**10. Microbial enzymes involved in PAHs degradation process**

As bacteria initiate PAHs degradation by the action of intracellular dioxygenases, oxygenase, dehydrogenase, phosphatases, dehalogenases, nitrilases, nitroreductases and lignolytic enzymes [33] (Table 1). Aromatic ring dioxygenases are multicomponent enzymes which consist of an electron transport chain containing a ferredoxin and a reductase and a terminal dioxygenase [99]. The best studied PAHs dioxygenase is naphthalene dioxygenase from *Pseudomonas putida* encoded by the NAH plasmid pDTG1 [100].

*al.* [48] identified bacteria growing in a PAHs contaminated area that produces biosurfactants that facilitate the solubilisation of PAHs and hence biodegradation by microbes. This property is also of interest because a number of biodegradative microbes exhibit positive chemotaxis towards the pollutants [44]. Therefore, the combined action of biosurfactant and chemotaxis can contribute to bacterial proliferation and to microbial spread in polluted soils, in order that

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Microbial degradation of contaminants in the rhizosphere provides a positive effect for the plant; the pollutant concentration is decreased in the area near the roots and the plant can grow better than those in contaminated areas [101]. Because of this mutual benefit it has been proposed that plants can select specific genotypes to be present in their roots. Experiments performed by Siciliano *et al.*[102] demonstrated that the presence of the alkane monooxygenase gene was more prevalent in endophytic and rhizosphere microbial communities than in those present in bulk soil contaminated with hydrocarbons. However, the results obtained when they studied the prevalence of the xylene monoxygenase or naphthalene dioxygenase genes were just the opposite, their presence was higher in bulk soil microbial communities than those near or on the plant. This suggests that if plants are influencing the rhizosphere, this effect is dependent on the contaminant. Some researchers also concluded that the effect depended on the type of the plant. This has led to the hypothesis that the effectiveness of rhizoremediation

strategies is related to the selection of the best plant bacterium pair in each case.

In a case study it was found that rhizospheric *Pseudomonas* sp. of *Calotropis* plant a good degrader for naphthalene (78.44 %) and anthracene (63.53 %) as determined by HPLC analysis. Thus, it can be concluded that rhizosphere of *Calotropis* sp. is a source of *Pseudomonas* sp. possessing potent PGP attributes, PAH degradation and biocontrol activities against phyto‐ pathogenic fungi. Further studies are under- way to confirm their effectiveness in field

A number of scientists established chemotaxis of PAHs degrading rhizosphere bacteria (*P. alcaligenes, P. stutzeri and P. putida*) to naphthalene, phenanthrene and root exudates [104]. Fascinatingly, the bacteria were repelled by anthracene and pyrene. The attraction of compe‐ tent bacteria to the root zone may improve bioavailability and increase PAHs degradation in the rhizosphere. Subjugation of the phenanthrene degrading activity of *P. putida* following exposure to root extracts and exudates recommended that enzyme induction may not occur during rhizodegradation of PAHs [92]. Genetically engineered plant microbial systems to improve the rhizoremediation techniques, in which the gene cloning of plants containing bacterial gene for the degradation of organic pollutants and of recombinant, root-colonizing bacteria (e.g. *P. fluorescens*) expressing degradative enzymes e.g. orthomonooxygenase for

The studies pertaining to the rhizospheric microorganism associated with specific plant are still missing in the available literature even though a lot of work has been reported on bioremediation. Many researchers have carried out work on plant growth promoting (PGP) activity of rhizosphere of different plants, but no information about rhizosphere community of specific plant, its molecular characterization and utilization in sustainable agriculture, biofertilization and ecorestoration is reported in the literature. Rhizoremediation can be

more ample zones can be cleaned [28].

conditions [103].

toluene degradation [25].


**Table 1.** Some important enzymes associated with bioremediation [26].

#### **11. Improvement in rhizoremediation**

Rhizoremediation process can be designed to improve in several aspects like bioavailability of contaminant molecules, expression and maintenance of genetically engineered plantmicro‐ bial systems and root exudates for the effectiveness of the process.

