Early career researchers benefit from inclusive, diverse and international collaborations: Changing how academic institutions utilize the seminar series.

Efforts to make research environments more inclusive and diverse are beneficial for the next generation of Great Lakes researchers. The global COVID-19 pandemic introduced circumstances that forced graduate programs and academic institutions to re-evaluate and promptly pivot research traditions, such as weekly seminar series, which are critical training grounds and networking opportunities for early career researchers (ECRs). While several studies have established that academics with funded grants and robust networks were better able to weather the abrupt changes in research and closures of institutions, ECRs did not. In response, both existing and novel partnerships provided a resilient network to support ECRs at an essential stage of their career development. Considering these challenges, we sought to re-frame the seminar series as a virtual collaboration for ECRs. Two interdisciplinary graduate programs, located in different countries (Windsor, Canada, and Detroit, USA) invested in a year-long partnership to deliver a virtual-only seminar series that intentionally promoted: the co-creation of protocols and co-led roles, the amplification of justice, equity, diversity and inclusion throughout all aspects of organization and representation, engagement and amplification through social media, the integration of social, scientific and cultural research disciplines, all of which collectively showcased the capacity of our ECRs to lead, organize and communicate. This approach has great potential for application across different communities to learn through collaboration and sharing, and to empower the next generation to find new ways of working together.

Investigating the microbial dynamics of microcystin-LR degradation in Lake Erie sand.

Cyanobacterial blooms and the associated hepatotoxins produced (e.g., microcystins, MCs) create a significant human health risk in freshwater lakes around the world, including Lake Erie. Though various physical and chemical treatment options are utilized, these are costly and their effectiveness decreases when other organics are present. Laboratory studies have identified a remediation option based on a mlr gene operon that can systematically degrade this toxin; however, studies on Lake Erie have been unable to amplify mlr genes from MC-degrading bacteria. These results suggest that either existing primers may be inefficient for broad identification of the mlr genes or that MC degradation genes and/or pathways may vary among bacterial taxa. To investigate the dynamics of the Lake Erie microbial community involved in the degradation of microcystin-LR (MC-LR), a flow-through column experiment using collected beach sand was conducted over a period of six weeks. Increasing concentrations of lake water spiked with MC-LR were continuously delivered to both biotic and abiotic (sterilized) sand columns, with influent and effluent MC-LR concentrations measured by LC-MS/MS. Despite the toxin concentrations far exceeding natural conditions during a bloom event (maximum dosage = 15.4 µg/L), MC-LR was completely removed within 21 h of contact time in the biotic columns. Stimulation of community taxa during the degradation process included Burkholderiaceae, Illumatobacteraceae, Pseudomonadaceae, Rhodocyclaceae and Nitrosomonadaceae. The overall results suggest several critical species may be required for the most complete and effective degradation of MC-LR.

Impacts of industrial atmospheric emissions on watershed export of dissolved ions in coastal streams: a Bayesian modeling approach.

Anthropogenic atmospheric emission and subsequent deposition of sulfur (S) has been linked to disrupted watershed biogeochemical processes through soil and surface water acidification. We investigated watershed-scale impacts of acidic deposition on tributary concentrations and watershed exports of major nutrients and ions for the Kitimat River Watershed, British Columbia. Since the 1950s, the Kitimat watershed had an aluminum smelting facility with substantial emissions at the river estuary. Emissions load the airshed overlying the watershed and potentially impact western tributaries leaving eastern tributaries available as reference. We assessed concentrations and export of key compounds in three reference and six potentially impacted tributaries and watersheds in 2015 and 2016. Sulfate (SO4), fluoride (F), nitrate (NO3), and chloride (Cl) were significantly higher in impacted tributaries. F concentrations exceeded the Canadian Council of Ministers of the Environment guideline for aquatic life in 83% of samples collected from impacted streams. Watershed export and associated uncertainty were determined by bootstrapped flow-stratified Beale's unbiased estimator. Impact of emissions on watershed export was modeled in a Bayesian approach to include variance in the export estimate to inform the uncertainty of model parameters. Export of SO4 and Ca increased significantly within 16 km and 8 km, respectively, toward the smelter emissions. The corresponding impacted area for SO4 and Ca was approximately 100 km2 and 45 km2, respectively. SO4 export is likely due to direct impacts of S deposition, with excess S being flushed from the watersheds. Ca export patterns likely result from indirect impacts of S deposition on soil chemistry and flushing of Ca. These impacts may contribute to effects within tributaries on benthic stream communities and regionally important juvenile Pacific salmon.

