Factors that affect water column hydrocarbon concentrations have minor impacts on microbial responses following simulated diesel fuel spills.

Effects of season and mixing on hydrocarbon concentrations and the microbial community response was explored in a series of mesocosm experiments simulating surface spills of diesel into coastal waters. Mixing of any amount contributed to hydrocarbons entering the water column, but diesel fuel composition had a significant effect on hydrocarbon concentrations. Higher initial concentrations of aromatic hydrocarbons resulted in higher water column concentrations, with minimal differences among seasons due to high variability. Regardless of the concentrations of hydrocarbons, prokaryotes increased and there were higher relative abundances of hydrocarbon affiliated bacteria with indications of biodegradation within 4 d of exposure. As concentrations decreased over time, the eukaryote community shifted from the initial community to one which appeared to be composed of organisms with some resilience to hydrocarbons. This series of experiments demonstrates the wide range of conditions under which natural attenuation of diesel fuel is an effective response.

Microbial community response to simulated diluted bitumen spills in coastal seawater and implications for oil spill response.

Oil spills in coastal waters can have devastating impacts on local ecosystems, from the microscopic base through to mammals and seabirds. Increasing transport of diluted bitumen has led to concerns about how this novel product might impact coastal ecosystems. A mesocosm study determined that the type of diluent and the season can affect the concentrations of hydrocarbons entering the water column from a surface spill. Those same mesocosms were sampled to determine whether diluent type and season also affected the microbial response to a surface spill. Overall, there were no differences in impacts among the three types of diluted bitumen, but there were consistent responses to all products within each season. Although microbial abundances with diluted bitumen rarely differed from unoiled controls, community structure in these organisms shifted in response to hydrocarbons, with hydrocarbon-degrading bacteria becoming more abundant. The relative abundance of heterotrophic eukaryotes also increased with diluted bitumen, with few photosynthetic organisms responding positively to oil. Overall shifts in the microbial communities were minimal relative to spills of conventional oil products, with low concentrations of hydrocarbons in the water column. Oil spill response should focus on addressing the surface slick to prevent sinking or stranding to minimize ecosystem impacts.

Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Potential for Microbially Mediated Natural Attenuation of Diluted Bitumen on the Coast of British Columbia (Canada).

Western Canada produces large amounts of bitumen, a heavy, highly weathered crude oil. Douglas Channel and Hecate Strait on the coast of British Columbia are two water bodies that may be impacted by a proposed pipeline and marine shipping route for diluted bitumen (dilbit). This study investigated the potential of microbial communities from these waters to mitigate the impacts of a potential dilbit spill. Microcosm experiments were set up with water samples representing different seasons, years, sampling stations, and dilbit blends. While the alkane fraction of the tested dilbit blends was almost completely degraded after 28 days, the majority of the polycyclic aromatic hydrocarbons (PAHs) remained. The addition of the dispersant Corexit 9500A most often had either no effect or an enhancing effect on dilbit degradation. Dilbit-degrading microbial communities were highly variable between seasons, years, and stations, with dilbit type having little impact on community trajectories. Potential oil-degrading genera showed a clear succession pattern and were for the most part recruited from the "rare biosphere." At the community level, dispersant appeared to stimulate an accelerated enrichment of genera typically associated with hydrocarbon degradation, even in dilbit-free controls. This suggests that dispersant-induced growth of hydrocarbon degraders (and not only increased bioavailability of oil-associated hydrocarbons) contributes to the degradation-enhancing effect previously reported for Corexit 9500A.IMPORTANCE Western Canada hosts large petroleum deposits, which ultimately enter the market in the form of dilbit. Tanker-based shipping represents the primary means to transport dilbit to international markets. With anticipated increases in production to meet global energy needs, the risk of a dilbit spill is expected to increase. This study investigated the potential of microbial communities naturally present in the waters of a potential dilbit shipping lane to mitigate the effects of a spill. Here we show that microbial degradation of dilbit was mostly limited to n-alkanes, while the overall concentration of polycyclic aromatic hydrocarbons, which represent the most toxic fraction of dilbit, decreased only slightly within the time frame of our experiments. We further investigated the effect of the oil dispersant Corexit 9500A on microbial dilbit degradation. Our results highlight the fact that dispersant-associated growth stimulation, and not only increased bioavailability of hydrocarbons and inhibition of specific genera, contributes to the overall effect of dispersant addition.

Chemical dispersants enhance the activity of oil- and gas condensate-degrading marine bacteria.

Application of chemical dispersants to oil spills in the marine environment is a common practice to disperse oil into the water column and stimulate oil biodegradation by increasing its bioavailability to indigenous bacteria capable of naturally metabolizing hydrocarbons. In the context of a spill event, the biodegradation of crude oil and gas condensate off eastern Canada is an essential component of a response strategy. In laboratory experiments, we simulated conditions similar to an oil spill with and without the addition of chemical dispersant under both winter and summer conditions and evaluated the natural attenuation potential for hydrocarbons in near-surface sea water from the vicinity of crude oil and natural gas production facilities off eastern Canada. Chemical analyses were performed to determine hydrocarbon degradation rates, and metagenome binning combined with metatranscriptomics was used to reconstruct abundant bacterial genomes and estimate their oil degradation gene abundance and activity. Our results show important and rapid structural shifts in microbial populations in all three different oil production sites examined following exposure to oil, oil with dispersant and dispersant alone. We found that the addition of dispersant to crude oil enhanced oil degradation rates and favored the abundance and expression of oil-degrading genes from a Thalassolituus sp. (that is, metagenome bin) that harbors multiple alkane hydroxylase (alkB) gene copies. We propose that this member of the Oceanospirillales group would be an important oil degrader when oil spills are treated with dispersant.

