Metal-Microbial Interactions in Toronto Sunnyside Beach: Impact on Water Quality and Public Health
Assessing recreational water quality requires a fundamental understanding of metal-microbial interactions and the key biogeochemical processes occurring in urban public beaches. Metals play an important role in the distribution and virulence (e.g. resistance) of microorganisms in water systems. In turn, microorganisms have a significant influence on metal cycling, thus affecting metal mobility, bioavailability and toxicity in the aquatic environment. Bacteria adhere to floc, small suspended mineral-bacterial aggregates, in aquatic systems resulting in high-density floc-associated bacterial biofilm communities. These nanoparticulate bacterial microhabitats are important environmental sinks for metals and potential reservoirs for antibiotic resistant and pathogenic bacteria. The objectives of this study are to identify and quantify (1) metal distributions among suspended floc, bed sediment and water-column aqueous compartments (2) important biogeochemical processes influencing metal cycling and (3) linkages between floc metals and the occurrence of floc associated antibiotic resistant bacteria and pathogens across a series of variably contaminated aquatic systems. Results of this project will provide new diagnostic indicators of pathogens in recreational water systems and aid in the development of public health policies to improve water quality and reduce water borne infectious disease. Here, results will be presented assessing the metal and microbial community dynamics in samples collected from Toronto's Sunnyside Beach (May 13 and August 20), an urban public beach on Lake Ontario. Water column, floc and bed sediments near and offshore were analyzed for physico-chemical characteristics and metal concentrations. Floc were imaged using DAPI and FISH to assess microbial community structure. Results to date, characterizing the linkages amongst bacteria, metal contaminant concentrations and sediment partitioning and system physico-chemical conditions will be discussed.
Examining trace metal contamination in an unanthropogenically impacted lake in Algonquin Park: implications for environmental bacterial communities and antibiotic resistance
Identifying the biogeochemical processes influencing the interactions amongst trace metals, microbial communities, pathogenicity and antibiotic resistance (ABR) is key to predicting the emergence, dissemination and maintenance of ABR in the environmental arena. The co-selection of heavy metal resistance and ABR has been documented in metal-contaminated environments. However, as yet, little research has been conducted assessing the metal status of 'pristine' area lakes and associated environmental bacterial communities. As part of a larger project evaluating metal-bacterial-ABR-pathogen interactions, a field survey of 6 variably contaminated aquatic systems was conducted in the summer of 2008, including Brewer Lake -a highly organic, circumneutral, Fe stained lake in Algonquin Park. To our knowledge, this study is the first to assess metal concentrations for the suite of Ag, As, Cd, Co, Cu, Ni, Pb, Se, Zn amongst the water column, suspended floc and bed sediments for this lake. The characterization and sampling protocol included 1) in situ characterization of overlying water column physicochemical parameters and 2) collection of water samples, suspended flocs (by field flow centrifugation) and bed sediment samples (by core; surficial and at depth) for subsequent metal analysis. Floc- and sediment-associated metals were partitioned into 6 operationally defined solid matrix fractions by sequential extraction: the exchangeable (loosely bound); carbonate; reducible amorphous Fe/Mn hydrous oxides; reducible crystalline Fe/Mn oxides; and residual fractions. Results indicate that the partitioning of metals between solid (floc, sediments) and dissolved compartments is largely element- dependent. Mean total metal concentrations in the sediments ranged from nM (Ag,Se, Cd) to μM (As, Co, Cu, Ni, Pb, Zn) with only Cu and Co (nM) and Zn ( μM) being detected in the water column. However in all cases floc-associated metal concentrations were an order of magnitude greater than in any other compartment (with the exception of Cd), indicating the role of floc as a metal-sink in this system. Moreover, within the floc, metal retention and affinities for the solid matrix fractions were in general different than those observed within the bed sediments (surficial and at depth) indicating differing suspended vs bed sediment controls in metal sequestration. These results will be discussed in the context of metal distributions within Brewer Lake as well as the possible implications for microbial community and ABR dynamics.