'Nitrification kinetics and microbial community dynamics of attached biofilm in wastewater treatment'.

A comparative bench-scale and field site analysis of BioCord was conducted to investigate seasonal microbial community dynamics and its impact on nitrogen removal in wastewater. This was assessed using metabolite (NO3 -) stable isotope analysis, high-throughput sequencing of the 16S rRNA gene, and RT-qPCR of key genes in biological treatment representing nitrification, anammox, and denitrification. Bench-scale experiments showed an increase in nitrifiers with increasing ammonia loading resulting in an ammonia removal efficiency up to 98 ± 0.14%. Stable isotope analysis showed that 15ɛ and δ18ONO3 could be used in monitoring the efficiency of the enhanced biological nitrification. In the lagoon field trials, an increase in total nitrogen promoted three principle nitrifying genera (Nitrosomonas, Nitrospira, Candidatus Nitrotoga) and enhanced the expression of denitrification genes (nirK, norB, and nosZ). Further, anaerobic ammonia oxidizers were active within BioCord biofilm. Even at lower temperatures (2-6°C) the nitrifying bacteria remained active on the BioCord.

Distinguishing point and non-point sources of dissolved nutrients, metals, and legacy contaminants in the Detroit River.

Water quality impacts to the Laurentian Great Lakes create bi-national issues that have been subject of investigation since the 1970s. However, distinguishing upgradient sources of nutrients, metals and legacy contaminants in rivers remains a challenge, as they are derived from multiple sources and flows typically vary throughout the region. These complications are especially pertinent in the Lake Huron to Lake Erie corridor and Detroit River. The Detroit River supplies 90% of the water to the western basin of Lake Erie (5300 m3/s) and is subject to a variety of co-occurring potential sources (e.g., agriculture, urbanization, and upgradient water bodies) of water quality indicators that limit source disaggregation. To find the source signal in the noise we used an integrative interpretation of dissolved chemical and isotopic parameters with sediment chemical, isotopic, and contaminant indicators. The approach combines archival data to distinguish point and non-point sources, and upgradient water bodies as sources of nutrients, metals and contaminants to the Detroit River and ultimately the western basin of Lake Erie. Persistent organic pollutants and metals cluster together as an urban group. Regional dissolved orthro-phosphate (PO4) in the water column also groups with urban point sources rather than agricultural sources. Urbanization as the primary source of PO4 in the Detroit River highlights the need for continued research on urban impacts and assessments of broader best management practices protecting Lake Erie.

Increased nutrient concentrations in Lake Erie tributaries influenced by greenhouse agriculture.

Greenhouse production of vegetables is a growing global trade. While greenhouses are typically captured under regulations aimed at farmland, they may also function as a point source of effluent. In this study, the cumulative impacts greenhouse effluents have on riverine macronutrient and trace metal concentrations were examined. Water samples were collected Bi-weekly for five years from 14 rivers in agriculturally dominated watersheds in southwestern Ontario. Nine of the watersheds contained greenhouses with their boundaries. Greenhouse influenced rivers had significantly higher concentrations of macronutrients (nitrogen, phosphorus, and potassium) and trace metals (copper, molybdenum, and zinc). Concentrations within greenhouse influenced rivers appeared to decrease over the 5-year study while concentrations within non-greenhouse influenced river remained constant. The different temporal pattern between river types was attributed to increased precipitation during the study period. Increases in precipitation diluted concentrations in greenhouse influenced rivers; however, non-influenced river runoff proportionally increased nutrient mobility and flow, stabilizing the observed concentrations of non-point sources. Understanding the dynamic nature of environmental releases of point and non-point sources of nutrients and trace metals in mixed agricultural systems using riverine water chemistry is complicated by changes in climatic conditions, highlighting the need for long-term monitoring of nutrients, river flows and weather data in assessing these agricultural sectors.

Determining Carbon Kinetic Isotope Effects Using Headspace Analysis of Evolved CO2.

Isotope ratio mass spectrometry (IRMS) provides accurate measurements of relative abundance of isotopes of heavy atoms for reactions that are subject to kinetic isotope effects (KIEs). The recent development of compound-specific isotope analysis (CSIA) allows the use of multiple time points that provide data for a rate plot as well as isotope ratios. Utilizing CSIA in enzymology presents opportunities for obtaining heavy atom KIEs in diverse areas.

