
consistent. For example, the complex forms of cyanide found in effluents from the
chemical manufacturing plants can be either 0 percent or 100 percent of the total cyanide
content. Generally, the cyanides in industrial effluents are predominantly in complexed
forms. Thiocyanate (SCN) is one of the most important cyanide complexes present in
industrial effluents, being found in some coal coking and coal conversion effluents and
its level ranges from 17–1500 mg L−1. Thiocyanate concentrations from 11 to 50 mg
CN− L−1 have been detected in effluents emanating from chemical manufacturing plants
(Kelada, 1989). From the preceding data, it is apparent that many industrial wastewater
effluents contain elevated cyanide concentrations.

4.  Effluent disposal standards of cyanide

Natural sources responsible for production of cyanide in environment include plants,
bacteria and fungi that synthesize and secrete it; however, industrial and agricultural
wastes are the most significant sources of cyanide in environment (Ubalua, 2010).
Cyanide as HCN, KCN, NaCN is produced on the industrial scale for its use in the
94  Bioremediation Current Research and Application

metal extraction, electroplating, polymer, steel, carbonization, organic chemicals,

pharmaceutical and agricultural product industries. All these industries and mining
operations and coal manufacturing plants produce solid wastes and waste water with
high cyanide content. Most of the cyanide occurring in environment is attributed to
metal finishing and mining industries (Dash et al., 2009). Electroplating industrial
wastes contain 0.5 percent to 20 percent cyanide (2011). According to International
Cyanide Management Institute, 1.1 million metric tons of cyanide is produced annually
worldwide. The release of cyanide from industries worldwide has been estimated to be
more than 14 million kg yr−1 (Ebbs, 2004). Such kind of waste if not properly disposed
or detoxified, can cause health hazards for human and animals as well as poisoning to
the fish and other aquatic organisms present in water bodies. To protect the environment
and water bodies, effluents containing cyanide from various industries must be treated
before discharging into the environment. Thus, many countries and environmental
protection agencies have imposed limiting standards for discharge of cyanide bearing
wastewater. The US Environmental Protection Agency (USEPA) has proposed a limit of
200 and 50 ppb for drinking and aquatic-biota waters, respectively, where total cyanide
refers to free and metal-complexed cyanides (Dash et al., 2009). The German and Swiss
regulatory agencies have set limit of 0.01 mg L−1 for cyanide in surface water and 0.5
mg  L−1 for sewages. In Mexico, disposal limit for cyanide has been set as 0.2 mg L −1
(Parga et al., 2003). In India, Central Pollution Control Board (CPCB) has minimal
national standard (MINAS) limit for cyanide in effluent as 0.2 mg L−1 (Dash et al., 2009;
Kumar et al., 2014). In view of these considerations, cyanide recovery and/or removal
is necessary to contain the concentrations of cyanides below regulatory limits.

5.  Degradation pathways for cyanides

Microorganisms follow different biochemical pathways to understand and survive in
various stresses they come across. Sometimes, the same microbial species can employ
different strategic pathways for the degradation of toxic pollutants. Five different
pathways for the degradation of cyanide and cyano-group containing compounds have
been identified (Dash et al., 2009, Ebbs, 2004). The pathways and the enzymes involved
in the metabolism of cyanides are listed in Figure 4.2 and are discussed in proceeding
section.

5.1  Oxidative Pathway

The degradation of cyanide compounds via the oxidative pathway utilizing NADPH
as well as an additional carbon source leads to its conversion to ammonia and carbon
dioxide. There are two oxidative pathways involving two enzymes. The first pathway
converts cyanide to cyanate, with cyanase and then conversion of cyanate to ammonia
and carbon dioxide, which is bicarbonate dependent. Cyanases have been identified in
numerous microorganisms (bacteria, fungi), plants and animals (Dash et al., 2009, Ebbs,
2004). The other pathway involves direct conversion of cyanide to ammonia and carbon
dioxide catalysed by a cyanide dioxygenase. It has been proposed that this pathway is
dependent on the cofactor pterin and the formation of cyanohydrins as an important
step for cyanide degradation via the oxygenase-mediated pathways (Ebbs, 2004).
 Microbial Remediation of Cyanides  95

Pseudomonas putida utilizes the oxidative pathway for degradation of cyanide. This
microorganism is well suited to industrial applications as it is able to survive at high pH
up to 11.5 in 780 mg F-CN/L cyanide. P. putida has been able to degrade thiocyanate,
resulting in the formation of sulphite as well as sulphate, tetrathionate, trithionate and
thiosulphate (Dash et al., 2009; Gupta et al., 2010; Luque-Almagro et al., 2011).

