**3. Phytoremediation**

Together with the microorganisms participate on the elimination of xenobiotics green plants. Green plants use for the xenobiotics elimination four different strategies: extraction of contaminants from soil and water (mostly heavy metals), uptake and detoxication, in some cases even degradation (organic pollutants), volatilization (organic compounds and some metaloids Se and As) and stimulation of microbial degradation in rhizosphere or by endo‐ phytic microorganisms. All this processes are called phytoremediation (Figure 17.), the use of green plants for removal or transformation of pollutants from the environment [76, 77].

controversial because it does not reduce contamination, only transfer pollutants from soil to

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 17

Rhizodegradation is stimulation of degrading microorganisms in plant rhizosphere. This stimulation can be done by support of proliferation or by creation of conditions suitable for

Phytodegradation is a process when pollutants are taken up into the plant body, transformed and eliminated by plant metabolism. Phytodegradation can be considered also as a process in which contamination is reduced by enzymes released by the plant into the soil. Phytodegra‐

Plant metabolism of xenobiotics is in many aspects similar to the mammalian metabolism, therefore plants are called green liver of the planet. Uptake of xenobiotics is done by roots, from roots are then xenobiotics transported by xylem to the aboveground (harvestable) parts of plant, where xenobiotics are transformed to the non-phytotoxic metabolites. Metabolisation of xenobiotics in plant body is done in three phases. First is transformation followed by

better survival of microorganisms with degradation activity [47, 76].

**Figure 17.** Schematic presentation of phytoremediation processes, adapted from [47].

conjugation and the last phase is sequestration in plant tissues.

dation is most suitable for organic pollutants [47].

the air [47, 76].

Phytoextraction is an accumulation of substances from the environment to the plant biomass. Precondition for the successful application of this process is that the plant is capable to take up contaminant by roots and ideally transport it to the aboveground parts, where contaminant is deposited in relatively high concentrations (more than 1 g per 1 kg of plant biomass). The disadvantage of this process is the possibility to re-release of contaminants from plant biomass, and their introduction into the food chain in the case of animals grazing on such plants [47, 76-78].

Phytovolatilization is uptake of pollutant by plant, which is followed by pollutant conversion to the volatile form and release in to the atmosphere [78, 79]. This type of phytoremediation is

**Figure 17.** Schematic presentation of phytoremediation processes, adapted from [47].

degradation pathway (Figure 1.). How was proved by accumulation of hydroxylated biphen‐

Acylchloride can be also irreversibly bound on catechol or chlorocatechol-2,3-dioxygenase, the first enzyme in the *meta*- cleavage pathway. Its inactivation leads to catechols or chlorocate‐

Another potentially dangerous intermediate is protoanemonine, substance with antibiotic properties. Protoanemonine is formed in the modified *ortho*- cleavage pathway from muconic or chloromuconic acid. The main effect of protoanemonine is growth inhibition of degrading

The presence of a mixture of CBAs can also cause inhibition or activation of CBA degradation. Strain *Burkholderia cepacia* JHR22 can degrade 2-CBA, 3-CBA, 4-CBA and 3,5-CBA [44] when present individually. This strain loses 2-CBA degradation ability when 2-CBA is present in mixture with 2,3-CBA or 3,4-CBA. On the other hand addition of 2,5-CBA or 2,6-CBA had no effect on 2-CBA degradation. When was 2-CBA added with 2,4-CBA strain *Burkholderia cepacia* JHR22 in addition to the 2-CBA degradation can cometabolically degrade 2,4-CBA. Catechols or chlorocatechols are also potential inhibitors of bacterial CBAs degradation. If these intermediates of CBAs degradation are not enough quickly degraded they can be subject to the auto-oxidation or enzymatic polymerization with formation of brown or black pigment [73, 74]. The presence of this pigment inhibits CBAs degradation by affecting the shape of bacterial cells which may consequently lead to their death as in case of strain *Pseudomonas*

Together with the microorganisms participate on the elimination of xenobiotics green plants. Green plants use for the xenobiotics elimination four different strategies: extraction of contaminants from soil and water (mostly heavy metals), uptake and detoxication, in some cases even degradation (organic pollutants), volatilization (organic compounds and some metaloids Se and As) and stimulation of microbial degradation in rhizosphere or by endo‐ phytic microorganisms. All this processes are called phytoremediation (Figure 17.), the use of green plants for removal or transformation of pollutants from the environment [76, 77].

