Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Anoxygenic photosynthesis and iron-sulfur metabolic potential of Chlorobia populations from seasonally anoxic Boreal Shield lakes.

Aquatic environments with high levels of dissolved ferrous iron and low levels of sulfate serve as an important systems for exploring biogeochemical processes relevant to the early Earth. Boreal Shield lakes, which number in the tens of millions globally, commonly develop seasonally anoxic waters that become iron rich and sulfate poor, yet the iron-sulfur microbiology of these systems has been poorly examined. Here we use genome-resolved metagenomics and enrichment cultivation to explore the metabolic diversity and ecology of anoxygenic photosynthesis and iron/sulfur cycling in the anoxic water columns of three Boreal Shield lakes. We recovered four high-completeness and low-contamination draft genome bins assigned to the class Chlorobia (formerly phylum Chlorobi) from environmental metagenome data and enriched two novel sulfide-oxidizing species, also from the Chlorobia. The sequenced genomes of both enriched species, including the novel "Candidatus Chlorobium canadense", encoded the cyc2 gene that is associated with photoferrotrophy among cultured Chlorobia members, along with genes for phototrophic sulfide oxidation. One environmental genome bin also encoded cyc2. Despite the presence of cyc2 in the corresponding draft genome, we were unable to induce photoferrotrophy in "Ca. Chlorobium canadense". Genomic potential for phototrophic sulfide oxidation was more commonly detected than cyc2 among environmental genome bins of Chlorobia, and metagenome and cultivation data suggested the potential for cryptic sulfur cycling to fuel sulfide-based growth. Overall, our results provide an important basis for further probing the functional role of cyc2 and indicate that anoxygenic photoautotrophs in Boreal Shield lakes could have underexplored photophysiology pertinent to understanding Earth's early microbial communities.

Estimating cumulative wastewater treatment plant discharge influences on acesulfame and Escherichia coli in a highly impacted watershed with a fully-integrated modelling approach.

Wastewater treatment plant (WWTP) discharge is often considered a principal source of surface water contamination. In this study, a three-dimensional fully-integrated groundwater-surface water model was used to simulate the transport characteristics and cumulative loading of an artificial sweetener (acesulfame) and fecal indicator bacteria (Escherichia coli) from WWTPs within a 6800 km2 mixed-use, highly impacted watershed in Ontario, Canada. The model, which employed 3.5 × 106 computational nodes and 15 layers, facilitated a comprehensive assessment of groundwater-surface water interactions under high and low flow conditions; processes typically not accounted for in WWTP cumulative effects models. Simulations demonstrate that the model had significant capacity in reproducing the average and transient multi-year groundwater and surface water flow conditions in the watershed. As a proxy human-specific conservative tracer, acesulfame was useful for model validation and to help inform the representation of watershed-scale transport processes. Using a uniform WWTP acesulfame loading rate of 7.14 mg person-1 day-1, the general spatial trends and magnitudes of the acesulfame concentration profile along the main river reach within the watershed were reproduced; however, model performance was improved by tuning individual WWTP loading rates. Although instream dilution and groundwater-surface water interactions were strongly dependent on flow conditions, the main reach primarily consisted of groundwater discharge zones. For this reason, hydrodynamic dispersion in the hyporheic zone is shown as the predominant mechanism driving acesulfame into near-stream shallow groundwater, while under high flow conditions, the simulations demonstrate the potential for advective flushing of the shallow groundwater. Regarding the cumulative impact of the WWTPs on E. coli concentrations in the surface flow system, simulated transient E. coli levels downstream of WWTPs in the watershed were significantly lower than observed values, thus highlighting the potential importance of other sources of E. coli in the watershed.

Contemporary limnology of the rapidly changing glacierized watershed of the world's largest High Arctic lake.

Glacial runoff is predicted to increase in many parts of the Arctic with climate change, yet little is known about the biogeochemical impacts of meltwaters on downstream freshwater ecosystems. Here we document the contemporary limnology of the rapidly changing glacierized watershed of the world's largest High Arctic lake (Lake Hazen), where warming since 2007 has increased delivery of glacial meltwaters to the lake by up to 10-times. Annually, glacial meltwaters accounted for 62-98% of dissolved nutrient inputs to the lake, depending on the chemical species and year. Lake Hazen was a strong sink for NO3--NO2-, NH4+ and DOC, but a source of DIC to its outflow the Ruggles River. Most nutrients entering Lake Hazen were, however, particle-bound and directly transported well below the photic zone via dense turbidity currents, thus reinforcing ultraoligotrophy in the lake rather than overcoming it. For the first time, we apply the land-to-ocean aquatic continuum framework in a large glacierized Arctic watershed, and provide a detailed and holistic description of the physical, chemical and biological limnology of the rapidly changing Lake Hazen watershed. Our findings highlight the sensitivity of freshwater ecosystems to the changing cryosphere, with implications for future water quality and productivity at high latitudes.

