**3. Bioaugmentation, biostimulation or bioattenuation on PAHs removal**

In the past decades, prominent microorganisms have been obtained and isolated, as consortia or individual strains, able to grow using aromatic compounds as the only carbon and energy source. These microorganisms have been used for PAHs' degradation in soil by bioaugmen‐ tation, as it is mentioned in the following section.

#### **3.1. Bioattenuation**

that have two or more fused aromatic rings arranged in a linear, angular or cluster array. PAHs are found ubiquitously in the environment and are present in oil‐based fuels. Currently, PAHs had become increasingly important because they are considered as emerging contaminants by their high risk to humans and the environment. According to the US Environmental Protection Agency (EPA), they are toxic, mutagenic and carcinogenic, and are priority to be eliminated from the environment. There are several biological alternatives to eliminate PAHs and this chapter focuses on developments in bioaugmentation, biostimulation and bioatten‐

Methods to remove PAHs have been classified as physicochemical, chemical and biological, and are briefly described in **Table 1**. Among biological techniques, bioremediation is consid‐ ered a viable technology, environmental friendly and inexpensive that uses the metabolic diversity of some microorganisms to degrade and decrease the concentration of toxic com‐

Solvent extraction Treatment with two or more solvents, either alone or within mixtures to extract PAHs. Chemical oxidation Different types of oxidants such as Fenton's reagent, ozone, potassium permanganate,

Photocatalytic oxidation‐reaction is used to destroy PAHs in presence of UV light.

It is applied mainly to treat soils with low permeability and contaminated with heavy metals. In addition, co‐contaminated soils with organic pollutants can also be treated.

However, PAHs removal can also occur through synergistic interaction in the rhizosphere

hydrogen peroxide are used to oxidize PAHs.

Thermal technology PAHs can be either destroyed or volatilized by the use of high temperatures.

(plant and microorganism).

**Table 1.** Technologies suitable to remove PAHs from contaminated soils.

microorganisms or by their enzymes.

Phytoremediation Plants are commonly used to extract and sequester heavy metals from contaminated soil.

Biological remediation Mineralization or biotransformation of toxic organic compounds either by specialized

Biological removal or (biodegradation) is a process carried out by aerobic organisms mainly indigenous microorganisms and commonly it reaches the mineralization of toxic compounds to inorganic forms (CO2 and H2O). However, anaerobically PAHs biodegradation under denitrifying and sulphate‐reducing conditions has been well recognized [1]. The aerobic biodegradation mechanism of PAHs begins with the initial oxidation step, either where two atoms of oxygen are incorporated into the aromatic ring to form *cis*‐dihydrodiol or where monooxygenases enzymes are involved in the first initial oxidation to form *trans*‐dihydrodiols. Otherwise, bioremediation can be conducted in two ways: (1) ex situ that is held off the

uation for the removal of PAHs.

330 Soil Contamination - Current Consequences and Further Solutions

pounds.

Photocatalist degradation

Electrokinetic remediation

**Technology Purpos**

**2. Remediation and biodegradation technologies**

It relies on natural processes to dissipate contaminants through biological transformation, during which the indigenous microbial populations degrade recalcitrants or xenobiotics compounds based on their metabolic processes. Bioattenuation includes a variety of chemical, physical and biological processes that reduce the mass, toxicity, volume or concentration of contaminants. These processes include aerobic and anaerobic biodegradation, sorption, volatilization, and chemical or biological stabilization, transformation of contaminants. The time is not a limiting factor and usually is applied on sites with low concentration of contam‐ inants, where no other remedial techniques are applicable.

In order to reveal that bioattenuation occurs in remote areas consistently and continuously, deep‐sea sediments of Artic Ocean were collected in the summer of 2010; the PAHs composi‐ tions were examined and the 16 EPA‐priority PAHs were from 2.0 to 41.6 ng g‐1 dry weight, among them, phenanthrene was relatively abundant in all sediments. The 16S rRNA gene of the total environmental DNA revealed potential degraders. Meanwhile, 40 PAH‐degrading bacteria were isolated though enrichment culture, of which *Cycloclasticus* and *Pseudomonas* showed the best degradation capability under low temperatures. Based on the 16S rDNA library and isolation of strains, the author suggested that bacteria of *Cycloclasticus, Pseudomo‐ nas, Pseudoalteromonas, Halomonas, Marinomonas* and *Dietzia* play the most important role in PAH mineralization in situ [3].

In terrestrial environments, where the biodegradation of a mixture of PAHs (fluorene, phenanthrene and pyrene) in mangrove sediments chronically exposed to industrial discharge, livestock and household waste and wastewater was revealed, the bioattenuation favoured the removal of fluorene and phenanthrene up to 99% while pyrene removal (98%) was only improved by adding salt medium as a nutrient supplement [4]. Besides, the bioattenuation was effective in the removal of total petroleum hydrocarbons (TPHs) and high molecular weight PAH residuals after applying a pilot‐scale biopile remediation treatment, by properly enhancing their catabolic capacities with the addition of lignocellulosic substrate as a biosti‐ mulant [5].

