Keywords

12.1 Introduction

Pyridine and its derivatives are important representatives of heterocyclic compounds. Pyridine ring is a major constituent of natural alkaloids, coenzymes such as nicotinamide, nicotine, cavadin etc. (Bernstein et al. 1966; Houghton and Cain 1972). These compounds are produced by plants and animals and are used as insecticides and pesticides. Pyridine compounds such as 2-hydroxypyridine-1-oxide are also reported to have fungicidal activity against Microsporum infections and 2,6-dichloro-4-phenylpyridine-3,5-di-carbonitrile is active against Alternaria, Venturia inaequalis, Cladosporium fulvum and Plasmospora. Pyridine is widely employed as a solvent in industry and laboratory. It is used as a denaturant in alcohol and antifreeze mixtures, as a solvent for paint, rubber and polycarbonate resins and as an intermediate in the manufacture of herbicides like paraquat, diquat and picloram which are widely used in agriculture (Calderbank 1968; Maya 1981). It is also used as a solvent and intermediate in the preparation of vitamins and drugs, dyes, textile water repellents and flavouring agents in food. Pyridine is produced from coal and recovery from coke-oven gases and coal tar middle oil. It can also be produced synthetically from the vapour phase reaction of acetaldehyde and ammonia, with formaldehyde and methanol (Jori et al. 1983).

12.1.1 Risk Associated with Exposure to Pyridine

The greatest potential exposure to pyridine and its derivatives is in the workplace, where these chemicals are manufactured or used to synthesize other chemicals. Occupational exposure, usually by inhalation or dermal absorption, may occur ­during their production or use as chemical intermediates. The U.S. Environment Protection Agency (EPA) estimated that 2,49,000 persons were occupationally exposed to pyridine. According to the National Institute for Occupational Safety and Health (NIOSH) 8 h, time weighted average permissible exposure level for pyridine is 5 ppm (16 mg/m3) and a concentration of 3,600 ppm is immediately dangerous to life. A concentration of 10 ppm becomes objectionable to unaccustomed individual and a concentration above 5 ppm leads to olfactory fatigue. EPA regulates pyridine as a toxic waste under the Resource Conservation and Recovery Act (RCRA); a maximum pyridine concentration of 5.0 mg per litre of leachate is allowed using analysis determined by the Toxicity Characteristic Leaching Procedure. Pyridine is listed as a chemical known to the State of California to cause cancer under the safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition 65).

12.1.2 Fate of Pyridine in Various Environments

Pyridine exists in atmosphere as vapour. The estimated atmospheric lifetime of pyridine is 23–46 days. In atmosphere, it gets photo degraded by hydroxyl radicals in the troposphere. A large fraction of atmospheric pyridine vapour phase would trend to dissolve in water vapour due to its high water solubility. Much of the atmospheric pyridine is removed by precipitation and if it is dissolved in water, it does not volatilize readily into the atmosphere. The volatility and sorption of pyridine and its derivatives in water varies considerably and is pH dependent. At concentrations less than 20 mg/L, pyridine degradation was virtually completed in 8 days or less. Many of the pyridine compounds, both natural and synthetic, are ultimately degraded therefore their concentrations do not increase substantially in the soil environment. The carbon and nitrogen skeleton of pyridine is thus mineralized and are recycled. Such recycling is essential because continued addition of compounds, which are completely resistant to degradation, can soon lead to the accumulation of concentrations that are potentially toxic or otherwise unacceptable.

12.1.3 Pyridine Derivatives – Alkyl Pyridines

Alkylpyridines are toxic environmental pollutants commonly found in many surface and ground waters near synthetic liquid fuel industries where they are extensively used to produce many chemical intermediates, solvents, paints etc., (Riley et al. 1981; Stuermer et al. 1982; Turney and Goerlitz 1990). They are reported to be found in more concentrations than pyridine and its other derivatives. Despite their occurrence and toxicity, degradation of these chemicals is poorly studied (Sims and O’Loughlin 1989; Kaiser et al. 1996). There have been numerous laboratory studies on alkylpyridines (Shukla 1974, 1975; Korosteleva et al. 1981; Feng et al. 1994) their degradation by many microorganisms (Kost and Modyanova 1978; Shukla 1974, 1984; Sims and O’Loughlin 1989 and Ronen and Bollag 1995) under both aerobic and anaerobic conditions (Rogers et al. 1985; Kaiser and Bollag 1991).

