Quantitative assessment, molecular docking and novel metabolic pathways reveal the interaction mechanisms between norfloxacin biodegradation and environmental implications.

Norfloxacin (NOR) is widely used in medicine and animal husbandry, but its accumulation in the environment poses a substantial threat to ecological and human health. Traditional physical, chemical, and rudimentary biological methods often fall short in mitigating NOR contamination, necessitating innovative biological approaches. This study proposes an engineered bacterial consortium found in marine sediment as a strategy to enhance NOR degradation through inter-strain co-metabolism of diverse substrates. Strategically supplementing the engineered bacterial consortium with exogenous carbon sources and metal ions boosted the activity of key degradation enzymes like laccase, manganese peroxidase, and dehydrogenase. Iron and amino acids demonstrated synergistic effects, resulting in a remarkable 70.8% reduction in NOR levels. The innovative application of molecular docking elucidated enzyme interactions with NOR, uncovering potential biodegradation mechanisms. Quantitative assessment reinforced the efficiency of NOR degradation within the engineered bacterial consortium. Four metabolic routes are herein proposed: acetylation, defluorination, ring scission, and hydroxylation. Notably, this study discloses distinctive, co-operative metabolic pathways for NOR degradation within the specific microbial community. These findings provide new ways of understanding and investigating the bioremediation potential of NOR contaminants, which may lead to the development of more sustainable and effective environmental management strategies.

Characterization of hydrocarbon degraders from Northwest Passage beach sediments and assessment of their ability for bioremediation.

Global warming-induced sea ice loss in the Canadian Northwest Passage (NWP) will result in more shipping traffic, increasing the risk of oil spills. Microorganisms inhabiting NWP beach sediments may degrade hydrocarbons, offering a potential bioremediation strategy. In this study, the characterization and genomic analyses of 22 hydrocarbon-biodegradative bacterial isolates revealed that they contained a diverse range of key alkane and aromatic hydrocarbon-degradative genes, as well as cold and salt tolerance genes indicating they are highly adapted to the extreme Arctic environment. Some isolates successfully degraded Ultra Low Sulfur Fuel Oil (ULSFO) at temperatures as low as -5 °C and high salinities (3%-10%). Three isolates were grown in liquid medium containing ULSFO as sole carbon source over 3 months and variation of hydrocarbon concentration was measured at three time points to determine their rate of hydrocarbon biodegradation. Our results demonstrate that two isolates (Rhodococcus sp. R1B_2T and Pseudarthrobacter sp. R2D_1T) possess complete degradation pathways and can grow on alkane and aromatic components of ULSFO under Arctic conditions. Overall, these results demonstrate that diverse hydrocarbon-degrading microorganisms exist in the NWP beach sediments, offering a potential bioremediation strategy in the events of a marine fuel spill reaching the shores of the NWP.

Assessment of prokaryotic communities in Southwestern Atlantic deep-sea sediments reveals prevalent methanol-oxidising Methylomirabilales.

Continental slopes can play a significant contribution to marine productivity and carbon cycling. These regions can harbour distinct geological features, such as salt diapirs and pockmarks, in which their depressions may serve as natural sediment traps where different compounds can accumulate. We investigated the prokaryotic communities in surface (0-2 cm) and subsurface (18-20 or 22-24 cm) sediments from a salt diapir and pockmark field in Santos Basin, Southwest Atlantic Ocean. Metabarcoding of 16 samples revealed that surface sediments were dominated by the archaeal class Nitrososphaeria, while the bacterial class Dehalococcoidia was the most prevalent in subsurface samples. Sediment strata were found to be a significant factor explaining 27% of the variability in community composition. However, no significant difference was observed among geomorphological features. We also performed a metagenomic analysis of three surface samples and analysed the highest quality metagenome-assembled genome retrieved, which belonged to the family CSP1-5, phylum Methylomirabilota. This non-methanotrophic methylotroph contains genes encoding for methanol oxidation and Calvin Cycle pathways, along with diverse functions that may contribute to its adaptation to deep-sea habitats and to oscillating environmental conditions. By integrating metabarcoding and metagenomic approaches, we reported that CSP1-5 is prevalent in the sediment samples from Santos Basin slope, indicating the potential importance of methanol metabolism in this region. Finally, using a phylogenetic approach integrating 16S rRNA sequences assigned to Methylomirabilota in this study with those from a public database, we argued that CSP1-5 public sequences might be misclassified as Methylomirabilaceae (the methanotrophic clade) and, therefore, the role of these organisms and the methanol cycling could also be neglected in other environments.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Best available technique for the recovery of marine benthic communities in a gravel shore after the oil spill: A mesocosm-based sediment triad assessment.