5.2  Reductive Pathway

A very limited literature available on cyanide-degrading enzymes that utilize reductive
pathway. Klebsiella oxytoca has been reported to degrade cyanide via the reductive
pathway. In this pathway cyanide is ultimately converted to methane and ammonia
(Chen et al., 2008).

5.3  Hydrolytic Pathway

The hydrolytic mechanism for cyanide degradation is widely utilized by microorganisms.
It offers many advantages over other routes of cyanide detoxification. This route involves
direct cleavage of triple bond between the carbon and nitrogen atom, destroying the
cyano moiety. To date, there are five enzymes that utilize the hydrolytic pathway
to degrade free cyanide and nitriles. Cyanidase, cyanide hydratase, nitrilase, nitrile
hydratase and thiocyanate hydrolase have been identified as the main enzymes in
the direct conversion of cyanide and nitriles to carbon dioxide and ammonia, with
formamide being an intermediate in the reaction (Bhalla et al., 2012; Gupta et al., 2010).
Cyanide hydratase is mainly of fungal origin and cyanidase is mainly of bacterial origin.
These two enzymes are highly specific to cyanide as a substrate. Nitrilase and nitrile
hydratase are mainly involved in the degradation of nitriles. These two enzymes are
specific to their substrate, but their specificity is lower than that of cyanidase and cyanide
hydratase. Nitrilase and nitrile hydratase are responsible for the conversion of aliphatic
and aromatic nitriles to carboxylic acid and formamide, with the formamide being
converted to ammonia through the action of an amidase enzyme. The biodegradation
of thiocyanate is mainly catalyzed by thiocyanate hydrolase to form sulphate, ammonia
and carbon dioxide (Gupta et al., 2010).

5.4  Substitution or Transfer Pathway

The first of the assimilative pathways is the transfer pathway. In this pathway, a transfer
of sulphur species from a source (normally thiosulfate) is donated to the cyanide ion,
thereby forming thiocyanate. This pathway involves cyanide assimilation with subsequent
microbial growth due to the provision of an extra nitrogen source. The enzymes that
catalyze the transfer of the sulphur species belong to sulfurtransferase family. Two
enzymes are of particular interest in this pathway: rhodanase and mercaptopyruvate
sulfurtransferase (Dash et al., 2009; Gupta et al., 2010; Bhalla et al., 2012).
Rhodanases are highly conserved and are currently regarded as one of the
mechanisms involved for cyanide degradation in various species. The activity of the
enzyme is regulated by phosphate ions and divalent ions that interact with the active site
96  Bioremediation Current Research and Application

of the enzyme (Gupta et al., 2010). The rhodanases are able to function at their optimum
temperatures between 35 to 55°C and at a pH range of 8.5 to 11.5.
The mercaptopyruvate sulfurtranferase enzyme catalyses a two-step reaction, where-
by an enzyme-sulphur intermediate is formed in the first reaction. The intermediate fur-
ther reacts with free cyanide and the enzyme transfers the sulphur moiety to the cyanide
ion, thus forming thiocyanate as an end product (Gupta et al., 2010). The thiocyanate that
is formed in this pathway can be further degraded by thiocyanate hydratase.

Cyanide Degradation

Oxidative pathway Synthesis pathway

HCN Æ CO2 + NH3

Reductive pathway Substitution pathway
 Cyanoalanine synthase
 Cyanase  g-cyano-a-aminobutyric
 Cyanide dioxygenase HCN Æ CH3 + NH3  Rhodanase acid synthase
∑ Bicarbonate and  Mercaptopyruvate
sulfurtransferase
NADPH dependent

Hydrolytic pathway
HCN Æ CO2 + NH3
HCN Æ HCOOH + NH3
HCN Æ HCOOH2
 Cyanidase
 Cyanide hydratase (Formamide as
intermediate)
 Nitrilase
 Nitrile hydratase (Amide as product)
 Thiocyanate hydrolase