Phytoextraction is an accumulation of substances from the environment to the plant biomass. Precondition for the successful application of this process is that the plant is capable to take up contaminant by roots and ideally transport it to the aboveground parts, where contaminant is deposited in relatively high concentrations (more than 1 g per 1 kg of plant biomass). The disadvantage of this process is the possibility to re-release of contaminants from plant biomass, and their introduction into the food chain in the case of animals grazing on such plants [47,

Phytovolatilization is uptake of pollutant by plant, which is followed by pollutant conversion to the volatile form and release in to the atmosphere [78, 79]. This type of phytoremediation is

yls during PCBs degradation by strain *Pseudomonas testosteroni* B-356 [16].

chols accumulation and therefore to the inhibition of CBAs degradation [71].

microorganisms[72].

16 Applied Bioremediation - Active and Passive Approaches

*fluorescens* [75].

76-78].

**3. Phytoremediation**

controversial because it does not reduce contamination, only transfer pollutants from soil to the air [47, 76].

Rhizodegradation is stimulation of degrading microorganisms in plant rhizosphere. This stimulation can be done by support of proliferation or by creation of conditions suitable for better survival of microorganisms with degradation activity [47, 76].

Phytodegradation is a process when pollutants are taken up into the plant body, transformed and eliminated by plant metabolism. Phytodegradation can be considered also as a process in which contamination is reduced by enzymes released by the plant into the soil. Phytodegra‐ dation is most suitable for organic pollutants [47].

Plant metabolism of xenobiotics is in many aspects similar to the mammalian metabolism, therefore plants are called green liver of the planet. Uptake of xenobiotics is done by roots, from roots are then xenobiotics transported by xylem to the aboveground (harvestable) parts of plant, where xenobiotics are transformed to the non-phytotoxic metabolites. Metabolisation of xenobiotics in plant body is done in three phases. First is transformation followed by conjugation and the last phase is sequestration in plant tissues.

During transformation phase xenobiotics are subjected to enzymatic hydrolysis, oxidation or reduction. Reactive groups exposed or generated in transformation stage react with moieties such as glucuronate or glutathione in conjugation phase. Soluble conjugates are transported in the final stage to the vacuole or incorporated into the cell wall, thus preventing disturbing of the cell function by them [80]. Some water plants can transport conjugates outside the plant [47].

removed 100% of the mentioned CBAs. Culture of black nightshade with the same efficiency metabolized 3-CBA, 4-CBA and 2,3,5-CBA. Culture of horseradish metabolized other tested

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 19

Siciliano and Germida [83] tested the ability of 16 different kinds of grasses grow in soil contaminated with 2-CBA (816 mg/kg). Only five of them had this ability, namely it were grasses *Bromus inermis*, *Agropyron intermedium*, *Bromus biebersteinii*, *Agropyron riparum* a *Elymus dauricus*. Only four last mentioned species were also able to metabolize 2-CBA from soil with

From these examples of CBAs metabolisation in plants is evident, that this process is not only influenced by the structure and concentration of CBA, but it is also influenced by plant species

There are known several mechanisms for the promotion of the contamination removal from soil by plants. One of these mechanisms is a non-specific support of growth and proliferation of microbial communities by plants [84]. Support of microbial activity in the rhizosphere leading to the protection of plants from the effects of contamination and increasing the speed of xenobiotics elimination [85]. Another mechanism is specific support of degrading microor‐ ganisms in the rhizosphere of plants [86, 87], secretion of enzymes capable of catalyzing the

Plants can promote the growth of microorganisms in the rhizosphere by excretion of root exudates into the soil [89]. Exudates may contain various ions, free oxygen, water, enzymes (e.g. laccase, peroxidase, dehalogenase, nitroreductase), substances of the saccharide nature to facilitate root growth and diverse mixes of primary and secondary metabolites. Organic compounds in exudates are divided into two groups. One is for substances with low molecular weights such as amino acids, organic acids, sugars, phenolic compounds and other secondary metabolites and second is for substances of higher molecular weight such as proteins and

