soil remediation and plants || improving phytoremediation of soil polluted with oil hydrocarbons in...

547Soil Remediation and Plants. http://dx.doi.org/10.1016/B978-0-12-799937-1.00019-XCopyright © 2015 Elsevier Inc. All rights reserved.

Improving Phytoremediation of Soil Polluted with Oil Hydrocarbons in Georgia

Gia Khatisashvili,* Lia Matchavariani† and Ramaz Gakhokidze‡

*Durmishidze Institute of Biochemistry and Biotechnology at Agricultural University of Georgia, Laboratory of Biological Oxidation, Tbillisi, Georgia; †Department of Soil Geography Faculty of Exact & Natural Sciences, Tbilisi State University of Iv. Javakhishvili, Tbillisi, Georgia; ‡Department of Bioorganic Chemistry, Faculty of Exact & Natural Sciences, Tbilisi State University of Iv. Javakhishvili, Tbillisi, Georgia

INTRODUCTION

One of the main sources of environmental contamination is determined by petroleum production, transportation, refining and accidental waste (Korte et al., 1992). In Georgia, which is the transit route of different goods transportation between East and West, the Baku–Supsa and Baku–Tbilisi–Ceyhan oil pipelines in particular pose great danger of contamination with oil hydrocarbons and require the creation of a special ecological technology of environmental remediation and protection.

Due to the great power of natural detoxification processes, interest in the ecological potential of microorganisms and plants has increased in the last two decades (Arthur and Coats, 1998; Salt et al., 1998; Tsao, 2003b; Kvesitadze et al., 2006). Microorganisms that transform organics play an important role in maintaining the ecological balance in various ecosystems and, due to their high degradation and transformation powers, are successfully used for sewage and soil purification. Plants actively participate in soil and air remediation processes. Plants and microorganisms, together or individually, mainly through their pow-erful oxidative enzyme systems, are capable of remediating environments pol-luted by a wide spectrum of contaminants.

Phytoremediation is a unique clean-up strategy (Tsao, 2003b; Kvesitadze et al., 2006). The realization of phytoremediation technologies implies the plant-ing of a contaminated area with one or more specific, previously selected species of plants having the potential to extract contaminants from the soil. The treatment continues by harvesting the plants and composting or incinerating them. To create a truly effective phytoremediation system, all components of the system should

Chapter 19

548 Soil Remediation and Plants

be thoroughly analyzed. The major constitutive component of such a system is obviously the plant. The goal of plant selection is to choose a definite plant spe-cies with appropriate characteristics. A survey of site vegetation should be under-taken to determine which species of plants would have the best growth on the contaminated site, taking into account the ability of the plants to accumulate and degrade the contaminants (Korte et al., 2000; Kvesitadze et al., 2001, 2006).

Evidence that plant roots and the rhyzosphere-associated microbial commu-nity are capable of enhancing the degradation of petroleum chemicals in soils pro-vides a potentially important approach for the in situ treatment of contaminated sites. Vegetation may act to immobilize water-soluble contaminants, increase their stability in soil structure, and create a favorable environment for degrada-tive microorganisms. Before phytoremediation can be practically or efficiently employed, more research is needed to reveal the basic mechanisms involved.

The combined impact of plant and microbes of soil pollutants is numerous, and many attempts have been made to manipulate enhancing contaminant deg-radation. The approach we searched for in our investigation refers to rhizoaug-mentation, which is the addition of hydrocarbon-degrading microorganisms to soil with the intention of them associating with rhizospheres of plants involved in remediation (Tsao, 2003a; Kirk et al., 2005; Shaw and Burns, 2007). How-ever, we have enhanced the aims of such rhizoaugmentation by using not only simple hydrocarbon-degrading microorganisms, but with those which demon-strate an ability to produce biosurfactants (Shin et al., 2006).

The rhyzosphere is a unique environment where soil conditions are much different than in bulk soil (Burgmann et al., 2005). Loading of carbon substrates from root exudates can provide a potentially attractive environment for bacteria as an initiating agent among hardly consumed petroleum hydrocarbons. Under-standing how root exudates influence soil populations that have the ability to degrade different petroleum hydrocarbons may help our understanding of the phytoremediation strategy.

The main idea of the work carried out is to create a database for development of a novel approach for the ecological safety of oil pipelines and the rehabilita-tion of oil-polluted sites through biological treatment. The approach is based on a combined application of plant and microbial hydrocarbons degradation potential for remediation and long-term protection of the environment.

The success of this work should promote the solution of existing problems of hydrocarbon contamination in soil and water reservoirs. First, an ecologi-cal preservation strategy for oil sources near oil reservoirs and along pipelines will be created, to prevent hydrocarbons spreading. Second, the fundamental problem of the metabolic mechanisms of hydrocarbon degradation (including that of aromatic compounds) by plants and microorganisms will be thoroughly investigated. Third, commercially valuable technologies and recommendations to preserve the environment from hydrocarbons will be created. The results will promote the preservation of the ecology along existing pipelines and those under construction in Georgia.

549Chapter | 19 Improving Phytoremediation of Soil Polluted

CHARACTERIZATION OF SOIL TYPES

The oil pipelines (Baku–Supsa and Baku–Tbilisi–Ceyhan), crossing over the most part of the territory of Georgia pass through 23 different types of soils, which are characterized by various physico-chemical and biological characteristics.