Figure 4.2  Various pathways and the enzymes involved in cyanide degradation
O
O
H Amidase
e H NH3
as +
drat NH2
hy OH
nide
C ya O Formamide Formic acid Ammonia
H2
HC∫N
Cyanide C 2H
ya 2O
nid
ed
ihy
dra
ta O
se
H + NH3
OH
Formic acid Ammonia

Figure 4.3  Mechanism of cyanide hydrolysis by cyanide hydratase (CHT) and cyanide dihydratase
(CynD). Cyanide hydratase produces formamide, requiring an amidase for the subsequent hydrolysis
to formic acid and ammonia. Cyanide dihydratase hydrolyses cyanide to formic acid and ammonia.
 Microbial Remediation of Cyanides  97

5.5  Synthesis Pathway

The synthesis pathway is an assimilative pathway and normally results in the synthesis
of amino acids, such as b-cyanoalanine and g-cyano-a-aminobutyric acid by using
amino acid precursors which react with cyanide. The b-cyanoalanine and g-cyano-a-
aminobutyric acid are structurally homologous to each other. The synthesis of amino
acids from cyanide is catalysed by two enzymes, mainly cyanoalanine synthase and
g-cyano-a-aminobutyric acid synthase. The cyanoalanine synthase belongs to the lyase
enzyme family and catalyses the reaction between free cyanide and L-cysteine to form
b-cyanoalanine and hydrogen sulphide as products. During this process, there is neither
direct requirement for oxygen or NADPH, nor carbon dioxide is produced (Dash et al.,
2009; Gupta et al., 2010). The g -cyano-a-aminobutyric acid synthase requires phosphate
to function and it is induced by glutamate or glycine. Once the g-cyano-a-aminobutyric
acid is synthesised, it is slowly converted to glutamate, an amino acid.

6.  Treatment processes for cyanide waste

There are various methods for detoxification of cyanide. Non-biological and biological
methods for the treatment of cyanide contaminated soil and water are well documented
(Dash et al., 2009). Initially chemical and physical treatments were more popular, but
presently biological treatments are gaining much attention due to various advantages
over non-biological processes.

6.1  Natural Degradation of Cyanide

Cyanide compounds undergo numerous natural degradation reactions. Adsorption,
biodegradation, oxidation, volatilization and photodecomposition are common types
of reactions that cause detoxification of cyanides. Different mechanisms of natural
cyanide degradation occur which are dependent on number of variables, such as pH,
water chemistry, dissolved oxygen concentration and temperature. The volatilization of
cyanide from the wastewater is mainly dependent on the pH of the solution (Luque-
Almagro et al., 2011).
However, majority of the cyanides are degraded through natural decomposition,
but the metal-complexed cyanides are resistant to degradation due to the instability.
Ultraviolet and visible radiations cause the dissociation of metallic cyanides, especially
in the presence of increased dissolved oxygen. However, wastewater containing cyanide
and metal cyanides is often found in a stagnant form, limiting exposure of metal cyanides
to sunlight, thus limiting their biodegradation. Therefore, it is realized that chemical,
physical, and, biological degradation strategies have to be employed in combination to
reduce these limitations.

6.2  Chemical Treatment

The majority of processes used for remediation of cyanide convert it into one or less
toxic forms through an oxidation reaction. Sulphur dioxide/air method is a common
98  Bioremediation Current Research and Application

process developed by ‘The International Nickel Company (INCO)’ more than two
decades ago. This process uses SO2 or a derivative along with air in the presence of
a soluble copper catalyst and it causes oxidation of cyanide to the less toxic cyanate.
The SO2/air and the hydrogen peroxide processes, which are both catalyzed by copper,
are the most successful of the non-biological processes available so far. The hydrogen
peroxide treatment process is similar to the SO2/air process, but hydrogen peroxide
is used in place of SO2 and air. The hydrogen peroxide process is primarily used for
solutions, whereas the SO2/air process can be used in both the treatments of slurry and
solutions (Mudder et al., 2004). Various chemical methods which are in place for cyanide
treatment are listed in Table 4.3.

