Ghosh and Mukherji compared the effects of two nonionic surfactants and rhamnolipid biosurfactant at 10X CMC on mineralization of pyrene in liquid culture and found that the rhamnolipid amendment resulted in decreased mineralization compared to the unamended control due to the preferential degradation of the biosurfactant over pyrene, causing a decreased specific growth rate and pyrene utilization by P. aeruginosa. Bezza and Chirwa also observed a decrease in PAH degradation in the rhamnolipid and nutrient supplemented liquid culture treatments because of preferential microbial consumption of the biosurfactant. Most of the studies examining the preferential utilization of surfactants by PAHdegrading microorganisms were accomplished in liquid culture, and relatively few studies have examined this phenomenon in a soil matrix that would have a more realistic representation of  bio-remediation practices. For example, while nonionic surfactants are less likely to adsorb to soil surfaces compared to cationic surfactants due to the negative charge of the soil, some of the Brij-35 or rhamnolipid biosurfactant added to the mineralization assays was likely sorbed to the soil . Lladò et al. concluded that rhamnolipid biosurfactant applied to a microbial consortium resulted in increased PAH biodegradation in liquid culture; however, the same rhamnolipid biosurfactant application did not improve PAH biodegradation after 200 d when applied to a soil matrix. Maslin and Maier suggested that rhamnolipid biosurfactant was used as a preferential carbon source over phenanthrene by the native soil microbial populations when the rhamnolipid was present at higher concentrations. Deschênes et al. observed that the biodegradation of three-ringed PAHs was not affected by the addition of rhamnolipid biosurfactant at concentrations below and above the CMC; however,fodder system trays four-ringed PAHs anthracene, and chrysene had a 4–6 week lag phase before biodegradation commenced in aged PAH-contaminated soil.

The enumeration of viable M. vanbaalenii PYR- 1 CFUs in the rhamnolipid-amended treatments had a similar pattern compared to the treatments amended with glucose, an easily degraded monosaccharide, with substantial growth at the beginning of the incubation, followed by significant decrease in CFUs as the carbon substrate used for microbial growth was depleted. Yu et al. also observed rapid PAH-degrading bacteria growth using rhamnolipid biosurfactant up to 1000 mg L−1 , which resulted in a 40-fold increase in the maximum bacteria population as compared to the unamended control. Bezza and Chirwa concluded that the addition of a lipopeptide biosurfactant produced by Bacillus cereus SPL-4 resulted in a significant increase in the number of CFUs in PAHcontaminated soil during a 64-d incubation. While nontoxic to M. vanbaalenii PYR-1, Brij-35 at both surfactant concentrations showed increased resistance to degradation as compared to the rhamnolipid biosurfactant based upon viable M. vanbaalenii PYR-1 CFUs. In the Brij-35 10X CMC-amended culture, viable CFUs were significantly lower compared to the Brij- 35 0.1X CMC treatments after 14 d of incubation, potentially due to the limited bio-availability of the surfactant in the micellar phase at 10X CMC as compared to the monomeric surfactant phase at 0.1X CMC . In another study, Zhang et al. showed that the small radius size and structure characteristics of the micellized nonionic surfactants could prevent close contact between the microorganism and surfactant, resulting in limited degradation. Previous studies also showed preferential degradation of biosurfactants compared to synthetic surfactants by microorganisms. Zeng et al. compared the microbial degradation of a nonionic surfactant , cationic surfactant , anionic surfactant , and biosurfactant and observed that the rhamnolipid biosurfactant was the most readily biodegradable among all surfactants in the study. Mohan et al. compared the biodegradability of Triton X-100 and rhamnolipid biosurfactant as sole carbon sources to Vibriocyclotrophicus sp. nov., a PAH-degrading microorganism, and showed that the rhamnolipid was readily biodegradable under aerobic conditions while Triton X-100 was only partially biodegradable. Based on results from the 14C-pyrene mineralization experiment and the effect of surfactants on cell growth, the amendment of Brij-35 or rhamnolipid biosurfactant at the highest rate was not toxic to the PAH-degrading soil microorganisms.