Ecotoxicological effects of spilled oils are well documented, but study of recovery of marine benthic communities is limited. Long-term recovery of hard bottom communities during physical and biological remediations after a spill was monitored. A 60-day experiment was conducted using a mesocosm with monitoring of eight endpoints by use of the sediment quality triad (SQT). First, physical treatment of hot water + high pressure flushing maximally removed residual oils (max=93%), showing the greatest recovery among SQT variables (mean=72%). Physical cleanup generally involved adverse effects such as depression of the microphytobenthic community during the initial period. Next, biological treatments, such as fertilizer, emulsifier, enzyme and augmentation of the microbes, all facilitated removal of oil (max=66%) enhancing ecological recovery. Analysis of the microbiome confirmed that oil-degrading bacteria, such as Dietzia sp. and Rosevarius sp. were present. A mixed bioremediation, including fertilizer + multi-enzyme + microbes (FMeM) maximized efficacy of remediation as indicated by SQT parameters (mean=47%). Natural attenuation with "no treatment" showed comparable recovery to other remediations. Considering economic availability, environmental performance, and technical applicability, of currently available techniques, combined treatments of physical removal via hand wiping followed by FMeM could be most effective for recovery of the rocky shore benthic community.

Phospholipid fatty acid (PLFA) analysis as a tool to estimate absolute abundances from compositional 16S rRNA bacterial metabarcoding data.

Microbial biodiversity monitoring through the analysis of DNA extracted from environmental samples is increasingly popular because it is perceived as being rapid, cost-effective, and flexible concerning the sample types studied. DNA can be extracted from diverse media before high-throughput sequencing of the prokaryotic 16S rRNA gene is used to characterize the taxonomic diversity and composition of the sample (known as metabarcoding). While sources of bias in metabarcoding methodologies are widely acknowledged, previous studies have focused mainly on the effects of these biases within a single substrate type, and relatively little is known of how these vary across substrates. We investigated the effect of substrate type (water, microbial mats, lake sediments, stream sediments, soil and a mock microbial community) on the relative performance of DNA metabarcoding in parallel with phospholipid fatty acid (PLFA) analysis. Quantitative estimates of the biomass of different taxonomic groups in samples were made through the analysis of PLFAs, and these were compared to the relative abundances of microbial taxa estimated from metabarcoding. Furthermore, we used the PLFA-based quantitative estimates of the biomass to adjust relative abundances of microbial groups determined by metabarcoding to provide insight into how the biomass of microbial taxa from PLFA analysis can improve understanding of microbial communities from environmental DNA samples. We used two sets of PLFA biomarkers that differed in their number of PLFAs to evaluate how PLFA biomarker selection influences biomass estimates. Metabarcoding and PLFA analysis provided significantly different views of bacterial composition, and these differences varied among substrates. We observed the most notable differences for the Gram-negative bacteria, which were overrepresented by metabarcoding in comparison to PLFA analysis. In contrast, the relative biomass and relative sequence abundances aligned reasonably well for Cyanobacteria across the tested freshwater substrates. Adjusting relative abundances of microbial taxa estimated by metabarcoding with PLFA-based quantification estimates of the microbial biomass led to significant changes in the microbial community compositions in all substrates. We recommend including independent estimates of the biomass of microbial groups to increase comparability among metabarcoding libraries from environmental samples, especially when comparing communities associated with different substrates.

A metagenomic assessment of microbial communities in anaerobic bioreactors and sediments: Taxonomic and functional relationships.