Fate and Transport of Shale-derived, Biogenic Methane.

Natural gas extraction from unconventional shale gas reservoirs is the subject of considerable public debate, with a key concern being the impact of leaking fugitive natural gases on shallow potable groundwater resources. Baseline data regarding the distribution, fate, and transport of these gases and their isotopes through natural formations prior to development are lacking. Here, we define the migration and fate of CH4 and δ13C-CH4 from an early-generation bacterial gas play in the Cretaceous of the Williston Basin, Canada to the water table. Our results show the CH4 is generated at depth and diffuses as a conservative species through the overlying shale. We also show that the diffusive fractionation of δ13C-CH4 (following glaciation) can complicate fugitive gas interpretations. The sensitivity of the δ13C-CH4 profile to glacial timing suggests it may be a valuable tracer for characterizing the timing of geologic changes that control transport of CH4 (and other solutes) and distinguishing between CH4 that rapidly migrates upward through a well annulus or other conduit and CH4 that diffuses upwards naturally. Results of this study were used to provide recommendations for designing baseline investigations.

Branched pathways in the degradation of cDCE by cytochrome P450 in Polaromonas sp. JS666.

Compound specific isotope analysis (CSIA) is widely used to monitor contaminant remediation in groundwater. CSIA-based approaches that use enrichment (ε) values to assess degradative processes rely on the assumption that the contaminant being investigated will have an ε value that is constant and specific to a catalytic pathway of a microorganism. Distinct ε values have been reported for aerobic degradation of cis-dichloroethene (cDCE), which has led to a number of proposed degradation mechanisms; however, cytochrome P450 catalyzed oxidation is the only biochemical mechanism that has been established in Polaromonas sp. JS666. Using CSIA we measured the ε values for microbial oxidation of cDCE (-18.8‰±1.5‰) and 1,2-dichloroethane (1,2-DCA) (-16.6‰±0.9‰) in wild-type JS666 and the oxidation of cDCE (-13.5‰±2.3‰) from a recombinant E. coli strain expressing the cytochrome P450 enzyme from JS666. This study supports the hypothesis that cytochrome P450 catalyzes the initial step in the degradation pathway of both cDCE and 1,2-DCA and provides evidence that a single enzyme can catalyze multiple pathways with different products and distinct ε values for a single substrate. Therefore, in cases where the products of the reaction cannot, or have not been characterized, caution must be used when employing ε values to interpret mechanisms, pathways, and their applications to environmental contaminant remediation.

Measuring Concentrations of Dissolved Methane and Ethane and the 13 C of Methane in Shale and Till.

Baseline characterization of concentrations and isotopic values of dissolved natural gases is needed to identify contamination caused by the leakage of fugitive gases from oil and gas activities. Methods to collect and analyze baseline concentration-depth profiles of dissolved CH4 and C2 H6 and δ13 C-CH4 in shales and Quaternary clayey tills were assessed at two sites in the Williston Basin, Canada. Core and cuttings samples were stored in Isojars® in a low O2 headspace prior to analysis. Measurements and multiphase diffusion modeling show that the gas concentrations in core samples yield well-defined and reproducible depth profiles after 31-d equilibration. No measurable oxidative loss or production during core sample storage was observed. Concentrations from cuttings and mud gas logging (including IsoTubes® ) were much lower than from cores, but correlated well. Simulations suggest the lower concentrations from cuttings can be attributed to drilling time, and therefore their use to define gas concentration profiles may have inherent limitations. Calculations based on mud gas logging show the method can provide estimates of core concentrations if operational parameters for the mud gas capture cylinder are quantified. The δ13 C-CH4 measured from mud gas, IsoTubes® , cuttings, and core samples are consistent, exhibiting slight variations that should not alter the implications of the results in identifying the sources of the gases. This study shows core and mud gas techniques and, to a lesser extent, cuttings, can generate high-resolution depth profiles of dissolved hydrocarbon gas concentrations and their isotopes.

Diffusion sampler for compound specific carbon isotope analysis of dissolved hydrocarbon contaminants.

