Progress 05/15/19 to 05/14/23
Outputs
Target Audience:During the duration of the project, data evaluating the partitioning behavior of wastewater contaminants As, Hg, Se, and Cd in microalgae aquaculture was published in the peer-reviewed journal "Environmental Pollution". This data is now available to academic researchers, agriculturalists, and industrial practitioners to inform sustainable microalgae aquaculture practices. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has supported the training of one research faculty, two graduate students (one completed MS, one ongoing PhD), and two undergraduate students on various analytical methods for quantifying mercury and other metals, algae cultivation and harvesting, data analysis, and data dissemination via preparation of professional publications. How have the results been disseminated to communities of interest?To date results have been disseminated via 1 MS Thesis, 1 international technical conference, and 1 research article. In addition, this work (detailed in the achievements section of this report) will be included in the doctoral thesis of the PhD student. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Arthrospira maxima can yield 20-400 times more protein than sources such as corn, soybean and beef, drawing interest from the agricultural sector an alternative to traditional crops for sustainable protein production. Wastewater has been suggested as a medium for microalgae cultivation because of the reduced requirement for freshwater and the range of species that can be cultivated. However, a large body of literature investigating the use of algae for wastewater remediation reflects the capacity for microalgae to sorb or bioaccumulate contaminants. The degree or conditions under which A. maxima and Chlamydomonas reinhardtii may become potential vectors of contaminant transport from wastewater to consumers or workers as an agricultural crop is not well understood. Additionally, the potential for microalgae to volatilize toxic elements has not been previously investigated in the agriculturally-relevant strain A. maxima, although biotransformation and volatilization of toxic organic forms of mercury (Hg), arsenic (As), and selenium (Se) by other microorganisms has been observed. Consumption of wastewater contaminants Hg, As, Se, and Cd are associated with severe health effects, and are of particular concern. The overarching goal of this work was to investigate the partitioning of these elements between liquid, biomass and gas phases in A. maxima and C. reinhardtii cultures to provide evidence for health risks associated with cultivation of microalgae as a crop in wastewater on an industrial scale. To meet this goal, one agriculturally-relevant microalgae strain and one microalgae strain capable of producing dimethlyselenide were cultured in the presence of Hg, As, Se, and/or Cd. Each algae strain was grown in the presence of a single element (Hg, As, Se, or Cd) and in a mixture of all four elements (combination-element experiments), and the partitioning behavior of each element at the near the end of the algae's exponential growth was evaluated in the gas, biomass, and liquid phases. Microalgae were grown in sealed microcosms and microcosm headspace was purged through concentrated nitric acid traps to capture volatilized contaminants for analysis. Total recovery was confirmed by mass balance. In single-element C. reinhardtii experiments, an average of 55 ± 8% of added Hg was recovered in biomass while 34 ± 11% was recovered in the liquid phase (Fig. 1A). 27 ± 6% of the As mass balance was recovered in biomass and 71 ± 4% in the liquid (Fig. 2A) while only 3 ± 0% and 83 ± 1% of the Se was observed in biomass and liquid respectively (Fig. 3A). Cd was recovered almost entirely in the liquid phase (Fig. 4A), with a small recovery in the biomass phase. In combination-element C. reinhardtii microcosms, statistical differences in the % element recovered in biomass were apparent for Hg and Se when compared to biomass recoveries in Hg-only and Se-only microcosms. This difference was prominent for Hg; 85 ± 11% was recovered in combination-element experiments vs. 55 ± 8% in single-element experiments, with corresponding changes in the liquid phase (34 ± 11% in single-element microcosms to 8 ± 3% in combination-element microcosms). This indicates a meaningful shift in Hg partitioning from the liquid to biomass phases in the presence of other elements. Minimal Hg was recovered in the gas phase of biotic and abiotic microcosms. As and Se were not detected in the gas phase. However, As was detected above the MDL (0.005 ppb) in the gas phase of both biotic and abiotic microcosms. 48 ± 2% of the Hg in single-element microcosms were recovered in A. maxima biomass and 16 ± 1% in the liquid (Fig. 2B). No As or Se was observed in the A. maxima biomass of single-element microcosms, and mass balance recoveries in the liquid phase were 92 ± 2% of As, 90 ± 13% of Se, respectively, although Se was likely under detected due to method limitations. 