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

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]. 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

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

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

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

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

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

xenobiotics [77, 78, 81].

18 Applied Bioremediation - Active and Passive Approaches

CBAs in plant tissues.

pH of the culture medium [82].

have not demonstrated metabolisation.

**3.1. Plant metabolization of chlorobenzoic acids**

plants.

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

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 polysaccharides [90].

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 was even half than in the control without exudates.

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 is source of nutrients for plant e.g. nitrogen compounds. Microorganisms may also increase the solubility of minerals such as phosphorus, and thus make them more accessible for plants. Another mechanism of protection of plants by microorganisms is the synthesis of low molec‐ ular weight siderophores in addition to phytosiderophores. Siderophores bind the free iron in the rhizosphere of plants. Lack of free iron negatively affects the proliferation of fungal pathogens in the root system of plants [93, 94].The presence of microorganisms can also initiate the synthesis of plant hormone such as auxins, cytokinins and gibberellins, which promote the growth of plants [91, 95]. Equally important is the mechanism for reducing stress by reduction of stress hormone ethylene concentration in plant by synthesis of microbial enzyme 1 aminocyklopropan-1-carboxylate deaminase (ACC deaminase) [96].

consortium, thus *Elimus dauricus* and strains of *Pseudomonas aeruginosa* R75 and *Pseudomonas savastanoi* CB35 was able to remove 74% of 3-CBA from the soil. *Bromus biebersteinii* with strain *Alcaligenes* sp. BR60 remove 56% of 2,3-CBA from the soil and the same consortium was able to metabolize 61% of 2,3-CBA and 50% of 3-CBA if both CBAs were added together. A consortium consisting of *Elymus angitus* and strains *Pseudomonas aeruginosa* R75 and *Pseudo‐ monas savastanoi* CB35 eliminate 46% of 2,5-CBA from soil and from the mixture of 3-CBA, 2,3- CBA and 2,5-CBA this consortium has removed around 40% of all three CBAs from soil [99]. These results were obtained by testing a large number of combinations of grasses with bacterial inoculants. Most of the combinations did not show the positive impact on reducing the tested CBA or a mixture of CBAs from soil. Inoculants did not reduce CBA phytotoxicity and caused

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 21

The above examples show that the use of plants and microorganisms in bioremediation technologies has great potential. However, selection of suitable pair of plant and microorgan‐ ism is very complicated. We can assume that not only depend on the type of contamination, but it requires a deeper understanding of the principles of cooperation between this two

The work was supported by the grant GAČR 525/09/1058 and EU project MINOTAURUS (FP7-

, Martina Macková, Tomáš Macek and Kateřina Demnerová

Institute of Chemical Technology, Prague, Faculty of Food and Biochemical Technology, De‐

[1] Horvath RS Cometabolism of the herbicide 2trichlorobenzoate. Journal of Agricultur‐

[2] Holtze, M. S, Sørensen, S. R, & Sørensen, J. Aamand J Microbial degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil and subsurface

plant death in contaminated soil.

organisms.

**Acknowledgements**

KBBE-2010-4/265946).

**Author details**

Blanka Vrchotová\*

**References**

\*Address all correspondence to: vrchotob@vscht.cz

partment of Biochemistry and Microbiology, Prague, Czech Republic

al and Food Chemistry. (1971). , 19(2), 291-293.

During the life of the plant its growth may be influenced by microorganisms with one or more of these mechanisms. Influence is most evident in plants grown under stressful conditions.

In a consortium consisting of microorganisms and plants growing in the contaminated soil, in addition to the above mentioned mechanisms, can occur production of metabolites or inter‐ mediates of degradation no matter whether by plant or microorganism. These metabolites may be by the other partner further degraded and metabolized or they may be toxic. The study of the metabolism of both biological systems and their possible influence should lead to the obtaining of such a system consisting of plant - microorganisms, which increases speed and efficiency of the xenobiotics degradation process.

#### **4.1. Plant and microbial cooperation on chlorobenzoic acids remediation**

The issue of co-operation of plants and microorganisms on degradation of CBA is solved in several publications.

Haby and Crowley [84] compared the degradation capacity of soil from the rhizosphere of perennial ryegrass (*Lolium perenne*) with non-vegetated soil. Results showed that the degrada‐ tion rate of 3-CBA was at the beginning of the experiment accelerated by the rhizosphere soil, they also showed that in soil from the rhizosphere is increased amount of microorganisms [84].

Dittmann et al. [97] examined the ability of three months old pine seedlings (*Pinus sylvestris*) with mycorrhizal fungi *Suillus bovinus* to degrade 3-CBA. They found that 3-CBA is accumu‐ lated in the aboveground parts of the seedlings and removal efficiency of 3-CBA from the soil by a consortium was very low (around 25% for 4 weeks). In plant tissues were not detected elevated concentrations of Cl in comparisom with control plants grown in uncontaminated soil. Therefore they concluded that 3-CBA was not or only to a very small extent degraded by plants [97].

The aforementioned grass *Elimus dauricus*, together with microorganismus *Pseudomonas aeruginosa* R75 and *Pseudomonas savastanoi* CB35 was able to remove almost 50% of 2-CBA from soil during 56 days [98]. The consortium showed greater metabolic efficiency than nonvegetated soil.

In this work has been continued testing the degradation capacity of microorganisms and plants with other grasses and other CBAs or with a mixture of CBAs. The same composition of the consortium, thus *Elimus dauricus* and strains of *Pseudomonas aeruginosa* R75 and *Pseudomonas savastanoi* CB35 was able to remove 74% of 3-CBA from the soil. *Bromus biebersteinii* with strain *Alcaligenes* sp. BR60 remove 56% of 2,3-CBA from the soil and the same consortium was able to metabolize 61% of 2,3-CBA and 50% of 3-CBA if both CBAs were added together. A consortium consisting of *Elymus angitus* and strains *Pseudomonas aeruginosa* R75 and *Pseudo‐ monas savastanoi* CB35 eliminate 46% of 2,5-CBA from soil and from the mixture of 3-CBA, 2,3- CBA and 2,5-CBA this consortium has removed around 40% of all three CBAs from soil [99]. These results were obtained by testing a large number of combinations of grasses with bacterial inoculants. Most of the combinations did not show the positive impact on reducing the tested CBA or a mixture of CBAs from soil. Inoculants did not reduce CBA phytotoxicity and caused plant death in contaminated soil.

The above examples show that the use of plants and microorganisms in bioremediation technologies has great potential. However, selection of suitable pair of plant and microorgan‐ ism is very complicated. We can assume that not only depend on the type of contamination, but it requires a deeper understanding of the principles of cooperation between this two organisms.