The present study used metagenomic sequencing, metagenome assembly and physical-chemical analysis to describe taxonomically and functionally 3 anaerobic bioreactors treating manure (LI), brewery (BR) and cornmeal (CO) wastes, and an anaerobic estuarine sediment (ES). Proteobacteria, Firmicutes, Euryarchaeota and Bacteroidetes were the most abundant Phyla in all metagenomes. A bacteria/archaea ratio of 3.4 was found in the industrial full-scale anaerobic bioreactors BR and CO, while ratios greater than 10 were found for LI and ES. Canonical correspondence analysis showed that environmental variables such as chemical oxygen demand, lipid content, and ammonium nitrogen influenced the ordination of taxonomic groups. Mesotoga prima was linked to high-temperature conditions, particularly in the BR bioreactor, along with the presence of heat shock proteins genes. Likewise, the hydrogenotrophic methanogen, Methanoregula formicica, was associated with high ammonium concentration in LI bioreactor. The interactions of microbes with specific methanogenic pathways were identified using Clusters of Orthologous Groups (COG) functions, while metagenome-assembled genomes (MAGs) further confirmed relationships between taxa and functions. Our results provide valuable information to understand microbial processes in anaerobic environments.

Assessment of the tolerance to Fe, Cu and Zn of a sulfidogenic sludge generated from hydrothermal vents sediments as a basis for its application on metals precipitation.

A paramour factor limiting metal-microorganism interaction is the metal ion concentration, and the metal precipitation efficiency driven by microorganisms is sensitive to metal ion concentration. The aim of the work was to determine the tolerance of the sulfidogenic sludge generated from hydrothermal vent sediments at microcosms level to different concentrations of Fe, Cu and Zn and the effect on the microbial community. In this study the chemical oxygen demand (COD) removal, sulfate-reducing activity (SRA) determination, inhibition effect through the determination of IC50, and the characterization of the bacterial community´s diversity were conducted. The IC50 on SRA was 34 and 81 mg/L for Zn and Cu, respectively. The highest sulfide concentration (H2S mg/L) and % of sulfate reduction obtained were: 511.30 ± 0.75 and 35.34 ± 0.51 for 50 mg/L of Fe, 482.48 ± 6.40 and 33.35 ± 0.44 for 10 mg/L of Cu, 442.26 ± 17.1 and 30.57 ± 1.18 for 10 mg/L of Zn, respectively. The COD removal rates were of 71.81 ± 7.6, 53.92 ± 1.07 and 57.68 ± 10.2 mg COD/ L d for Fe (50 mg/L), Cu (40 mg/L) and Zn (20 mg/L), respectively. Proteobacteria, Firmicutes, Chloroflexi and Actinobacteria were common phyla to four microcosms (stabilized sulfidogenic and added with Fe, Cu or Zn). The dsrA genes of Desulfotomaculum acetoxidans, Desulfotomaculum gibsoniae and Desulfovibrio desulfuricans were expressed in the microcosms supporting the SRA results. The consortia could be explored for ex-situ bioremediation purposes in the presence of the metals tested in this work.

Bioplastic (poly-3-hydroxybutyrate) production by the marine bacterium Pseudodonghicola xiamenensis through date syrup valorization and structural assessment of the biopolymer.

Biobased degradable plastics have received significant attention owing to their potential application as a green alternative to synthetic plastics. A dye-based procedure was used to screen poly-3-hydroxybutyrate (PHB)-producing marine bacteria isolated from the Red Sea, Saudi Arabia. Among the 56 bacterial isolates, Pseudodonghicola xiamenensis, identified using 16S rRNA gene analyses, accumulated the highest amount of PHB. The highest PHB production by P. xiamenensis was achieved after 96 h of incubation at pH 7.5 and 35 °C in the presence of 4% NaCl, and peptone was the preferred nitrogen source. The use of date syrup at 4% (w/v) resulted in a PHB concentration of 15.54 g/L and a PHB yield of 38.85% of the date syrup, with a productivity rate of 0.162 g/L/h, which could substantially improve the production cost. Structural assessment of the bioplastic by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy revealed the presence of methyl, hydroxyl, methine, methylene, and ester carbonyl groups in the extracted polymer. The derivative products of butanoic acid estimated by gas chromatography-mass spectrometry [butanoic acid, 2-amino-4-(methylseleno), hexanoic acid, 4-methyl-, methyl ester, and hexanedioic acid, monomethyl ester] confirmed the structure of PHB. The present results are the first report on the production of a bioplastic by P. xiamenensis, suggesting that Red Sea habitats are a potential biological reservoir for novel bioplastic-producing bacteria.