Table 4.3 Methods for chemical remediation of cyanide

Removal of
Removal Methods Process Description
Free Cyanide Thiocyanate
Alkaline chlorination Cyanide containing waste is Yes Yes
initially treated with chlorine or
hypochlorite to produce, cyanogen
chloride which is then hydrolyzed
to form much less toxic cyanate
at pH > 11. Further chlorination
oxidizes the cyanate to ammonia,
carbon dioxide and nitrogen.
Hydrogen peroxide In the presence of soluble Cu2+ cata- Yes No
lyst CN– is oxidized to cyanate with
H2O­2 at pH 9.0 to 9.5
Ozonation At pH 10.0–12.0, ozone with an Yes Yes
electrode potential of +1.24 Volt
oxidizes cyanide to cyanate and
further hydrolyzed to bicarbonate
and nitrogen
SO2/Air (INCO) In the presence of soluble Cu2+ cata- Yes Yes
lyst CN– is oxidized to cyanate with
SO2 at pH 8.0 to 9.0

6.3  Biological Treatment

The exploitation of cyanides by a variety of taxa, as a mechanism to avoid production
or to inhibit competitors has led to the evolution of many enzymes that catalyze
degradation of a range of cyanide compounds (Cummings and Baxter, 2006). The
presence of cyanide in the environment causes additional problems, i.e., the formation
of extremely stable metal-cyanide complex that makes essential metals unavailable to
the organisms. Therefore, bacterial proliferation in the presence of cyanide requires
specific metal uptake systems. The strategy for iron uptake consists of the production
of organic compounds widely known as called siderophores which strongly bind iron
and are transported and assimilated by the microorganisms (Andrews et al., 2003).
 Microbial Remediation of Cyanides  99

The biological assimilation of cyanide requires the concurrence of three separate

processes, i.e., a cyanide resistance mechanism, a system for metal acquisition and a
cyanide assimilation pathway. These processes in conjunction with one another have
not been considered so far, however, a number of microorganisms that are able to
degrade cyanide and its metal complexes have been very well described (Harris and
Knowles; 1983; Raybuck, 1992; Barclay et al., 1998). Dumestre et al. (1997) observed
that some phytopathogenic fungi, like Fusarium solani are able to degrade cyanide, but
the degradation of cyanide by bacteria is advantageous since bacteria are more easily
manipulated both at biochemical and genetic levels. Harris and Knowles (1983) also
reported that Pseudomonas fluorescens NCIMB11764 is capable of growth on cyanide
(CN−/HCN) as the sole nitrogen source. Different microorganisms which are reported
to degrade/metabolize till date are listed in Table 4.4.

Table 4.4 Various microorganisms reported till date for cyanide degradation and their
sources of isolation

Name of Microorganisms Source References

Stemphylium loti Soil Fry and Millar, 1972
Fusarium lateritium Soil Fry and Millar, 1972,
Cluness et al., 1993
Achromobactor Soil Knowles, 1976
Bacillus cereus Soil Knowles, 1976
Chromobacterium violaceum Soil Knowles, 1976
Tetrahymena pyriformis Soil Knowles, 1976
Pseudomonas sp. Activated sludge White et al., 1988
Pseudomonas fluorescens NCIMB 11764 Kunz et al., 1992
Alcaligenes xylosoxidans Soil Ingovresen et al., 1991
Bacillus pumulis Waste water Meyers et al., 1991
Fusarium solani Alkaline waste Dumestre et al., 1997
water
Burkholderia cepacia Soil Ohta and Adjei, 1999
Pseudomonas stutzeri AK 61 Metal plating waste Watanbste et al., 1998
Fusarium solani Soil Barclay et al., 2002
Pseudomonas pseudoalcaligens Waste water Luque-Almagro et al., 2005
Pseudomonas flourescens NCIMB 11764 Waste water Kunz and Fernadez, 2005
Trichoderma sp. Soil Lynch and Ezzi, 2005
Contd.
100  Bioremediation Current Research and Application

Fusarium sp. Soil Lynch and Ezzi, 2005

Saragassum sp. Soil Igwe and Abia, 2006
Ascophyllum sp Soil Lynch and Ezzi, 2005
Rhizopus orrhizae Soil Lynch and Ezzi, 2005
Ecklonia radiata Soil Igwe and Abia, 2006
Pseudomonas aeuroginosa Soil Cipollone et al., 2006
Cryptococcus humicolus Full scale coke Park and Lee, 2008
wastewater
Rhizobium daejeonense Quan et al., 2005
Rhizopus oryzae (MTCC 2541) Wastewater Dash et al., 2009
Agrobacterium tumefaciens SUTS 1 Cassava wastewater Potivichayanon and
Kitleartpornpairoat, 2010
Klebesiella oxyotoca Soil Chen, 2008
Micromonospora braunna Garden soil Shete and Kapdnis, 2012
Bacillus sp. CN-22 Soil Wu et al., 2013
Serratia marcescens RL2b Soil Kumar et al., 2013
Aspergillus niger K10 Soil Rinágelová et al., 2014
Trichoderma harzianum VSL291 Jorge Rodríguez and lepe, 2015

