**2. Bacteria and nitrogen fixation microorganisms in bioremediation of contaminated soil**

Microorganisms have some potential as an effective and inexpensive mean to remediation of contaminated soils [13]. The successful application of bioremediation techniques (bioaugmen‐ tation, phytoremediation) is largely dependent on the some capacity of plant growth‐promot‐ ing microorganisms to efficiently colonize growing plants roots [14].

Bacteria are the class of microorganisms actively involved in the degradation of organic pollutants from contaminated sites especially from soils rhizosphere [13, 14]. A number of bacterial species are known to degrade PAHs (shown in **Table 1**). These bacteria very often are isolated from contaminated soil and have special potential to degradation of oil derivatives. The most carcinogenic and toxic from PAHs is benzo(a)pyrene. This hydrocarbon is a model contaminate in bioremediation study. Bacteria which can degrade benzo(a)pyrene grow well on alternative carbon source in liquid culture experiments [19–21].

Other authors [22] observed a 5 % decrease in benzo(a)pyrene concentration after 168 h during incubations with *Sphingomonas paucimobilis* strain of bacteria. They also noticed that resting cells of *S. paucimobilis* grown on nutrient agar supplemented with glucose resulted in signifi‐ cant evolution of 14 CO2 (28%), indicating higher hydroxylation and ring cleavage. Some


**Table 1.** Examples of bioremediation of organic contaminants in soil with bacteria species.

authors [14, 19] isolated 11 strains from a variety of contaminated sites (oil, motor oil, refinery derivatives) with the ability to degrade benzo(a)pyrene. Bacteria capable to PAHs degradation and using as the only source of carbon and energy belong to the main species *Pseudomonas*, *Agrobacterium*, *Bacillus*, *Burkholderia*, *Sphingomonas, and Phanerochaete chrysosporium* [23]. Other authors reported PAH degradation using other bacteria including *Rhodococcus* sp., *Mycobacte‐ rium*, and mixed culture of *Pseudomonas* and *Flavobacterium* species [20]. In study of Heitkamp et al. [24], the authors described about bacterial isolated from oil‐contaminated soil which was capable of mineralizing the pyrene. *Pseudomonas aeruginosa* isolated from a stream heavily polluted by a petroleum refinery was very effective in degradation of phenanthrene [25]. *Pseudomonas aeruginosa* actively grow over high doses of phenanthrene with complete removal of the pollutant in a period of 30 days of the experiment. Other authors report that *Mycobac‐ terium* species isolated from a PAH‐contaminated soil were able to utilize pyrene as the only sole source of carbon and energy (up to 60% of the pyrene added (0.5 mg ml‐1) within 8 days at 20°C of temperature) [26]. Some products of this degradation pathway were analyzed (Cis‐ 4,5‐pyrene dihydrodiol, 4‐5‐phenanthrene dicarboxylic acid, 1‐ hydroxy‐2‐naphthoic acid, 2‐ carboxybenzaldehyde, phthalic acid, and protocatechuic acid). In the study of Yuan et al. [11], the authors isolated strains of bacteria from a petrochemical waste which having the capacity of degrading acenaphthene, fluorene, phenanthrene, anthracene, and pyrene by 70–100% in a period of 40 days of the experiment. This bacteria belong to the *Pseudomons fluoresens* and *Haemophilus* species. Dean‐Ross et al. [15] isolated two bacterial strains (*Mycobacterium flavescens* and *Rhodococcus* spp.) from some sediments. This bacteria were found to be capable of PAH degradation (pyrene mineralization by *M. flavescens* and anthracene mineralization by *Rhodococcus species*) [27]. The study also proposed the degradation pathway of fluoranthene. In both strains, metabolism of fluoranthene occurred on the fused ring of fluoranthene molecule, producing 9‐fluorenone‐1‐carboxylic acid.

Microbial degradation is the mean to remove PAHs from contaminated soils, especially using strains of bacteria which are able to degrade PAHs and using them as a source of carbon and energy and fix free nitrogen such as the strains of *Azospirillum* spp. and *Pseudomonas stutzeri*. These strains are the diazotrophic bacteria capable of free nitrogen fixing, hydrocarbon degradation as an only source carbon, and energy and biosurfactant production. Bacteria of the genus *Pseudomonas* are known in the literature as the most active degraders of hydrocar‐ bons in natural biotopes of polluted sites and within biotechnological preparations [9, 10, 69].