Assessment of microbiota present on a Portuguese historical stone convent using high-throughput sequencing approaches.

The study performed on the stone materials from the Convent of Christ revealed the presence of a complex microbial ecosystem, emphasizing the determinant role of microorganisms on the biodecay of this built cultural heritage. In this case study, the presence of Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. seems to be responsible for colored stains and biofilm formation while Ulocladium sp., Cladosporium sp., and Dirina sp. may be related to structural damages. The implementation of high-throughput sequencing approaches on the Convent of Christ's biodecay assessment allowed us to explore, compare, and characterize the microbial communities, overcoming the limitations of culture-dependent techniques, which only identify the cultivable population. The application of these different tools and insights gave us a panoramic view of the microbiota thriving on the Convent of Christ and signalize the main biodeteriogenic agents acting on the biodecay of stone materials. This finding highlighted the importance of performing metagenomic studies due to the improvements and the reduced amount of sample DNA needed, promoting a deeper and more detailed knowledge of the microbiota present on these dynamic repositories that support microbial life. This will further enable us to perform prospective studies in quarry and applied stone context, monitoring biogenic and nonbiogenic agents, and also to define long-term mitigation strategies to prevent biodegradation/biodeterioration processes.

Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations.

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments.IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

Biodegradation potential assessment by using autochthonous microorganisms from the sediments from Lac Mégantic (Quebec, Canada) contaminated with light residual oil.

In July 2013, a fatal train derailment led to an explosion and fire in the town of Lac-Mégantic (LM), Quebec, and the crude oil contamination of regional surface water, soil, and sediment in the adjacent Lake Mégantic. This study investigated the degradation potential of the spilled crude oil by using the sediments from the incident site as the source of microorganisms. Two light crude oils (LM source oil and Alberta Sweet Mixed Blend (ASMB)) were tested at 22 °C for 4 weeks and 4 °C for 8 weeks, respectively. The post-incubation biological and chemical information of the samples were analysed. There was no marked difference in degradation efficacy and biological activities for both the LM and ASMB oils, although the biodegradation potential differed between the two incubations. Higher temperature favoured the growth of microorganisms, thus for the degradation of all petroleum hydrocarbons, except for some conservative biomarkers. The degradation of both oils followed the order of resolved components > total saturated hydrocarbons (TSH) > unresolved complex mixture (UCM) >total aromatic hydrocarbons (TAH). Normal alkanes were generally degraded more significantly than branched ones, and polycyclic aromatic hydrocarbons (PAHs). Degradation of polycyclic aromatic hydrocarbons (PAHs) and their alkylated congeners (APAHs) for both incubations generally decreased as the number of aromatic rings, and the degree of alkylation increased. This study showed that the LM sediments can biodegrade the petroleum hydrocarbons efficaciously if appropriate ambient temperatures are generated to favour the growth of autochthonous microorganisms.

Metabarcoding assessment of prokaryotic and eukaryotic taxa in sediments from Stellwagen Bank National Marine Sanctuary.

Stellwagen Bank National Marine Sanctuary (SBNMS) in the Gulf of Maine is a historic fishing ground renowned for remarkable productivity. Biodiversity conservation is a key management priority for SBNMS and yet data on the diversity of microorganisms, both prokaryotic and eukaryotic, is lacking. This study utilized next generation sequencing to characterize sedimentary communities within SBNMS at three sites over two seasons. Targeting 16S and 18S small subunit (SSU) rRNA genes and fungal Internal Transcribed Spacer (ITS) rDNA sequences, samples contained high diversity at all taxonomic levels and identified 127 phyla, including 115 not previously represented in the SBNMS Management Plan and Environmental Assessment. A majority of the diversity was bacterial, with 59 phyla, but also represented were nine Archaea, 18 Animalia, 14 Chromista, eight Protozoa, two Plantae, and 17 Fungi phyla. Samples from different sites and seasons were dominated by the same high abundance organisms but displayed considerable variation in rare taxa. The levels of biodiversity seen on this small spatial scale suggest that benthic communities of this area support a diverse array of micro- and macro-organisms, and provide a baseline for future studies to assess changes in community structure in response to rapid warming in the Gulf of Maine.