12.2 Critical Review

12.2.1 Pyridine

Though pyridine is considered as toxic, its concentrations in environment, ­particularly in soils are kept under check by many microorganisms. Corynebacterium sp. and Brevibacterium sp. isolated by Shukla (1973) and Nocardia strain Z1 isolated by Watson et al. (1974) reported degradation of pyridine. The organisms were unable to transform monohydroxylated pyridines. Hydroxylated pyridines were never detected as metabolites ruling out any role of hydroxylases in pyridine metabolism. Formic acid and ammonia were detected as products of degradation in the absence of any metabolic inhibitor. The degradation product of pyridine in the presence of semicarbazide as metabolic inhibitor was succinate semialdehyde. Brevibacterium sp. isolated by Shukla (1973) degraded pyridine and produced ­succinic acid semialdehyde and pyruvic acid when arsenate was added as metabolic inhibitor. The Micrococcus luteus reported by Sims et al. (1986) oxidized pyridine to give aliphatic intermediates. Watson and Cain (1975) studied pathway of pyridine degradation with Bacillus strain 4. This organism produced succinic acid semialdehyde as a product when semicarbazide was used as an inhibitor and in presence of cyanide it produced formic acid from the second carbon atom of this heterocyclic ring. Transformation of pyridine by Bacillus strain 4 could follow the pathway suggested by Watson and Cain 1975 (Fig. 12.1). Watson and Cain reported a Nocardia strain Z1 that could degrade pyridine resulting in accumulation of glutaric acid semialdehyde. The pathway for pyridine degradation proposed by the same authors is given in Fig. 12.2.

Fig. 12.1
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Biotransformation of pyridine by Bacillus strain 4 (Watson and Cain 1975)

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Fig. 12.2
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Pyridine biodegradation by Nocardia strain Z1 (Watson and Cain 1975)

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12.2.2 Hydroxypyridines

Microbial metabolism of hydroxypyridines and other pyridine derivatives are reported by isolating several pure cultures. An Arthrobacter sp. degraded 2-hydroxypyridine (Gupta and Shukla 1975), resulting in accumulation of a blue pigment, corresponding to 2, 3, 6-trihydroxypyridine, which eventually converted to maleamate, maleate and pyruvate. Production of similar pigment during 2-hydroxypiridine degradation with other Arthrobacter sp. was reported by Ensign and Rittenberg (1963). Kolenbrander and Weinberger (1977) reported 2-hydroxypyridine metabolism by Arthrobacter crystallopoietes, A. pyridinolis and A.viridescens all of which produced extra cellular crystalline pigment. Cell free extracts required reduced NAD, molecular oxygen as well as FMN, suggesting the role of monooxygenase in the degradation activity. The pigment produced was a monopotassium salt of 4, 5, 4′, 5′ tetrahydroxy-3, 3′ diazadiphenoquinone-(2, 2′), structurally related to indigoidine. The role of monooxygenase indicated in the metabolism of 2-hydroxypyridine by the three organisms. In this process plasmid, involvement was detected only in A. crystallopoietes and curing experiments supported the same. An Arthrobacter sp. was isolated from soil samples of Tarai jungles by aerobic enrichment culture technique in phosphate salt growth medium. This organism utilized pyrrolidine as sole source of carbon, nitrogen and energy for its growth (Ensign and Rittenberg 1963) and metabolized pyrrolidine through ∆-pyrroline, γ-aminobutyraldehyde (GABA), succinic semialdehyde and succinate. The enzymes responsible for pyrrolidine metabolism have been demonstrated in the cell free extracts of this organism.