Diazotrophic bacteria such as *Azospirillum* spp. and *Pseudomonas stutzeri* are also using in bioremediation of crude oil derivatives in soils naturally and artificially polluted [9, 10]. Gał‐ ązka et. al. reported the study with three soils artificially polluted with PAHs (anthracene, phenanthrene, and pyrene) at the doses of 100, 500, and 1000 mg kg‐1 d.m. of soil and diesel fuel at the doses of 0.1%, 0.5%, and 1% (v/v). In study was also used soil naturally contami‐ nated with crude oil (brown soil). Grasses were inoculated with the mixture of bacteria strains *Azospirillum* and *Pseudomonas stutzeri* and applied in the bioremediation process in the amount of 1 ml per 500 g of soil.. The amounts of anthracene, phenanthrene, and pyrene were determined in soils artificially polluted and Σ15 PAHs in soils artificially polluted with diesel fuel, as well as in brown soil aged polluted with crude oil. It was found that the inoc‐ ulation of plants with *Azospirillum* spp. and *Pseudomonas stutzeri* had a positive effect on bio‐ remediation process either in soils artificially polluted with PAHs (decrease from 25–60% of the primary concentration comparing to the control) or in soils polluted with diesel fuel (de‐ crease from 2–25%) [9, 10]. The slime of *Azospirillum* spp. and *Pseudomonas stutzeri* intro‐ duced to soil did not limit the development of indigenous bacteria consortia in the polluted soil; instead, progressive biodegradation of PAHs enabled major growth of total number of bacteria, *Actinomycetes* and their biological groups. The ability of *Azospirillum* spp. and *Pseu‐ domonas stutzeri*, populating rhizosphere and the inside of grass roots, to free nitrogen fixing and the use of PAHs (phenanthrene, anthracene, and phyrene) as the only source of carbon and energy suggests that in the future, after the series of detailed analysis, it will be possible to invent preparation based on these species, suitable for bioremediation of soils polluted with PAHs, with very limited supplementation of environment with nitrogen fertilizers. The successful results were observed (an important decrease in the content of PAHs in soils) in soil inoculated with *Azospirillum* and *Pseudomonas stutzeri* after grass growth (maize, mead‐ ow fescue). This processes were especially effective in calcareous rendzina artificially pollut‐ ed with PAHs and in soil long‐term contaminated with crude oil [28, 29].

authors [14, 19] isolated 11 strains from a variety of contaminated sites (oil, motor oil, refinery derivatives) with the ability to degrade benzo(a)pyrene. Bacteria capable to PAHs degradation and using as the only source of carbon and energy belong to the main species *Pseudomonas*, *Agrobacterium*, *Bacillus*, *Burkholderia*, *Sphingomonas, and Phanerochaete chrysosporium* [23]. Other authors reported PAH degradation using other bacteria including *Rhodococcus* sp., *Mycobacte‐ rium*, and mixed culture of *Pseudomonas* and *Flavobacterium* species [20]. In study of Heitkamp et al. [24], the authors described about bacterial isolated from oil‐contaminated soil which was capable of mineralizing the pyrene. *Pseudomonas aeruginosa* isolated from a stream heavily polluted by a petroleum refinery was very effective in degradation of phenanthrene [25]. *Pseudomonas aeruginosa* actively grow over high doses of phenanthrene with complete removal of the pollutant in a period of 30 days of the experiment. Other authors report that *Mycobac‐ terium* species isolated from a PAH‐contaminated soil were able to utilize pyrene as the only sole source of carbon and energy (up to 60% of the pyrene added (0.5 mg ml‐1) within 8 days at 20°C of temperature) [26]. Some products of this degradation pathway were analyzed (Cis‐ 4,5‐pyrene dihydrodiol, 4‐5‐phenanthrene dicarboxylic acid, 1‐ hydroxy‐2‐naphthoic acid, 2‐ carboxybenzaldehyde, phthalic acid, and protocatechuic acid). In the study of Yuan et al. [11], the authors isolated strains of bacteria from a petrochemical waste which having the capacity

