Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation.

Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ∼15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60-100%) and HMs removal (21-100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities. Most promising yields (∼100%) were obtained towards naphthalene, pyrene and lead. We propose these novel bacterial strains and related genetic repertoire to be further exploited for effective bioremediation of marine environments contaminated with both PAHs and HMs.

Calm and Frenzy: marine obligate hydrocarbonoclastic bacteria sustain ocean wellness.

According to current estimates, the annual volume of crude oil entering the ocean due to both anthropogenic activities and naturally occurring seepages reaches approximately 8.3 million metric tons. Huge discharges from accidents have caused large-scale environmental disasters with extensive damage to the marine ecosystem. The natural clean-up of petroleum spills in marine environments is carried out primarily by naturally occurring obligate hydrocarbonoclastic bacteria (OHCB). The natural hosts of OHCB include a range of marine primary producers, unicellular photosynthetic eukaryotes and cyanobacteria, which have been documented as both, suppliers of hydrocarbon-like compounds that fuel the 'cryptic' hydrocarbon cycle and as a source of isolation of new OHCB. A very new body of evidence suggests that OHCB are not only the active early stage colonizers of plastics and hence the important component of the ocean's 'plastisphere' but also encode an array of enzymes experimentally proven to act on petrochemical and bio-based polymers.

Metaproteomics and metabolomics analyses of chronically petroleum-polluted sites reveal the importance of general anaerobic processes uncoupled with degradation.

Crude oil is one of the most important natural assets for humankind, yet it is a major environmental pollutant, notably in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, in which the metabolic potential of indigenous microbial populations towards the large-scale chronic pollution is yet to be defined, particularly in anaerobic and micro-aerophilic sites. Here, we provide an insight into the microbial metabolism in sediments from three chronically polluted marine sites along the coastline of Italy: the Priolo oil terminal/refinery site (near Siracuse, Sicily), harbour of Messina (Sicily) and shipwreck of MT Haven (near Genoa). Using shotgun metaproteomics and community metabolomics approaches, the presence of 651 microbial proteins and 4776 metabolite mass features have been detected in these three environments, revealing a high metabolic heterogeneity between the investigated sites. The proteomes displayed the prevalence of anaerobic metabolisms that were not directly related with petroleum biodegradation, indicating that in the absence of oxygen, biodegradation is significantly suppressed. This suppression was also suggested by examining the metabolome patterns. The proteome analysis further highlighted the metabolic coupling between methylotrophs and sulphate reducers in oxygen-depleted petroleum-polluted sediments.

Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature.

Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.

Conversion of Uric Acid into Ammonium in Oil-Degrading Marine Microbial Communities: a Possible Role of Halomonads.

Uric acid is a promising hydrophobic nitrogen source for biostimulation of microbial activities in oil-impacted marine environments. This study investigated metabolic processes and microbial community changes in a series of microcosms using sediment from the Mediterranean and the Red Sea amended with ammonium and uric acid. Respiration, emulsification, ammonium and protein concentration measurements suggested a rapid production of ammonium from uric acid accompanied by the development of microbial communities containing hydrocarbonoclastic bacteria after 3 weeks of incubation. About 80 % of uric acid was converted to ammonium within the first few days of the experiment. Microbial population dynamics were investigated by Ribosomal Intergenic Spacer Analysis and Illumina sequencing as well as by culture-based techniques. Resulting data indicated that strains related to Halomonas spp. converted uric acid into ammonium, which stimulated growth of microbial consortia dominated by Alcanivorax spp. and Pseudomonas spp. Several strains of Halomonas spp. were isolated on uric acid as the sole carbon source showed location specificity. These results point towards a possible role of halomonads in the conversion of uric acid to ammonium utilized by hydrocarbonoclastic bacteria.

Bioremediation of Southern Mediterranean oil polluted sites comes of age.

