**2. Using enzymes and PGPRs for soil remediation**

Plant growth promoting rhizobacteria naturally exist at plant roots or they are used as inocula that are applied to the roots of plants to stimulate growth by changing the soil environment. PGPRs generally produce important substances for plants, facilitate the uptake of nutrients, and have a role in soil remediation. Soil remediation is an important process for plant health, in which soul pollutants, contaminants, or plant pathogens are reduced or eliminated.

Due to industrialization, soil is polluted together with water and air. The most encountered pollutions can be from organic substances such as total petroleum hydrocarbons (TPHs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated terphenyls (PCTs), perchloroethylene (PCE), trichloroethylene (TCE), and pesticides like atrazine and bentazon or from inorganic substances, which are mostly heavy metals, cadmium, chromium, copper, lead, mercury, zinc, and others.

In a study, different concentrations of TPH contamination was successfully reduced with the help of *Enterobactor cloacae* UW4 and *E. cloacae* CAL2 strains which are PGPRs. Aminocyclo‐ propane‐1‐carboxylate (ACC) deaminase helped the process by lowering the ethylene levels of TPH. PAHs are remediated by dehydrogenases (e.g., 1,2‐dihydroxy‐l,2‐dihydronaphthalene dehydrogenase), dioxygenases (e.g., 1,2‐dihydroxynaphthalene dioxygenase), and aldolases (e.g., cis‐2′‐hydroxybenzalpyruvate aldolase) produced by *Pseudomonas paucimobilis* Q1. PAHs include napthalene, accenaphtene, phenanthrene, fluoranthene, pyrene, benzo[a]pyrene, and others. PCBs are remediated with biphenyl dioxygenases. TCE is remediated with toluene ο‐ monooxygenase produced by recombinant *Pseudomonas fluorescens*. Biopolymers such as kraft and lignin or trinitrotoluene (TNT) are remediated with Mn‐dependent peroxidase (MnP) and lignin peroxidase (LiP).

There are a variety of insoluble substances, whether natural or synthetic, in origin and can be hydrolyzed by specific enzymes. Cellulose, chitin, keratin, Kraft pulp, and sewage sludge are examples of natural insoluble substances. Cellulose can be degraded by cellulase while chitin by chitinase, keratin by keratinase, Kraft pulp by both xylanase and β‐xylosidase, and sewage sludge by protease and phosphatase. For synthetic insoluble substances, nylon can be hydrolyzed by MnP, poly‐l‐lactic acid by depolymerase and alkaline protease, polyacry‐ late by cellobiose dehydrogenase, and polyurethane by esterase.

Fungi are the most common known yet not the sole producers for such enzymes. Many bacteria which are used as PGPRs can also produce them.

Chemical degradation of heavy metals is not possible, and other alternative methods should be used to relieve the soil from heavy metal accumulation. Alternative methods for remedia‐ tion of soil include immobilization, separation, extraction, and isolation of metals, as well as reduction of toxicity and mobility.

This section of the chapter focuses on selected enzymes such as ACC deaminase, phytase, and nitrogenase, which can be used in bioremediation of soil.

#### **2.1. 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase**

and flora becomes more imbalanced in its nutrients (becomes extremely salty, acidic, alkali, etc.). In particular, as a result of industrial activities, water gets polluted and drinking water becomes more inaccessible to humans. Again with industrial activities, greenhouse and other pollutant gases release to the atmosphere, which decreases the quality of the air, causing an

The rest of this article focuses on enzymes used for soil remediation as a special case of bioremediation via so‐called plant growth promoting rhizobacteria (PGPRs). As such, it represents one of the alternative tools for soil remediation, such as thermal soil remediation,

Plant growth promoting rhizobacteria naturally exist at plant roots or they are used as inocula that are applied to the roots of plants to stimulate growth by changing the soil environment. PGPRs generally produce important substances for plants, facilitate the uptake of nutrients, and have a role in soil remediation. Soil remediation is an important process for plant health, in which soul pollutants, contaminants, or plant pathogens are reduced or eliminated.

