Using weighted Principal Coordinates Analysis , the differences between the soil microbial communities of the two soils can be seen by the two distinct clusters, regardless of surfactant amendment . Due to their broad occurrence in numerous contaminated sites and diverse metabolic pathways for xenobiotic degradation, genera of the Sphingomonadaceae family such as Sphingomonas are considered effective PAH-degrading soil microorganisms . Bastida et al. evaluated PAH biodegradation in a semiarid petroleum-contaminated soil amended with compost and concluded that Sphingomonadales played a dominant role in the initial steps of PAH biodegradation, suggesting that Sphingomonadales were primarily responsible for the conversion of the aromatic hydrocarbons into cis-dihdyrodiol via dioxygenases as well as in the metacleavage pathway to catechol. Kaistobacter has only recently been linked with PAH degradation and their role in PAH biodegradation is still unclear; however, Li et al. utilized 13C-phenanthrene and stable isotope probing in activated sludge and suggested that Kaistobacter was among the primary native microorganisms responsible for phenanthrene degradation. Wang et al. utilized KEGG functional prediction and PICRUSt analysis of PAH-contaminated sediment and concluded that Kaistobacter contributed to the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, specifically, the process of metabolizing pyrene to 3,4-dihydroxyphenanthrene. Although Firmicutes and Proteobacteria phyla comprised a substantial proportion of the soil microbial community, the effects of Brij-35 and rhamnolipid surfactant application, particularly at the high rates, on soil microbial dynamics was apparent. In the pyrene-contaminated clay and sandy loam soils, the OTU numbers and Shannon diversity index were not different from the surfactant-amended treatments in both native and bio-augmented soil treatments,hydroponic bucket except for the addition of rhamnolipid at the high rate, which resulted in a dramatic decrease in OTU number and Shannon diversity index .
The Shannon diversity index of the Native+Pyrene clay and sandy loam soils decreased from 6.53 and 7.74 to 2.54 and 4.18, respectively, in the soils amended with rhamnolipid at the high rate. Notably, the abundance of Bacillus present after the 50-d incubation of the pyrene-contaminated clay soil, with or without bio-augmentation, was less than 2% when rhamnolipid bio-surfactant was amended at the high rate . In the native clay soil amended with rhamnolipid at the high rate, the most dominant genus was Mycoplana . The ability of Mycoplana to effectively use the rhamnolipid bio-surfactant as a carbon source likely resulted in a substantial decrease in the abundance of known PAH degraders, such as Bacillus, Sphingomonas, Kaistobacter, Mycobacterium, and Rhodococcus that were present in other soil treatments . Although it has been shown that some species of Mycoplana such as Mycoplana sp. MWVMB2 were capable of effective PAH biodegradation in soils contaminated with phenanthrene up to 200 mg kg-1 with or without the use of surfactants such as Span 80, Tween 20, cetyl trimethyl ammonium bromide, sodium dodecyl sulfate, and Triton X-100, the Mycoplana sp. that was the dominant genus identified in this study was not able to mineralize pyrene after 50 d . In contrast, the native sandy loam soil amended with rhamnolipid at the high rate did not follow this trend and Bacillus comprised approximately 58% of the genera relative abundance . It should be noted that at the end of the 50-d mineralization study, the native sandy loam amended with rhamnolipid at the medium or high rate was just commencing pyrene mineralization, suggesting that the rhamnolipid biosurfactant was potentially exhausted as a preferential carbon source by the soil microbes . A study by Wang et al. considered the influence of rhamnolipid biosurfactant, Tween 80, and sodium dodecyl benzenesulfonate at 5, 10, 50, 100, and 1,000 mg kg-1 on soil microbial dynamics and PAH biodegradation in aged PAH-contaminated soil. The researchers reapplied the surfactants after 42 d due to surfactant adsorption onto solid matrices as well as partial surfactant biodegradation based upon surfactant degradation results by Cserháti et al. . Wang et al. observed similar results to the sandy loam soil amended with rhamnolipid at the high rate in this study, with Bacillus abundance being three to five times as high as that of the other surfactant-amended PAH-contaminated soils.Additionally, the native sandy loam soil amended with rhamnolipid biosurfactant at the medium rate as well as the bioaugmented sandy loam soil amended with rhamnolipid at the high rate contained a substantially greater relative abundance of Pseudomonas compared to the unamended and bioaugmented sandy loam soil .