On development of an environmental protection strategy against contami-nation with petroleum hydrocarbons in the case of damage overflow, different adsorption features of soils should be taken into consideration. The risk level of total petroleum hydrocarbons (TPH) penetration into ground waters should also be estimated (Kvesitadze et al., 2008). To this end, the capability of soil samples to adsorb oil hydrocarbons was studied. The soil samples that represented 13 different types of soils were collected in Georgia from different sites along the Baku–Tbilisi–Ceyhan and Baku–Supsa Oil Pipelines (Figure 19.1).

The results showed that investigated soil types differently adsorb hydrocar-bons. In particular, they may be divided into three groups:

1. Soils from which oil hydrocarbons are washed out rapidly. Oil contamina-tion of such types of soils represents a danger to penetration of more than 70% of hydrocarbons into ground waters under the influence of atmospheric precipitation. Soils which belong to these types of soils along the pipelines passing through the territory of Georgia are as follows:

l Alluvial calcareous (Calcaric fluvisols); l Yellow brown forest (Chromic cambisols and stagnic alisols); l Cinnamonic calcareous (Calcaric cambisols and Calcic kastanozems); l Raw humus calcareous (Rendzic leptosols); l Cinnamonic (Eutric cambisols and Calcic kastanozems). 2. Soils which have average ability of TPH absorption. From these soils

50–70% of oil hydrocarbons are washed out by water. Such types of soils are: l Meadow grey cinnamonic (Calcic vertisols); l Grey cinnamonic (Calcic kastanozems); l Chernozems (Chernozems); l Brown forest weakly unsaturated (Eutric cambisols); l Cinnamonic light (Calcic kastanozems); l Black calcareous (Calcic vertisosls). 3. Soils distinguished by strong TPH adsorption ability. Contamination in such

types of soils is maintained over a long period of time at less than 50%. In the territory of Georgia such soil types are as follows:

l Mountain meadow soddy (Leptosols, cambisols and cryosols); l Brown forest podzolized (Dystric cambisols).

Thus, during oil contamination, considerable amounts of hydrocarbons might appear in groundwaters from soils of types Alluvial calcareous, Yellow brown forest, Cinnamonic calcareous, Raw humus calcareous and Cinnamonic under the influence of atmospheric precipitation.


SELECTION OF MICROORGANISMS

The use of microorganisms during soil bioremediation after oil spills and other contaminations is one of the most ecologically progressive approaches compared with physico-chemical treatments, which usually result in sharp disfunction of soil ecosystems. Plenty of bacteria, actinobacteria, microscopic fungi and yeasts are well-known degradants of petroleum hydrocarbons, At the present time the active search is on for the methods of intensification of biological degradation of oil hydrocarbons in ground and water. The microbiological method based on introduction of active oil- oxidizing microorganisms into contaminated environment is the most promising. For realization of this method it is necessary to select the most active microorganisms adapted to high concentrations of oil, and to develop the technology of their use by taking into account the complexity of soil properties and the level of pollution.

A large amount of hydrocarbon-degrading bacteria and fungi have been isolated from the contaminated and non-contaminated soils along the pipeline: 360 bacterial and 200 fungal strains were obtained and purified, and their taxonomic affiliations identified.

FIGURE 19.1 Baku–Supsa and Baku–Tbilisi–Ceyhan Oil Pipelines in the territory of Georgia. Sampling points of soils are indicated on the map. The types of soils are as follows: (1) Meadow grey cinnamonic (Calcic vertisols) (pH 5.2); (2) Grey cinnamonic (Calcic kastanozems) (pH 4.7); (3) Alluvial calcareous (Calcaric fluvisols) (pH 4.5); (4) Raw humus calcareous (Rendzic leptosols) (pH 6.0); (5) Chernozems (Chernozems) (pH 4.5); (6) Mountain meadow soddy (Leptosols, cambisols and cryosols) (pH = 4.0); (7) Brown forest podzolized (Dystric cambisols) (pH 4.5); (8) Brown forest weakly unsaturated (Eutric cambisols) (pH 5.0); (9) Cinnamonic (Eutric cambisols and Calcic kastanozems) (pH 5.5); (10) Cinnamonic calcareous (Calcaric cambisols and Calcic kastanozems) (pH 5.0); (11) Cinnamonic light (Calcic kastanozems) (pH 5.0); (12) Black calcare-ous (Calcic vertisosls) (pH 5.0); (13) Yellow brown forest (Chromic cambisols and stagnic alisols) (pH 5.0). For a colour version of this figure, please see the section at the end of this book.


As a result of screening, 20 strains of microorganisms from different taxonomic groups (bacteria, microscopic fungi and yeast) were selected accord-ing to their abilities to effectively assimilate oil hydrocarbons when grown on media containing crude oil or separate individual hydrocarbons (alkanes: C6H14, C8H18, C11H24, C13H28, C14H30, C15H32 and C16H34, arenes: benzene and toluene). The capability of selected strains and their consortia to utilize oil hydrocarbons was determined by their submerged cultivation in nutrient media with crude oil (3%) as the sole source of carbon. The chromatographic analysis has shown that for oil degradation the most efficient combination of bacterial cultures is the consortium consisting of Pseudomonas and Rhodococcus strains. Chromatographic analysis also reveals that hydrocarbon fractions to C17 are assimilated completely and heavier hydrocarbons remain in the incubation medium in minimal amounts (Figure 19.2).