Geochemical Interactions and Viral-Prokaryote Relationships in Freshwater Environments
Viral and prokaryotic abundances were surveyed throughout southern Ontario aquatic habitats to determine relationships with geochemical parameters in the natural environment. Surface water samples were collected from acid mine drainage in summer of 2007 and 2008 and from circum-neutral pH environments in October to November 2008. Site determination was based on collecting samples from various aquatic habitats (acid mine drainage, lakes, rivers, tributaries, wetlands) with differing bedrock geology (limestone and shale dominated vs granitic Canadian Shield) to obtain a range of geochemical conditions. At each site, measurements of temperature, pH, and Eh were conducted. Samples collected for microbial counts and electron imaging were preserved to a final concentration of 2.5 % (v/v) glutaraldehyde. Additional sample were filtered into 60 mL nalgene bottles and amber EPA certified 40 mL glass vials to determine chemical constituents and dissolved organic carbon (DOC), respectively. Water was also collected to determine additional physiochemical parameters (dissolved total iron, ferric iron, nitrate, sulfate, phosphate, alkalinity, and turbidity). All samples were stored at 4 °C until analysis. Viral and prokaryotic abundance was determined by staining samples with SYBR Green I and examining with a epifluorescence microscope under blue excitation. Multiple regression analysis using stepwise backwards regression and general linear models revealed that viral abundance was the most influential predictor of prokaryotic abundance. Additional predictors include pH, sulfate, phosphate, and magnesium. The strength of the model was very strong with 90 % of the variability explained (R2 = 0.90, p < 0.007). This is the first report, to our knowledge, of viruses exhibiting such strong controls over prokaryotic abundance in the natural environment. All relationships are positively correlated with the exception of Mg, which is negatively correlated. Iron was also noted as a contributor to prokaryotic abundance but given the elements strong multicollinearity with sulfate, iron was removed from the model (as sulfate acts more conservatively across the range of pH sampled, 2.5-9.0). Geochemical variables that have been reported to influence viral abundances under laboratory and field experiments (i.e. Ca2+, DOC, temperature) had minimal effect in the natural environment despite 2 to 3 orders of magnitude range in the data. However, log transformed viral abundance did revealed a significant relationship with pH (Pearson correlation coefficient of r = 0.70) when using principle component analysis. Prokaryotic abundance did not reveal significant correlations with geochemical parameters (all r < 0.38).
Microbial ecology of a novel sulphur cycling consortia from AMD: implications for acid generation
Recent work1 identified a novel microbial consortia consisting of two bacterial strains common to acid mine drainage (AMD) environments (autotrophic sulphur oxidizer Acidithiobacillus ferrooxidans and heterotrophic Acidiphilium spp.) in an environmental enrichment from a mine tailings lake. The two strains showed a specific spatial arrangement within an EPS macrostructure or "pod" allowing linked metabolic redox cycling of sulphur. Sulphur species characterisation of the pods using scanning transmission X-ray microscopy (STXM) indicated that autotrophic tetrathionate disproportionation by A. ferrooxidans producing colloidal elemental sulphur (S0) is coupled to heterotrophic S0 reduction by Acidiphilium spp. Geochemical modelling of the microbial sulphur reactions indicated that if they are widespread in AMD environments, then global AMD-driven CO2 liberation from mineral weathering have been overestimated by 40-90%1. Given the common co-occurrence of these two bacteria in AMD settings, the purpose of this study was to evaluate if these pods could be induced in the laboratory by pure strains and if so, whether their combined sulphur geochemistry mimicked the previous findings. Laboratory batch experiments assessed the development of pods with pure strain type cultures (A. ferrooxidans ATCC 19859 with mixotroph Acidiphilium acidophilum ATCC 738 or strict heterotroph Acp. cryptum ATCC 2158) using fluorescent in situ hybridization (FISH) imaging. The microbial sulphur geochemistry was characterized under autotrophic conditions identical to those used with the environmental AMD enrichment in which the pods were discovered. Results showed that the combined pure strain A. ferrooxidans and Acp. acidophilum form pods identical in structure to the AMD enrichment. To test the hypothesis that these pods form for mutual metabolic benefit, experiments were performed amending pure strain and AMD enrichment bacterial treatments with organic carbon and/or additional sulphur to assess whether or not pods formed and/or disassociated under non-competitive and/or non-nutrient limiting scenarios. The results of these experiments will be presented and their ecological and AMD sulphur geochemical implications discussed.