#### **3.2. Biostimulation**

Biostimulation is the addition of nutrients to a contaminated site in order to encourage the growth of naturally occurring chemical‐degrading microorganisms. Generally, inorganic additions of macro (as N, P, K) or micronutrients (as Mg, S, Fe, Cl, Zn, Mn, Cu, Na) are important to recover depleted soils by agricultural management systems or contaminated with PAHs, in order to improve the degradation activity of native or foreign microorganisms. Thus, the type and concentration of nutrient can play an important role in biodegradation of PAHs. Particularly, the effect of biostimulation on phenanthrene removal from contaminated soil via adding macro and/or micronutrients revealed that the optimal phenanthrene reduction resulted when a high level of macronutrient in the range of 67–87% and low level of micro‐ nutrient in the range of 12–32% were used with the nitrogen as the dominant macronutrient [6]. Other strategies had been implemented by the use of stable organic supplements such as compost, sewage sludge, manure, vermicompost, etc., as biostimulant nutrients to activate the catabolic potentials of microorganisms. The success of applying stable organic residuals may be a promissory technology due to the high content of essential nutrients and the harbouring of large quantities of diverse microorganisms that accelerates the biodegradation of some contaminants in soil. The biostimulation with compost achieves an improved removal of PAHs in an artificially contaminated agricultural soil [7]. The dissipation of phenanthrene, anthra‐ cene and benzo(a)pyrene in a spiked agricultural soil amended with manure and vermicom‐ post resulted in a transient effect in the removal of PAHs during the first 30 days [8]. Furthermore, it was observed that the inorganic nutrients or biosolid amendment have a similar effect on the degradation of phenanthrene and anthracene in an artificially contami‐ nated agricultural soil. Polyacrylamide, a flocculant used in wastewater treatment, was added in two different artificially contaminated soils, and the concentrations of phenanthrene and anthracene were removed rapidly in both soils (agricultural soil and alkaline‐saline soil) [9].

#### **3.3. Bioaugmentation**

It is defined as a technique for improvement of the removal capacity of contaminated areas by the introduction of specific competent strains or consortia of microorganisms to the contami‐ nated site, thus favouring the biodegradation process. In this way, a bacterial mixed culture was added to a PAHs (pyrene and benzo[a]pyrene)‐contaminated soil, and after the treatment, the mineralization rate of pyrene was about 36% (after 150 days), and benzo[a]pyrene 5% (after 70 days) [10]. Similar results were observed with *Scopulariopsis brevicaulis* PZ‐4 that was able to remove phenanthrene (60%), fluoranthene (62%), pyrene (64%) and benzo[a]pyrene (82%) in liquid medium after 30 days of incubation; while, in a PAH‐contaminated soil, PZ‐4 removed 77% of total PAHs and the highest removal of PAHs occurred for phenanthrene (89%) and benzo[a]pyrene (75%) after incubation for 28 days [11]. On the other hand, organic pollutant‐ contaminated soils are often co‐contaminated with heavy metals, and the success of applying a bioaugmentation treatment has been tested by some authors; for example, a bacterial consortium composed by 12 indigenous strains with different catabolic capacities (resistant to heavy metals, producer of surfactants and degraders of hydrocarbons) was added in a soil spiked with diesel oil and heavy metals (Pb and Zn) obtaining the total removal of diesel oil [12]. Consequently, the authors concluded that the entire indigenous community was pushed towards an effective bioremediation by the addition of the microbial consortium.

#### **3.4. Combinations and improvements in biodegradation techniques**

livestock and household waste and wastewater was revealed, the bioattenuation favoured the removal of fluorene and phenanthrene up to 99% while pyrene removal (98%) was only improved by adding salt medium as a nutrient supplement [4]. Besides, the bioattenuation was effective in the removal of total petroleum hydrocarbons (TPHs) and high molecular weight PAH residuals after applying a pilot‐scale biopile remediation treatment, by properly enhancing their catabolic capacities with the addition of lignocellulosic substrate as a biosti‐