Millions of Boreal Shield Lakes can be used to Probe Archaean Ocean Biogeochemistry.

Life originated in Archaean oceans, almost 4 billion years ago, in the absence of oxygen and the presence of high dissolved iron concentrations. Early Earth oxidation is marked globally by extensive banded iron formations but the contributing processes and timing remain controversial. Very few aquatic habitats have been discovered that match key physico-chemical parameters of the early Archaean Ocean. All previous whole ecosystem Archaean analogue studies have been confined to rare, low sulfur, and permanently stratified lakes. Here we provide first evidence that millions of Boreal Shield lakes with natural anoxia offer the opportunity to constrain biogeochemical and microbiological aspects of early Archaean life. Specifically, we combined novel isotopic signatures and nucleic acid sequence data to examine processes in the anoxic zone of stratified boreal lakes that are naturally low in sulfur and rich in ferrous iron, hallmark characteristics predicted for the Archaean Ocean. Anoxygenic photosynthesis was prominent in total water column biogeochemistry, marked by distinctive patterns in natural abundance isotopes of carbon, nitrogen, and iron. These processes are robust, returning reproducibly after water column re-oxygenation following lake turnover. Evidence of coupled iron oxidation, iron reduction, and methane oxidation affect current paradigms of both early Earth and modern aquatic ecosystems.

Use of an Artificial Sweetener to Identify Sources of Groundwater Nitrate Contamination.

The artificial sweetener acesulfame (ACE) is a potentially useful tracer of waste water contamination in groundwater. In this study, ACE concentrations were measured in waste water and impacted groundwater at 12 septic system sites in Ontario, Canada. All samples of septic tank effluent (n = 37) had ACE >6 µg/L, all samples of groundwater from the proximal plume zones (n = 93) had ACE >1 µg/L and, almost all samples from the distal plume zones had ACE >2 µg/L. Mean mass ratios of total inorganic nitrogen/ACE at the 12 sites ranged from 680 to 3500 for the tank and proximal plume samples. At five sites, decreasing ratio values in the distal zones indicated nitrogen attenuation. These ratios were applied to three aquifers in Canada that are nitrate-stressed and an urban stream where septic systems are present nearby to estimate the amount of waste water nitrate contamination. At the three aquifer locations that are agricultural, low ACE values (<0.02-0.15 µg/L) indicated that waste water contributed <15% of the nitrate in most samples. In groundwater discharging to the urban stream, much higher ACE values (0.2-11 µg/L) indicated that waste water was the likely source of >50% of the nitrate in most samples. This study confirms that ACE is a powerful tracer and demonstrates its use as a diagnostic tool for establishing whether waste water is a significant contributor to groundwater contamination or not.

Degradation of sucralose in groundwater and implications for age dating contaminated groundwater.

The artificial sweetener sucralose has been in use in Canada and the US since about 2000 and in the EU since 2003, and is now ubiquitous in sanitary wastewater in many parts of the world. It persists during sewage treatment and in surface water environments and as such, has been suggested as a powerful tracer of wastewater. In this study, longer-term persistence of sucralose was examined in groundwater by undertaking a series of three sampling snapshots of a well constrained wastewater plume in Canada (Long Point septic system) over a 6-year period from 2008 to 2014. A shrinking sucralose plume in 2014, compared to earlier sampling, during this period when sucralose use was likely increasing, provides clear evidence of degradation. However, depletion of sucralose from a mean of 40 µg/L in the proximal plume zone, occurred at a relatively slow rate over a period of several months to several years. Furthermore, examination of septic tank effluent and impacted groundwater at six other sites in Canada, revealed that sucralose was present in all samples of septic tank effluent (6-98 µg/L, n = 32) and in all groundwater samples (0.7-77 µg/L, n = 64). Even though sucralose degradation is noted in the Long Point plume, its ubiquitous presence in the groundwater plumes at all seven sites implies a relatively slow rate of decay in many groundwater septic plume environments. Thus, sucralose has the potential to be used as an indicator of 'recent' wastewater contamination. The presence of sucralose identifies groundwater that was recharged after 2000 in Canada and the US and after 2003 in the EU and many Asian countries.

Natural attenuation of septic system nitrogen by anammox.

On-site disposal of sewage in septic systems can lead to groundwater plumes with NO(3)(-)-N concentrations exceeding the common drinking water limit of 10 mg/L. Currently, denitrification is considered as the principal natural attenuation process. However, at a large seasonal-use septic system in Ontario (256 campsites), a suboxic zone exists where nitrogen removal of up to 80% occurs including removal of NH(4)(+)-N. This zone has both NO(3)(-)-N and NH(4)(+)-N at >5 mg/L each. In the distal NH(4)(+)-rich zone, NH(4)(+)-N concentrations (8.1 ± 8.0 mg/L) are lower than in the proximal zone (48 ± 36 mg/L) and NH(4)(+)-N is isotopically enriched (concentration-weighted mean δ(15)N of +15.7‰) compared to the proximal zone (+7.8‰). Furthermore, δ(15)N-NH(4)(+) isotopic enrichment increases with depth in the distal zone, which is opposite to what would result if nitrification along the water table zone was the mechanism causing NH(4)(+) depletion. Bacterial community composition was assessed with molecular (DNA-based) analysis and demonstrated that groundwater bacterial populations were predominantly composed of bacteria from two Candidatus genera of the Planctomycetales (Brocadia and Jettenia). Together, these data provide strong evidence that anaerobic ammonium oxidation (anammox) plays an important role in nitrogen attenuation at this site.

