*2.2.2. N2 fixing bacteria*

fundamental mechanism for N2 fixation which is carried out by the nitrogenase enzyme

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

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

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

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

When optimum requirements are provided, the overall stoichiometry for the reaction where

with an overall negative enthalpy of reaction which is ΔH0 = ‐45.2 kJ mol‐1 NH3 and a very high

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.

system.

supplied to reduce N2 to NH3.

be found below:

N2 + 8H+ + 8e‐

characteristics of these individual metalloproteins.

222 Soil Contamination - Current Consequences and Further Solutions

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

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

of the MoFe‐protein, is reduced by an electron transfer.

activation energy which is EA = 230–420 kJ mol‐1.

nitrogenase reduces the N2 to NH3 can be summarized as [47]:

 + 16MgATP ➔ 2NH3 + H2 + 16MgADP + 16Pi

Several bacteria fix nitrogen, a short list is given in **Table 6**. Rhizobium bacteria, listed as the first kind as "symbiotic bacteria," is typically linked to leguminous plants, frankia, or cyano‐ bacteria with nonlegume plants. The "nonsymbiotic" second kind (also referred as "free‐ living" bacteria), exist either in water or soil. Examples of the nonsymbiotic N2 fixing bacteria are cyanobacteria (blue‐green algae, *Anabaena*, and *Nostoc)* and genera such as *Azotobacter*, *Beijerinckia*, and *Clostridium*. The third kind typically is found around roots of the plant rhizosphere and stream the fixed nitrogen to the plant. This group is typically referred as "associative nitrogen fixation" bacteria and includes *Azospirillum*, *Klebsiella sp.*, *Azotobacter paspali*, and *Alcaligenes*. The fourth kind is "endophytic nitrogen fixation" linked with cereal grasses such as sugarcane and includes *Azoarcus sp.* and *Burkholderia sp.*


**Table 6.** Plant growth promoting rhizobacteria (PGPR) and their relationship to hosts [67].

The abundantly available PGPRs are diazotrophs and can fix N2 via the biological nitrogen fixation, this characteristic is not the main mechanism with which they promote growth to their host plant. The plant growth stimulations primarily occur due to bacteria's enzymatic activities such as nitrogenase.

#### **2.3. Phytase**

Phosphorus (P) is an essential element for plants to grow and develop. Although P is found in soil both as insoluble inorganic and organic forms, it is unavailable for plants [49]. In soil, there are phosphate‐solubilizing bacteria (PSB) which can turn the insoluble inorganic phosphates in organic acids, into an available form. Therefore, these microorganisms have been generally studied to improve the growth properties and yield of crops. Despite being the most abundant form of phosphates in soil (10–50% of total P) [50, 51], phytates should be hydrolyzed by phytases (myo‐inositol‐hexakisphosphate‐phosphohydrolases) to be con‐ sumed by the plants [52, 53].

Phytic acid (myo‐inositol hexa‐phosphate, IP6) has six phosphate groups. It is present mainly in plant‐based nutrients, particularly in cereals and legumes. Phytic acid is thought to be a major stock component for plant germination and growth [54]. IP6 forms a vigorous structure called "chelating agent" by its six P groups and this structure plays a role in binding minerals such as Ca2+, Mg2+, Fe3+, and Zn2+. Presence of phytates may also have a negative effect on digestion of protein [55, 56], starch [57], and lipids [58]. Endogenous phytases in most seeds of higher plants may degrade the IP6 partly to produce penta‐, tetra‐, or tri‐phosphate compounds through food processing and digestion [59].

Phytases are the enzymes which catalyze the degradation reaction of phytate which is the primary reserve form of P in plants. Phytases are a different type of phosphatases and they can hydrolyze phytate to a set of lower phosphate esters of myo‐inositol and phosphate. Phytates are present in wide range of living things including plants and microorganisms. In the last decade, the number of researches, which focuses on how to lower the phytate levels found in animal feed by improving the enzymatic reaction of phytases, has been increased [60– 62].

A great deal of phytases assumes broad specificity to substrates and can therefore hydrolyze different phosphorylated compounds, irrespective of their similarity to phytic acid, including phosphorylated sugars (e.g., G6P). In contrast, few phytases, e.g., the one from Bacillus sp. and few other bacteria and fungi, e.g., *Aspergillus* sp., are characterized to be highly specific to phytic acid and/or to the class of protein tyrosine phosphatase‐PTP‐like pyhtases.

### *2.3.1. Pathways of phytic acid dephosphorylation*

Pythase degrade phytic acid at various rates and order. The mechanism of hydrolysis is reported to be step‐wise, the product of each step is the substrate of the subsequent one. Depending on the mechanism, this enzyme is recognized having three subclasses: 3‐phytase (EC 3.1.3.8), 4‐phytase (EC 3.1.3.26), and 5‐phytase (EC3.1.3.72), each class depending on the position of the first phosphate hydrolyzed. Note that, phytases are mostly able to hydrolyze five out of six available phosphates.