SELECTION OF PLANTS

The assessment of plant detoxification potential is determined by the rate and depth of contaminant uptake from the soil, how they accumulate in the plant cell, and the degree of their transformation to regular cell metabolites. To select the best plants for a particular phytoremediation task, ideally many plant characteristics should be made available. Firstly, the actual phytoremediation-related characteristics of the candidate plants should be established, notably (Kvesitadze et al., 2006):

l Their overall ability to take up and degrade contaminants existing in the soil or groundwater.

l Their ability to accumulate organic and inorganic contaminants in their cells and intracellular spaces.

l Their excretion of exudates to stimulate the multiplication of soil microorgan-isms and secretion of enzymes participating in the initial transformations of the contaminants.

l The existence, within the cells, of contaminant-degrading or conjugating enzymes (oxidases, reductases, transferases, esterases, etc.).

l Their high resistance against contaminants, that is that the plants’ growth and metabolism is not adversely affected by the contaminants.

l Their root system (main and fibrous); the range of root depths of the plants. l Whether the plants are endemic and non-agricultural. l Their tolerance to salty soil (halophilicity). l Their appropriate adaptediveness (to warm or cold conditions). l Their growth rate.

Plants have the capacity to uptake organic pollutants and to subsequently metabolize or transform them into less toxic metabolites. Once taken up and translocated, the organic chemicals generally undergo three transformation stages: (1) chemical modification (oxidations, reductions, hydrolysis, etc.); (2)


conjugation (with glutathione, carbohydrates, amino acids, etc.); and (3) com-partmentalization in vacuoles and / or the cell wall (Sandermann, 1994; Burken, 2003; Kvesitadze et al., 2006). Reactions occurring during all three detoxification processes are enzymatic in nature. In the absence of xenobiotics, these enzymes catalyze other reactions typical for plant cell regular metabolism (Kvesitadze et al., 2006). Phytoremediation abilities of plants, besides anatomical and physi-ological characteristics of plants, significantly depend on high activity of these

FIGURE 19.2 GS diagrams of crude oil hydrocarbons after incubation with strain from genera Rhodococcus. (A) Control variant: incubation without bacterial strain. (B) Test variant: incubation with bacterial strain.


enzymes and their induction by chemical agents (Sandermann, 1994; Kvesitadze et al., 2001).

Hydrocarbon degradation in plants proceeds mainly through a multistage oxidation pathway. The initial and rate-limiting stage of such transformation is hydroxylation. As a result of hydroxylation nonpolar hydrocarbon molecules get a polar functional (hydroxyl) group and become easily accessible for fur-ther oxidative degradation. The following oxidative metal-containing enzymes catalyze hydroxylation: cytochrome P450 containing monooxygenases (EC 1.14.14.1), peroxidases (EC 1.11.1.7) and phenoloxydases (EC 1.14.18.1) (Marrs, 1996; Kvesitadze et al., 2001, 2006; DeRidder et al., 2002).

Chemical modification of organic pollutants is a process whereby a molecule of a hydrophobic organic xenobiotic acquires a hydrophilic functional group (hydroxyl, amino, carboxyl, etc.) as a result of enzymatic transformations (Kvesitadze et al., 2006). Due to the functional group the polarity and reactivity of the toxicant molecule is enhanced. This promotes an increase of its affin-ity to enzymes, catalyzing further transformation (conjugation or further deep oxidation). Further, xenobiotic oxidative degradation proceeds to standard cell metabolites and mineralization to CO2 (Chrikishvili et al., 2006). Through this pathway the plant cell not only fully detoxifies the xenobiotic but also utilizes its carbon atoms for intracellular biosynthetic and energetic needs. The totality of such transformations is the essence of the detoxification process.

Conjugation is a process where a xenobiotic is chemically coupled to cell endogenous compounds (proteins, peptides, amino acids, organic acids, mono-, oligo- and polysaccharides, lignin, etc.) by formation of peptide, ether, ester, thioether or other bonds of a covalent nature (Kvesitadze et al., 2006). Inter-mediates of xenobiotic transformations or xenobiotics already bearing func-tional groups capable of reacting with intracellular endogenous compounds are susceptible to conjugation. The formation of conjugates leads to the enhance-ment of the hydrophilicity of organic contaminants, and consequently to an increase in their mobility. Such characteristics further simplify compartmenta-tion of the transformed toxic compounds. Being in conjugated form, a xenobi-otic in the plant cell is kept apart from vital processes and is therefore rendered harmless for the plant (Burken et al., 2003).

A group of enzymes, called glutathione S-transferases (GSTs) (EC 2.5.1.18) have wide specificity and couple electrophilic xenobiotics and their metabolites with the reduced tripeptide glutathione (GSH) (Marrs, 1996; DeRidder et al., 2002). In plants, a large and diverse gene family encodes the GSTs. GSTs facili-tate the reaction between the functional group of the contaminant intermedi-ates and the SH-group of the glutathione cysteine residue. They participate in the conjugation of a wide spectrum of toxic compounds and their metabolites. As a consequence, the toxicant is bound to intracellular compounds via a cova-lent bond to the sulphur atom (Marrs, 1996).