Sulfur Isotopic Fractionation During Dissimilatory Sulfate Reduction from the Perspective of an Entire Microbial Metabolism
Whether in the investigation of the most ancient life on Earth, examination of surface oxidation properties across geological timescales, or the estimation of microbial metabolism in inaccessible environments, dissimilatory sulfate reduction (DSR) constrains biogeochemical processes in a variety of spatial and temporal scales. Pioneering work in the 1970s established the importance of DSR to biogeochemical processes and its potential as a geochemical tracer, and models for biological controls of DSR were published from empirical results of in vitro microbial cultures. Recent efforts have expanded upon this body of work and further extended toward multiple sulfur isotopes and through the more precise definition of the biological processes themselves. Resulting from these recent efforts is an rigorous description of DSR of the sulfur metabolism of sulfate-reducing bacteria. However, despite these efforts, the exact mechanisms of DSR within the scope of a complex system such as microbial metabolism remain incomplete and obscure. We will be presenting ongoing work coupling together recent mathematical models of isotopic fractionation with a flux-oriented, genomically-derived software model of the metabolism of Desulfovibrio vulgaris, a patent sulfate-reducing bacterium. Our presentation will explore the effects on isotopic fractionation throughout the sulfate reduction pathway of D. vulgaris by a multitude of separate and distinct biological pathways within the bacterial metabolism. Further, we will be discussing both the pitfalls and promise of such an approach and its implications for future research.
Defluoridation by Bacteriogenic Iron Oxides: Sorption Studies
At concentrations above 1 mg/L, fluoride in drinking water can lead to dental and skeletal fluorosis, a disease that causes mottling of the teeth, calcification of ligaments, crippling bone deformities and many other physiological disorders that can, ultimately, lead to death. Conservative estimates are that fluorosis afflicts tens of millions of people worldwide. As there is no treatment for fluorosis, prevention is the only means of controlling the disease. While numerous defluoridation techniques have been explored, no single method has been found to be both effective and inexpensive enough to implement widely. Our research began in India, with a large-scale geochemical study of the groundwater in a fluoride-contaminated region of Orissa. Having developed a better understanding of the geochemical relationships that exist between fluoride and other parameters present in an affected area, as well as the complex relationships that arise among those parameters that can impact the presence of fluoride, we began investigating certain remediation scenarios involving iron oxides. A common approach to remediation involves the partitioning of fluoride from groundwater by sorption onto a variety of materials, one of the most effective of which is iron oxide whose surface area acts as a scavenger for fluoride. In the presence of iron oxidizing bacteria, the oxidation rate of iron has been shown to be ∼6 times greater than in their absence; fluoride should, therefore, be removed from an aqueous environment by bacteriogenic iron oxides (BIOS) much more quickly than by abiotic iron oxides. Most recently, sorption studies have been conducted using both BIOS and synthetic hydrous ferric oxides in order to compare the behavior between biotic and abiotic sorbents. These studies have provided sorption isotherms that allow comparison of fluoride removed by sorption to BIOS versus synthetic iron oxides. Sorption affinity constants have also been determined, which allow for the prediction of fluoride removal in a wide variety of groundwater systems. Sorption isotherms and affinity constants show the use of BIOS to be a promising technique for the remediation of fluoride in groundwater.
Fossilization of Iron-Oxidizing Bacteria at Hydrothermal Vents: a Useful Biosignature on Mars?
Iron oxidizing bacteria are ubiquitous in marine and terrestrial environments on Earth, where they often display distinctive cell morphologies and are commonly encrusted by minerals, especially bacteriogenic iron oxides and silica. Putative microfossils of iron oxidizing bacteria have been found in jaspers as old as 490Ma and microbial iron oxidation may be an ancient metabolic pathway. In order to investigate the usefulness of mineralized iron oxidizing bacteria as a biosignature, we have examined mineral samples collected from relict hydrothermal systems along Explorer Ridge, NE Pacific Ocean. In addition, microaerophilic, neutrophilic iron oxidizing bacteria, isolated from Pacific hydrothermal vents, were grown in a Fe-enriched seawater medium at constant pH (6.5) and oxygen concentration (5 percent) in a controlled bioreactor system. Both natural samples and experimental products were examined with a combination of variable pressure scanning electron microscopy (SEM), field emission gun SEM, and in some cases by preparing samples with a focused ion beam (FIB) milling system. Natural seafloor samples display abundant