Due to industrialization, soil is polluted together with water and air. The most encountered pollutions can be from organic substances such as total petroleum hydrocarbons (TPHs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated terphenyls (PCTs), perchloroethylene (PCE), trichloroethylene (TCE), and pesticides like atrazine and bentazon or from inorganic substances, which are mostly heavy metals, cadmium,

In a study, different concentrations of TPH contamination was successfully reduced with the help of *Enterobactor cloacae* UW4 and *E. cloacae* CAL2 strains which are PGPRs. Aminocyclo‐ propane‐1‐carboxylate (ACC) deaminase helped the process by lowering the ethylene levels of TPH. PAHs are remediated by dehydrogenases (e.g., 1,2‐dihydroxy‐l,2‐dihydronaphthalene dehydrogenase), dioxygenases (e.g., 1,2‐dihydroxynaphthalene dioxygenase), and aldolases (e.g., cis‐2′‐hydroxybenzalpyruvate aldolase) produced by *Pseudomonas paucimobilis* Q1. PAHs include napthalene, accenaphtene, phenanthrene, fluoranthene, pyrene, benzo[a]pyrene, and others. PCBs are remediated with biphenyl dioxygenases. TCE is remediated with toluene ο‐ monooxygenase produced by recombinant *Pseudomonas fluorescens*. Biopolymers such as kraft and lignin or trinitrotoluene (TNT) are remediated with Mn‐dependent peroxidase (MnP) and

There are a variety of insoluble substances, whether natural or synthetic, in origin and can be hydrolyzed by specific enzymes. Cellulose, chitin, keratin, Kraft pulp, and sewage sludge are examples of natural insoluble substances. Cellulose can be degraded by cellulase while chitin by chitinase, keratin by keratinase, Kraft pulp by both xylanase and β‐xylosidase, and sewage sludge by protease and phosphatase. For synthetic insoluble substances, nylon can be hydrolyzed by MnP, poly‐l‐lactic acid by depolymerase and alkaline protease, polyacry‐

late by cellobiose dehydrogenase, and polyurethane by esterase.

air sparging, encapsulation, chemical oxidation, stabilization, and soil washing.

increase in public health and waste management problems.

218 Soil Contamination - Current Consequences and Further Solutions

**2. Using enzymes and PGPRs for soil remediation**

chromium, copper, lead, mercury, zinc, and others.

lignin peroxidase (LiP).

PGPRs help plant growth and development directly and indirectly. In case of direct stimula‐ tion, it fixes the nitrogen present in the air, produces the phytohormones necessary for plants and enables uptake of some metals including iron and soluble phosphate. The indirect stimulation covers biocontrol actions, i.e., mediating fight with plant pathogens. Both direct and indirect mechanisms operate via specific enzymes. An important enzyme is 1‐aminocy‐ clopropane‐1‐carboxylate deaminase (ACC‐deaminase) that plays a well‐described role in plant hormone and ethylene regulation (an important stress inducer in plants).

It has been extensively reported that ACC deaminase is found in numerous microbial species of Gram‐negative and Gram‐positive bacteria, rhizobia, endophytes, and fungi. Also the biochemical and physical aspects of ACC deaminase have been investigated broadly by numerous researchers. **Table 4** summarizes both the plant growth promoting microorganisms and results of the relevant studies.


**Table 4.** Biochemical characterization of 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase from selected microorganisms [65].

There are several mechanisms in which the ACC deaminase concurrently catalyzes the reac‐ tion where ACC breaks down to α‐ketobutyrate and ammonia along with the regulation of ethylene production which under stress conditions inhibits the plant growth [36]. When plants were treated with bacteria producing ACC deaminase, relatively extensive root growth was observed due to presence less ethylene [37, 38] and improved resistance to various stress‐ es was reported [37, 39]. Therefore, using PGPRs which are showing ACC deaminase activity and genetic manipulation of other microorganism to express ACC deaminase genes to stimu‐ late plant grown and development, under either normal or stress conditions, is now a hot topic in biotechnology [39, 40].

#### *2.1.1. Mode of action of bacterial ACC deaminase*

The model which explains the mode of action of PGPR containing ACC deaminase is given in detail in [41]. They extensively investigated the competition between ACC deaminase with a low affinity for ACC and ACC oxidase. ACC oxidase is the plant enzyme that has a high affinity for ACC, and it decreases plant's endogenous ethylene concentration. They suggested that there is a relation between ACC deaminase and ACC oxidase in the system and the ACC deaminase level must be at least 100‐ to 1000‐fold greater than the ACC oxidase level for the biological activity of PGPR to be able to decrease plant ethylene levels.