Conjugation is not the most successful pathway of xenobiotic detoxifica-tion from the ecological point of view. Plant remains, containing the conjugated


contaminants, actually become the toxicant carrier. Typically 70% or more of the absorbed xenobiotics accumulate in plants in the form of conjugates (Kvesitadze et al., 2001). This fact must be taken into account when considering the ultimate ecological fate of xenobiotics. Conjugates of toxic compounds are especially hazardous on insertion into the food chain: enzymes of the digestive tract of warm-blooded animals can hydrolyze conjugates and release the xenobiotics or products of their partial transformation, which in some cases, due to increased reactivity, are more toxic than the initial xenobiotics. Therefore, it is highly desirable that plants applied to phytoremediation have a phenomenal capability to accomplish deep enzymatic degradation of xenobiotics. The selection of such plants, or the promotion of gene expression of enzymes participating in plant detoxification processes are the basic strategies of modern phytoremediation technologies.

Thus, the main biocemical criteirion for selection of plant phytoremediators with regard to contamination with oil hydrocarbons is high activities of basic oxidative enzymes (cytochrome P450-containing monooxygenase, peroxidase and phenolox-idase) participating in oil hydrocarbons degradation. During the selection of such plants, 12 species (grasses, shrubs and trees) which are well adapted to almost every type of soil that exists along the oil pipelines in Georgia, are tested: evergreens – tri-foliate orange (Poncirus trifoliate) and privet (Ligustrum sempervirens); deciduous plants – poplar (Populus canadensis) and mulberry (Morus alba); annual plants – maize (Zea mays), rye-grass (Lolium multiflorum), alfalfa (Medicago sativa) and soybean (Glycine max), chickpea (Cicer arietinum), chickling vetch (Lathyrus sativum), mung bean (Vigna radiata) and pea (Pisum sativum).

According to the results, the following conclusions can be drawn:

l Evergreens privet and trifoliate orange are respectively distinguished by high phenoloxidase and peroxidase activities. Highest monooxygenase activity is characteristic for rye-grass and poplar.

l Alfalfa and chickpea possess a stronger pool of oxidative enzymes, especially hemoproteins, and mung bean prevails in phenoloxidase activity.

l For oxidative enzymes benzene is a much stronger indicator (maximum level of induction degree is 185–200%) than octane (145–160%).

l Investigated oxidative enzymes were induced by the action of penetrated hydrocarbons and if the plant possesses a stronger pool of oxidative enzymes the induction degree of these enzymes is comparatively low.

l Two types of changes of oxidative enzyme activities are observed: (i) specific activation of certain oxidase (in alfalfa, mung bean and soybean) or (ii) equal induction of all oxidative enzymes (in rye-grass, chickling vetch, chickpea and pea). In the first case oxidative metabolism of hydrocarbons in roots is preferably realized via the monooxygenase pathway, while oxidation via phe-noloxidase is more active in leaves. In the second case, the oxidation of hydro-carbons is carried out via inter-replacement of haemoproteins (cytochrome P450-containing monooxigenase and peroxidase) and copper-containing (phenoloxidase) oxidative enzymes.


l Oil hydrocarbons with open chains, as well as with aromatic ring, undergo oxidative degradation via participating of following plant enzymes: peroxidase, phenoloxidase, cytochrome P450-containing monooxygenase. Oxidative metabolism of benzene and octane in roots of tested plants is real-ized via participation of monooxygenase and peroxidase, and in leaves by phenoloxidase.

l Trifoliate orange, privet, rye-grass, alfalfa and soybean, according to content of oxidizing enzymes and their tolerance towards high concentrations of oil hydro-carbons, are serviceable for phytoremediation of soils polluted by oil spills.

In addition, the effects of petroleum hydrocarbons on plant cell ultrastruc-ture and enzymes of basic metabolism, such as nitrogen assimilation and energy generation, were studied.

The understanding of the oil biodegradation process after a spill is becoming increasingly important. Bacterial pathways of hydrocarbon metabolism, for exam-ple, are reasonably well delineated (Ensley and Gibson, 1983; Bartha, 1986). A number of comprehensive reviews have addressed microbial-based bioremedia-tion (Lee et al., 1988; Lee and Banks, 1993; Sims et al., 1989; Huesemann, 1994). Degradation of oil by bacteria and fungi may be limited by a lack of sufficient oxygen and nutrients, given that hydrocarbon metabolism is primarily an aero-bic process (Dibble and Bartha, 1979; Bartha, 1986; Leahy and Colwell, 1990; Pritchard and Costa, 1991; Ferro et al., 1997; Siciliano and Germida, 1998).

Several studies have demonstrated that spilled oil degrades more rapidly on vegetated soils than on soils lacking vegetation (Lee and Banks, 1993; Schwab and Banks, 1994; Qiu et al., 1997; Lin and Mendelssohn, 1998; Wiltse et al., 1998; Banks et al., 2000), but the mechanisms of this acceleration have not been clearly defined. Organic pollutants penetrated into plant cells cause significant changes across a whole range of intracellular metabolic processes. This is first manifested in the activation of inductive processes directed to the synthesis of enzymes and enzymatic systems participating in xenobiotics detoxification. As a result of the progressive oxidation of the xenobiotics, standard cellular intermediates are formed (Kvesitadze et al., 2006).

