Bioremediation of Xenobiotics

Use of pesticides has benefited the modem society by improving the quantity and quality of the worlds' food production. Gradually, pesticide usage has become an integral part of modern agriculture system. Many of the artificially made complex compounds i.e. xenobiotics persist in environment and do not undergo biological transformation. Microorganisms play an important role in degradation of xenobiotics, and maintaining of steady state concentrations of chemicals in the environment. The complete degradation of a pesticide molecule to its inorganic components that can be eventually used in an oxidative cycle removes its potential toxicity from the environment. However, there are two objectives in relation to biodegradation of xenobiotics: (i) how biodegradation activity arises, evolves and transferred among the members of soil microflora, and (ii) to device bioremediation methods for removing or detoxifying high concentration of dangerous pesticide residues (Gupta and Mukerji, 1998).

The characters of pesticide degradation of microorganisms are located on plasmids and transposons, and are grouped in clusters on chromosome. Understanding of the characters provides clues to the evolution of degradative pathways and makes the task of gene manipulation easier to construct the genetically engineered microbes capable of degrading the pollutants.

Microbial Degradation of Xenobiotics
Biodegradation of pesticides occurs by aerobic soil microbes. Pesticides are of wide varieties of chemicals e.g. chlorophenoxyalkyl caboxylic acid, substituted ureas, nitrophenols, tri-azines, phenyl carbamates, orga-nochlorines, organophosphates, etc. Duration of persistence of herbicides and insecticides in soil is given in Table 21.2. Otganophos-phates (e.g. diazion, methyl par-athion and parathion) are perhaps the most extensively used insecticides under many agricultural systems. Biodegradation through hydrolysis of p-o-aryl bonds by Pseudomonas diminuta and Flavobacterium are considered as the most significant steps in the detoxification of organophospho-rus compounds. Organomercurials (e.g. Semesan, Panodrench, Panogen) have been practiced in agriculture since the birth of fungicides. Several species of Aspergillus, Penicillium and Trichoderma have been isolated from Semesan-treated soil. Moreover, they have shown ability to grow over 100 ppm of fungicide in vitro. The major fungicides used in agriculture are water soluble derivatives such as Ziram, Ferbam, Thiram, etc. All these are degraded by microorganisms.
Pentachlorophenol (PCP) is a broad spectrum biocide which has been used as fungicide, insecticide, herbicide, algicide, disinfectant and antifouling agent. Bioreactors containing alginate immobilized + Polyurethane foam immobilized PCP degrading Flavobacterium (ATCC39723) cells have been used to remove PCP from contaminated water. Absorption of PCP by Polyurethane immobilized matrix plays a role in reducing the toxicity of PCP. Flavobacterium removed and detoxified PCP (Zhong -Cheng, 1994). In other experiment P. chrysosporium enzyme (ligninase) has been found to dehalogenate PCP. Steps of PCP degradation has been shown in Fig. 21.7.

Table 21.2. Duration of persistence of insecticides and herbicides in soil. Source : Madigan et al. (1997). DDT (l,l,l-trichlofo-2,2-bis (p-chlorophenyl) ethane) is an insecticide that persists in soil for four years. Degradation pathway of DDT involves an initial dechlorination of the trichloromethyl group to form 1,1-dichloro 2-ethane which then undergoes further dechlorination, oxidation and decarboxylation to form bis methane. Subsequent cleavage of one of the normal aromatic rings yields p-chlorophenyl acetic acid, which may also undergo ring cleavage. Microorganisms associated with DDT degradation are Aspergillus flavus, Fusarium oxysporum, Mucor aternans, P. chrysosporium, Trichoderma viride, etc. Environmental factors including pH, temperature, bioavailability, nutrient supply and oxygen availability affect biodegradation of pesticides.

Gene Manipulation of Pesticide-degrading Microorganisms
Day-by-day the number of xenobiotic-degrading microorganisms is increasing. However, pesticide-degrading genes of only a few microorganisms have been characterized. Most of genes responsible for catabolic degradation are located on the chromosomes, but in a few cases these genes are found on plasmids or transposons. Chakrabarty and Gunsalus (1971) found that camphor degrading genes of Pseudomonas putida are located on plasmid. Soon after, naphthalene (NAH) degrading plasmid was isolated. Discovery of these genes made it possible to construct a new genetically engineered strain of P. putida that alone was potent to degrade camphor, naphthalene, xylene, toluene, octanes and hexanes. For detailed discussion, see Abatement of pollution.
For the first time pesticide degradation through plasmid mediated genetically engineered microorganism was reported by Chakrabarty et al (1981). Nagata et al (1993) have also cloned and sequenced two genes involved in early steps of Y-HCH degradation in UT26. The linAgene encodes Y-HCH dehydrochlorinase which converts Y-HCH to 1,2,4TCB via Y-PCCH and 1,4-TCDN. The linBgene encodes 1,4-TCDN chlorohydrolase which converts 1,4-TCDN to 2,4-DDOL via 2,4,5-DNOL. This gene is a member of the haloalkanedehalogenase family with a broad range specificity for substrate. The genetically engineered P. putida comprises of both linA and linB genes (Nagata et al, 1993).

The pod gene, initially isolated from Flavobacterium sp. ATCC27551 and Pseudomonas diminuta, has been well characterized. It is associated for degradation of pesticides such as parathion, methylparathion, etc. Sims et al (1990) transferred a recombinant DNA plasmid containing pod gene into a fungus, Gliocladium virens which expressed at low level. G. virens is a useful soil saprophyte which has shown a strong mycoparasitic activity against many fungal pathogens. Strains of G. virens are potential for use in the bioremediation of contaminated soil. Optimization of pod expression in bioremedially useful organism such as P. chrysosporium, G. virens, etc. holds a great promise in lessening the pesticide pollution.