Tracking bacterial pollution at a marine wastewater outfall site - A case study from Norway.

Coastal marine environments are increasingly affected by anthropogenic impacts, such as the release of sewage at outfall sites and agricultural run-off. Fecal pollution introduced to the sea through these activities poses risks of spreading microbial diseases and disseminating antibiotic resistant bacteria and their genes. The study area of this research, Bore beach, is situated between two such point sources, an outfall site where treated sewage is released 1 km off the coast and a stream that carries run-off from an agricultural area to the northern end of the beach. In order to investigate whether and to what extent fecal contamination from the sewage outfall reached the beach, we used microbial source tracking, based on whole community analysis. Samples were collected from sea water at varying distances from the sewage outfall site and along the beach, as well as from the sewage effluent and the stream. Amplicon sequencing of 16S rRNA genes from all the collected samples was carried out at two time points (June and September). In addition, the seawater at the sewage outfall site and the sewage effluent were subject to shotgun metagenomics. To estimate the contribution of the sewage effluent and the stream to the microbial communities at Bore beach, we employed SourceTracker2, a program that uses a Bayesian algorithm to perform such quantification. The SourceTracker2 results suggested that the sewage effluent is likely to spread fecal contamination towards the beach to a greater extent than anticipated based on the prevailing sea current. The estimated mixing proportions of sewage at the near-beach site (P4) were 0.22 and 0.035% in June and September, respectively. This was somewhat below that stream's contribution in June (0.028%) and 10-fold higher than the stream's contribution in September (0.004%). Our analysis identified a sewage signal in all the tested seawater samples.

Abundance and diversity of n-alkane and PAH-degrading bacteria and their functional genes - Potential for use in detection of marine oil pollution.

Monitoring environmental status through molecular investigation of microorganisms in the marine environment is suggested as a potentially very effective method for biomonitoring, with great potential for automation. There are several hurdles to that approach with regards to primer design, variability across geographical locations, seasons, and type of environmental pollution. Here, qPCR analysis of genes involved in the initial activation of aliphatic and aromatic hydrocarbons were used in a laboratory setup mimicking realistic oil leakage at sea. Seawater incubation experiments were carried out under two different seasons with two different oil types. Degenerate primers targeting initial oxygenases (alkane 1-monooxygenase; alkB and aromatic-ring hydroxylating dioxygenase; ARHD) were employed in qPCR assays to quantify the abundance of genes essential for oil degradation. Shotgun metagenomics was used to map the overall community dynamics and the diversity of alkB and ARHD genes represented in the microbial community. The amplicons generated through the qPCR assays were sequenced to reveal the diversity of oil-degradation related genes captured by the degenerate primers. We identified a major mismatch between the taxonomic diversity of alkB and ARHD genes amplified by the degenerate primers and those identified through shotgun metagenomics. More specifically, the designed primers did not amplify the alkB genes of the two most abundant alkane degraders that bloomed in the experiments, Oceanobacter and Oleispira. The relative abundance of alkB sequences from shotgun metagenomics and 16S rRNA-based Oleispira-specific qPCR assay were better signals for oil in water than the tested qPCR alkB assay. The ARHD assay showed a good agreement with PAHs degradation despite covering only 25% of the top 100 ARHD genes and missing several abundant Cycloclasticus sequences that were present in the metagenome. We conclude that further improvement of the degenerate primer approach is needed to rely on the use of oxygenase-related qPCR assays for oil leakage detection.

Metatranscriptomic Analysis of Oil-Exposed Seawater Bacterial Communities Archived by an Environmental Sample Processor (ESP).