12.2.3 -Picolinate

Shukla and Kaul (1973) reported 2-picolinate (pyridine-2-carboxylate) ­biotransformation by Bacillus sp. resulting in the accumulation of 6-hydroxy picolinic acid, 3, 6-­dihydroxy picolinic acid and 2, 5-dihydroxy pyridine. Resting cell suspensions of this organism metabolized 2-picolinate to pyruvate and 6-hydroxy picolinate with sodium arsenate as a metabolic inhibitor. A Gram-negative coccus reported by Shukla et al. (1977) transformed 2-picolinate to 6-hydroxy picolinate.

12.2.4 Alkylpyridines (Picolines)

Alkylpyridines or picolines are methylated pyridines, which are considered more toxic than their parent compound pyridine. Their toxicity even lies in position of alkyl group. They are more commonly present in environment than pyridine. However, because of their toxicity they are reported to be more resistant to ­microbial attack than pyridine. Presence of alkyl group in the ring (in particular 2-picoline) makes the compound selective for only few microorganisms (Shukla 1974, 1975; Korosteleva et al. 1981; Feng et al. 1994). Studies on biodegradation of alkylpyridines were carried out under aerobic and anaerobic conditions.

12.2.5 Biodegradation of Alkylpyridines Under Aerobic Condition

Biodegradations of alkylpyridines were reported by many bacterial and fungal species. Shukla (1974) reported an Arthrobacter sp. that degrades 2-picoline and also utilizes 2-ethylpyridine or piperidine as alternate growth substrates. Chromatographic and U.V examination of the fermented broth and methanol extraction of freeze-dried broth failed to show the presence of any metabolite. Even identification of intermediate metabolites using metabolic inhibitors like sodium azide, sodium fluoride, sodium arsenate etc., failed to show any intermediate metabolites. However, a pale yellow pigment having a broad absorption band around 435 nm was reported to be released into the broth during later stages of fermentation that eventually identified as riboflavin. The pathway reported for 2-picoline degradation has some undetected intermediate metabolites (compounds mentioned in parenthesis) as given in Fig. 12.3. O’Loughlin et al. (1995) with an Arthrobacter sp. reported similar findings. This organism was able to utilize 2-picoline and 2-ethylpyridine as primary carbon and energy sources. It also utilized 2-, 3-, 4-hydroxyl benzoate, gentisic acid, proto catechuic acid and catechol. Degradation of 2-picoline was accompanied by overproduction of riboflavin. Lee et al. (2001) reported Gordonia nitida LE31 that could degrade 3-methylpyridine and 3-ethylpyridine. No cyclic intermediates were found, but formic acid was identified as a metabolite. In this degradation ­pathway, 3-methylpyridine and 3-ethylpyridine were degraded through the enzyme system that catalyzed the cleavages of C2–C3 of heterocyclic rings. Coryneform bacteria group reported by Shukla (1975) degraded 2-ethylpyridine, 2, 4-lutidine and 2, 4, 6-collidine. The bacterium also used 2-picoline as a growth substrate after a lag period of about 48 h. However, no metabolites of these pathways were reported. Lee et al. (2001) carried out biodegradation of 3-methylpyridine and 3-ethylpyridine in their laboratory using Gordonia nitida LE31. Cells of Gordonia nitida LE31 grown on 3-methylpyridine degraded 3-ethylpyridine without a lag time and vice versa. Cyclic intermediates were not detected, but formic acid was identified as a metabolite. The pathway proposed by them was novel involving C2–C3 ring cleavage ­during biodegradation of 3-methylpyridine and 3-ethylpyridine.

Fig. 12.3
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Biodegradation of 2-picoline by Arthrobacter species (Shukla 1974) – A. N-acetyl γ-aminobutyric acid; B. γ-aminobutyric acid; C. Succinic semialdehyde; D. 1,4-dihydro-α-­picoline; E. Maleic semialdehyde