Industrially generated waste water contains free cyanide, in addition to cyano-metal

complexes, rendering it more poisonous (Huertas et al., 2006). Inspite of cyanide toxicity,
there are organisms that are able to survive in its presence and some of them are able to
utilize it as a nitrogen source. At present physicochemical treatments are also available
for these compounds, but they are expensive and also present some limitations. Since
cyanide is a natural biodegradable compound, it is therefore technically suggested that
biological treatments may be a better alternative for its management in environment.

7.  Enzymes responsible for cyanide degradation

There are five enzymes reported so far which could be utilized potentially for detoxifying
cyanide containing wastes. These enzymes are discussed in subsequent sections.

7.1  Cyanase (EC 4.2.1.104)

Cyanase is an enzyme that catalyzes the decomposition of cyanate into carbon dioxide
and ammonia through oxidative pathway (Fig. 4.2). In this pathway, bicarbonate acts
as the nucleophilic reactant that attacks and breaks down the cyanate to ammonia with
 Microbial Remediation of Cyanides  101

carbamate as an unstable intermediate (Luque-Almagro et al., 2011). The cyanase (cynS)

and carbonic anhydrase genes are often clustered together probably for this reason.
Three physiological roles have been attributed to cyanase activity in microrganisms, (i)
assimilation of nitrogen, (ii) detoxification of cyanate, and (iii) regulation of metabolism.
As this enzyme catalyzes the direct formation of ammonium from cyanate, so it enables
some bacteria to utilize cyanate as a nitrogen source. All heterotrophic bacteria capable of
cyanate assimilation endow cyanase activity (Luque-Almagro et al., 2011). This enzyme
has also been reported from cyanobacteria (Harano et al., 1997) and plants (Aichi et al.,
1998). Cyanase from Escherichia coli B has been reported to catalyze the decomposition
of cyanate in a bicarbonate-dependent reaction to give carbamate, which spontaneously
decarboxylates to carbon dioxide (Luque-Almagro et al., 2011)
The role of cyanase in detoxification of cyanate is based on the toxicity of cyanate at
relatively low concentrations (Luque-Almagro et al., 2011). This toxicity is largely due to
the reactivity of isocyanate, which is in equilibrium with cyanate. It carbamoylates some
nucleophilic groups of proteins (Pieniazek and Gwozdzinski, 2003). Cyanate has been
shown to be a strong competitive inhibitor of nitrate, and due to its oxidative nature,
cyanate also reactivates the reductively inactivated form of the enzyme.

7.2  Rhodanase (Thiosulfate: Cyanide Sulfurtransferases; EC 2.8.1.1)

This enzyme catalyzes the transfer of a sulphur atom from a suitable donor (e.g.,
thiosulfate) to cyanide, yielding the less toxic thiocyanate (Westley et al., 1983; Cipollone
et al, 2007; Oyedeji et al., 2013). Rhodanases have been purified from a number of
sources and consist of two structurally similar domains containing either a catalytic
Cys or an inactive Asp residue (Bordo and Bork, 2002). The functions of rhodanases
have not yet been defined, though these enzymes are implicated to act as facilitators
of cyanide detoxification in animals (Aminlari et al., 2002; Oyedeji et al., 2013). Due to
high conservation of rhodanases in the three evolutionary lineages (Bordo and Bork,
2002), it seems that these enzymes contribute to cyanide detoxification also in bacteria.
Rhodanases have been identified in a variety of bacterial species, including Escherichia
coli (Ray et al., 2000), and P. aeruginosa (Ryan and Tilton, 1977). The high sequence
homology between Azotobacter vinelandii and Pseudomonas aeruginosa RhdA rhodanases
predicts that these two enzymes belong to the same subfamily (Cippolone et al., 2006),
sharing structural features with mitochondrial bovine rhodanase (Bordo and Bork,
2002). Pseudomonas aeruginosa and Bacillus brevis exhibited appreciably high rhodonase
activity (Oyedeji et al., 2013).
The optimum temperature and pH for the activity of enzyme from P. aeruginosa were
50°C and 6.0, respectively while the optimum temperature and pH for the activity of
enzyme from B. brevis were 40°C and 7.0, respectively. The pH range for the activity of
P. aeruginosa and B. brevis rhodanase were 5.5 to 9.5 and 5.5 to 9.0, respectively (Oyedeji
et al., 2013). These results are in agreement with results reported for rhodanases from
different sources. Sorbo (1953) reported an optimum temperature of 50°C for bovine
102  Bioremediation Current Research and Application