Selection of bacteria, which are able to produce biosurfactants in the rhizosphere of the plants, is an interesting alternative to improve the removal efficiency [85]. In this context Kuiper *et* *al.* [48] identified bacteria growing in a PAHs contaminated area that produces biosurfactants that facilitate the solubilisation of PAHs and hence biodegradation by microbes. This property is also of interest because a number of biodegradative microbes exhibit positive chemotaxis towards the pollutants [44]. Therefore, the combined action of biosurfactant and chemotaxis can contribute to bacterial proliferation and to microbial spread in polluted soils, in order that more ample zones can be cleaned [28].

**10. Microbial enzymes involved in PAHs degradation process**

*Pseudomonas putida* encoded by the NAH plasmid pDTG1 [100].

340 Applied Bioremediation - Active and Passive Approaches

As bacteria initiate PAHs degradation by the action of intracellular dioxygenases, oxygenase, dehydrogenase, phosphatases, dehalogenases, nitrilases, nitroreductases and lignolytic enzymes [33] (Table 1). Aromatic ring dioxygenases are multicomponent enzymes which consist of an electron transport chain containing a ferredoxin and a reductase and a terminal dioxygenase [99]. The best studied PAHs dioxygenase is naphthalene dioxygenase from

**Enzyme Target pollutant** Aromatic dehalogenase Chlorinated aromatics (DDT, PCBs etc.)

Dehalogenase Chlorinated solvents and Ethylene

Laccase Oxidative step in degradation of explosives

Carboxyl esterases Xenobiotics Cytochrome P450 Xenobiotics (PCBs)

Glutathione s-transferase Xenobiotics Peroxygenases Xenobiotics Peroxidases Xenobiotics

N-glucosyl transferases Xenobiotics

N-malonyl transferases Xenobiotics

O-glucosyl transferases Xenobiotics O-malonyl transferases Xenobiotics Peroxdase Phenols Phosphatase Organophosphates

**Table 1.** Some important enzymes associated with bioremediation [26].

bial systems and root exudates for the effectiveness of the process.

**11. Improvement in rhizoremediation**

Nitrilase Herbicides Nitroreductase Explosives (RDX and TNT)

O-demethylase Alachlor, metalachor

Rhizoremediation process can be designed to improve in several aspects like bioavailability of contaminant molecules, expression and maintenance of genetically engineered plantmicro‐

Selection of bacteria, which are able to produce biosurfactants in the rhizosphere of the plants, is an interesting alternative to improve the removal efficiency [85]. In this context Kuiper *et* Microbial degradation of contaminants in the rhizosphere provides a positive effect for the plant; the pollutant concentration is decreased in the area near the roots and the plant can grow better than those in contaminated areas [101]. Because of this mutual benefit it has been proposed that plants can select specific genotypes to be present in their roots. Experiments performed by Siciliano *et al.*[102] demonstrated that the presence of the alkane monooxygenase gene was more prevalent in endophytic and rhizosphere microbial communities than in those present in bulk soil contaminated with hydrocarbons. However, the results obtained when they studied the prevalence of the xylene monoxygenase or naphthalene dioxygenase genes were just the opposite, their presence was higher in bulk soil microbial communities than those near or on the plant. This suggests that if plants are influencing the rhizosphere, this effect is dependent on the contaminant. Some researchers also concluded that the effect depended on the type of the plant. This has led to the hypothesis that the effectiveness of rhizoremediation strategies is related to the selection of the best plant bacterium pair in each case.

In a case study it was found that rhizospheric *Pseudomonas* sp. of *Calotropis* plant a good degrader for naphthalene (78.44 %) and anthracene (63.53 %) as determined by HPLC analysis. Thus, it can be concluded that rhizosphere of *Calotropis* sp. is a source of *Pseudomonas* sp. possessing potent PGP attributes, PAH degradation and biocontrol activities against phyto‐ pathogenic fungi. Further studies are under- way to confirm their effectiveness in field conditions [103].

A number of scientists established chemotaxis of PAHs degrading rhizosphere bacteria (*P. alcaligenes, P. stutzeri and P. putida*) to naphthalene, phenanthrene and root exudates [104]. Fascinatingly, the bacteria were repelled by anthracene and pyrene. The attraction of compe‐ tent bacteria to the root zone may improve bioavailability and increase PAHs degradation in the rhizosphere. Subjugation of the phenanthrene degrading activity of *P. putida* following exposure to root extracts and exudates recommended that enzyme induction may not occur during rhizodegradation of PAHs [92]. Genetically engineered plant microbial systems to improve the rhizoremediation techniques, in which the gene cloning of plants containing bacterial gene for the degradation of organic pollutants and of recombinant, root-colonizing bacteria (e.g. *P. fluorescens*) expressing degradative enzymes e.g. orthomonooxygenase for toluene degradation [25].