Pseudomonas are known PAH-degrading soil microorganisms and have been shown to effectively degrade PAHs such as naphthalene, phenanthrene, pyrene, and anthracene in crude-oil contaminated soils. The PAH biodegradation by Pseudomonas was also shown to be enhanced in the presence of surfactants such as Tween 80, Triton 100, and rhamnolipid biosurfactant . Cébron et al. used DNA stable isotope probing in 13C-phenanthrene-contaminated soil to assess the effects of ryegrass root exudates on PAH biodegradation and concluded that Pseudomonas sp. was one of the few soil microorganisms activated by the root exudates because the easily degradable carbon source addition provided by the root exudates favored the development of fast-growing rstrategists and copiotrophic soil microorganisms belonging to Gammaproteobacteria . Rhamnolipid biosurfactant, which is composed of a β-hydroxy fatty acid connected by the carboxyl moiety to a rhamnose sugar molecule, has the potential to also be utilized by Pseudomonas as an easily degradable carbon source similar to root exudates . Colores et al. investigated the effect of Witconol SN70 nonionic surfactant on the soil microbial community as well as the biodegradation of hexadecane and concluded that Pseudomonas populations in the soil could utilize both the surfactant and hexadecane for growth, which could have important implications on remediation efforts. The effect of rhamnolipid at the high rate can also be seen using weighted PCoA, where the treatments in both soils clustered separate of the other unamended and surfactant-amended treatments . Additionally, Brij-35 surfactant at the high rate resulted in a cluster separate from the unamended and surfactants amended at the low and medium rates, which were clustered together, indicating no substantial difference in the soil microbial communities . The amendment of either surfactant at various rates, except rhamnolipid at the high rate, to the sandy loam soil resulted in an increase in Brevibacillus abundance compared to the unamended native or bioaugmented sandy loam soil . Wei et al. evaluated Brevibacillus in liquid culture spiked with pyrene and showed that Brevibacillus was able to degrade 57% of pyrene as the sole energy and carbon source; however, these findings have yet to be repeated in a soil system and warrant future research,stackable planters as the increased abundance of Brevibacillus may be attributable to growth due to surfactant degradation in the sandy loam soil and may have important implications for surfactant-enhanced bio-remediation.
The addition of Brij-35 at the low rate to the native or bio-augmented sandy loam soil resulted in a dramatic increase in Bacillus compared to the unamended native or bio-augmented sandy loam . The greater Bacillus abundance in the native sandy loam soil amended with Brij-35 at the low rate may have contributed to the increased pyrene mineralization compared to the native unamended sandy loam soil . The inoculation of PAH-degrading bacteria in a wide range of contaminated soils has been successfully implemented for the removal of priority PAH pollutants and continues to be a promising remediation method due to its low cost, lack of secondary pollution, and environmental safety . The bio-augmentation of M. vanbaalenii PYR-1, an isolate from an oil-contaminated estuary of the Gulf of Mexico, Redfish Bay, near Aransas Pass, has previously been shown to significantly enhance the initiation and rate of PAH mineralization in both PAH-contaminated soils compared to the native soils.The effectiveness of the bioaugmentation of M. vanbaalenii PYR-1 on pyrene mineralization was evident in the unamended, Brij-35 amended at all rates, and rhamnolipid biosurfactant amendment at the low rate, in both soil systems . Mycobacterium vanbaalenii PYR-1 has been studied in detail with respect to the molecular genetics of PAH degradation and has been shown to encode PAH ringhydroxylating oxygenases nidAB/nidA3B3, which are utilized in the oxidation of HMW PAHs such as pyrene . Additionally, M. vanbaalenii PYR-1 has a complex and very hydrophobic rigid cell envelope that is enriched in mycolic acids and the mycolic acid wall monolayer acts as a biosurfactant to enhance PAH solubility and bio-degradation . Because of these characteristics, M. vanbaalenii PYR-1 is considered an excellent candidate for bio-augmentation in PAH-contaminated soils. The 16S rRNA gene analysis was used in this study to determine if M. vanbaalenii PYR-1 was capable of successfully acclimating after the introduction in both soil systems with or without the addition of the surfactants at different rates. As shown in Figs. 3.2 and 3.3, the bio-augmentation of M. vanbaalenii PYR-1 in all clay and sandy loam soil treatments, except for rhamnolipid at the high ratein both soils and rhamnolipid at the medium rate in the sandy loam soil, resulted in an increase in Mycobacterium compared to the native treatments. This increase in Mycobacterium was especially evident when comparing the Native+Pyrene and PYR- 1+Pyrene treatments . Additionally, LEfSE software was used to determine which soil microorganisms were differentially abundant between the bioaugmented and native soil systems. The abundance of Mycobacterium was found to be significantly greater in the bio-augmented soil treatments compared to the native soil treatments . Functions of different OTUs and prediction of the functional composition of the metagenome in both soils was accomplished using the 16S rRNA gene data, Greengenes reference database, KEGG pathways, and PICRUSt to evaluate the effectiveness of surfactant addition as well as the bio-augmentation of M. vanbaalenii PYR-1 on pyrene mineralization.