Despite the fact that collateral biochemical processes accompanying the detoxification process in plants are not well investigated, there are quite a few examples in the literature indicating that the activities of the enzymes partici-pating in different regular cellular processes are also influenced by xenobiotics that have penetrated into the cell. These xenobiotics may, furthermore, affect the activity of the regulatory enzymes involved in the tricarboxylic acid cycle and in the process of cell main metabolism (Kvesitadze et al., 2006).

The revelation of plant response to xenobiotics, expressed as cell structure–function deviations, characteristic to each plant species, enables plant resistance to contaminated environment to be revealed and allows the estimation of the prospects of their application in phytoremediation technologies.

The destructive changes in the ultrastructure of alfalfa and rye-grass root cells under the influence of octane as a typical oil hydrocarbon have been


investigated. Under the influence of octane, the root cells of rye-grass were completely destroyed (Figure 19.3A).

To reveal measurable declines in plant cell homeostasis after the influence of oil hydrocarbons, activities of enzymes [glutamate dehydrogenase (GDH), malate dehydrogenase (MDH) and glutamine synthetase (GS)] were inves-tigated. GDH catalyzing reversible reaction – oxidative deamination of L- glutamate and reductive amination of 2-oxoglutarate and thus connecting nitrogen metabolism with the tri-carboxylic acid cycle, occupies the central position in cell metabolism. GS plays an important role in the incorporation of inorganic nitrogen in a form of ammonium into amino acids and proteins. This enzyme catalyzes ammonium assimilation in the process of glutamine synthe-sis. The significance of GDH and GS is conditioned by the fact that the products of the reactions catalyzed by these enzymes are the donors of amino groups for biosynthesis of other amino acids. GDH and GS play key roles in maintaining the balance of carbon and nitrogen in plant cells (Miflin and Habash, 2002). MDH, participating in the tricarboxylic acid cycle, is involved in the processes of respiration and energy exchange.

The obtained results show that octane caused induction of GDH in all grassy plants; however, the rate of induction decreased parallel to the increase of the hydrocarbon concentration. Octane at a concentration of 1 mM caused induction of GDH in privet, trifoliate and white mulberry shoots. Higher con-centration (10 mM) caused corresponding further increase in GDH activity

(A) (B)

(C) (D)

FIGURE 19.3 Changes in ultrastructure of rye-grass (A) and alfalfa (B, C and D) root cells under the action of 0.1 mM octane solution.


in leaves of privet and white mulberry shoots but not in trifoliate orange. 100 mM octane was characterized by its inhibitory effect on GDH activity in all plants. Octane had an inductive effect on MDH in almost all plants and all tested concentrations, indicating the intensification of the tricarboxylic acid cycle.

Activation of GDH at low hydrocarbon concentrations indicates participa-tion of the enzymes in plant defence mechanisms, namely, in intensification of amino acid catabolism, leading to energy generation which is in demand in a cell under contamination stress. The induction of MDH in plants exposed to increasing octane concentrations indicates the intensification of the tricar-boxylic acid cycle, probably for further oxidation of xenobiotic oxidation intermediates.

Assessment of declinations from the normal range of metabolic processes under the influence of oil hydrocarbons allows elaboration of plant selection cri-teria for their application in phytoremediation of sites polluted by hydrocarbons.

DETERMINATION OF THE DEGREE OF OXIDATIVE DEGRADATION OF HYDROCARBONS

The metabolism of [1–6 14С] benzene in alfalfa and rye-grass has been investi-gated. For this aim, the radioactivity of low- and high-molecular compounds of plants after uptake of [1–6 14С] benzene by plants is detected. The results have shown that in both plants the main part of uptaken benzene (about 80–85%) is metabolized to low-molecular compounds and 6–9% undergoes deep oxida-tion with cleavage of aromatic ring to form 14СO2. The rest part of 14С label is detected in high-molecular compounds.

To determine the level of oxidative degradation of benzene, application of [1–6 14C] benzene was carried out. As seen from the obtained results among organic acids, fumaric, muconic and succinic acids are metabolites of oxidative degradation in plants. The presence of the acids, which are standard cell metab-olites, indicates to the cleavage of the benzene aromatic ring in the process of oxidative transformation. Apart from organic acids, amino acids with aro-matic rings, tyrosine and phenylalanine, are also dominantly formed in plants after benzene degradation. The identification of alanine, glycine and asparagine among 14С-labelled amino acids suggest that uptaken benzene undergoes deep oxidation by oxidative enzymes of plant tissues.

According to the obtained data the inferred scheme of benzene metabolism in plants is the following:

OH OHOH

OO

Benzene Phenol Catechol o-Quinone


CC

O OHOOH

COOH

COOH

CO2

Cis-cis-muconic acid

Fumaricacid

Tricarboxylic Acid Cycle

As seen from the above scheme, benzene after gradual hydroxylation to phenol, catechol and o-quinone is oxidized with cleavage of the aromatic ring and forms cis-cis-muconic acid, which can form fumaric acid and, thus, be incorporated into the general metabolism of organic acids in plant cells.