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada.

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.

Toxicogenomic effects of nano- and bulk-TiO2 particles in the soil nematode Caenorhabditis elegans.

The toxicity and toxicogenomics of selected anatase and rutile nanoparticles (NP) and bulk titanium dioxide (TiO2) particles were evaluated in the soil nematode Caenorhabditis elegans. Results indicated that bulk or nano-TiO2 particles were slightly toxic to soil nematode C. elegans, as measured by reproduction EC50 values ranging from 4 to 32 mg/L. Whole-genome microarray results indicated that the regulation of glutathione-S-transferase gst-3, cytochrome P450 cypp33-c11, stress resistance regulator scl-1, oxidoreductase wah-1 and embryonic development pod-2 genes were significantly affected by nano-sized and bulk-TiO2 particles. More specifically, it was determined that anatase particles exerted a greater effect on metabolic pathways, whereas rutile particles had a greater effect on developmental processes. The up-regulation of the pod-2 gene corroborated the phenotypic effect observed in the reproduction test. Our results demonstrated that C. elegans is a good genomic model for nano-TiO2 toxicity assessment.

Analysis of the temporal expression of Trichoplusia ni single nucleopolyhedrovirus genes following transfection of BT1-Tn-5B1-4 cells.

Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a 134,394-bp double-stranded DNA group II Nucleopolyhedrovirus, is pathogenic to the lepidopteran T. ni. TnSNPV transcription is temporally regulated and divided into three promoter sequence-dependent classes (early, late and very late genes). A viral oligonucleotide DNA microarray containing all potential (144) viral genes of TnSNPV was designed to investigate global viral gene expression during cell infection. Total BT1-Tn-5B1-4 cellular mRNAs extracted between 0 and 72 h posttransfection with TnSNPV genomic DNA were hybridized to the microarray. Initial average expression of early genes was detected between 12 and 24 h posttransfection while late genes were mainly detected between 24 and 72 h posttransfection. The microarray expression profiling data verified many computer predicted promoter assignments. K-means clustering was used to sort the 144 genes based on their temporal expression pattern similarities. This clustering resulted in the confirmation and temporal class assignment of previously unidentified genes and promoters.

Dysregulated inflammatory response to Candida albicans in a C5-deficient mouse strain.

Experimental infection of inbred mouse strains with Candida albicans provides a good model system to identify host genetic determinants that regulate onset of, response to, and ultimate outcome of disseminated candidiasis. The A/J mouse strain is exquisitely sensitive to infection with C. albicans, while the C57BL/6J strain is relatively resistant, as measured by survival following intravenous injection of Candida blastospores. This differential susceptibility is caused by an A/J-specific loss-of-function mutation in the C5 component of the complement pathway. C5 plays several critical roles in host response to infection, including target lysis and phagocyte recruitment. Therefore, to determine which of its functions were required for host resistance to candidiasis, a detailed comparative analysis of pathophysiology and host response to acute C. albicans infection was conducted in A/J and C57BL/6J mice. C5-sufficient C57BL/6J mice were found to succumb late in infection due to severe kidney pathology, typified by fungal replication and robust neutrophil-based inflammatory response associated with extensive tissue damage. In contrast, A/J mice were moribund within 24 h postinfection but displayed little if any kidney damage despite an inability to mobilize granulocytes and a high fungal load in the kidney. Rather, C5 deficiency in A/J mice was associated with higher levels of circulating cytokines tumor necrosis factor alpha, interleukin-6, monocyte chemotactic protein 1 (MCP-1), MCP-5, and eotaxin in response to C. albicans. Transfer of the C5-defective allele from A/J onto a C57BL/6J genetic background in recombinant congenic strain BcA17 recapitulated the phenotypic aspects of the susceptibility of A/J mice to C. albicans, confirming the causative role of C5 deficiency in the dysregulated cytokine response.

Gene expression in HL60 granulocytoids and human polymorphonuclear leukocytes exposed to Candida albicans.

Candida albicans is an opportunistic human pathogen causing both superficial and disseminated diseases. It is a dimorphic fungus, switching between yeast and hyphal forms, depending on cues from its microenvironment. Hyphae play an important role in the pathogenesis of candidiasis. The host's response to Candida infection is multifaceted and includes the participation of granulocytes as key effector cells. The aim of this investigation was to study host gene expression during granulocyte-Candida interaction. Effector cells were generated by the granulocytic differentiation of HL60 cells. The resulting cell population was shown to be morphologically and functionally equivalent to granulocytes and is therefore referred to as HL60 granulocytoids for the purposes of this study. Gene expression profiles were determined 1 h after hosts were infected with C. albicans. Three Candida-granulocytoid ratios were chosen to reflect different degrees of HL60 granulocytoid inhibition of C. albicans. The data demonstrate that at the high pathogen-host ratio, C. albicans modulated the HL60 granulocytoid's response by downregulating the expression of known antimicrobial genes. In addition, looking at the expression of a large number of genes, not all of which have necessarily been implicated in candidastatic or candidacidal mechanisms, it has been possible to describe the physiological response of the HL60 granulocytoid to an infectious challenge with C. albicans. Finally, some of the observed changes in HL60 granulocytoid gene expression were investigated in freshly isolated human polymorphonuclear leukocytes infected with C. albicans. Similar changes were seen in these primary human cells, lending support to the validity of this model.