Therefore, it may be concluded that in this study, the rhamnolipid biosurfactant was preferentially used by the PAH-degrading soil microorganisms and only after the eventual depletion of the biosurfactant, the mineralization of 14C-pyrene in both soils would start. This wasconsistent with the observation that the lag time of pyrene mineralization increased with increasing rhamnolipid amendment rate .Compared to the native sandy loam soil treatments, the native clay soil treatments had a significantly shorter lag period in the unamended and all surfactant-amended treatments, except for rhamnolipid at the high rate . The differences between the clay and sandy loam soil treatments in 14C-pyrene mineralization by the native microbes may be attributed to the higher TOC in the clay soil. The quantity and quality of soil organic matter has been previously shown to regulate the soil microbial community diversity and activity, as organic matter acts as energy and nutrient sources for heterotrophic soil microorganisms, stimulating microbial biomass growth and activity . Additionally, the finer silt and clay fractions could provide more sites for bacteria or organic matter attachment, leading to a larger microbial biomass in the clay aggregate and better utilization of 14C-pyrene . Bioaugmentation of M. vanbaalenii PYR-1 significantly increased the mineralization of 14C-pyrene in both PAH-contaminated soils . Because of the rapid mineralization, bioaugmentation of M. vanbaalenii PYR-1 in soils with or without surfactant resulted in a shortened lag period before 14C-pyrene mineralization commenced as compared to all corresponding soil treatments without the bacterial augmentation . There were no differences in the length of lag period between the clay and sandy loam bioaugmentation treatments, except for the medium rate rhamnolipid treatment where the clay soil had a shorter lag period than the sandy loam soil . Additionally, bioaugmentation of M. vanbaalenii PYR-1 in both soils resulted in an increased biodegradation rate in all soil treatments as compared to the native soils, except for the rhamnolipid-amended treatments at medium and high rates . The bioaugmentation of M. vanbaalenii PYR-1 in the clay soil treatments resulted in greater biodegradation rates compared to the bioaugmented sandy loam soil treatments, except for treatments amended with rhamnolipid biosurfactant at themedium and high rates . The addition of Brij-35 at the low and medium rates in the bioaugmented clay soil treatments resulted in a higher biodegradation rate, with the rate constant at 3.88 d−1 and 4.55 d−1 , respectively, as compared to the bioaugmented clay soil without the surfactant . However, after 25 d of incubation, the microbes in the native clay soil amended with Brij-35 surfactant at all three levels had undergone rapid 14C-pyrene mineralization and were not significantly different as compared to the bioaugmented clay soil treatments amended with Brij-35 .

This suggests that the bioaugmentation of PAH degrading microbes may not be necessary for treating PAH-contaminated soils in environments where the native soil microbial community is capable of degrading PAHs and in situations where PAHs are readily bioavailable. After 50 d of incubation, there were no significant differences between the unamended bioaugmented soil treatments, and the bioaugmented soil amended with Brij-35 at all levels . This observation may be partially attributed to the production of surface-active trehalose containing glycolipids by M. vanbaalenii PYR-1 . It is worth noting that the mixture of the biologically produced glycolipids by M. vanbaalenii PYR-1 with the rhamnolipid biosurfactant or Brij-35 surfactant did not result in increased 14C-pyrene mineralization. In a previous study, mixtures of synthetic surfactants such as sodium dodecyl sulfate, Tween-80, Triton X-100, and Brij-35 had a synergistic effect,aeroponic tower garden system causing a lower CMC as well as a significant PAH solubility enhancement in the mixed surfactant systems, leading to increased biodegradation of phenanthrene . Mycobacterium vanbaalenii PYR-1 has been extensively studied in pure cultures for the elucidation of mechanisms of PAH degradation; however, few studies have examined PAH biodegradation by M. vanbaalenii PYR-1 in contaminated soils where microbe survival and growth may be a limiting factor due to various environmental and microbial variables such as microbial competition with native soil populations, nutrient availability, moisture content, soil pH, and soil temperature . In the current study, the survival and PAH-degradation enhancement of M. vanbaalenii PYR-1 bioaugmentation can be clearly seen in the unamended and rhamnolipid-amended treatments by the significant decrease in lag period and total 14C-pyrene mineralization . Ghaley et al. also observed rapid PAH mineralization in pyrene-contaminated soils bioaugmented with M. vanbaalenii PYR-1 with a similar lag period and total pyrene mineralization . A similar Mycobacterium sp. also exhibited effective degradation potential for PAHs, resulting in enhanced 14C-pyrene mineralization in three different petroleum contaminated soils . Polycyclic aromatic hydrocarbons are a class of fused-ring aromatic compounds and 16 PAH compounds are designated as priority pollutants by the U.S. EPA because of their known or suspected toxicity and genotoxicity as well as frequent environmental occurrence . Bioremediation, the utilization of microorganisms to biologically degrade hazardous organic compounds to levels below concentration limits established by regulatory authorities, is considered an effective technique to remediate soils contaminated with PAHs . In a 2007 U.S. EPA report on contaminant treatment technologies, 37 out of 145 PAH remediation projects bio-remediation applications . However, biological treatment of PAHcontaminated soils is dependent upon the presence and degradation activity of soil microbes capable of transforming the priority pollutants .

