Synergistic Effect of Rhamnolipids and Inoculation on the Bioremediation of Petroleum-Contaminated Soils by Bacterial Consortia.

Crude oil is a serious soil pollutant, requiring large-scale remediation efforts. Bacterial consortia in combination with rhamnolipids can be an effective bioremediation method. However, the underlying mechanisms and associated changes in soil bacterial composition remain uncharacterized. Therefore, this study sought to evaluate the effectiveness of rhamnolipids in petroleum hydrocarbon removal, and the associated bacterial community dynamics during bioremediation of petroleum-contaminated soils. Contaminated soils were subjected to natural attenuation, bioremediation with rhamnolipids, bioremediation with bacterial consortia, or bioremediation with bacterial consortia supplemented with rhamnolipids (BMR). High-throughput sequencing of bacterial sample partial 16S rRNA sequences was performed. Additionally, the n-alkanes and aromatic fractions were analyzed by gas chromatography-mass spectroscopy. The results showed that rhamnolipid supplementation increased the rate and extent of total petroleum hydrocarbon biodegradation to a maximum of 81% within 35 days. Further, phylogenetic analysis revealed that the bacterial community was composed of 14 phylotypes (similarity level = 97%). Actinobacteria and Proteobacteria were the two core phyla in all samples, accounting for 63-89%, but Proteobacteria was the most dominant phylum in the BMR sample (~ 53%). Among the top 20 genera, Pseudomonas, Pseudoxanthomonas, Cavicella, Mycobacterium, Rhizobium, and Acinetobacter were more abundant in BMR samples compared to other samples. Predicted functional profiles revealed that rhamnolipid addition also induced changes in gene abundance related to hydrocarbon metabolic pathways. This study provided comprehensive insights into the synergistic effect of rhamnolipids and bacterial consortia for altering bacterial populations and specific functional traits, which may serve to improve bacteria-mediated petroleum hydrocarbon biodegradation in contaminated soils.

Binding interaction of a ring-hydroxylating dioxygenase with fluoranthene in Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1 can efficiently utilize fluoranthene as its sole carbon source, and the initial reaction in the biodegradation process is catalyzed by a ring-hydroxylating dioxygenase (RHD). To clarify the binding interaction of RHD with fluoranthene in the strain DN1, the genes encoding alpha subunit (RS30940) and beta subunit (RS05115) of RHD were functionally characterized through multi-technique combination such as gene knockout and homology modeling as well as molecular docking analysis. The results showed that the mutants lacking the characteristic alpha subunit and/or beta subunit failed to degrade fluoranthene effectively. Based on the translated protein sequence and Ramachandran plot, 96.5% of the primary amino-acid sequences of the alpha subunit in the modeled structure of the RHD were in the permitted region, 2.3% in the allowed region, but 1.2% in the disallowed area. The catalytic mechanism mediated by key residues was proposed by the simulations of molecular docking, wherein the active site of alpha subunit constituted a triangle structure of the mononuclear iron atom and the two oxygen atoms coupled with the predicted catalytic ternary of His217-His222-Asp372 for the dihydroxylation reaction with fluoranthene. Those amino acid residues adjacent to fluoranthene were nonpolar groups, and the C7-C8 positions on the fluoranthene ring were estimated to be the best oxidation sites. The distance of C7-O and C8-O was 3.77 Å and 3.04 Å respectively, and both of them were parallel. The results of synchronous fluorescence and site-directed mutagenesis confirmed the roles of the predicted residues during catalysis. This binding interaction could enhance our understanding of the catalytic mechanism of RHDs and provide a solid foundation for further enzymatic modification.