Stable oxygen isotopic fractionation during photolytic O(2) consumption in stream waters.

Oxygen (O(2)) is required for life in higher organisms, however, processes such as respiration, the oxidation of reduced inorganic species, and the photolytic breakdown of dissolved organic matter (DOM) decrease the O(2) concentrations in aquatic systems. Filtered, inoculated, and sterile samples of stream waters from Ontario, Canada, were incubated in natural sunlight to examine the effects of photolysis of DOM, respiration, and abiotic reactions on O(2) consumption and delta(18)O of dissolved oxygen (delta(18)O-O(2)). Oxygen consumption rates in the light were up to an order of magnitude greater than in the dark, suggesting light-mediated processes controlled O(2) consumption. Rates of O(2) loss were the same for each treatment (i.e. filtered, inoculated, and sterile) indicating that photolysis was the dominant O(2) consuming process over respiration in these incubations. O(2) consumption rates were different between streams, even when normalized to the change in dissolved organic carbon (DOC), signifying that DOM photolability varied among streams. During DOM breakdown to CO(2), the lighter (16)O isotopomer was preferentially consumed. Fractionation factors observed for photolysis, respiration, and abiotic reactions ranged between 0.988 and 0.995, and were similar in both the light and in the dark incubations in all streams. These fractionation factors are not a function of O(2) consumption rates, and are outside the range published for respiration (0.975-0.982). In current models of O(2) and delta(18)O-O(2), photolysis and respiration are not considered separately and the isotopic fractionation during respiration that is measured in the dark is used in the light. In these incubations, DOM degradation and abiotic reactions are important O(2) consuming and delta(18)O-O(2) fractionating processes. Current models of O(2) and delta(18)O-O(2) incorporate photolysis of DOM and other abiotic processes into the respiratory component of O(2) consumption, thereby overestimating respiration and underestimating photosynthesis to respiration ratios. Consequently, photolysis and abiotic reactions should be considered separately, particularly in shallow aquatic systems with high DOC.

Geochemical stability of phosphorus solids below septic system infiltration beds.

Review of 10 mature septic system plumes in Ontario, revealed that phosphorus (P) attenuation commonly occurred close to the infiltration pipes, resulting in discrete narrow intervals enriched in P by a factor of 2-4 (. MSc thesis, Dept. Earth Sci., University of Waterloo, Waterloo, Ont.; Ground Water 36 (1995) 1000; J. Contam. Hydrol. 33 (1998) 405). Although these attenuation reactions appeared to be sustainable under present conditions, the potential for remobilization of this P mass, should geochemical conditions change, is unknown. To test the stability of these P solids, dynamic flow column tests were carried out using sediments from three of the previously studied sites (Cambridge, Langton and Muskoka) focusing on sediments from the 'High-P' and underlying (Below) zones. Tests were continued for 166-266 pore volumes (PVs), during which time varying degrees of water saturation were maintained. During saturated flow conditions, relatively high concentrations of PO4 were eluted from the Cambridge and Langton High-P zones (up to 4 and 9 mg/l P, respectively), accompanied by elevated concentrations of Fe (up to 1.4 mg/l) and Mn (up to 4 mg/l) and lower values of Eh (<150 mV). The Below zones from Cambridge and Langton, however, maintained lower concentrations of P (generally<2 mg/l), Fe (<0.2 mg/l) and Mn (<1 mg/l) and maintained higher Eh (>250 mV) during saturated flow conditions. During unsaturated flow, P and Fe declined dramatically in the High-P zones (P<1 mg/l, Fe<0.2 mg/l), whereas concentrations remained about the same during saturated and unsaturated flow in the Below zones. This behavior is at least partly attributed to the development of reducing conditions during saturated flow in the High-P zones, leading to reductive dissolution of Fe (III)-P solids present in the sediments. Reducing conditions did not develop in the Below zones apparently because of lower sediment organic carbon (OC) contents (0.03-0.04 wt.%) compared to the High-P zones (0.2-0.65 wt.%). At the Muskoka site, where the sediments were noncalcareous, low values of P (<0.2 mg/l) were maintained in both the High-P and Below columns and reducing conditions did not develop. Results indicate the possibility of remobilizing P accumulated below septic system infiltration beds should conditions become more reducing. This could occur if sewage loading patterns change, for example when a seasonal use, lakeshore cottage is converted to a permanent dwelling.