**5. Organic matter content and particle size: sorption or sequestering; how could they affect the bioavailability?**

Soil is composed of organic and inorganic components separated by pores containing water or air. The interactions between hydrocarbons and mineral surfaces (clay, silt and sand) are only significant when the organic matter content is <0–1%. Thus, organic matter is very important in the fate and behaviour of organic contaminants in soil. The soil organic matter can be divided into two types: soft carbon (rubbery), which is defined as expanded and flexible structures with humic and fulvic acids as component with reversible sorption, and hard carbon (glassy), defined as rigid and condensed structures with humin, kerogen and pyrogenic carbon as commonly identified components, which are involved in irreversible sequestration [23]. Therefore, the organic matter content can directly affect the bioavailability of contaminants to microorganisms by sorption or sequestration mechanisms, and thus the success of bioreme‐ diation technologies can be hindered. The effect of organic matter on the degradation of PAH was studied in [24], and it was found that microbial activity was influenced by the amount of organic matter in the soil by either nutrient limitation or PAHs sequestration. In addition, microbial activities developed in humic acid were much higher than those developed in humin (aged organic matter), demonstrating that humin is able to sequester organic contaminant in a stronger way. In another study, it was demonstrated that a high content of organic carbon in the soil produces a low degradation rate of PAHs by indigenous microorganisms [25], indicating The sequestration of PAHs by organic carbon is the major mechanism for the accumulation of PAH in soils. On the other hand, it has been proposed that humic acids promote degradation of aromatic compounds by changing pore size and the structure of the soil [26]. It has been well known that the mineral complexes also affect the bioavailability of some contaminants because they could be involved in sorption phenomena (adsorption and desorption). Different bioremediation techniques were applied to a clay soil artificially contaminated with diesel oil and the removal rate of PAH was depending on adsorption and desorption phenomena [27]. Additionally, the soil organic matter presents different sorption properties due to its biochemical contents, which include substances such as polysaccharides, lipids, lignin, proteins, humic substances, kerogen and black carbon.

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

Bioremediation is influenced by abiotic factors such as temperature, humidity, pH, aeration, nutrient content, redox potential and soil type; however, interaction of biotic factors such as competition, predation and biological factors also play a major role in the success of this technique [20]. Some studies have shown that the microorganisms added for degrading contaminants at laboratory level were not able to mineralize, survive or compete with the native microorganisms when they were introduced into foreign environments, probably due to susceptibility to toxins or predators in the environment, due to the preferential use of easily assimilated organic compounds or due to slow motion throughout the inner porous soil that harbours the contaminant [21]. To facilitate the adaptation of microorganisms added to a soil, the following criteria must be considered: contaminant‐availability for microorganisms; microbial activity; survival of microorganisms in the foreign environment; and environmental conditions such as nutrient availability, water content and pore size of the aggregates [20]. On the other hand, when a population is introduced into a foreign site, it tends to decrease with time due to the abiotic and biotic factors mentioned above, and thus the treatment can be adjusted either by adding more specialized microorganisms or by using immobilized bacteria [22]. The introduction of a microorganism in an environment is complex and its permanence may be only temporarily, depending on the ability of the microorganism to adapt to environ‐ mental conditions. The strategy to isolate indigenous microorganisms and incorporate them into the environmental is a viable alternative; however, this technique does not always produce

**4. Limiting factors for a successful biological remediation**

the expected results, suggesting that the above factors play an important role.

**could they affect the bioavailability?**

**5. Organic matter content and particle size: sorption or sequestering; how**

Soil is composed of organic and inorganic components separated by pores containing water or air. The interactions between hydrocarbons and mineral surfaces (clay, silt and sand) are only significant when the organic matter content is <0–1%. Thus, organic matter is very

degradation in the soil.

334 Soil Contamination - Current Consequences and Further Solutions

The particle size of the aggregates, the shape and the interconnections amongst the pores of a soil are physical factors that determine the microbial colonization, since they have effect on air diffusion and water infiltration. The association of soil organic matter with secondary minerals, such as clay and amorphous oxides, form complex organomineral aggregates which partici‐ pates in the soil structure. Furthermore, it has been observed that PAHs distribution in soil depends mainly on the hydrophobicity of the PAH and their affinity towards microcompart‐ ments of the aggregates [28]. It is known that as time goes on in a contaminated soil, the contaminants diffuse into hydrophobic areas (ageing), reducing the bioavailability to the microorganisms and thus slowing down their removal. Some authors suggest that biodegra‐ dation and removal of contaminants become difficult with ageing of soil; moreover, the rate of desorption of PAH decreases, persisting even in the presence of indigenous microorganism degraders [29]. Bioavailability of anthracene in freshly and aged spiked agricultural soil were studied by its removal efficiency. The 72% of anthracene was removed in freshly spiked soil, while only 34% was degraded in aged soil [30]. However, in experiments conducted in [31], the lack of response of microorganisms to some contaminants is not related to a limited bioavailability, but rather is related to microbial factors, such as lack of co‐metabolic substrates or insufficient numbers of hydrocarbon‐degrading populations. Besides, it was found that biostimulation with inorganic nutrients and terminal electron acceptors did not improve the removal of PAHs in freshly spiked soil with phenanthrene or pyrene [32]. Moreover, total

biodegradation extent was evident in ageing but not in freshly spiked soil, which was consid‐ ered to be the result of the adaptation of indigenous bacteria *P. aeruginosa* by entering a stationary phase during the time of ageing (200 days) and by the subsequent production of surfactants. On the other hand, it was suggested that ageing of the soil is not the main parameter influencing PAH‐availability level, but the complexity of the organic constituents (i.e. coal tar, pitch, soot or coke) influence overall PAH availability in soil [33]. In addition, some bioremediation studies have evidenced the importance of the physicochemical param‐ eters of organic contaminants on the availability to microorganisms, which have effect on the biodegradation rate [27]. Soil properties and the indigenous microbial population affect the level of biodegradation; therefore, a detailed study on soil properties such as physicochemical and biological parameters must be performed to select the bioremediation technique.