filamentous forms often resembling, in both size and shape, the twisted stalks of Gallionella and the elongated filaments of Leptothrix. Generally, these filamentous features are 1-5 microns in diameter and up to several microns in length. Some samples consist entirely of low- density, porous masses of silica encrusted filamentous forms. Presumably, these masses were formed by a rapid precipitation by the influx of silica-rich fluids into a microbial mat dominated by bacteria with filamentous morphologies. The presence of rare, amorphous (unmineralized) filamentous matter rich in C and Fe suggests that these bacteria were iron oxidizers. There is no evidence that sulfur oxidizers were present. Filamentous features sectioned by FIB milling show internal material within semi-hollow tubular-like features. Silica encrustations also show pseudo-concentric growth bands. In the bioreactor cultures, constant conditions led to abundant microbial growth and formation of an iron oxyhydroxide precipitate, either in direct association with the cells or within the growth medium. This suggests that not all of the iron precipitation is biogenic in origin. Cells typically show a filamentous morphology reminiscent of the mineral-encrusted forms observed in the natural samples. Continuing work includes high-resolution TEM observations of cultured organisms, examination of 2-year long in situ seafloor incubation experiments, and bioreactor silicification experiments in order to better understand the roles of iron and silica in the fossilization process. Microaerophilic iron oxidation could have existed on the early Earth in environments containing small amounts of oxygen produced either by locally concentrated photosynthetic microorganisms (e.g., cyanobacteria) or abiotically, as proposed for the subsurface of the Fe-dominated Rio Tinto (Spain) basin system. By analogy, similar subsurface or near-surface microaerophilic environments could have existed on Mars in the past. The distinctive morphologies and mineralization patterns of iron oxidizing bacteria could be a useful biosignature to search for on Mars. Deposits and biogenic features similar to those described here could theoretically be identified on Mars with existing imaging and analytical technologies. Therefore, future missions to Mars should target ancient hydrothermal systems, some of which have been putatively identified already.
Astrobiological and Planetary Exploration Implications of Microbial Ichnofossils in Terrestrial Basaltic Glasses
Over the past decade, studies have demonstrated that terrestrial basaltic glass in pillow rims and hyaloclastites are suitable microbial habitats. Microbes rapidly begin colonizing the glassy surfaces along fractures and cracks that have been exposed to water. Microbial colonization of basaltic glass leads to the alteration and modification of the rocks to produce characteristic granular and/or tubular bioalteration textures. The early precipitation of sub-micron titanite grains within the biologically etched alteration structures serves as an agent for preservation that may persist for geologically extended periods of time in the absence of later penetrative deformation. These microbial alteration structures have been observed in several Archean greenstone belts including the Abitibi greenstone belt (2.7 Ga), Pilbara craton (3.35 Ga), and the Barberton greenstone belt (3.5 Ga). Archean subaqueous volcanic rocks provide an excellent analogue for a potential habitat for possible early Martian life, given that basaltic rocks are a major component of the Martian crust. A wide variety of recent evidence strongly suggests the long-term existence of abundant liquid water on ancient Mars. Recent orbiter, lander, and rover missions have found evidence for the presence of transient liquid water on Mars, perhaps persisting to the present day. Beyond Mars, other solar system bodies, notably Europa, Enceladus, and other icy satellites, may well host subaqueous basaltic glasses. We will explore the implications of the newly discovered geological record of basaltic glass bioalteration and basaltic glass as a microbial habitat for planetary exploration and astrobiology.
Possible Biofilms in Earth's Oldest Known Tidal Environment (> 3.7 Ga Isua Greenstone Belt, Greenland)
Earth's earliest biosphere is known from carbon and sulfur isotopes in rocks of Eoarchean (3.85 to 3.6 Ga) ages. However, fossils or biogenic structures of this antiquity have not been found. We have identified a preserved sandy tidal flat from the > 3.7 Ga Isua Greenstone Belt (IGB), Greenland, in which quartzite and metapelite rocks define original tidal sedimentary beds. On several bedding planes, flat clasts are preserved that in geometry and dimension strikingly resemble pieces of biofilm ripped off from their parent benthic community. Such biofilms are common in similar shallow-marine environments that have been described in siliciclastic deposits of early Archean to Recent ages., The preserved clasts in the IGB have sulfur isotope signals of possible biogenic origin and suggest that by 3.7 Ga biofilms may have covered portions of the ancient seafloor. If so, the clasts are the oldest known fossils preserved in Earth history.