Mediterranean Sea is facing a very high risk of oil pollution due to the high number of oil extractive and refining sites along the basin coasts, and the intense maritime traffic of oil tankers. All the Mediterranean countries have adopted severe regulations for minimizing pollution events and bioremediation feasibility studies for the most urgent polluted sites are undergoing. However, the analysis of the scientific studies applying modern 'meta-omics' technologies that have been performed on marine oil pollution worldwide showed that the Southern Mediterranean side has been neglected by the international research. Most of the studies in the Mediterranean Sea have been done in polluted sites of the Northern side of the basin. Those of the Southern side are poorly studied, despite many of the Southern countries being major oil producers and exporters. The recently EU-funded research project ULIXES has as a major objective to increase the knowledge of the bioremediation potential of sites from the Southern Mediterranean countries. ULIXES is targeting four major polluted sites on the coastlines of Egypt, Jordan, Morocco and Tunisia, including seashore sands, lagoons, and oil refinery polluted sediments. The research is designed to unravel, categorize, catalogue, exploit and manage the diversity and ecology of microorganisms thriving in these polluted sites. Isolation of novel hydrocarbon degrading microbes and a series of state of the art 'meta-omics' technologies are the baseline tools for improving our knowledge on biodegradation capacities mediated by microbes under different environmental settings and for designing novel site-tailored bioremediation approaches. A network of twelve European and Southern Mediterranean partners is cooperating for plugging the existing gap of knowledge for the development of novel bioremediation processes targeting such poorly investigated polluted sites.

Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study.

Diversity of indigenous microbial consortia and natural occurrence of obligate hydrocarbon-degrading bacteria (OHCB) are of central importance for efficient bioremediation techniques. To investigate the microbial population dynamics and composition of oil-degrading consortia, we have established a series of identical oil-degrading mesocosms at three different locations, Bangor (Menai Straits, Irish Sea), Helgoland (North Sea) and Messina (Messina Straits, Mediterranean Sea). Changes in microbial community composition in response to oil spiking, nutrient amendment and filtration were assessed by ARISA and DGGE fingerprinting and 16Sr RNA gene library analysis. Bacterial and protozoan cell numbers were quantified by fluorescence microscopy. Very similar microbial population sizes and dynamics, together with key oil-degrading microorganisms, for example, Alcanivorax borkumensis, were observed at all three sites; however, the composition of microbial communities was largely site specific and included variability in relative abundance of OHCB. Reduction in protozoan grazing had little effect on prokaryotic cell numbers but did lead to a decrease in the percentage of A. borkumensis 16S rRNA genes detected in clone libraries. These results underline the complexity of marine oil-degrading microbial communities and cast further doubt on the feasibility of bioaugmentation practices for use in a broad range of geographical locations.

The environmental and biosecurity characteristics of livestock carcass disposal methods: A review.

Livestock mortalities represent a major waste stream within agriculture. Many different methods are used throughout the world to dispose of these mortalities; however within the European Union (EU) disposal options are limited by stringent legislation. The legal disposal options currently available to EU farmers (primarily rendering and incineration) are frequently negatively perceived on both practical and economic grounds. In this review, we assess the potential environment impacts and biosecurity risks associated with each of the main options used for disposal of livestock mortalities in the world and critically evaluate the justification for current EU regulations. Overall, we conclude that while current legislation intends to minimise the potential for on-farm pollution and the spread of infectious diseases (e.g. transmissible spongiform encephalopathies, bacterial pathogens), alternative technologies (e.g. bioreduction, anaerobic digestion) may provide a more cost-effective, practical and biosecure mechanism for carcass disposal as well as having a lower environmental footprint. Further social, environmental and economic research is therefore warranted to assess the holistic benefits of alternative approaches for carcass disposal in Europe, with an aim to provide policy-makers with robust knowledge to make informed decisions on future legislation.

A mesocosm study of the changes in marine flagellate and ciliate communities in a crude oil bioremediation trial.