Biostimulation is the addition of nutrients to a contaminated site in order to encourage the growth of naturally occurring chemical‐degrading microorganisms. Generally, inorganic additions of macro (as N, P, K) or micronutrients (as Mg, S, Fe, Cl, Zn, Mn, Cu, Na) are important to recover depleted soils by agricultural management systems or contaminated with PAHs, in order to improve the degradation activity of native or foreign microorganisms. Thus, the type and concentration of nutrient can play an important role in biodegradation of PAHs. Particularly, the effect of biostimulation on phenanthrene removal from contaminated soil via adding macro and/or micronutrients revealed that the optimal phenanthrene reduction resulted when a high level of macronutrient in the range of 67–87% and low level of micro‐ nutrient in the range of 12–32% were used with the nitrogen as the dominant macronutrient [6]. Other strategies had been implemented by the use of stable organic supplements such as compost, sewage sludge, manure, vermicompost, etc., as biostimulant nutrients to activate the catabolic potentials of microorganisms. The success of applying stable organic residuals may be a promissory technology due to the high content of essential nutrients and the harbouring of large quantities of diverse microorganisms that accelerates the biodegradation of some contaminants in soil. The biostimulation with compost achieves an improved removal of PAHs in an artificially contaminated agricultural soil [7]. The dissipation of phenanthrene, anthra‐ cene and benzo(a)pyrene in a spiked agricultural soil amended with manure and vermicom‐ post resulted in a transient effect in the removal of PAHs during the first 30 days [8]. Furthermore, it was observed that the inorganic nutrients or biosolid amendment have a similar effect on the degradation of phenanthrene and anthracene in an artificially contami‐ nated agricultural soil. Polyacrylamide, a flocculant used in wastewater treatment, was added in two different artificially contaminated soils, and the concentrations of phenanthrene and anthracene were removed rapidly in both soils (agricultural soil and alkaline‐saline soil) [9].

It is defined as a technique for improvement of the removal capacity of contaminated areas by the introduction of specific competent strains or consortia of microorganisms to the contami‐ nated site, thus favouring the biodegradation process. In this way, a bacterial mixed culture was added to a PAHs (pyrene and benzo[a]pyrene)‐contaminated soil, and after the treatment, the mineralization rate of pyrene was about 36% (after 150 days), and benzo[a]pyrene 5% (after 70 days) [10]. Similar results were observed with *Scopulariopsis brevicaulis* PZ‐4 that was able to remove phenanthrene (60%), fluoranthene (62%), pyrene (64%) and benzo[a]pyrene (82%)

mulant [5].

**3.2. Biostimulation**

332 Soil Contamination - Current Consequences and Further Solutions

**3.3. Bioaugmentation**

Some reports showed that the addition of microorganisms (bioaugmentation) or nutrients (biostimulation) either individually or combined have negligible effects on the removal of PAHs at field or microcosm level. In this manner, the effect of applying bacterial or fungus consortium to artificially contaminated forest soil with a mixture of PAHs reported that bioaugmentation did not improve the removal of naphthalene, phenanthrene, anthracene and pyrene as compared to bioattenuation [13]. However, successful approaches were achieved when nutrients and microorganisms were added simultaneously [14] or successively during the treatment [15]. Therefore, some modifications have been made in bioremediation techni‐ ques to improve the removal efficiency of PAHs. A strategy is the use of carriers and the results obtained are promising. Biocarriers have particular characteristics that allow microbial survival by providing a temporary nutrition medium and a protective niche. Immobilization of cells also avoids protozoan grazing and promotes a slow release of cells from the biocarriers, prolonging their degrading activity. Encapsulated *Pseudomonas aeruginosa* strains effectively removed PAHs only in the soil bioaugmented with nutrients, moisture and oxygen supplies [16]. Another modification to bioremediation technique is the dose of the inoculum. It has been seen that the use of several doses of the inoculum improves the removal of contaminants in comparison with a single dose. Thus, the inoculation of two doses in different times of a specialized bacterial consortium, able to degrade alkanes and PAHs, improved the overall removal of TPHs above 30% [15]. The repeated inoculation of *Arthrobacter* sp. to an artificially contaminated soil improved the removal of phenanthrene as compared to one dosage [17].

The addition of compounds with similar characteristics to the contaminants can stimulate indigenous microorganisms of the soils suggested that the ability of indigenous microorgan‐ isms to remove a particular contaminant could be enhanced by the presence of other contam‐ inants or by the repeated exposure to the contaminant of concern, which favours the selection of specific microorganisms with desired specific metabolic capabilities. Additionally, the effect of adding various types of chlorophenols at different concentrations on the indigenous population from a calcareous agricultural soil without a previous history of exposure to such contaminants helped microorganisms to survive and stay alive during the treatment even in the presence of a more toxic compound [18]. On the other hand, knowledge of the physico‐ chemical properties of soil is important to establish and design the best strategy bioremedia‐ tion. The response of indigenous microorganisms in an artificially contaminated agricultural soil was studied, and it was faster during the removal of phenanthrene than fluoranthene. This difference was attributed to the physicochemical properties of both contaminants and the specific metabolic capacity showed by the microorganisms at the onset of the experiment [19]. PAHs‐contaminated soil has a negative impact on the stability of an ecosystem, therefore the physicochemical properties of a contaminated soil and its associated microbial community should be considered to ensure the success of bioremediation. The knowledge of these parameters will avoid conflicting reactions between the different techniques of bioremedia‐ tion. Therefore, it is necessary to conduct assays of the combinations of techniques at labora‐ tory level to determine the synergistic effects and to achieve improvements in the PAHs degradation in the soil.