The problem of the use of the cooperation in bioremediation is that the increase in the number of microorganisms in the rhizosphere of plants does not always mean increased degradation of the contamination. An example of this behavior is experiment when exudates of perennial ryegrass (*Lolium Perence*) were used in the degradation of phenanthrene [89]. Results showed that despite the increased number of microorganisms caused by addition of exudates, no increased phenanthrene degradation was observed. Phenanthrene degradation in this case

Just as plants can affect the growth of microorganisms, microorganisms can also affect the growth and survival of plants in the contaminated areas. Microorganisms can help plants by production of protective biofilms or antibiotics acting as a protection against organisms potentially pathogenic for plants [91, 92]. In addition, the activity of certain microorganisms

CBAs with 30-60% efficiency [10].

efficiency from 32 to 42% in 60 days [83].

and plant capacity to "resist" the toxicity of CBA.

conversion of contaminants in the soil by plants [88].

was even half than in the control without exudates.

polysaccharides [90].

**4. Plant and microbial cooperation on bioremediation**

Using plants for decontamination has many advantages. It is a cost-saving process. After planting the area, are the costs for growing and harvesting biomass relatively low, and the obtained biomass can also be used to produce heat or electricity. It is aesthetically pleasing process positively accepted by the public. It causes minimal disruption to the environment. It is also applicable to the areas with trace or low pollution and is usable for a wide range of xenobiotics [77, 78, 81].

Phytoremediation has also drawbacks. The main disadvantage is that phytoremediation compared with physico-chemical methods is slower. It should take a few years to eliminate pollution and during the process most of the decontaminated areas can not be used for commercial purposes [78]. Therefore, the goal of the phytoremediation study is to increase the efficiency and speed of this process, by the understanding and improvement of the mechanism of phytoremediation and metabolic pathways involved in the conversion of xenobiotics by plants.

#### **3.1. Plant metabolization of chlorobenzoic acids**

About the CBAs metabolism in plants it is known, in comparison with microbial metabolism, very little. Although works have been published that deal with the CBA metabolisation in plants, it was not yet clearly demonstrated that plants degrade CBAs and not just accumulate CBAs in plant tissues.

Deavers et al. [82] investigated the metabolism of 4-CBA by cells of willow (*Salix viminalis*) under sterile conditions, as well as by whole plants. Cells were able to remove 65% of 4-CBA from the media during 360 hours with an initial concentration of 4-CBA 50 mg/l. For the whole willow plants, the concentration 50 mg/l of 4-CBA was toxic, and therefore the loss of 4-CBA was only in the range of 10-30%. When was initial concentration of 4-CBA reduced to 5 mg/l, willow plants managed during 305 hours to remove 70-90% of 4-CBA in dependence on the pH of the culture medium [82].

Other uses of the plants for metabolisation of CBAs has been described in plant tissue cultures of black nightshade (*Solanum nigrum*), tobacco (*Nicotiana tabacum*), horseradish (*Armoracia rusticana*) and alfalfa (*Medicago sativa*) [10]. In this case, the two initial concentrations were tested, 200 and 50 mg/l, and 11 different CBAs. From tested plant species the best ability to metabolize CBAs demonstrated black nightshade and horseradish. For *in vitro* grown cells of tobacco and alfalfa was the concentration of CBAs 200 mg/l toxic. So that named plant cells have not demonstrated metabolisation.

Tissue cultures of black nightshade and horseradish were within 14 days able to remove from the medium 90% of 2-CBA and about 30% of 2,3-CBA, 2,4-CBA and 2,5-CBA at an initial concentration of 200 mg/l, if the initial concentration was reduced to 50 mg/l this cell cultures removed 100% of the mentioned CBAs. Culture of black nightshade with the same efficiency metabolized 3-CBA, 4-CBA and 2,3,5-CBA. Culture of horseradish metabolized other tested CBAs with 30-60% efficiency [10].

Siciliano and Germida [83] tested the ability of 16 different kinds of grasses grow in soil contaminated with 2-CBA (816 mg/kg). Only five of them had this ability, namely it were grasses *Bromus inermis*, *Agropyron intermedium*, *Bromus biebersteinii*, *Agropyron riparum* a *Elymus dauricus*. Only four last mentioned species were also able to metabolize 2-CBA from soil with efficiency from 32 to 42% in 60 days [83].

From these examples of CBAs metabolisation in plants is evident, that this process is not only influenced by the structure and concentration of CBA, but it is also influenced by plant species and plant capacity to "resist" the toxicity of CBA.