**Bacteria Plant Contaminant Role of bacteria Ref.**

Tall fescue Polycyclic aromatic

Meadow fescue Maize Winter rye

88 Soil Contamination - Current Consequences and Further Solutions

*Enterobactor cloacae* Tall fescue Total petroleum

hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs)

Hydrocarbons (TPHs)

biphenyls(PCBs)

**Table 1.** Examples of bioremediation of organic contaminants in soil with bacteria species.

Wheat Trichloroethylene (TCE) Degraded TCE with toluene

Crude oil

Alfalfa Polychlorinated

Wheat Crude oil Promoted development of wheat

root system enhanced level

Promoted plant growth under stress

Promoted development of plant root system enhanced level of oil

Increased plant tolerance to PAHs

More effectively metabolized PCBs

Promoted plant growth in the presence of environmental contaminants such as TPHs

and PAH degradation

o‐monooxygenase

with bph gene cloned

Arabidopsis PCBs Utilized plant secondary metabolites [18]

Increased plant tolerance to PAHs [15]

[14]

[9, 10]

[14]

[16]

[17]

of oil degradation

*Azospirillum lipoferum*

*Azospirillum brasilense*

*Azospirillum* spp. *Pseudomonas stutzeri*

*Pseudomonas fluorescens*

*Pseudomona fluorescens*

*Pseudomonas putida*

#### **2.1. Bacterial diversity in soil contaminated with PAHs**

Soil microorganisms play a big roles in various biogeochemical cycles and are responsible for the cycling of organic compounds especially oil derivatives and polycyclic aromatic hydro‐ carbons. Also they influence above‐ground ecosystems by contributing to plant nutrition, plant health, soil structure, and soil fertility. Our knowledge on soil microbial diversity is limited in part by our inability to study soil microorganisms. It is known that in 1 g of soil there are 1030 different soil microorganisms [30]. Only 1% of this soil bacterial population can be cultured by classical methods. About 99% is unknown, and this group of microorganism is possible to measure only in using molecular methods [31, 32].

Various molecular methods have been used to study soil bacterial communities. Many biotic and abiotic factors play a big role to changes in microbial diversity (contamination, anthropo‐ genic activities, plant growth). It is not known how changes in microbial community structure influence ecosystem functions. Study of microorganisms function is the need for reliable and accurate mechanisms of understanding their diversity and taxonomic [33–35].

Typically, diversity studies include the relative diversities of communities across a gradient of stress, disturbance, or other biotic or abiotic difference [35]. It is difficult with current techni‐ ques to study true diversity since we do not know what is present and we have no way of determining the accuracy of our extraction or detection methods. Species diversity consists of species richness, the total number of species present, species evenness, and the distribution of species [32].

Methods to measure microbial diversity in soil can be categorized into two groups: bio‐ chemical‐based techniques and molecular‐based techniques. But more common for studying microbial diversity in soil contaminated with polycyclic aromatic hydrocarbons are the mo‐ lecular methods.

#### **2.2. Limitations of molecular methods to study bacterial diversity in contaminated soils**

Molecular techniques based on polymerase chain reaction (PCR) have been used to overcome the limitations of culture‐based methods; however, they are not without their own limitations [32, 34].

Soil microorganisms (especially bacteria) are located between soil aggregates. There is a very big problem with separating these from micro‐ and macro‐components of soil struc‐ ture. The study bacterial biodiversity requires isolated genomic DNA from bacterial cells [35]. This process is dependent on bacterial cells (gram‐negative or gram‐positive bacterial cells). Gram‐negative cells would be lysed when the cell extraction is sensitive, but the gram‐positive cells may be lysed in stronger conduction, but in this case DNA may be dis‐ integrated [32]. The special method of DNA or RNA extraction from bacterial cells used can also bias biodiversity studies. The harsh and drastic DNA extraction methods (bead beat‐ ing) can shear the nucleic acids, leading to some problems in subsequent PCR detection products [36]. With soil samples, it is necessary to remove some inhibitory substances (ful‐ vic acids, humic acids). These substances can be coextracted and can strongly interfere with subsequent PCR and analysis. Second step of analysis can lead to loss of DNA or RNA in‐ hibitory of PCR. The most popular in bacterial biodiversity studies are primers which tar‐ geted typical regions coding genes present in all organisms such as 16S rRNA or ITS (internal transcribed spacer). This genes have well‐defined regions for taxonomic classifica‐ tion of bacteria and are not subject to horizontal transfer and have sequence databases avail‐ able to researchers.