liver rhodanase, Ezzi et al (2003) had reported a wide temperature optimum of 35 to

55°C for rhodanase of different strains of Trichoderma while Okonji et al. (2011) obtained
an optimum temperature of 50°C for mudskipper liver rhodanase. The values obtained
for the rhodanase from the two isolated bacterial strains of Pseudomonas aeruginosa
and Bacillus brevis underscore their versatility in detoxification of cyanide at varying
physicochemical conditions (Oyedeji et al., 2013).

7.3  Cyanide Hydratase (EC 4.2.1.66)

Cyanide hydratase is an intracellular, inducible enzyme, having cyanide and
metallocyanides as inducers. Cyanide undergoes hydrolysis to formamide by cyanide
hydratase (formamide hydrolyase EC 4.2.1.66) which in turn is converted to formic acid
and ammonia by amidase. A variety of fungal species are known to degrade cyanide
through the action of cyanide hydratases, a specialized subset of nitrilases. Fry and
Millar (1972) first reported this enzyme in the fungus Stemphylium loti, a pathogen of the
cyanogenic plant, bird’s foot trefoil (Lotus corniculatus). The native molecular mass of the
S. loti enzyme was not determined, but as it is eluted in the void volume of a Sephadex
G-200 column, thus it is thought to be of very large size. This enzyme has been found
in many fungal species, but not in bacteria or plants (Wang et al., 1999; Rinágelová
et al., 2014). The cyanide hydratases are much more substrate specific than nitrilases
with HCN, being the most effective substrate for all the enzymes. The enzymes from
Fusarium solani and Fusarium oxysporum N-10 can hydrolyse a metal-cyano complex,
tetracyanonickelate (II) (TCN) (Barclay et al., 1998; Yanase et al., 2000) but the activity
in F. oxysporum N-10 was very low (Yanase et al., 2000). Fusarium oxysporum N-10
cyanide hydratase also has some activity with a number of aliphatic nitriles showing
best activity with acrylonitrile, methacrylonitrile and crotononitrile (Yanase et al., 2000).
Induction of enzyme activity for cyanide hydratase has been described for a number of
cyanide-degrading fungi, including the strain of F. solani (Fry and Millar, 1972; Wang
and Vanetten, 1992; Cluness et al., 1993; Barclay et al., 1998; Sexton and Howlett, 2000).
The product of nitrile hydrolysis is the corresponding amide, which indicates that the
enzyme is catalysing a monohydrolysis of the substrate and therefore the reaction is
a nitrile hydratase reaction rather than a nitrilase reaction. Nolan et al. (2003) have
reported that the cyanide hydratase of F. lateritium has low nitrilase activity with
benzonitrile, propionitrile and acetonitrile. Potential cyanide degrading actinomycetes,
identified as Micromonospora braunna was isolated and acclimatized in the minimal
medium containing 1000 ppm cyanide. The detection of formamide in the culture
broth confirmed the cyanide hydratase activity. An activity of 76.66 enzyme units have
been detected in the production broth supplemented with dextrose as carbon source
and potassium cyanide as sole nitrogen source. The enzyme cyanide hydratase was
extracted and its reaction parameters have been determined. The kinetic studies have
revealed the Km value as 33mM and Vmax as 39 µmoles ml−1 min−1 (Shete and Kapadnis,
2012). Cyanide hydratase has been characterized from Gloeocercospora sorghi, Fusarium
 Microbial Remediation of Cyanides  103