#### *2.3.2. Phytase and plant growth promotion*

There are several microorganisms in rhizosphere which interact with plant roots and affect plant nutrition in different ways. Direct effects of these microorganisms are altering the uptake and availability of plant nutrition. Indirect effects include promoting plant growth. For instance, in a study phytate was used as the unique source of phosphate to grow *Trifolium subterraneum*, as a result secretion of phytase in a very low grade from plant roots was observed. Following *A. niger* phytase was added to the medium and liberation of sufficient phosphates was observed. This step enables *T. subterraneum* seedlings to grow and plants supplied with inorganic phosphorus.


**Table 7.** Microorganisms expressing (extracellularly) phytase and their affectees of the resulting enzyme [68].

Since fungi hydrolyze several organic phosphorus compounds efficiently, they are considered as sufficient utilizers of organic phosphorous which can beneficial to the plant growth. Therefore, fungi which produce phytase and phosphatases were applied to seeds as inoculant, for effective use of phytate phosphorus in soil [63]. For instance, *Chaetomium globosum* is a fungus which produces phosphatase and phytase was used as the inoculation agent for wheat and pearl millet crops [64]. As a result, a remarkable progress in plant biomass, root length, plant phosphate concentration, seed and straw yield, and seed P content was obtained after inoculation with the fungus. A brief summary of phytase sources and their host plants can be found on **Table 7**.

### **3. Conclusion**

hydrolyzed by phytases (myo‐inositol‐hexakisphosphate‐phosphohydrolases) to be con‐

Phytic acid (myo‐inositol hexa‐phosphate, IP6) has six phosphate groups. It is present mainly in plant‐based nutrients, particularly in cereals and legumes. Phytic acid is thought to be a major stock component for plant germination and growth [54]. IP6 forms a vigorous structure called "chelating agent" by its six P groups and this structure plays a role in binding minerals such as Ca2+, Mg2+, Fe3+, and Zn2+. Presence of phytates may also have a negative effect on digestion of protein [55, 56], starch [57], and lipids [58]. Endogenous phytases in most seeds of higher plants may degrade the IP6 partly to produce penta‐, tetra‐, or tri‐phosphate

Phytases are the enzymes which catalyze the degradation reaction of phytate which is the primary reserve form of P in plants. Phytases are a different type of phosphatases and they can hydrolyze phytate to a set of lower phosphate esters of myo‐inositol and phosphate. Phytates are present in wide range of living things including plants and microorganisms. In the last decade, the number of researches, which focuses on how to lower the phytate levels found in animal feed by improving the enzymatic reaction of phytases, has been increased [60–

A great deal of phytases assumes broad specificity to substrates and can therefore hydrolyze different phosphorylated compounds, irrespective of their similarity to phytic acid, including phosphorylated sugars (e.g., G6P). In contrast, few phytases, e.g., the one from Bacillus sp. and few other bacteria and fungi, e.g., *Aspergillus* sp., are characterized to be highly specific to

Pythase degrade phytic acid at various rates and order. The mechanism of hydrolysis is reported to be step‐wise, the product of each step is the substrate of the subsequent one. Depending on the mechanism, this enzyme is recognized having three subclasses: 3‐phytase (EC 3.1.3.8), 4‐phytase (EC 3.1.3.26), and 5‐phytase (EC3.1.3.72), each class depending on the position of the first phosphate hydrolyzed. Note that, phytases are mostly able to hydrolyze

There are several microorganisms in rhizosphere which interact with plant roots and affect plant nutrition in different ways. Direct effects of these microorganisms are altering the uptake and availability of plant nutrition. Indirect effects include promoting plant growth. For instance, in a study phytate was used as the unique source of phosphate to grow *Trifolium subterraneum*, as a result secretion of phytase in a very low grade from plant roots was observed. Following *A. niger* phytase was added to the medium and liberation of sufficient phosphates was observed. This step enables *T. subterraneum* seedlings to grow and plants supplied with

phytic acid and/or to the class of protein tyrosine phosphatase‐PTP‐like pyhtases.

sumed by the plants [52, 53].

224 Soil Contamination - Current Consequences and Further Solutions

62].

compounds through food processing and digestion [59].

*2.3.1. Pathways of phytic acid dephosphorylation*

five out of six available phosphates.

inorganic phosphorus.

*2.3.2. Phytase and plant growth promotion*

Soil pollution is an important problem affecting millions of individuals, and surely this effect is not restricted to humans. Therefore, sustainable methods that are suitable for large‐scale methods, to remediate soil, become increasingly interesting for both fundamental and applied research. In particular, using biological systems (microbes and enzymes produced by these plants) has shown considerable progress. This needs to be applied in different agro climatic zones of the world.

A key element in these remediation methods is the fundamental (underlying principles) and executive (application principles) understanding of the microbe‐plant interaction, that may be physical, chemical, and biological. This will further draw attention to generating engineered agro‐lands, as mass production of these organisms and enzymes also economically interesting.

Despite important progress made in, particularly for PGPRs, growth conditions, enzyme portfolio vis‐a‐vis to soil remediation, and other (symbiotic) interaction with plants, the research is still at its infancy, especially about the interaction with plant roots and other bacteria.

A still unexplored aspect is the molecular engineering of these microbes and/or plants that would enhance the efficiency of these organisms for soil remediation. This has a large potential, as some PGPR can increase plant tolerance to degraded soil and other extreme conditions such as heavy metal contamination and increased salinity.