**2.1. Bacterial diversity in soil contaminated with PAHs**

90 Soil Contamination - Current Consequences and Further Solutions

measure only in using molecular methods [31, 32].

species [32].

lecular methods.

[32, 34].

Soil microorganisms play a big roles in various biogeochemical cycles and are responsible for the cycling of organic compounds especially oil derivatives and polycyclic aromatic hydro‐ carbons. Also they influence above‐ground ecosystems by contributing to plant nutrition, plant health, soil structure, and soil fertility. Our knowledge on soil microbial diversity is limited in part by our inability to study soil microorganisms. It is known that in 1 g of soil there are 1030 different soil microorganisms [30]. Only 1% of this soil bacterial population can be cultured by classical methods. About 99% is unknown, and this group of microorganism is possible to

Various molecular methods have been used to study soil bacterial communities. Many biotic and abiotic factors play a big role to changes in microbial diversity (contamination, anthropo‐ genic activities, plant growth). It is not known how changes in microbial community structure influence ecosystem functions. Study of microorganisms function is the need for reliable and

Typically, diversity studies include the relative diversities of communities across a gradient of stress, disturbance, or other biotic or abiotic difference [35]. It is difficult with current techni‐ ques to study true diversity since we do not know what is present and we have no way of determining the accuracy of our extraction or detection methods. Species diversity consists of species richness, the total number of species present, species evenness, and the distribution of

Methods to measure microbial diversity in soil can be categorized into two groups: bio‐ chemical‐based techniques and molecular‐based techniques. But more common for studying microbial diversity in soil contaminated with polycyclic aromatic hydrocarbons are the mo‐

**2.2. Limitations of molecular methods to study bacterial diversity in contaminated soils**

Molecular techniques based on polymerase chain reaction (PCR) have been used to overcome the limitations of culture‐based methods; however, they are not without their own limitations

Soil microorganisms (especially bacteria) are located between soil aggregates. There is a very big problem with separating these from micro‐ and macro‐components of soil struc‐ ture. The study bacterial biodiversity requires isolated genomic DNA from bacterial cells [35]. This process is dependent on bacterial cells (gram‐negative or gram‐positive bacterial cells). Gram‐negative cells would be lysed when the cell extraction is sensitive, but the gram‐positive cells may be lysed in stronger conduction, but in this case DNA may be dis‐ integrated [32]. The special method of DNA or RNA extraction from bacterial cells used can also bias biodiversity studies. The harsh and drastic DNA extraction methods (bead beat‐ ing) can shear the nucleic acids, leading to some problems in subsequent PCR detection products [36]. With soil samples, it is necessary to remove some inhibitory substances (ful‐ vic acids, humic acids). These substances can be coextracted and can strongly interfere with subsequent PCR and analysis. Second step of analysis can lead to loss of DNA or RNA in‐

accurate mechanisms of understanding their diversity and taxonomic [33–35].

Many authors [32, 34, 36, 37] discussed some issues surrounding differential PCR amplification including different affinities of primers to templates, different copy numbers of target genes, hybridization efficiency, and primer specificity. In addition, some sequences with lower G+C content are thought to separate more efficiently in the denaturing step of polymerase chain reaction and therefore could be preferentially amplified [32, 34]. There are known a few important points in optimalization of PCR such as amplification including different affinities of primers to templates, different copy numbers of target genes, hybridization efficiency, and primer specificity. The above discusses a few limitations of molecular‐based methods, which can influence the analysis and interpretation of their community analysis. Molecular‐based methods provide valuable information about the microbial community as opposed to only culture‐based techniques.