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Biodegradation of pyridine, 2-picoline and other pyridine derivatives was studied in our laboratory. Screening of industrial soil samples for isolating efficient degrading microbial consortia by enrichment culturing technique obtained 9 bacterial isolates where bacilli dominated over cocci. Among the isolates, an efficient bacterial isolate was identified by morphological observations as Gram positive, motile and endospore forming bacillus (Madhusudan Reddy et al. 2006). Biochemical and genetic methods identified the bacterium as Bacillus cereus. 16S rRNA gene sequence of this bacterium was unique from reported Bacillus cereus and its ability to metabolize 2-picoline efficiently designated this bacterium as a novel strain. Hence a strain name Bacillus cereus GMHS was given to this isolate and its 16S rRNA gene sequence is deposited at GENBANK under accession number DQ 351239 (gi no. 84620825) (Madhusudan et al. 2009). Ammonia was accumulated during biodegradation of 2-picoline indicating ring cleavage. No other intermediate metabolites were detected under normal conditions. However, when sodium azide and sodium arsenate were used as metabolic inhibitors, 2-hydroxypicoline and 6-hydroxypicolinic acid were accumulated as intermediate metabolites (Madhusudan Reddy et al. 2009a). A 11 kb plasmid was isolated from Bacillus cereus GMHS and role of this plasmid in 2-picoline degradation is evidenced by plasmid curing with acridine orange and hexammine ruthenium (III) chloride. Transformation of this plasmid into E.coli DH5α resulted in transfer of degradation character, where untransformed cells were sensitive to 2-pcoline (Madhusudan Reddy et al. 2009b). Biodegradations of most of the aromatic heterocyclics are carried out by a group of efficient oxygenase enzymes (Williams and Sayers 1994). These enzymes produce oxygenated metabolites from these heterocyclics. 2-Picoline degradation was studied in our laboratory and the role of these enzymes was detected using Gibb’s reagent. This reagent detects oxygenated metabolites that are formed during biodegradation. Toluene dioxygenase (TODA) was reported for its extended activity in utilizing 4-picoline as substrate for growth (Takeshi et al. 2001). Therefore, this enzyme was used during our study to know its affinity towards 2-picoline and some other selected pyridine derivatives. For this a three dimensional (3D) model for TODA was generated and refined (Fig. 12.4). Active sites on this enzyme were identified and from those sites, the one that is more stable was used to dock with the substrates such as 2-picoline, toluene, 2-hydroxypicoline, picolinic acid and 6-hydroxypicolinic acid (Figs. 12.5 and 12.6). For this study bioinformatics tools such as SYBYL, PROCHECK and GOLD etc., were used. From the results of this study, it was observed that 2-picoline is most preferred substrate next to toluene than other pyridine derivatives (Tables 12.1 and 12.2). This also provided information regarding possibility of evolution of toluene dioxygenase to accept 2-picoline as substrate and possibility of TODA like dioxygenase enzyme system in Bacillus cereus GMHS that has a key role in 2-picoline degradation (Madhusudan Reddy et al. 2008). The proposed pathway for biodegradation of 2-picoline by Bacillus cereus GMHS is shown in Fig. 12.7.

Fig. 12.4
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Three dimensional (3D) model for toluene dioxygenase TODA (Energy minimizing and low energy confirmation obtain the structure over the last 1,000 fs of MD simulation. The α-helix is represented in red and β-sheet in yellow) (Madhusudan Reddy et al. 2008)

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Fig. 12.5
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Possible binding sites (active sites) of toluene dioxygenase enzyme (Madhusudan Reddy et al. 2008) (Site 1 represented by purple. Site 2 represented by violet color. Ste 3 represented by white color. Site 4 represented by cyan color. Site 5 represented by red orange color. Site 6 represented by yellow color. Site 7 represented by magenta color, Site 8 represented by green blue color. Site 9 represented by green color. Site 10 represented by cyan color. Site 11 represented by magenta color. Site 12 represented by orange color. Site 13 represented by white color. Site 14 represented by yellow color)

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Fig. 12.6
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Docking study of toluene dioxygenase (TODA) with selected substrates (substrates are represented in red color) (Madhusudan Reddy et al. 2008)

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Table 12.1 Hydrogen bonds and hydrophobic interactions between the substrate and active site residues of toluene dioxygenase (TODA) using molecular operating environment (MOE) (Madhusudan Reddy et al. 2008)
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Table 12.2 The total energy (Etotal), electrostatic energy (Eele), steric energy (Este), of the best-docked conformations of TODA with substrates (Madhusudan Reddy et al. 2008)
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Fig. 12.7
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Proposed pathway for biodegradation of 2-picoline by Bacillus cereus GMHS. 1&2 are identified hydroxyl intermediates of the pathway (Madhusudan et al. 2009)