Assessment of environmental gene tags linked with carbohydrate metabolism and chemolithotrophy associated microbial community in River Ganga.

The microbial community mediated biogeochemical cycles play important role in global C-cycle and display a sensitive response to environmental changes. Limited information is available on microbial composition and functional diversity controlling biogeochemical cycles in the riverine environment. The Ganga River water and sediment samples were studied for environmental gene tags with reference to carbohydrate metabolism, photoheterotrophy and chemolithotrophy using high throughput shotgun metagenomic sequencing and functional annotation. The diversity of environmental gene tags specific microbial community was annotated against reference sequence database using Kaiju taxonomic classifier. The metagenomic analyses revealed that the river harbored a broad range of carbohydrate and energy metabolism genes. The in-depth investigation of metagenomic data revealed that the enzymes associated with reverse TCA cycle, Calvin-Benson cycle enzyme RuBisCO, starch and sucrose metabolism genes were highly abundant. The enzymes associated with sulfur metabolism such as EC:2.7.7.4 (sulfate to ammonium per sulfate), EC:1.8.1.2, EC:1.8.7.1 (sulfite to H2S) were prevalent in both the class of samples. The principal component analysis of the functional profiles revealed that the water and sediment samples were clustered distinctly suggesting that both the sites had variable abundance of functional genes and associated microbiota. The taxonomic classification showed abundance of Proteobacteria, Actinobacteria and Bacteroidetes phyla. Also, the metagenomic study showed the presence of purple sulfur bacteria viz. Thiodictyon, Nitrosococcus and purple non-sulfur bacteria viz. Bradyrhozobium and Rhodobacter. The study demonstrates that the Ganga River microbiome has prevalence of functional genes involved in carbohydrate anabolism and catabolism, and CO2 fixation with great prospects in cellulose and sulfide degrading enzyme production and characterization.

Radiative Energy Budgets in a Microbial Mat Under Different Irradiance and Tidal Conditions.

Irradiance and temperature variations during tidal cycles modulate microphytobenthic primary production potentially by changing the radiative energy balance of photosynthetic mats between immersion and emersion and thus sediment daily net metabolism. To test the effect of tidal stages on the radiative energy budget, we used microsensor measurements of oxygen, temperature, and scalar irradiance to estimate the radiative energy budget in a coastal photosynthetic microbial mat during immersion (constant water column of 2 cm) and emersion under increasing irradiance. Total absorbed light energy was higher in immersion than emersion, due to a lower reflectance of the microbial mat, while most (> 97%) of the absorbed light energy was dissipated as heat irrespective of tidal conditions. During immersion, the upward heat flux was higher than the downward one, whereas the opposite occurred during emersion. At highest photon irradiance (800 µmol photon m-2 s-1), the sediment temperature increased ~ 2.5 °C after changing the conditions from immersion to emersion. The radiative energy balance showed that less than 1% of the incident light energy (PAR, 400-700 nm) was conserved by photosynthesis under both tidal conditions. At low to moderate incident irradiances, the light use efficiency was similar during the tidal stages. In contrast, we found an ~ 30% reduction in the light use efficiency during emersion as compared to immersion under the highest irradiance likely due to the rapid warming of the sediment during emersion and increased non-photochemical quenching. These changes in the photosynthetic efficiency and radiative energy budget could affect both primary producers and temperature-dependent bacterial activity and consequently daily net metabolism rates having important ecological consequences.

Retrospective eDNA assessment of potentially harmful algae in historical ship ballast tank and marine port sediments.