The studies pertaining to the rhizospheric microorganism associated with specific plant are still missing in the available literature even though a lot of work has been reported on bioremediation. Many researchers have carried out work on plant growth promoting (PGP) activity of rhizosphere of different plants, but no information about rhizosphere community of specific plant, its molecular characterization and utilization in sustainable agriculture, biofertilization and ecorestoration is reported in the literature. Rhizoremediation can be successfully used for restoration of contaminated sites by choosing right type of plant cultivar with right rhizobacteria or by inoculating efficient rhizobacterial strains on plant seeds [34]. Bacteria inhabiting the rhizosphere of a suitable plant may be used as 'bacterial injection system' in soils for effective growth promotion and rhizoremediation.

[7] Wakeham, S.G., Schaffner, C. and Giger, W. Polycyclic aromatic hydrocarbons in re‐ cent lake sediments II compound having anthropogenetic origins. Geochimica et

Rhizoremediation: A Promising Rhizosphere Technology

http://dx.doi.org/10.5772/56905

343

[8] Freeman, D. J. and Cattell, F. C. R. Wood burning as a source of atmospheric polycy‐ clic aromatic hydrocarbons. Environmental Science and Technology 24, (1990),

[9] Oviasogie, P. O., Ukpebor, E. E. and Omoti, U. Distribution of polycyclic aromatic hydrocarbons in rural agricultural wetland soils of the Niger Delta Region. African

[10] Kanaly, R. A. and Harayama,S. Biodegradation of high molecular weight polycyclic aromatic hydrocarbons by bacteria. Journal of Bacteriology 182, (2000), 2059-2067.

[11] Jones, K. C., Stratford, J. A., Waterhouse, K. S., Furlong, E. T., Giger, W., Hites, R. A., Schaffner, C. and Johbston, A. E. Increases in the polynuclear aromatic hydrocarbon content of an agricultural soil over the last century. Environmental Science and Tech‐

[12] Cerniglia, C. E. and Heitkamp, M. A. Microbial degradation ofpolycyclic aromatic hydrocarbons in the aquatic environment. In: Varanasi, U. (Ed.), metabolism of poly‐ cyclic aromatic hydrocarbons in the aquatic environment. CRC Press, Boca Raton,

[13] Nishioka, M., Chang, H.C., Lee, V. Structural characteristics of polycyclic aromatic hydrocarbon isomers in coal tars and combustion products. Environmental Science

[14] Lijinsky, W. The formation and occurence of polynuclear aromatic hydrocarbons as‐

[15] Walter, U., Beyer, M., Klein, J. and Rehm, H. J. Degradation of pyrene by Rhodococ‐

[16] Ramadah, T., Alfheim, I., Rustad, S. and Olsen, T. Chemical and biological characteri‐ sation of emissions from small residential stoves burning wood and charcoal. Che‐

[17] Juhasz A. L. and Naidu R. Bioremediation of high molecular weight polycyclic aro‐ matic hydrocarbons: A review of the microbial degradation of benzo[a]pyrene. Inter‐

[18] Guoa, H., Leea, S.C., Hoa, K.F., Wangb, X.M. and Zou, S.C. Particle associated poly‐ cyclic aromatic hydrocarbons in urban air of Hong Kong. Atmospheric Environment

[19] Shiaris, M. P. and Jambard, S. D. Polycyclic aromatic hydrocarbons in surficial sedi‐ ments of Boston Harbour, MA, USA.Marine Pollution Bulletin 17, (1986), 469-472.

sociated with food. Mutation Research 259, (1991), 251-262.

national Biodeterioration & Biodegradation 45, (2000), 57-88.

cus sp. Applied Microbiology and Biotechnology 34, (1991), 671-676.

Cosmochimica Acta 44, (1980), 403-413.

Journal of Biotechnology 5, (2006), 1415-1421.

1581-1585.

nology 23, (1989), 95-101.

and Technology 20, (1986), 1023-1027.

mosphere 11, (1982), 601-611.

37, (2003), 5307-5317.

(1989), pp. 42-64.