The use of natural marine bacteria as "oil sensors" for the detection of pollution events can be suggested as a novel way of monitoring oil occurrence at sea. Nucleic acid-based devices generically called genosensors are emerging as potentially promising tools for in situ detection of specific microbial marker genes suited for that purpose. Functional marker genes are particularly interesting as targets for oil-related genosensing but their identification remains a challenge. Here, seawater samples, collected in tanks with oil addition mimicking a realistic oil spill scenario, were filtered and archived by the Environmental Sample Processor (ESP), a fully robotized genosensor, and the samples were then used for post-retrieval metatranscriptomic analysis. After extraction, RNA from ESP-archived samples at start, Day 4 and Day 7 of the experiment was used for sequencing. Metatranscriptomics revealed that several KEGG pathways were significantly enriched in samples exposed to oil. However, these pathways were highly expressed also in the non-oil-exposed water samples, most likely as a result of the release of natural organic matter from decaying phytoplankton. Temporary peaks of aliphatic alcohol and aldehyde dehydrogenases and monoaromatic ring-degrading enzymes (e.g., ben, box, and dmp clusters) were observed on Day 4 in both control and oil-exposed and non-exposed tanks. Few alkane 1-monooxygenase genes were upregulated on oil, mostly transcribed by families Porticoccaceae and Rhodobacteraceae, together with aromatic ring-hydroxylating dioxygenases, mostly transcribed by Rhodobacteraceae. Few transcripts from obligate hydrocarbonoclastic genera of Alcanivorax, Oleispira and Cycloclasticus were significantly enriched in the oil-treated exposed tank in comparison to control the non-exposed tank, and these were mostly transporters and genes involved in nitrogen and phosphorous acquisition. This study highlights the importance of seasonality, i.e., phytoplankton occurrence and senescence leading to organic compound release which can be used preferentially by bacteria over oil compounds, delaying the latter process. As a result, such seasonal effect can reduce the sensitivity of genosensing tools employing bacterial functional genes to sense oil. A better understanding of the use of natural organic matter by bacteria involved in oil-biodegradation is needed to develop an array of functional markers enabling the rapid and specific in situ detection of anthropogenic pollution.

Insights into the Potential of the Atlantic Cod Gut Microbiome as Biomarker of Oil Contamination in the Marine Environment.

BACKGROUND: Microorganisms are widespread in all environments, including in and on animal bodies. The gut microbiome has an essential influence on fish health, and is affected by several persistent and harmful organic and inorganic contaminants. Considering the shifts in gut microbiota composition observed in those studies, we hypothesized that certain microbial groups in the gut can serve as indicators of pollution. To test this hypothesis, we explored the possibility of identifying key microbial players that indicate environmental contamination. METHODS: Published 16S rRNA gene amplicon sequencing data generated from the gut microbiota of Atlantic cod caught in geographically different Norwegian waters were used for bacterial diversity comparison. RESULTS: Different microbiomes were identified between the northern Norway and southern Norway samples. Several bacterial genera previously identified as polycyclic aromatic hydrocarbon degraders were present only in the samples collected in the southern Norway area, suggesting fish contamination with oil-related compounds. CONCLUSIONS: The results contribute to the identification of bacterial taxa present in the Atlantic cod gut that indicate fish exposure to contaminants in the marine environment.

Implementing Morpholino-Based Nucleic Acid Sensing on a Portable Surface Plasmon Resonance Instrument for Future Application in Environmental Monitoring.

A portable surface plasmon resonance (SPR) instrument was tested for the first time for the detection of oligonucleotide sequences derived from the 16S rRNA gene of Oleispira antarctica RB-8, a bioindicator species of marine oil contamination, using morpholino-functionalized sensor surfaces. We evaluated the stability and specificity of morpholino coated sensor surfaces and tested two signal amplification regimes: (1) sequential injection of sample followed by magnetic bead amplifier and (2) a single injection of magnetic bead captured oligo. We found that the sensor surfaces could be regenerated for at least 85 consecutive sample injections without significant loss of signal intensity. Regarding specificity, the assay clearly differentiated analytes with only one or two mismatches. Signal intensities of mismatch oligos were lower than the exact match target at identical concentrations down to 200 nM, in standard phosphate buffered saline with 0.1 % Tween-20 added. Signal amplification was achieved with both strategies; however, significantly higher response was observed with the sequential approach (up to 16-fold), where first the binding of biotin-probe-labeled target oligo took place on the sensor surface, followed by the binding of the streptavidin magnetic beads onto the immobilized targets. Our experiments so far indicate that a simple coating procedure in combination with a relatively cost-efficient magnetic-bead-based signal amplification will provide robust SPR based nucleic acid sensing down to 0.5 nM of a 45-nucleotide long oligo target (7.2 ng/mL).

Gastrointestinal microbial community changes in Atlantic cod (Gadus morhua) exposed to crude oil.