26 ± 8% of the Cd mass balance was observed in the biomass of single-element experiments with 71 ± 5% observed in the liquid phase (Fig. 4B). In A. maxima, less Cd was recovered in the biomass phase of combination-element microcosms (13 ± 4%) compared to single-element microcosms (26 ± 8%). A small amount of Hg was consistently recovered in the gas phase of A. maxima microcosms, which was determined to be due to abiotic volatilization, and thus did not include MeHg. While wastewaters can vary widely in contaminant concentration, these results demonstrate it is possible for sizable concentrations of these elements to be associated with C. reinhardtii and A. maxima biomass under the experimental growth conditions of this study. Thus, consumption of microalgae biomass as a protein source may lead to contaminant exposure if wastewaters containing Hg, As, Se, and Cd are used in growth medium. Differences in medium composition with corresponding differences in As and Se partitioning between the cultured strains detailed in this study suggest it may be possible to mitigate As and Se toxicity risks to consumers through manipulation of phosphate and sulfate concentrations in growth medium. Wastewater aquaculture remains a viable option for sustainable food production, but results presented in this work suggest that wastewaters containing Hg, As, Se, and/or Cd should be used cautiously, as consumption of biomass is a potential route of exposure. Hg was evident as the element of greatest concern under these culturing conditions given the large portion associated with A. maxima and C. reinhardtii biomass. This study supports A. maxima as a preferable microalgae strain for wastewater aquaculture.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lown, L., Vernaz, J. E., Dunham-Cheatham, S. M., Gustin, M. S., & Hiibel, S. R. (2023). Phase partitioning of mercury, arsenic, selenium, and cadmium in Chlamydomonas reinhardtii and Arthrospira maxima microcosms. Environmental Pollution, 329, 121679.
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Progress 05/15/21 to 05/14/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:No major changes occurred in the reporting period. The use of Chlamydomonas reinhardtii as the second algae species evaluated let to several minor changes in our experimental setup, primarily associated with maintaining culture sterility over the extended run times of the experiments. Because the Chlamydomonas media is at a neutral pH (not buffered at an elevated pH like the Spirulina maxima media), opportunistic bacteria and/or yeast that were able to contaminate the flasks were able to survive and grow during the experiments. Although every effort was taken to use aeseptic technique and maintain sterility to the best of our ability, ultimately the need for aeration and sampling prevents the microcosms from being sealed during the experiment. Through improvements in experimental setup (moved completely to a clean bench) and sampling protocols (reduced frequency with positive pressure maintained), the contamination issues have been minimized, and the microcosm experiments with C. reinhardtii are expected to be completed in early Fall 2022. The challeges described in the previous report and this one regarding the experimental setup, as well as the slowed rate of progress during the height of the COVID pandemic, have resulted in request for a no-cost extension to complete the project. The NCE has been granted and will extend the project through May 2023, which will result in an additional annual report (this report) and the final project report being submitted in 2023. What opportunities for training and professional development has the project provided?The project has supported the training of one research faculty, two graduate students (one completed MS, one ongoing PhD), and two undergraduate students on various analytical methods for mercury and other metal quantification, algae cultivation and harvesting, data analysis, and data dissemination via preparation of professional publications. How have the results been disseminated to communities of interest?To date results have been disseminated via 1 MS Thesis and 1 international technical conference. In addition, a peer-reviewed manuscript capturing the results from both algae strains is in preparation. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the microcosm experiments for individual metal(oid)s and a combination of all four will be completed with Chlamydomonas reinhardtii. We expect that the remaining PhD student will complete the experimental work with C. reinhardtii and will finish the peer-reviewed manuscript detailing the results from both algae species. She may also pursue a "methods" paper detailing the setup, sampling, and analysis for the microcosm systems developed in the project.
Impacts
What was accomplished under these goals?
