SUMMARY: Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H₂S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers on complex microbial communities of an explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias towards Alphaproteobacteria. The primer pair 341f/785r showed also in silico the highest coverage of the domain Bacteria (96.1 %) with no obvious bias towards the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.

Widespread pollution of terrestrial ecosystems with petroleum hydrocarbons (PHCs) has generated a need for remediation and, given that many PHCs are biodegradable, bio- and phyto-remediation are often viable approaches for active and passive remediation. This review focuses on phytoremediation with particular interest on the interactions between and use of plant – associated bacteria to restore PHC polluted sites. Plant-associated bacteria include endophytic, phyllospheric and rhizospheric bacteria, and cooperation between these bacteria and their host plants allows for greater plant survivability and treatment outcomes in contaminated sites. Bacterially-driven PHC bioremediation is attributed to the presence of diverse suites of metabolic genes for aliphatic and aromatic hydrocarbons, along with a broader suite of physiological properties including biosurfactant production, biofilm formation, chemotaxis to hydrocarbons, and flexibility in cell-surface hydrophobicity. In soils impacted by PHC contamination, microbial bioremediation generally relies on the addition of high-energy electron acceptors (e.g. oxygen) and fertilization to supply limiting nutrients (e.g. nitrogen, phosphorous, potassium) in the face of excess PHC carbon. As an alternative, the addition of plants can greatly improve bioremediation rates and outcomes as plants provide microbial habitats, improve soil porosity (thereby increasing mass transfer of substrates and electron acceptors), and exchange limiting nutrients with their microbial counterparts. In return, plant-associated microorganisms improve plant growth by reducing soil toxicity through contaminant removal, producing plant growth promoting metabolites, liberating sequestered plant nutrients from soil, fixing nitrogen, and more generally establishing the foundations of soil nutrient cycling. In a practical and applied sense, the collective action of plants and their associated microorganisms is advantageous for remediation of PHC contaminated soil in terms of overall cost and success rates for in situ implementation in a diversity of environments. Mechanistically, there remain biological unknowns that present challenges for applying bio- and phyto-remediation technologies without having a deep prior understanding of individual target sites. In this review, evidence from traditional and modern omics technologies is discussed to provide a framework for plant-microbe interactions during PHCs remediation. The potential for integrating multiple molecular and computational techniques to evaluate linkages between microbial communities, plant communities and ecosystem processes is explored with an eye on