Putative Bioalteration Textures Hosted Within Impact Melt Glasses From the Ries Crater, Germany
Impact cratering is a ubiquitous geological process on solid bodies. Any hypervelocity impact into a H2O- rich target has the potential to generate hydrothermal systems . Recent research has suggested that such impact-induced environments may be conducive to microbial colonization [e.g., 2]. Bioalteration of terrestrial basaltic glasses produces characteristic tubular and granular aggregate textures. Such bioalteration textures preserved in Archean greenstone belts constitute one of the oldest records of life on Earth . Our examination of glasses from the Ries crater in Germany has revealed tubular textures with remarkably similar morphologies to those seen in volcanic glasses. The hyperthermophilic root of the 16S phylogenic tree of life suggests an essential role for thermophilic environments in the origin or the early evolutionary history of life on Earth. Previous work has associated primitive life on Earth with submarine volcanic activity suggesting that submarine hydrothermal settings may have played an essential role in the origin of life [e.g., 4]. Impact-induced hydrothermal systems share many characteristics with submarine volcanic hydrothermal systems including the presence of chemical and thermal energy for microbial metabolism. Interestingly, the Late Heavy Bombardment period, during which life purportedly arose on Earth, was characterized by a high impact flux. Thus, impact-generated habitats were likely much more common on Earth than submarine hydrothermal systems suggesting the former as a more statistically probable habitat for the origin of life. Here we present preliminary data characterizing the putative bioalteration structures hosted within the Ries impact glasses. Establishing the biogenecity of the alteration structures observed in these glasses may have significant astrobiological implications: impact glasses share many similarities with volcanic glasses, however, fundamental differences make impact glasses unique geochemical systems . The bulk compositions of impact melts are diverse, reflecting heterogeneities in the target lithologies. Furthermore, impact melts often display heterogeneity on multiple scales. Given the probable ubiquity of impact glasses in hydrothermal settings throughout the Solar System, it is important to understand the biological components and potential of such systems. Impact derived endolithic habitats are being considered as possible locations for life on early Earth  and on the surface of other planets such as Mars . Understanding the geomicrobiology of impact craters on Earth is critical in furthering the search for life on Mars. Studies constraining the biogeochemistry of impact craters may not only yield insight into early life and the origin of life on Earth, but furthermore, may comprise a potential habitat for life and past life on other terrestrial planets such as Mars. References:  M.V. Naumov (2005) Geofluids, 5, 165-184.  C.S. Cockell, P. Lee (2002) Biological Reviews, 77, 279-310.  Banerjee et. al. (2007) Geochim. Cosmochim. Acta. 71, A58.  Staudigel et al. 2008. ES Rev. 89(3-4) 156-178.  Osinski G. R. (2003) MAPS 38(11), 1641-1667.  F. Westall, R.L. Folk (2003) Precambrian Res. 126.  Cockell C. S., et al. (2005) MAPS 40(12), 1901-1914
Preliminary Mineralogical and Geological Characterization of the Lost Hammer Perennial Spring, Axel Heiberg Island, Nunavut
Understanding past potential hydrological processes is fundamental in the search for past life on Mars. Despite the lack of liquid water on the Martian surface today, there is evidence of past upwelling and evaporation of groundwater [e.g., 1]. Cold, saline, perennial spring systems, in which water would have been able to flow to the Martian surface year round, may be of particular interest. Analogous systems on Earth are pertinent to better understand how they might have functioned, and possibly preserved biosignatures, on Mars. Several sets of cold saline springs have been documented in the region surrounding the McGill Arctic Research Station on Axel Heiberg Island, NU; these represent the highest latitude perennial springs on Earth, flow through 600 m of permafrost, and are not associated with any volcanic heat sources. Primitive life thrives in these springs year round . Here, first results of mineralogical analyses and geological field observations are presented for the Lost Hammer spring site. Spring deposits cover an area approximately 150 m x 30 m. The main vent is roughly 2.5 m tall and 3 m in diameter, and is covered in a layer several mm thick of a very fine, white, powdery mineral, overlying several cm of fine grey material. Preliminary XRD analysis has revealed the mineralogy of the white material to be thenardite (a dehydrated Na-sulphate; original mirabitite suspected) and halite, with trace amounts of quartz. The grey material is interpreted to be predominantly thenardite, mirabitite, and halite, with traces of other minerals. Hard white crusts on dried channel beds are thenardite and halite, and thicker crusts on pebbles are composed of halite and gypsum. Refs:  Allen, C.C, and Oehler, D.Z. 2008, A Case for Ancient Springs in Arabia Terra, Mars: Astrobiology, 8: 1093-1112.  Perreault, N.N. et al. 2007, Characterization of the Prokaryotic Diversity in Cold Saline Perennial Springs of the Canadian High Arctic: Appl. Environ. Microbiol., 73: 1532-1543.