Protozoan grazers play an important role in controlling the density of crude-oil degrading marine communities as has been evidenced in a number of microcosm experiments. However, small bioreactors contain a low initial titre of protozoa and the growth of hydrocarbon-depleting bacteria is accompanied by the fast depletion of mineral nutrients and oxygen, which makes microcosms rather unsuitable for simulating the sequence of events after the oil spill in natural seawater environment. In the present study, the population dynamics of marine protozoan community have been analysed in a 500 l mesocosm experiment involving bioaugmented oil booms that contained oil sorbents and slow-release fertilisers. A significant increase in numbers of marine flagellates and ciliates on biofilms of oil-degrading microbes was microscopically observed as early as 8 days after the start of the experiment, when protozoa exhibited a population density peak making up to 3,000 cells ml(-1). Further, the protozoan density varied throughout the experiment, but never dropped below 80 cells ml(-1). An 18S rRNA gene-based fingerprinting analysis revealed several changes within the eukaryotic community over the whole course of the experiment. Initial growth of flagellates and small ciliates was followed by a predominance of larger protozoa. According to microscopic observations and SSU rRNA molecular analyses, most predominant were the ciliates belonging to Euplotidae and Scuticociliatia. This is the first study to characterise the eukaryotic communities specifically in a large-scale oil bioremediation trial using both microscopy-based and several molecular techniques.

Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borkumensis SK2.

Alcanivorax borkumensis strain SK2 is a cosmopolitan hydrocarbonoclastic marine bacterium, with a specialized metabolism adapted to the degradation of petroleum oil hydrocarbons. Transposon mutagenesis was used for functional genome analysis of Alcanivorax SK2 to reveal the genetic basis of other environmentally relevant phenotypes, such as biofilm formation, adaptation to UV exposure, and to growth at either low temperature or high salinity. Forty-eight relevant transposon mutants deficient in any one of these environmentally responsive functions were isolated, and the corresponding genes interrupted by the mini-Tn5 element were sequenced using inverse PCR. Several cross connections between different phenotypes (e.g. biofilm and UV stress; biofilm and UV and osmoadaptation) on signal transduction level have been revealed, pointing at complex and tightly controlled cellular interactions involving oxygen as a primary messenger and cyclic-di-GMP as a secondary messenger required for Alcanivorax responses to environmental stresses. These results provide insights into bacterial function in a complex marine environment.

Crude oil-induced structural shift of coastal bacterial communities of rod bay (Terra Nova Bay, Ross Sea, Antarctica) and characterization of cultured cold-adapted hydrocarbonoclastic bacteria.

For preliminary screening of human impact on Antarctic coasts, the compositions of microbial communities were analyzed in seawater at two sites located in the Terra Nova Bay of Antarctica (Ross Sea) by a combination of 16S rRNA gene sequencing and culture techniques. The bacterial community in the sample from the Rod Bay site, located at the proximity to the Italian Station, was characterized by a high abundance of 16S rRNA gene sequences belonging to the microflora typically found in soil and freshwater environments. In contrast, the seawater sample from the Adelie Cove station, a pristine reference site, contained 16S rRNA gene sequences typically found in marine areas affected by algal blooms and sea ice decay. The addition of crude oil to the Rod Bay seawater sample rapidly induced a shift in the composition of the bacterial community with appearance of novel taxonomic groups and a dramatic increase in the relative abundance of gamma-Proteobacteria sequences, whereas no significant changes were detected in the bacterial community of the Adelie Cove sample under the same conditions. Bacteria-exhibiting features with potential interest for industrial and environmental applications were isolated from the Rod Bay oil-enriched sample. In particular, hydrocarbon-degrading, cold-adapted bacteria were selectively enriched, isolated and screened for their ability to synthesize polyunsaturated fatty acids. Twenty two bacterial strains were isolated from the oil enrichment culture and identified. Eighteen isolates were found to be members of gamma-Proteobacteria, while the remainder were representatives of alpha-Proteobacteria, CFB and high G + C divisions.