Compound Specific Isotope Analysis (CSIA) is widely utilized to study the fate of organic contaminants in groundwater. To date, however, no method is available to obtain CSIA samples at a fine (cm) spatial scale across the sediment-surface water interface (SWI), a key boundary for discharge of contaminated groundwater to surface water. Dissolved contaminants in such discharged zones undergo rapid temporal and spatial changes due to heterogeneity in redox conditions and microbial populations. The compatibility of a passive sediment pore water sampler ("peeper") to collect 40 mL samples for CSIA of benzene, toluene, monochlorobenzene, and 1,2-dichlorobenzene at field-relevant concentrations (0.1-5 mg L(-1)) was evaluated in laboratory experiments. Results demonstrate that physical diffusion across the polysulfone membrane does not alter the carbon isotope values (±0.5‰). Measured δ(13)C values also remain invariant despite significant adsorption of the compounds on the peeper material, an effect which increased with higher numbers of chlorine atoms and sorption coefficient (Koc) values. In addition, isotope equilibrium between the peeper chamber and the sediment pore water occurred in less than a day, indicating the peeper method can be used to provide samples for CSIA analysis at fine spatial and temporal sampling resolutions in contaminated sediments.

Distinct carbon isotope fractionation during anaerobic degradation of dichlorobenzene isomers.

Chlorinated benzenes are ubiquitous organic contaminants found in groundwater and soils. Compound specific isotope analysis (CSIA) has been increasingly used to assess natural attenuation of chlorinated contaminants, in which anaerobic reductive dechlorination plays an essential role. In this work, carbon isotope fractionation of the three dichlorobenzene (DCB) isomers was investigated during anaerobic reductive dehalogenation in methanogenic laboratory microcosms. Large isotope fractionation of 1,3-DCB and 1,4-DCB was observed while only a small isotope effect occurred for 1,2-DCB. Bulk enrichment factors (εbulk) were determined from a Rayleigh model: -0.8 ± 0.1 ‰ for 1,2-DCB, -5.4 ± 0.4 ‰ for 1,3-DCB, and -6.3 ± 0.2 ‰ for 1,4-DCB. εbulk values were converted to apparent kinetic isotope effects for carbon (AKIE) in order to characterize the carbon isotope effect at the reactive positions for the DCB isomers. AKIE values are 1.005 ± 0.001, 1.034 ± 0.003, and 1.039 ± 0.001 for 1,2-DCB, 1,3-DCB, and 1,4-DCB, respectively. The large difference in AKIE values between 1,2-DCB and 1,3-DCB (or 1,4-DCB) suggests distinct reaction pathways may be involved for different DCB isomers during microbial reductive dechlorination by the methanogenic cultures.

Carbon kinetic isotope effects reveal variations in reactivity of intermediates in the formation of protonated carbonic acid.

Kinetic evidence suggests that acid-catalyzed decarboxylation reactions of aromatic carboxylic acids can occur by a hydrolytic process that generates protonated carbonic acid (PCA) as the precursor of CO2. Measurements of reaction rates and carbon kinetic isotope effects (CKIE) for decarboxylation of isomeric sets of heterocyclic carboxylic acids in acidic solutions reveal that C-C cleavage to form PCA is rate-determining with significant variation in the magnitude of the observed CKIE (1.018-1.043). Larger values are associated with the more reactive member in each isomeric pair. This variation is consistent with stepwise mechanisms in which C-C cleavage is competitive with C-O cleavage, leading to reversion to the protonated reactant to varying degrees with an invariant intrinsic CKIE for C-C cleavage. Thus, the relative barriers to reversion and formation of PCA control the magnitude of the observed CKIE in a predictable manner that correlates with reactivity. Application of the proposed overall mechanism reveals that carboxylation reactions in acidic solutions will proceed by way of initial formation of PCA.

Pressure-monitored headspace analysis combined with compound-specific isotope analysis to measure isotope fractionation in gas-producing reactions.

RATIONALE: Processes that lead to pressure changes in closed experimental systems can dramatically increase the total uncertainty in enrichment factors (ε) based on headspace analysis and compound-specific isotope analysis (CSIA). We report: (1) A new technique to determine ε values for non-isobaric processes, and (2) a general approach to evaluate the experimental error in calculated ε values. METHODS: ε values were determined by monitoring the change in headspace pressure from the production of CO2 in a decarboxylation reaction using a pressure gauge and measuring the δ(13) C values using CSIA. The statistical error was assessed over shorter reaction progress intervals to evaluate the impact of experimental error on the total uncertainty associated with calculated ε values. RESULTS: As an alternative to conventional compositional analysis, calculation of CO2 produced during the reaction monitored with a pressure gauge resulted in rate constants and ε values with improved correlation coefficients and confidence intervals for a non-isobaric process in a closed system. Further, statistical evaluation of the ε values as a function of reaction progress showed that uncertainty in data points for reaction progress (f) at late stages of the reaction can have a significant impact on the reported ε value. CONCLUSIONS: Pressure-monitored headspace analysis reduces the uncertainty associated with monitoring the reaction progress (f) based on estimating substrate removal and headspace dilution during sampling. Statistical calculations over shorter intervals should be used to evaluate the total error for reported ε values.