BACKGROUND: The expansion of offshore oil exploration increases the risk of marine species being exposed to oil pollution in currently pristine areas. The adverse effects of oil exposure through toxic properties of polycyclic aromatic hydrocarbons (PAHs) have been well studied in Atlantic cod (Gadus morhua). Nevertheless, the fate of conjugated metabolites in the intestinal tract and their effect on the diversity of intestinal microbial community in fish is less understood. Here, we investigated the intestinal microbial community composition of Atlantic cod after 28 days of exposure to crude oil (concentration range 0.0-0.1 mg/L). RESULTS: Analysis of PAH metabolites in bile samples confirmed that uptake and biotransformation of oil compounds occurred as a result of the exposure. Various evidence for altered microbial communities was found in fish exposed to high (0.1 mg/L) and medium (0.05 mg/L) concentrations of oil when compared to fish exposed to low oil concentration (0.01 mg/L) or no oil (control). First, altered banding patterns were observed on denaturing gradient gel electrophoresis for samples pooled from each treatment group. Secondly, based on 16S rRNA sequences, higher levels of oil exposure were associated with a loss of overall diversity of the gut microbial communities. Furthermore, 8 operational taxonomic units (OTUs) were found to have significantly different relative abundances in samples from fishes exposed to high and medium oil concentrations when compared to samples from the control group and low oil concentration. Among these, only one OTU, a Deferribacterales, had increased relative abundance in samples from fish exposed to high oil concentration. CONCLUSIONS: The results presented herein contribute to a better understanding of the effects of oil contamination on the gut microbial community changes in fish and highlight the importance of further studies into the area. Our findings suggest that increased relative abundance of bacteria belonging to the order Deferribacterales may be indicative of exposure to oil at concentrations higher than 0.05 mg/L.

Wastewater characterisation by combining size fractionation, chemical composition and biodegradability.

The potential for resource recovery from wastewater can be evaluated based on a detailed characterisation of wastewater. In this paper, results from fractionation and characterisation of two distinct wastewaters are reported. Using tangential flow filtration, the wastewater was fractionated into 10 size fractions ranging from 1 kDa to 1 mm, wherein the chemical composition and biodegradability were determined. Carbohydrates were dominant in particulate size fractions larger than 100 µm, indicating a potential of cellulose recovery from these fractions. While the particulate size fractions between 0.65 and 100 µm show a potential as a source for biofuel production due to an abundance of saturated C16 and C18 lipids. Both wastewaters were dominated by particulate (>0.65 µm), and oligo- and monomeric (<1 kDa) COD. Polymeric (1-1000 kDa) and colloidal (1000 kDa-0.65 µm) fractions had a low COD content, expected due to degradation in the sewer system upstream of the wastewater treatment plant. Biodegradation rates of particulate fractions increase with decreasing size. However, this was not seen in polymeric fractions where degradation rate was governed by chemical composition. Analytical validation of molecular weight and particle size distribution showed below filter cut-off retention of particles and polymers close to nominal cut-off, shifting the actual size distribution.

Naphthalene biodegradation in temperate and arctic marine microcosms.

Naphthalene, the smallest polycyclic aromatic hydrocarbon (PAH), is found in abundance in crude oil, its major source in marine environments. PAH removal occurs via biodegradation, a key process determining their fate in the sea. Adequate estimation of PAH biodegradation rates is essential for environmental risk assessment and response planning using numerical models such as the oil spill contingency and response (OSCAR) model. Using naphthalene as a model compound, biodegradation rate, temperature response and bacterial community composition of seawaters from two climatically different areas (North Sea and Arctic Ocean) were studied and compared. Naphthalene degradation was followed by measuring oxygen consumption in closed bottles using the OxiTop(®) system. Microbial communities of untreated and naphthalene exposed samples were analysed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. Three times higher naphthalene degradation rate coefficients were observed in arctic seawater samples compared to temperate, at all incubation temperatures. Rate coefficients at in situ temperatures were however, similar (0.048 day(-1) for temperate and 0.068 day(-1) for arctic). Naphthalene biodegradation rates decreased with similar Q10 ratios (3.3 and 3.5) in both seawaters. Using the temperature compensation method implemented in the OSCAR model, Q10 = 2, biodegradation in arctic seawater was underestimated when calculated from the measured temperate k1 value, showing that temperature difference alone could not predict biodegradation rates adequately. Temperate and arctic untreated seawater communities were different as revealed by pyrosequencing. Geographic origin of seawater affected the community composition of exposed samples.

Estimation of hydrocarbon biodegradation rates in marine environments: a critical review of the Q10 approach.

Offshore oil & gas industry is moving exploration and production activities into Arctic and deep water regions. Governmental regulations require environmental impact assessments before operations to evaluate the possible effects of accidental oil releases. These are often performed by numerical fate models, like the Oil Spill Contingency and Response (OSCAR) model, which has become an industry standard in Norway. In this model, biodegradation rates are adjusted to local conditions by temperature compensation according to a Q10 approach. Q10 is the multiplier by which rates of enzymatic reactions increase at a 10 °C temperature rise. Herein, this Q10 approach implemented in the OSCAR model is investigated based on published data and novel obtained results. Overall, biodegradation rate predictions calculated by temperature compensation are found to be questionable, and choosing one universal Q10 value is considered not feasible. The high variation in Q10 values is herein attributed to indirect effects of temperature.