**Table 5.** Inoculation with plant growth promoting rhizobacteria, containing 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase and subsequent physiological changes in plants [66].

Indole‐3‐acetic acid (IAA), which is synthesized from tryptophan and other small molecules and secreted by PGPR, gets absorbed on the seed or root surface of the plants [42, 43]. Plants take up a part of the newly synthesized IAA; with the endogenous plant association IAA can stimulate plant cell proliferation and elongation. By the way, IAA induces the activity of the ACC synthetase enzyme to turn S‐adenosylmethionine (SAM) into ACC [44]. It seems from the model outlined in [41] that a considerable amount of ACC might be leaked from plant roots and received by the microbes in soil or hydrolyzed by the microbial enzyme ACC deaminase to provide ammonia and α‐ketobutyrate. Soil microorganisms containing ACC deaminase enzyme encourage plants to synthesize more ACC than the plant would otherwise need. The excess ACC would leak into the rhizosphere. Uptake and afterwards hydrolysis processes of ACC by the microorganisms reduces the level of ACC outside the plant [41]. In order to keep the balance of ACC between the internal and external ACC levels, more ACC flows into the rhizosphere. This cycle provides the microorganisms with a perfect source of nitrogen (ACC), and hereby, ACC deaminase containing microorganisms grow rapidly around the plant roots when compared to the other soil microorganism. With this action, while ACC level is decreas‐ ing in the plant, biosynthesis of the stress hormone ethylene is also inhibited [41]. Therefore, when a plant is inoculated with ACC deaminase containing microorganisms more root growth would be observed. ACC deaminase containing bacteria and the physiological effects of the latter have been described in **Table 5**.

#### **2.2. Nitrogenase**

was observed due to presence less ethylene [37, 38] and improved resistance to various stress‐ es was reported [37, 39]. Therefore, using PGPRs which are showing ACC deaminase activity and genetic manipulation of other microorganism to express ACC deaminase genes to stimu‐ late plant grown and development, under either normal or stress conditions, is now a hot topic

The model which explains the mode of action of PGPR containing ACC deaminase is given in detail in [41]. They extensively investigated the competition between ACC deaminase with a low affinity for ACC and ACC oxidase. ACC oxidase is the plant enzyme that has a high affinity for ACC, and it decreases plant's endogenous ethylene concentration. They suggested that there is a relation between ACC deaminase and ACC oxidase in the system and the ACC deaminase level must be at least 100‐ to 1000‐fold greater than the ACC oxidase level for the

*Brassica campestris Methylobacterium fujisawaense* Bacterium promoted root elongation in canola.

*Brassica napus Enterobacter cloacae* A significant increase in the root and shoot lengths

*Dianthus caryophyllus*  L.*Azospirillum brasilense* Cd1843 Inoculated cuttings produced longest roots. *Glycine max Pseudomonas cepacia* Rhizobacterium caused an early soybean growth.

*Vigna radiata* L. *Pseudomonas putida* The ethylene production was inhibited in

*Zea mays* L. *Pseudomonas* sp. Bacterium caused root elongation in maize. *Brassica campestris Methylobacterium fujisawaense* Bacterium promoted root elongation in canola.

**Table 5.** Inoculation with plant growth promoting rhizobacteria, containing 1‐aminocyclopropane‐1‐carboxylate (ACC)

elongation.

was observed.

inoculated cuttings.

parameters of maize.

Inoculation increased agronomic

Bacterial inoculation enhanced root and shoot

Inoculated plant demonstrated more vigorous growth than the control (uninoculated).

Bacterium enhanced nodulation in plants.

Bacterium promoted nodulation in mung bean.

biological activity of PGPR to be able to decrease plant ethylene levels.

*DUC2*, *Bacillus globisporus DUC3*

in biotechnology [39, 40].

*2.1.1. Mode of action of bacterial ACC deaminase*

220 Soil Contamination - Current Consequences and Further Solutions

*Brassica campestris Bacillus circulans DUC1*, *Bacillus firmus*

*Bacillus pumilus Pseudomonas* sp. *Variovorax paradoxus*

*Pisum sativum* L. *Rhizobium leguminosarum bv. viciae* 128C53K

*Zea mays* L. *Enterobacter sakazakii* 8MR5

*Bradyrhizobium* sp.

*Pseudomonas sp.* 4MKS8 *Klebsiella oxytoca* 10MKR7

deaminase and subsequent physiological changes in plants [66].

