# Bioaugmentation Treatment of a PAH-Polluted Soil in a Slurry Bioreactor
## 3 Results and Discussion
### Evolution of Bacterial Quantity and Diversity during the Bioslurry Treatments  
The plate count numbers of the culturable bacteria showed low variation during biostimulation (Treatment A; data not shown). In the _Rhodococcus_ biostimulation-bioaugmentation experiment (Treatment B), there was a decrease in the early stages from \(10^{8}\) to \(10^{7}\) CFU/g at seven days. However, the values increased to \(3.0\times 10^{7}\) after 15 days, suggesting possible competition between the augmentation strain used (_Rhodococcus_) and the native bacteria. This finding correlates with the lower rates of PAH degradation observed in the early times compared with the other reactors (Figure 3). In contrast, the addition of _Pseudomonas_ (Treatment C) was accompanied by a slight increase of more than \(10^{8}\) CFU/g at three days and the simultaneous disappearance of PAHs (Figure 3). From this time point, the number of bacteria decreased, reaching around \(3.0\times 10^{7}\) CFU/g at the end of the treatment. Figure 4: Total ion chromatogram (TIC) of the contaminants mixture of the study soil at the beginning (top) and after 15 days of treatment (bottom; control treatment). (1) Indene; (2) Methyl-phenol; (3) Cresols; (4) Naphthalene; (5) Quinoline; (6) C1-Alkylnaphthalenes; (7) Biphenyl; (8) C2-Alkylnaphthalenes; (9) Acenaphthene; (10) Dibenzofuran; (11) Fluorene; (12) Tetramethyl-biphenyl; (13) Dibenzothiophene; (14) Phenanthrene; (15) Anthracene; (16) Carbazole; (17) C1-Alkylanthracenes and phenanthrenes; (18) Dimethoxy-dimethylbiphenyl; (19) Fluoroanthene; (20) Pyrene; B[a]P; (21) C1-Alkylpyrenes; (22) Benzo[a] Anthracene; (23) Chrysene; (24) Bis(2-ethylhexyl)phthalate (DEHP); (25) Benzo[a]pyrene and other five- and six-ring PAHs; * procedure contaminant. The analysis of the soil culturable bacteria of the bioreactor experiments also provided useful additional information on their capacity to degrade PAHs. Twenty predominant aerobic bacterial colonies with distinct morphologies (colour, texture, size, etc.) were isolated from the bioreactor at various time points. The RFLP profiles obtained after the digestion of the 16S rDNA showed the presence of 10 distinct ribotypes (Table 4). Phylogenetic affiliation was assigned by sequencing the 16S rRNA genes and carrying out a subsequent comparison with the sequences deposited in the GenBank DNA. _Microbacterium_ (class Actinobacteria) has been described to degrade PAHs, as has _Diaphorobacter_ (belonging to the class Betaproteobacteria, family Comamonadaceae) and _Pseudoxanthomonas_ (phylum Proteobacteria, family Xanthomonadaceae), which have been reported as pyrene degraders. Various species of the genus _Olivibacter_ (phylum Bacteroidetes, class Sphingobacteriaceae) degrade hydrocarbons and can be found in contaminated soils and waste [55]. _Rhizobium_ belongs to the Rhizaceae family, of the class Alphaproteobacteria, which can degrade phenanthrene, alone or in consortium with _Pseudomonas_ and other bacteria [56; 57]. Figure 5: Single ion mode (SIM) chromatogram of ion (_m/z_): 57 representative of alkanes of the study soil at the beginning (top) and after 15 days of treatment (bottom; control treatment). Ci—n-alkanes of the “i” carbons chain. Pr—Pristane; Ph—Phytane; Hp—Hopanes; DEHP—Bis(2-ethylhexyl)phthalate \({}^{*}\) procedure contaminant. As usually occurs in hydrocarbon-enriched environments [27], phylum Proteobacteria of the class Gammaproteobacteria and specifically the _Pseudomonas_ genera were predominant in the culturable bacteria isolated. Various PAH degrader species of _Pseudomonas_ have been isolated from batch and bioslurry experiments, and strains of _Pseudomonas_, _Rhodococcus_, and _Burkholderia_ species have been successfully used for pyrene degradation in microcosm experiments with contaminated soil [22]. The efficiency of _P. stutzeri_ for bioaugmentation in PAH bioremediation is also supported by a previous study conducted at the bioreactor scale [58].