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12.2.6 Biodegradation of Alkyl Pyridines Under Anaerobic Condition

Kaiser et al. (1993), reported a mixed culture that could transform 3-picoline and 4-picoline under anaerobic conditions with sulfate as an electron acceptor. The 3-picoline degrading culture contained at least three types of bacterial forms where two of them were short rods and long rods. They were reported to be non-motile and spore forming bacteria. The third culture had a streptococcal shape and was non-motile. 4-Picoline degrading culture also contained three types of organisms with one organism as elliptical rods, pointed ends and central spore. Second culture had small coccobacilli and third bacterium was of vibroid shape. 3-Picoline under ­sulfate reducing conditions was completely degraded to CO2 and NH3. 4-Picoline was first transformed to 2-Hydroxy-4-picoline, which after 60 days accumulated sulphide which was completely mineralized after 90 days.

Comparative transformation rates of pyridine derivatives: Existence of a heterocyclic in the environment depends on their structure, concentration, physical and chemical properties. (Grbic-Galic 1990). Carboxylic substituents at any ­position in the pyridine ring greatly stimulated degradation than any other substituents (Naik et al. 1972). Chloropyridines are more resistant to transformation. The transformation rates of pyridine derivatives reported by Sims and Sommers (1985, 1986) is as follows:

Pyridinecarboxylic acids  >  Monohydroxypyridines  >  Methylpyridines  >  amin­opyridines  >  chloropyridines.

12.3 Conclusion

This chapter concludes that pyridine and its derivatives are very important chemicals that are used in making many important chemical intermediates and their ever increasing usage finds them in increasing concentrations in various effluents released by the industries. Though these chemicals are toxic to humans, their concentrations can be kept under check by various microorganisms that are very efficient in mineralizing or transforming them to non toxic compounds. Hence employing microorganisms like Arthrobacter, Nocardia, Micrococcus, Bacillus sp. etc. at the contaminated sites can render the sites free from accumulation of toxic chemicals. In-silico study mentioned in this chapter using bioinformatics tools can help in understanding the interactions at enzyme level thereby managing these toxic environments in a better way is possible.

12.3.1 Future Perspectives

Every year around 1,000–1,500 new chemicals are manufactured with perhaps 60,000 chemicals in daily use. Most of these are organic chemicals and pesticides. These compounds due to their extensive use as chemical intermediates are resulting in amassing higher concentration in the environment as recalcitrant. If their concentration is not checked in the environment, every pollutant that was released to the environment would still be here to haunt us. Fortunately, there are many reactions that check their concentrations such as chemical hydrolysis, photo degradation, volatilization, sorption, and most important and economic process bioremediation. Over the last few years, a number of companies have been established already to develop and commercialize biodegradation technologies. Existence of such companies now has become economically justifiable, because of burgeoning costs of ­traditional treatment technologies, increasing public resistance to such traditional technologies, accompanied by increasingly stringent regulatory requirements. The interest of commercial businesses in utilizing micro-organisms to detoxify effluents, soils, etc. is reflected in “bioremediation” having become a common buzzword in waste management. Companies specializing in bioremediation will need to develop a viable integration of microbiology and systems engineering.

Globally with the ever increasing population, the demand for requirement of various man made products also increases proportionately that poses threat in release and accumulation of toxic effluents in the environment. Hence, carrying a serious research to isolate potential microbial candidates is a vital step that can check the concentrations in the environment. Critical study of microbial environments especially environments where most of the uncultured micro-organisms are prevailing and using theses microbes, employing techniques like recombinant DNA technology, novel techniques like bioinformatics and nanotechnology tools, managing the contaminated sties would be easy and safe. Construction of gene cassettes responsible for metabolism of toxic chemicals and expression of these cassettes in easily cultivable and routine bacteria like E.coli that do not demand expensive ­nutrients or process to metabolize the toxic chemicals will definitely have advantage. These results can assure us promising improvements in managing the contaminated environments thereby helping us to live in a better environment devoid of toxic pollutants.