Similarly, in the works to determine the level of oxidative degradation of cyclohexane, the experiments were carried out by applying of [1-14C] cyclohexane. The radioactive preparation of cyclohexane was added to seed-lings of maize and after 48 h of incubation, the individual components of the fractions organic acids and amino acids were isolated. As a result, the insertion of cyclohexane radioactive label in organic acids is 10 times more intense than in amino acids. Succinic acid is the major metabolite of cyclohexane among organic acids, and in amino acids 14С is distributed equally. The presence of the acids, which are standard cell metabolites, suggests cleavage of the cyclohexane ring in the process of oxidative transformation.

According to the obtained results and literature data (Wagner et al., 2002) the inferred scheme of cyclohexane metabolism in plants is the following:

O

OH

OHOH C

C

O OHOOH

Cyclohexane Adipinic acid

Cyclohexene-2-ol-1

Cyclohexene-2-on-1

Cyclohexene-3-diol-1,2

COOH

COOH

CO2

COOH

COOH

Fumaricacid

Tricarboxylic Acid Cycle

Succinicacid

As seen from the scheme, in the initial stage, cyclohexane undergoes oxida-tion to unsaturated cyclic intermediates with oxo- or hydroxyl-groups. In the next


stage of metabolism these intermediates form cyclohexene-3-diol-1,2, which is oxidized with cleavage of the carbonic cycle and is transformed into adipinic acid. This metabolite can form fumaric acid and, thus, may be incorporated into the general metabolism of organic acids in plant cells.

Summing up the possible pathways of the studied hydrocarbons oxidative degradation, it can be concluded that hydroxylation is the initial stage of exog-enous arenes and cycloalkanes oxidative degradation in plants, followed by deep oxidation of intermediates to standard endogenous compounds and car-bon dioxide. It could be supposed that the oil hydrocarbons, penetrated into tested plants, preferably undergo oxidative degradation, which is realized via the cytochrome P450-containing monooxygenase and / or peroxidase pathways in roots, and via phenoloxidase in leaves. The products of bacterial oxidation of oil hydrocarbons, penetrated into tested plants undergo subsequent oxidative degradation preferably via cytochrome P450-containing monooxygenase and peroxidase, and they are also detoxified via conjugation with the reduced form of tripeptide glutathione, catalyzed by glutathione S-transferase.

REVELATION OF PLANT–MICROBIAL INTERACTION

Initially, suspension of microorganisms was inoculated in soils. Plants were sown after 35 days of incubation with bacterial strains (in the case of micro-scopic fungi, after 10 days). After 3 weeks from sowing plants, the seeds’ germination abilities (the correlation between the number of germinated and sown seeds) and plants raw biomass were estimated.

The results show that contamination of soil decreased plant growth in all cases, but the presence of the bacterial consortium in contaminated soils significantly stimulates the germination of seeds and accumulation of plant biomass. In experiments where microscopic fungi were used, similar results were obtained, but in some cases fungi inhibited the growth of plants, in par-ticular: maize did not germinate on the soil treated by selected strains from genera of Chaetomium; and rye-grass revealed weak growth on the soil treated by selected strains from genera of Aspergillus versicolor.

MODEL EXPERIMENTS

The joint action of the plant–microbial consortium for phytoremediation of soils polluted with oil hydrocarbons has been studied in model experiments. Four plant species (alfalfa, maize, rye-grass and soybean) together with a bacte-rial consortium (consisting of selected strains of Rhodococcus, Pseudomonas and Mycobacterium) and separate microscopic fungi (Strains of Aspergillus, Chaetomium and Trichoderma viride) were tested on soils contaminated by oil hydrocarbons with long chains (C30–C60). In tested variants, the levels of soil contamination with TPH were equal 44,000, 70,000, 96,000, 113,000 and 142,000 ppm.


Initially, the suspension of microorganisms was inoculated in soils. Plants were sown after 30–35 days of incubation with bacterial consortium (in the case of microscopic fungi, after 10 days). During the experiment the soil samples were analyzed for TPH content. Results are presented in Figures 19.4, 19.5, 19.6, 19.7, 19.8 and 19.9.

The obtained results show that the effect of soil cleaning depends on the initial contamination of soil. The use of microorganisms over 2 months without plants caused a decrease of TPH content in contaminated soil by:

l 60–75% in soil with 44,000 ppm initial contamination (Figures 19.6A, 19.7A, 19.8A and 19.9A).

l 40–50% in soil with 70,000 ppm initial contamination (Figures 19.6B, 19.7B, 19.8B and 19.9B).

l 40–45% in soil with 96,000 ppm initial contamination (Figures 19.4). l 35–40% in soil with 142,000 ppm initial contamination (Figures 19.5).

The effect of soil cleaning of microorganisms is enhanced by 15–25% as a result of the use of plants as phytoremediators (Figures 19.4–19.9). In tested variants, soy-bean is revealed to have the highest phytoremediation ability. It has been established that rye-grass, soybean and alfalfa, together with the selected bacterial consortium and microscopic fungi, are the best tools for phytoremediation of soils, polluted with oil hydrocarbons. A bacterial consortium consisting of strains of Rhodococcus and Pseudomonas is the best remediation agent among tested microorganisms.