Microalgal bloom events can cause major ecosystem disturbances, devastate local marine economies, and endanger public health. Therefore, detecting and monitoring harmful microalgal taxa is essential to ensure effective risk management in waterways used for fisheries, aquaculture, recreational activity, and shipping. To fully understand the current status and future direction of algal bloom distributions, we need to know how populations and ecosystems have changed over time. This baseline knowledge is critical for predicting ecosystem responses to future anthropogenic change and will assist in the future management of coastal ecosystems. We explore a NGS metabarcoding approach to rapidly identify potentially harmful microalgal taxa in 63 historic and modern Australian marine port and ballast tank sediment samples. The results provide a record of past microalgal distribution and important baseline data that can be used to assess the efficacy of shipping guidelines, nutrient pollution mitigation, and predict the impact of climate change. Critically, eDNA surveys of archived sediments were able to detect harmful algal taxa that do not produce microscopic fossils, such as Chattonella, Heterosigma, Karlodinium, and Noctiluca. Our data suggest a potential increase in Australian harmful microalgal taxa over the past 30 years, and confirm ship ballast tanks as key dispersal vectors. These molecular mapping tools will assist in the creation of policies aimed at reducing the global increase and spread of harmful algal taxa and help prevent economic and public-health problems caused by harmful algal blooms.

Molecular methods for cost-efficient monitoring of HAB (harmful algal bloom) dinoflagellate resting cysts.

Cyst abundance and identity are essential for understanding and predicting blooms, and for assessing the dispersal of toxic target dinoflagellate species by natural or human mediated ways, as with ballast waters. The aim of this study was to apply rapid, specific and sensitive qPCR assays to enumerate toxic dinoflagellate cysts in sediment samples collected from Adriatic harbours. The molecular standard curves of various target species allowed obtaining the rDNA copy number per cyst. The analytical sensitivity for specific standard curves was determined to be 2 or 10 rDNA copies per reaction. The abundance varied in the range of 1-747 dinoflagellate cysts g-1 dry weight. The assays showed greater sensitivity as compared to counts by light microscopy. This qPCR method revealed a powerful tool for the quantification of cysts from toxic dinoflagellate resting stages in sediment samples from Adriatic ports.

An assessment of the microbial community in an urban fringing tidal marsh with an emphasis on petroleum hydrocarbon degradative genes.

Small fringing marshes are ecologically important habitats often impacted by petroleum. We characterized the phylogenetic structure (16S rRNA) and petroleum hydrocarbon degrading alkane hydroxylase genes (alkB and CYP 153A1) in a sediment microbial community from a New Hampshire fringing marsh, using alkane-exposed dilution cultures to enrich for petroleum degrading bacteria. 16S rRNA and alkB analysis demonstrated that the initial sediment community was dominated by Betaproteobacteria (mainly Comamonadaceae) and Gammaproteobacteria (mainly Pseudomonas), while CYP 153A1 sequences predominantly matched Rhizobiales. 24 h of exposure to n-hexane, gasoline, dodecane, or dilution culture alone reduced functional and phylogenetic diversity, enriching for Gammaproteobacteria, especially Pseudomonas. Gammaproteobacteria continued to dominate for 10 days in the n-hexane and no alkane exposed samples, while dodecane and gasoline exposure selected for gram-positive bacteria. The data demonstrate that small fringing marshes in New England harbor petroleum-degrading bacteria, suggesting that petroleum degradation may be an important fringing marsh ecosystem function.

QMEC: a tool for high-throughput quantitative assessment of microbial functional potential in C, N, P, and S biogeochemical cycling.

Microorganisms are major drivers of elemental cycling in the biosphere. Determining the abundance of microbial functional traits involved in the transformation of nutrients, including carbon (C), nitrogen (N), phosphorus (P) and sulfur (S), is critical for assessing microbial functionality in elemental cycling. We developed a high-throughput quantitative-PCR-based chip, Quantitative microbial element cycling (QMEC), for assessing and quantifying the genetic potential of microbiota to mineralize soil organic matter and to release C, N, P and S. QMEC contains 72 primer pairs targeting 64 microbial functional genes for C, N, P, S and methane metabolism. These primer pairs were characterized by high coverage (average of 18-20 phyla covered per gene) and sufficient specificity (>70% match rate) with a relatively low detection limit (7-102 copies per run). QMEC was successfully applied to soil and sediment samples, identifying significantly different structures, abundances and diversities of the functional genes (P<0.05). QMEC was also able to determine absolute gene abundance. QMEC enabled the simultaneous qualitative and quantitative determination of 72 genes from 72 samples in one run, which is promising for comprehensively investigating microbially mediated ecological processes and biogeochemical cycles in various environmental contexts including those of the current global change.

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future.