By analyzing soil functional genes, contributions of different bacteria involved in the biodegradation of PAHs were assessed. For instance, according to the “Xenobiotics biodegradation and metabolism” list on the KEGG website , pyrene and phenanthrene can be degraded to 3,4-dihydroxyphenanthrene via “Polycyclic aromatic hydrocarbon degradation”, which can then be further metabolized into the TCA cycle via “Naphthalene degradation” and “Benzoate degradation” . These KEGG pathways include numerous predicted PAH-degradation-related KOs, such as PAH oxygenase large subunit , PAH oxygenase small subunit , and extradiol dioxygenase and determine whether bioaugmentation or surfactant addition had any significant effect on these genes and thus, PAH biodegradation in the two soils. Upon analysis of the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, M. vanbaalenii PYR-1 bioaugmentation in both soils significantly increased the KOs associated with the PAH biodegradation pathway compared to the native soils . The same trend of bioaugmentation of M. vanbaalenii PYR-1 resulted in increased PAH-biodegradation-related KOs in the “Naphthalene degradation” and “Benzoate degradation” compared to the native soil systems . Additionally, PICRUSt was utilized to assess which taxa contributed to the PAH-related KOs. For example, M. vanbaalenii PYR-1 substantially contributed to PAH oxygenase large and small unit; however, other soil microbes in addition to M. vanbaalenii PYR-1 contributed to the increased extradiol dioxygenase in the bioaugmented soils compared to the native soils . These results were in agreement with Niepceron et al. who evaluated phenanthrene biodegradation potential by assessing the PAH-ring hydroxylating dioxygenase sequences in PAH-contaminated soil and showed that PAH-RHDα was closely related to either Burkholderia or Mycobacterium. Wang et al. also used PICRUSt to investigate the successions of bacterial communities in PAH-contaminated soils undergoing bio-remediation and concluded that bacteria in the Mycobacterium genus contributed substantially to functional genes in all PAH-degradation pathways for metabolizing pyrene to the TCA cycle.Target shooting is an increasingly popular recreational sport with over approximately 100,000 shooting ranges worldwide . In the United States , there are an estimated 12,000 shooting ranges, consisting of 9,000 civilian and 3,000 military shooting ranges . Over 18 million adults participated in any type of clay target shooting on these civilian shooting ranges in 2014, which was a 3.6% increase compared to participants in 2012 . Accordingly, there have been vast numbers of clay targets used for these outdoor clay target shooting activities. Baer et al. determined that clay target use in the U.S. since 1970 has averaged approximately 560 million targets/year. Until recently, clay targets were composed of approximately 67- 70% clay or dolomitic limestone, 30-32% coal tar or petroleum pitch used as a binding agent, and fluorescent paint . The coal tar or petroleum pitch binding agent is a large source of PAHs with concentrations up to 3,000-40,000 mg/kg clay target . Each clay target used in these outdoor shooting activities weighs approximately 100 g each and spread into fragments of various sizes when shot.