*Brassica napus Alcaligenes* sp.

*Vigna radiata* L. *Pseudomonas* sp.

Proteins, nucleic acids, and most of the other biomolecules contain reduced nitrogen as the complementary component. Therefore, obtaining the metabolically consumable form of nitrogen is necessary for all organisms to grow and survive. Earth's atmosphere is rich in elemental dinitrogen, N2, but it is actually inert at room temperature in the absence of an appropriate catalyst. The reduction of N2 into ammonia is a good example for this situation. However, the activation energy which is necessary for reduction of N2 into ammonia is very high even though thermodynamically advantageous. This has been evidently demonstrated in the industrial fixation of nitrogen by the Haber‐Bosch process. This process allows forma‐ tion of NH3 from N2 only if temperature is between 300 and 500°C and pressure is higher than 300 atm with Fe‐based catalysts in the environment.

Despite the abundance of N2, obstacle of chemically using this source reveals a problem but nature has already figured it out via the process called biological nitrogen fixation (BNF), for example, the reduction of N2 to the metabolically consumable form of ammonia. While 60% of the fixed nitrogen is provided by BNF, unfortunately, in the nature, only a few numbers of microorganisms called diazotrophs are able to carry out this process [45]. Hence, the presence of diazotrophs is a major necessity for organisms to generate their own nitrogenous monomers which are used for the synthesis of nucleic acids, proteins, etc. via different biochemical pathways.

Diazotrophs are spread across a wide range of habitats. While they can be found in free forms, they also can be associated with various plants. Despite this difference, they all use the same fundamental mechanism for N2 fixation which is carried out by the nitrogenase enzyme system.

Nitrogenase contains two metalloprotein components: (i) the homodimeric Fe‐protein: acting as a reductase which has a high reducing power and is responsible for the providing of electrons and (ii) the heterotetrameric MoFe‐protein: a nitrogenase which utilizes the electrons supplied to reduce N2 to NH3.

The rate‐determining step in the overall nitrogenase enzyme kinetics is built on the complex‐ ation of Fe‐protein and MoFe‐protein [46]. Although the definitive structural properties of the nitrogenase complex are unknown, some possible properties can be determined by the characteristics of these individual metalloproteins.

#### *2.2.1. ATP hydrolysis and electron transfer in the nitrogenase system*

In the overall reaction which explains the electron flow during the nitrogenase activity, electrons are introduced by Fe‐protein and leave the system as reduced products. Although the intermediate steps have not been experimentally validated, there is a "consensus" model which suggests the order of compounds that electrons follow. The suggested occurrence can be found below:

Fe‐protein ➔ P‐cluster pair ➔ MoFe‐cofactor ➔ substrate

Degradation of substrate by nitrogenase is done via three elementary electron transfer reactions. In the first basic reaction Fe‐proteins are reduced by electron carries (i.e., flavodoxin, ferrodoxin, or dithionite). Second reaction is a MgATP‐dependent process where a single electron moves from Fe‐protein to MoFe‐protein. Third, the substrate, bound to the active site of the MoFe‐protein, is reduced by an electron transfer.

When optimum requirements are provided, the overall stoichiometry for the reaction where nitrogenase reduces the N2 to NH3 can be summarized as [47]:

N2 + 8H+ + 8e‐  + 16MgATP ➔ 2NH3 + H2 + 16MgADP + 16Pi

with an overall negative enthalpy of reaction which is ΔH0 = ‐45.2 kJ mol‐1 NH3 and a very high activation energy which is EA = 230–420 kJ mol‐1.

Mainly nitrogenase is responsible for N2 reduction to NH3 while simultaneously catalyzing the reduction reactions of protons and other small unsaturated molecules (i.e., acetylene, cyanide) [48]. With this property, nitrogenase can be considered as a hydrogenase with an ATP‐ dependent evolution activity. Uptake hydrogenase can play an important role in energy saving via recycling H2 released by nitrogenase. Furthermore, uptake hydrogenase allows some organisms such as *A. lipoferum*, *Derxia gummosa*, and *P. diazotrophicus* to grow chemolithoau‐ totrophically even under N2‐fixing conditions. Electron donor limitation can improve expres‐ sion of the uptake hydrogenase. Like nitrogenase, hydrogenase activity is sensitive to oxygen.