Large carbon isotope fractionation during biodegradation of chloroform by Dehalobacter cultures.

Compound specific isotope analysis (CSIA) has been applied to monitor bioremediation of groundwater contaminants and provide insight into mechanisms of transformation of chlorinated ethanes. To date there is little information on its applicability for chlorinated methanes. Moreover, published enrichment factors (ε) observed during the biotic and abiotic degradation of chlorinated alkanes, such as carbon tetrachloride (CT); 1,1,1-trichloroethane (1,1,1-TCA); and 1,1-dichloroethane (1,1-DCA), range from -26.5‰ to -1.8‰ and illustrate a system where similar C-Cl bonds are cleaved but significantly different isotope enrichment factors are observed. In the current study, biotic degradation of chloroform (CF) to dichloromethane (DCM) was carried out by the Dehalobacter containing culture DHB-CF/MEL also shown to degrade 1,1,1-TCA and 1,1-DCA. The carbon isotope enrichment factor (ε) measured during biodegradation of CF was -27.5‰ ± 0.9‰, consistent with the theoretical maximum kinetic isotope effect for C-Cl bond cleavage. Unlike 1,1,1-TCA and 1,1-DCA, reductive dechlorination of CF by the Dehalobacter-containing culture shows no evidence of suppression of the intrinsic maximum kinetic isotope effect. Such a large fractionation effect, comparable to those published for cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) suggests CSIA has significant potential to identify and monitor biodegradation of CF, as well as important implications for recent efforts to fingerprint natural versus anthropogenic sources of CF in soils and groundwater.

Protonated carbonic acid and reactive intermediates in the acidic decarboxylation of indolecarboxylic acids.

Elucidation of the mechanism for decarboxylation of indolecarboxylic acids over a wide range of solution acidity reveals the importance of protonated carbonic acid (PCA) as a reaction intermediate. In concentrated acid, the initial addition of water to the carboxyl group of the indolecarboxylic acid leads to a hydrated species that is capable of releasing PCA upon rate-determining carbon-carbon bond cleavage. The overall process is catalytic in water and acid, implicating PCA as a potential carboxylating reagent in the microscopic reverse reaction.

Monitoring biodegradation of ethene and bioremediation of chlorinated ethenes at a contaminated site using compound-specific isotope analysis (CSIA).

Chlorinated ethenes are commonly found in contaminated groundwater. Remediation strategies focus on transformation processes that will ultimately lead to nontoxic products. A major concern with these strategies is the possibility of incomplete dechlorination and accumulation of toxic daughter products (cis-1,2-dichloroethene (cDCE), vinyl chloride (VC)). Ethene mass balance can be used as a direct indicator to assess the effectiveness of dechlorination. However, the microbial processes that affect ethene are not well characterized and poor mass balance may reflect biotransformation of ethene rather than incomplete dechlorination. Microbial degradation of ethene is commonly observed in aerobic systems but fewer cases have been reported in anaerobic systems. Limited information is available on the isotope enrichment factors associated with these processes. Using compound-specific isotope analysis (CSIA) we determined the enrichment factors associated with microbial degradation of ethene in anaerobic microcosms (ε = -6.7‰ ± 0.4‰, and -4.0‰ ± 0.8‰) from cultures collected from the Twin Lakes wetland area at the Savannah River site in Georgia (United States), and in aerobic microcosms (ε = -3.0‰ ± 0.3‰) from Mycobacterium sp. strain JS60. Under anaerobic and aerobic conditions, CSIA can be used to determine whether biotransformation of ethene is occurring in addition to biodegradation of the chlorinated ethenes. Using δ(13)C values determined for ethene and for chlorinated ethenes at a contaminated field site undergoing bioremediation, this study demonstrates how CSIA of ethene can be used to reduce uncertainty and risk at a site by distinguishing between actual mass balance deficits during reductive dechlorination and apparent lack of mass balance that is related to biotransformation of ethene.