Additionally,  bio-remediation efficacy for PAH-contaminated soil may also be limited by the bioavailability of soil-bound PAHs due to their physical and chemical properties, resulting in low aqueous solubility and high solid-water distribution ratios that promote PAH accumulation in the solid phases of the terrestrial environment . Surfactant-amended bio-remediation has been proposed to increase the rate of PAH desorption from the soil to the aqueous phase through micellar solubilization and/or by direct modification of the soil matrix . In recent years, interest inand feasibility of biosurfactant-enhanced bio-remediation has increased because rhamnolipid biosurfactants offer several advantages over their synthetic counterparts such as increased stability in pH and temperature extremes and environmental compatibility, while still offering similar PAH desorption effects . Under situations where PAH bioavailability is limited, surfactants and biosurfactants have been shown to increase transport of PAHs from the soil matrix into the aqueous phase, resulting in increased bioavailability to PAH-degrading microorganisms and enhance PAH biodegradation . However, surfactants may also negatively affect the PAH-degrading activity of the soil microbial community in PAH-contaminated soils . For example, some surfactants, especially anionic surfactants, can bind to peptides, enzymes, and DNA and change the biological function of soil microorganisms . Conversely, surfactants can also be preferentially utilized as carbon and energy sources for growth by PAH-degrading microorganisms, resulting in a decrease in metabolism of the target contaminant, thus inhibiting PAH biodegradation . However, the effects of surfactant amendments on the PAH degradation capacities of the soil microbial community has been rarely examined. A better understanding of the soil microbial community dynamics in response to amendment of different surfactants is valuable for successful surfactant-enhanced  bio-remediation of PAH-contaminated soils. We previously reported that amendment of the synthetic surfactant Brij-35 enhanced pyrene mineralization in soils, while amendment of rhamnolipid biosurfactant significantly inhibited the mineralization and also increased the lag period before mineralization commenced . As the differences in pyrene degradation likely resulted from changes in the soil microbial communities due to surfactant application, we hypothesized that the PAH-degrading soil microorganisms that maintained a high relative abundance could be identified by analysis of 16S rRNA gene sequences. The objectives of this study were to evaluate the effects of Brij-35 and rhamnolipid amendment on the native soil microbes associated with PAH mineralization and to determine whether such effects were dose dependent. The 16S rRNA gene highthroughput sequencing and phylogenetic investigation of communities by reconstruction of unobserved states were employed to analyze the shifts in soil microbial taxa due to the presence of surfactants and to assess the bacterial species and functional genes responsible for PAH biodegradation.For each sample, 2 g soil was spiked with 10 mg kg-1 pyrene according to Brinch et al. . An additional 8 g soil was added to the treated soil and then mixed over a 5-d period. Following pyrene spiking, 10 mL sterilized minimal basal salts solution was added to the native soil treatment, followed by the addition of 10 mL surfactant solution resulting in initial amendment rates of 14, 140, and 1,400 µg g-1 for rhamnolipid biosurfactant and 21.6, 216, and 2,160 µg g-1 for Brij-35, respectively.