Innovative Culturomic Approaches and Predictive Functional Metagenomic Analysis: The Isolation of Hydrocarbonoclastic Bacteria with Plant Growth Promoting Capacity

Innovative culturomic approaches were adopted to isolate hydrocarbonoclastic bacteria capable of degrading diesel oil, bitumen and a selection of polycyclic aromatic hydrocarbons (PAH), e.g., pyrene, anthracene, and dibenzothiophene, from a soil historically contaminated by total petroleum hydrocarbons (TPH) (10,347 ± 98 mg TPH/kg). The culturomic approach focussed on the isolation of saprophytic microorganisms and specialist bacteria utilising the contaminants as sole carbon sources. Bacterial isolates belonging to Pseudomonas, Arthrobacter, Achromobacter, Bacillus, Lysinibacillus, Microbacterium sps. were isolated for their capacity to utilise diesel oil, bitumen, pyrene, anthracene, dibenzothiphene, and their mixture as sole carbon sources. Pseudomonas, Arthrobacter, Achromobacter and Microbacterium sps. showed plant growth promoting activity, producing indole-3-acetic acid and expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. In parallel to the culturomic approach, in the microbial community of interest, bacterial community metabarcoding and predictive functional metagenomic analysis were adopted to confirm the potentiality of the isolates in terms of their functional representativeness. The combination of isolation and molecular approaches for the characterisation of a TPH contaminated soil microbial community is proposed as an instrument for the construction of an artificial hydrocarbonoclastic microbiota for environmental restoration.

Isolation and Study of Biodegradation Capability of Hydrocarbonoclastic Bacteria from Industrial Waste Lubrication Oil Contaminated Sites

A research was carried out on Isolation and study of biodegradation capability of hydrocarbonoclastic bacteria from industrial waste lubricant oil contaminated sites. 12 different bacterial cultures are isolated from the soil samples collected from liquid effluent dump site of black galaxy granite, chimakurthy, prakasam Dist, and auto motive lubrication oil replacement garages, Auto Nagar, Vijayawada. Among the 12isolates 10 isolates were found to have capability of degrading used industrial lubrication oil VG 320 collected from NSL textiles LTD, Guntur. Among the isolates tested the isolate no BG4 obtained from liquid effluent dump site gave the maximum biodegradation potential of 18.78mm2followed by BG7 of 15.36, BG1 of 14.07and BG8 of 10.56 mm2 and the isolate no AL1 from automotive lubrication oil replacement garage soil sample found to be 9.03mm2 degradation potential. These isolates were identified based on physical and biochemical characters as Pseudomonas, Bacillus, Proteus, Flavobacterium and Enterococcussps respectively. For the future, our study will be focused on several data like the species of isolates, the optimal activity of isolates to degrade several Industrial lubricant oils.

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L–1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes.

Effects of Dispersants and Biosurfactants on Crude-Oil Biodegradation and Bacterial Community Succession

This study evaluated the effects of three commercial dispersants (Finasol OSR 52, Slickgone NS, Superdispersant 25) and three biosurfactants (rhamnolipid, trehalolipid, sophorolipid) in crude-oil seawater microcosms. We analysed the crucial early bacterial response (1 and 3 days). In contrast, most analyses miss this key period and instead focus on later time points after oil and dispersant addition. By focusing on the early stage, we show that dispersants and biosurfactants, which reduce the interfacial surface tension of oil and water, significantly increase the abundance of hydrocarbon-degrading bacteria, and the rate of hydrocarbon biodegradation, within 24 h. A succession of obligate hydrocarbonoclastic bacteria (OHCB), driven by metabolite niche partitioning, is demonstrated. Importantly, this succession has revealed how the OHCB Oleispira, hitherto considered to be a psychrophile, can dominate in the early stages of oil-spill response (1 and 3 days), outcompeting all other OHCB, at the relatively high temperature of 16 °C. Additionally, we demonstrate how some dispersants or biosurfactants can select for specific bacterial genera, especially the biosurfactant rhamnolipid, which appears to provide an advantageous compatibility with Pseudomonas, a genus in which some species synthesize rhamnolipid in the presence of hydrocarbons.

A Comparison between Chemical and Natural Dispersion of a North Sea Oil-spill

ABSTRACT The application of dispersants to an oil-slick is a key remediation tool and thus understanding its effectiveness is vital. Two in situ oil slicks were created in the North Sea (off the coast of The Netherlands), one left to natural processes whilst dispersant (Slickgone NS) was applied to the other. GC-MS analysis of seawater from the surface slick, and at 1.5 and 5 m below the slick, revealed only two samples with measurable hydrocarbons (221 ± 92 μg ml−1 seawater), from the surface of the “Slickgone Dispersed” oil-slick ~25.5 hours after oil-slick formation, which was likely due to environmental conditions hindering sampling. Additionally, 16S rRNA gene quantitative PCR and amplicon analysis revealed extremely limited growth of obligate hydrocarbonoclastic bacteria (OHCB), detected at a relative abundance of <1×10-6 %. Furthermore, the Ecological Index of Hydrocarbon Exposure (EIHE) score, which quantifies the proportion of the bacterial community with hydrocarbon-biodegradation potential, was extremely low at 0.012 (scale of 0 – 1). This very low abundance of hydrocarbon-degrading bacteria at the time of sampling, even in samples with measurable hydrocarbons, could potentially be attributed to nutrient limitation (~25.5 hours after oil-slick creation total inorganic nitrogen was 3.33 μM and phosphorus was undetectable). The results of this study highlight a limited capacity for the environment, during this relatively short period, to naturally attenuate oil.

Evaluation of Polycyclic Aromatic Hydrocarbon Pollution From the HMS Royal Oak Shipwreck and Effects on Sediment Microbial Community Structure

Despite many shipwrecks containing oil there is a paucity of studies investigating their impact on surrounding environments. This study evaluates any potential effect the World War II shipwreck HMS Royal Oak is having on surrounding benthic sediments in Scapa Flow, Scotland. HMS (Her Majesty’s Ship) Royal Oak sank in 1939, subsequently leaked oil in the 1960s and 1990s, and is estimated to still hold 697 tonnes of fuel oil. In this study, sediments were analysed, over a 17.5 cm depth profile, along a 50–950 m cruciform transect away from the shipwreck. Analysis of polycyclic aromatic hydrocarbons (PAHs) revealed low concentrations (205.91 ± 50.15 μg kg–1 of dry sediment), which did not significantly differ with either distance from the shipwreck or sediment depth. PAH concentrations were well below the effects-range low (ERL) for the OSPAR (Oslo/Paris convention for the Protection of the Marine Environment of the North-East Atlantic) maritime area. The average Pyrogenic Index, in sediments around HMS Royal Oak, was 1.06 (±0.34), indicating PAHs were pyrogenic rather than petrogenic. Moreover, analysis of sediment microbiomes revealed no significant differences in bacterial community structure with distance from the shipwreck, with extremely low levels of obligate hydrocarbonoclastic bacteria (OHCB; 0.21% ± 0.54%). Both lines of evidence suggest that sampled sediments are not currently being impacted by petrogenic hydrocarbons and show no long-term impact by previous oil-spills from HMS Royal Oak.

Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.