FIGURE 19.4 The dynamics of decrease of TPH content in soil contaminated with oil hydro-carbons during incubation with bacterial consortium (strains of Rhodococcus, Pseudomonas and Mycobacterium) and plants. The bacterial suspension was inoculated in the soil at the beginning of the experiment. On the 27th day of incubation, plants were sown in separate samples of soil (indi-cated by arrow). Initial degree of contamination, 96,000 ppm of TPH. Temperature, 20–25 C. For a colour version of this figure, please see the section at the end of this book.


FIGURE 19.5 The dynamics of decreasing TPH content in soil contaminated with oil hydro-carbons during incubation with bacterial consortium (strains of Rhodococcus, Pseudomonas and Mycobacterium) and plants. The bacterial suspension was inoculated in the soil at the beginning of the experiment. On the 35th day of incubation, plants were sown in separate samples of soil ( indicated by arrow). Initial degree of contamination: 142,000 ppm of TPH. Temperature: 20–25°C. For a colour version of this figure, please see the section at the end of this book.

FIGURE 19.6 TPH content in soils contaminated with oil hydrocarbons after treating with bacte-rial consortium (strains of Rhodococcus and Pseudomonas) and sowing with plants. The suspen-sion of bacteria was inoculated in the soil at the beginning of the experiment. On the 31st day of incubation, the plants were sown in separate samples of soil. Initial degree of contamination: (A) 44,000 ppm; (B) 70,000 ppm of TPH. Total time of incubation: 2 months; temperature: 20–25°C. Sample variants: (1) contaminated soil at the beginning of the experiment; (2) soil treated with bacterial consortium; (3) soil treated with bacterial consortium and sown with rye-grass; (4) soil treated with bacterial consortium and sown with alfalfa; (5) soil treated with bacterial consor-tium and sown with soybean.

FIGURE 19.7 TPH content in soils contaminated with oil hydrocarbons after treating with a strain of Aspergillus and sowing with plants. The suspension of fungi was inoculated in the soil at the beginning of the experiment. On the 31st day of incubation, the plants were sown in separate samples of soil. Initial degree of contamination: (A) 44,000 ppm; (B) 70,000 ppm of TPH. Total time of incubation: 2 months; temperature: 20–25°C. Sample variants: (1) contaminated soil at the beginning of the experiment; (2) soil treated with a strain of Aspergillus; (3) soil treated with a strain of Aspergillus and sown with rye-grass; (4) soil treated with a strain of Aspergillus and sown with alfalfa; (5) soil treated with a strain of Aspergillus and sown with soybean.

FIGURE 19.8 TPH content in soils contaminated with oil hydrocarbons after treating with a strain of Trichoderma and sowing with plants. The suspension of fungi was inoculated in the soil at the beginning of the experiment. On the 31st day of incubation, the plants were sown in separate samples of soil. Initial degree of contamination: (A) 44,000 ppm; (B) 70,000 ppm of TPH. Total time of incubation: 2 months; temperature: 20–25°C. Sample variants: (1) contaminated soil at the begin-ning of the experiment; (2) soil treated with a strain of Trichoderma; (3) soil treated with a strain of Trichoderma and sown with rye-grass; (4) soil treated with a strain of Trichoderma and sown with alfalfa; (5) soil treated with a strain of Trichoderma and sown with soybean.


FIGURE 19.9 TPH content in soils contaminated with oil hydrocarbons after treating with a strain of Chaetomium and sowing with plants. The suspension of fungi was inoculated in the soil at the beginning of the experiment. On the 31st day of incubation, the plants were sown in separate samples of soil. Initial degree of contamination: (A) 44,000 ppm; (B) 70,000 ppm of TPH. Total time of incubation: 2 months; temperature: 20–25°C. Sample variants: (1) contaminated soil at the begin-ning of the experiment; (2) soil treated with a strain of Chaetomium; (3) soil treated with a strain of Chaetomium and sown with rye-grass; (4) soil treated with a strain of Chaetomium and sown with alfalfa; (5) soil treated with a strain of Chaetomium and sown with soybean.

The chromatographic analysis of soil samples (Figures 19.10 and 19.11) shows that in the case of 70,000 ppm initial contamination in the beginning of remediation the ratio between ‘light’ and ‘heavy’ petroleum hydrocarbons (C8–15 : C16–35) equals 95 : 5 (Figure 19.10A); and after remediation this ratio is decreased to 53 : 47 (Figure 19.10B). In the case of 44,000 ppm initial con-tamination, this effect is expressed most clearly: the ratio is decreased from 87 : 13 (Figure 19.11A) to 6 : 94 (Figure 19.11B). These results indicate that the assimilation of C8–15 hydrocarbons takes place mostly during bioremediation.