lateritium and Leptosphaeria maculans (Wang and Vanetten, 1992; Cluness et al., 1993;
Sexton and Howlett, 2000). Cyanide hydratase activity has also been demonstrated in
cell-free extracts of the fungus Fusarium oxysporum, which could use the metal cyanide
K2Ni(CN)4 as a sole source of nitrogen (Yanase et al., 2000). Determination of amino acid
sequences of the cyanide hydratases from these fungi has revealed that the proteins of
F. lateritium and G. sorghi are 75 percent identical, whereas L. maculans shows 77 percent
and 82 percent homology to G. sorghi and F. lateritium, respectively (Cluness et al., 1993;
Sexton and Howlett, 2000).
A cyanide hydratase from Aspergillus niger K10 was expressed in E. coli and purified.
Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and
fumaronitrile. Vmax and Km for HCN are 6.8 mmol min−1 mg−1 protein and 109 mM,
respectively. Both cyanide hydratase and nitrilase activities have disappeared in
truncated enzyme variants missing in 18–34 C-terminal residues. This enzyme exhibits
the highest activity at 45°C and pH 8–9; it is unstable at temperature above 35°C and
at pH below 5.5. The operational stability of the whole-cell catalyst was examined
in continuous stirred membrane reactors with 70 mL working volume. The catalyst
exhibited a half-life of 5.6 h at 28°C. A reactor loaded with an excess of the catalyst
has been used to degrade 25 mM KCN. A conversion rate of over 80 percent has been
recorded for 3 days (Rinagelova et al., 2014).

7.4  Cyanide Dihydratase/Cyanidase (EC 3.5.5.1)

As there are a range of organisms that are able to metabolize cyanide, likewise there are
a variety of enzymes that have been shown to convert cyanide to non-toxic products.
The cyanide-degrading nitrilases differ from many of those enzymes in that they
are able to hydrolyze cyanide without the addition of any cofactors or secondary
substrates. Ingvorsen et al. (1991) reported an organism that Alcaligenes xylosoxidans
subsp. denitrificans strain DF3 that catalyses the hydrolysis of HCN to formate without
forming formamide as a free intermediate. This enzyme was named as cyanidase
(Cyn D). Enzymes catalyzing the direct hydrolysis of HCN to formate have also been
identified in Bacillus pumilus C1 (Meyers et al., 1993), P. fluorescens NCIMB 11764 (Kunz
et al., 1992) and P. stutzeri AK61 (Watanabe et al., 1998). All the CDH enzymes are highly
specific to cyanide.
Pseudomonas fluorescens NCIMB 11764 (Kunz et al., 1992) metabolizes HCN by a
number of different routes. The major activity of this organism under aerobic conditions
is due to an NADH-dependent cyanide oxygenase, which produces CO2 and NH3.
This enzyme is unrelated to the nitrilase/cyanide hydratase family of enzymes. The P.
fluorescens also produces formamide and formic acid, the amounts of which depend on
growth conditions. Kunz et al. (1992) proposed that P. fluorescens contain both cyanide
hydratase and cyanide dihydratase activities, but as the enzymes have not been purified
it is also possible that this organism contains a single enzyme with both activities or that
104  Bioremediation Current Research and Application

formic acid is considered as product of formamidase activity. The cyanide dihydratase

of Bacillus pumilus has been reported to be an 18-subunit spiral structure by three-
dimensional reconstruction of electron micrographs of negatively stained material at
its optimum pH 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed
helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the
fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was
cloned, and the recombinant enzyme was readily expressed at high levels in E. coli.
Purification of the recombinant enzyme was facilitated by the addition of a C-terminal
six-histidine affinity purification tag. The tagged recombinant enzyme has Km and Vmax
values similar to those for the native enzyme. This was the first cyanide dihydratase
from a gram-positive bacterium to be sequenced, and it was the first description of
the structure of any member of this enzyme class. The putative amino acid sequence
shares over 80 percent identity to the other sequenced cyanide dihydratase that of
the gram-negative Pseudomonas stutzeri strain AK61, and was similar to a number of
other bacterial and fungal nitrilases. This sequence similarity suggested that the novel
short spiral structure may be typical of these enzymes. In addition, an active cyanide
dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned
and expressed. This revealed that cynD, the gene coding for the cyanide dihydratase,
was not unique to the C1 strain of B. pumilus (Thuku et al., 2007).
The details of the mechanism of cyanide degradation have not been elucidated, but
evidence exists that a thioimidate intermediate is formed by a nucleophilic attack of the
active site cysteine on the hydrogen cyanide. This is then hydrolyzed with the formation
of ammonia and an acyl intermediate in the case of CynD or formamide in the case of
the cyanide hydratases. Presumably, subtle changes in the detailed configuration of the
active site determine whether the ammonia or the formamide is a better leaving group
(Jandhyala et al., 2005), thereby defining whether the enzyme is a cyanide dihydratase
or a cyanide hydratase (Dent et al., 2009).