Thus, conducted model experiments have shown that after the process of remediation implemented by applying plants and microorganisms, heavy frac-tion of oil hydrocarbons that do not undergo phytoremediation still reside in the soil. It can be concluded that efficiency of phytoremediation significantly decreases when utilization of contaminants by microorganisms and plants is complicated by small mobility, and, correspondingly, by low bioavailability of pollutant molecules. Similar cases arise during long-term contamination of soils with crude oil when light hydrocarbons become volatile, and components with a long-chain form a resinous mass and become extremely difficult to remove. In this case, it is necessary to use agents able to increase mobility and solubility of such hydrocarbons, which then will be effectively destroyed by microorgan-isms and easily extracted by plants. The feasible solution to this problem can be the application of surface-active substances, the most promising of which


FIGURE 19.11 GC diagrams of hexane extracts of soil samples in beginning (A) and after (B) remediation process. Contaminated soil (initial contamination, 44,000 ppm of TPH) is treated by bacterial consortium (strains of Rhodococcus and Pseudomonas). Duration of remediation: 2 months; temperature: 20–25°C.

FIGURE 19.10 GC diagrams of hexane extracts of soil samples in beginning (A) and after (B) remediation process. Contaminated soil (initial contamination, 70,000 ppm of TPH) is treated by bacterial consortium (strains of Rhodococcus and Pseudomonas). Duration of remediation: 2 months; temperature: 20–25°C.


are biogenic surfactants (biosurfactants). They are not inferior to the synthetic ones by their efficiency and, at the same time, are environmentally friendly. Due to their physico-chemical properties (desorption of hydrophobic substances from the soil, their solubilization, reduction of surface and interfacial tension of solutions) and biological activity, biosurfactants may increase the degree of biodegradation of contaminants.

Application of natural biosurfactants – surface-active substances of micro-biological origin – can become the real solution to this specified problem of phytoremediation. Microbial surfactants are biologically active compounds characterized by high efficiency, biodegradability and low toxicity. They are able to emulsify resin mass of heavy oil hydrocarbons, increase their bio-availability and improve their transport to plant cells. Parameters of their superficial and interphase tension of their solutions, ability to form stable fine-grained emulsion of water phase with hydrophobic compounds (hydrocarbons, including heavy fractions of oil, vegetable oils, fats) testify to high activity of biosurfactants (Desai and Banat, 1997; Bognolo, 1999). Due to all of the above, biosurfactants increase permeability of toxic compounds to plant cells ( Vasileva-Tankova et al., 2001; Sotirova et al., 2008). In addition, surfactants are capable of raising bioavailability of pollutants and regulate their transport in plant cells. Some types of biosurfactants, for example, rhamnolipids, possess metal-chelating properties that enable their application in phytoextraction of heavy metals (Maier et al., 2001; Juwarkar et al., 2007). All these properties of biosurfactants should considerably increase the efficiency of application in phytoremediation technologies for cleaning soils contaminated with long-chain oil components.

Based on the above, we decided to test the use of various biosurfactants in phytoremediation technology. For this reason, model phytoremediation experi-ments were carried out.

In the model experiments the following objects were used:

l The soil artificially contaminated with raw oil (the level of initial contamina-tion by TPH equals 27,500 ppm) – as the object of phytoremediation.

l Bacterial consortium that consist of strains of Pseudomonas and Bacillus – as bioremediation agents.

l Biosurfactants preparations: rhamnolipids, trehalose lipids and rhamnolipid biocomplex PS (they were used separately) – as agents to increase the effi-ciency of the phytoremediation process.

l Alfalfa – as plant phytoremediator.

The mass of the contaminated soil samples was 7.5 kg. Suspension of bacteria (1.2 l in each soil sample) and solutions of biosurfactants (100 mg / 500 ml in each soil samples) were inoculated in the soil at the beginning of the experi-ment. On the 14th day from inoculation plants were sown in soil samples. The experiment continued for 3 months (from 21 June to 21 September, 2011) in greenhouse conditions.


The obtained results (Figure 19.12) show that, as a result of abiotic and biotic factors action (volatilization of hydrocarbons and their assimilation by aboriginal microflora), TPH content in contaminated soil was decreased by 45% over 3 months. The use of plants (without bacterial consortium and biosurfactants) caused a decrease in contamination of 63%. Plants and bac-teria jointly are capable of assimilating 82% of hydrocarbons. The appli-cation of biosurfactants raises the intensity of the bioremediation process additionally and in cases of biopreparations of trehalose lipids and thamno-lipids, the maximum cleaning effects (correspondingly by 96 and 98%) are achieved.

Thus, as a result of research performed, effective phytoremediation technol-ogy for cleaning soils polluted with oil hydrocarbons has been developed. This technology can be used to eliminate the results of pollution caused by acciden-tal oil spills that can be a result of oil transportation through the pipelines in Georgia.

FIGURE 19.12 Phytoremediation of soil artificially contaminated with crude oil by using of plants (alfalfa), bacterial consortium (Pseudomonas sp. 6R67 + Bacillus sp. 3Zu9) and biosur-factants (Rhamnolipids, complex of biosurfactants and Trehalose lipids; concentration – 0.01%). Sample variants: (1) control (contaminated soil without plants, bacteria and biosurfactants); (2) plants (contaminated soil with plants; without bacteria and biosurfactants); (3) plants + bacteria (contaminated soil with plants and bacteria; without biosurfactants); (4) plants + bacteria + complex of biosurfactants (contaminated soil with plants, bacteria and biosurfactants); (5) plants + bacteria + trehalose lipids (contaminated soil with plants, bacteria and biosurfactants); (6) plants + bacteria + rhamnolipids (contaminated soil with plants, bacteria and biosurfactants).


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