7.5  Cyanide Monooxygenase

This enzyme was reported in P. fluorescens NCIMB 11764 that grows at concentrations of
CN equivalent to 300 mM (Kunz et al., 1992). For CN utilization, it has to be enzymatically
converted to ammonia, which is then assimilated by established pathways (Kunz et al.,
1992). In P. fluorescens NCIMB 11764, this occurs by a novel enzymatic mechanism in
which CN is cleaved oxidatively to give formate and ammonia via reactions involving
formamide as an intermediate (Table 4.5). The responsible enzyme, variously described
as cyanide oxygenase (CNO), is located in the cytosolic fraction of cells induced with CN
and requires both reduced pyridine nucleotide (NADH) and a source of reduced pterin
as a cofactor (Fernandez et al., 2004). Formate is further oxidized by a soluble formate
dehydrogenase (FDH), simultaneously elevated in CN-induced cells.
 Microbial Remediation of Cyanides  105

Table 4.5 Some enzymes involved in cyanide degradation

Name of Enzyme Reaction Catalyzed Nature of Microorganisms

Enzyme Class Reaction
Cyanase EC 4.2.1.104 HCN + HCO3– Æ CO2 + NH3 Oxidative Cyanobacteria, E. coli

Cyanide HCN + O2 + H+ + NADPH Æ Oxidative Pseudomonas sp.

mono- HOCN + NADP+ + H2O Æ
oxygenase HCOOH + NH3

Cyanide HCN + O2 + H+ + NADPH Æ Oxidative Pseudomonas

dioxygenase CO2 + NH3 + NADP+ fluorescens, Bacillus
cereus, Bacillus
pumillus

Cyanide EC 4.2.1.66 HCN + H2O Æ HCONH2 Hydrolytic Pathogenic fungi

hydratase

Cyanidase EC 3.5.5.1 HCN + 2H2O Æ HCOOH + NH3 Hydrolytic Alcaligenes

xylosoxidans
Rhodanase EC 2.8.1.1 HCN + S2O32– Æ HCNS + SO32– Substitution Thiobacillus
transfer denitrificans, Bacillus
subtilis, Bacillus
stearothermophilus

8.  Conclusion and future prospects

Due to the extensive use of cyanide, its level in the environmental is much higher than
the permissible limits. Microorganisms possess various detoxifying mechanisms to cope
with high concentrations of cyanide. Although there are various processes (physical
and chemical) reported to eliminate cyanide from environment yet these methods are
inefficient in removing cyanide from the environment. Biological treatment of cyanide
wastes provides a good alternative for remediation of cyanide from industrial effluents.
There are a number of advantages of using biological agents to detoxify cyanide wastes.
A number of cyanide degrading enzymes have been reported from microbes which are
responsible for conversion of cyanide to non toxic products, but the exact structures
and functioning of these enzymes are yet to be elucidated. The literature survey and
the information reviewed above clearly revealed that comparatively less work has been
done on the microbial decomposition of cyanides and related compounds. Information
on the biochemical and molecular characteristics of various enzymes involved in
microbial metabolism of cyanides is also scanty. Beside this, the maximum level of
cyanide that can be tolerated and degraded by the microbes reported hitherto is up to
106  Bioremediation Current Research and Application

12 mM. Therefore, it will be worthwhile to isolate cyanide degrading microbes from

less explored habitats with higher levels of cyanide tolerance and degradation potential,
and such microorganisms will have immense scope for bioremediation of cyanide
contaminated soil/water. Studies involving characterization of cyanide metabolizing
pathway vis-à-vis purification and molecular characterization of enzymes involved in
cyanide decomposition in microbes will be interesting to develop microbial consortia for
effective degradation of cyanide and to devise cell/enzyme based biosensors for rapid
detection of cyanide in water or food samples.

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