The microcosm experiments for Spirulina maxima grown in the presence of all four metal(oid)s - As, Cd, Hg, and Se - individually and combined were completed. Highlights of these experimental results are provided below. It was found that the difference in biomass recovery between the Hg-only test and combined metal(loid) tests was not statistically significant (p = 0.275), indicating that the presence of Cd, Se, and As in the concentrations tested did not affect the bioaccumulation of Hg in S. maxima. The majority of the initial Hg in experimental flasks was recovered in S. maxima biomass in the both Hg-only test and combined metal(loid) tests (Figure 1). Insignificantly different (p > 0.05) amounts of Hg were recovered in the liquid phase of the experimental flasks of the Hg-only test and combined metal(loid) tests, indicating that the uptake of liquid-phase Hg was not affected by the presence of other metal(loid)s in the growth medium. The presence of growing S. maxima significantly (p < 0.01) decreased the amount of Hg recovered from the HNO3 rinse of flasks in the Hg-only test and combined metal(loid) tests, suggesting that Hg preferentially sorbs to S. maxima over glass. Moreover, the simultaneous presence of Cd, Se, and As in the combined metal(loid) tests significantly (p = 0.001) increased the Hg recovered from the HNO3 rinse of flasks compared to the Hg-only test. Figure 1: Hg phase partitioning in Hg-only (individual) and combined metal(loid) tests in the presence of growing S. maxima. Recoveries are reported as percentages of the initial Hg mass. Bars represent average recovery of replicate (n = β (2), γ (3), δ (4), or ε (6)), and symbols represent calculated recovery from individual samples. A relatively greater fraction of Cd was found in S. maxima biomass in the Cd-only test compared to combined metal(loid) tests (Figure 2), but the difference was not statistically significant (p = 0.274). In the Cd-only and combined metal(loid) tests, the majority of the recovered Cd was found in the liquid phase of the treated flasks and the liquid phase of the abiotic control flasks. In the Cd-only test, a significant difference (p = 0.006) in liquid-phase recovery was observed when comparing experimental flasks to abiotic control flasks, but the difference may be attributed to Cd sorption to biomass, as the sum of Cd recovered in the liquid and biomass phases of experimental flasks was not significantly different (p = 0.293) than the Cd recovered in the liquid of abiotic control flasks. In the combined metal(loid) test, a significant difference (p = 0.034) in recovered Cd in the liquid phase was observed between experimental and abiotic control flasks that was not similarly explained by sorption or uptake. Thus, the presence of growing S. maxima affected the partitioning of Cd into the liquid phase when other metal(loid)s were present. A significantly different (p = 0.036) fraction of Cd was recovered in the liquid phase between experimental flasks of the Cd-only and the combined metal(loid) tests. Trace fractions of Cd were recovered in the gas phase, though this was possibly due to interferences caused by the condensation of effluent vapor from the flasks, as the HNO3 trap volume was observed to increase during bubbling. Figure 2: Cd phase partitioning in Cd-only (individual) and combined metal(loid) tests in the presence of growing S. maxima. Recoveries are reported as percentages of the initial Cd mass. Bars represent average recovery of replicate (n = β (2), δ (4), or ε (6)), and symbols represent calculated recovery from individual samples. Se was not recovered in S. maxima biomass or the gaseous effluent of the Se-only or combined metal(loid) tests (Figure 3). Se was only recovered in the liquid phase of all tests. Fractions of Se recovered from the HNO3 rinse of flasks were not significantly different (p > 0.05). The absence of Se bioaccumulation and potential volatilization may be attributed to the sulfate concentration in the growth medium. The decrease of selenate accumulation in microalgae by ambient sulfate concentrations has been documented in the literature. Total recoveries met the pre-defined 90% limit for a successful mass balance experiment. The discrepancy between reported recoveries and 100% recovery was possibly due to the proximity of Se concentrations of the samples to the limit of detection. Though Se was not recovered in biomass or gaseous effluent, Se may have been recovered if analysis was unobscured by a lower limit of detection relative to sample concentrations. Figure 3: Se phase partitioning in Se-only (individual) and combined metal(loid) tests in the presence of growing S. maxima. Recoveries are reported as percentages of the initial Se mass. Bars represent average recovery of replicate (n = β (2), γ (3), δ (4), or ε (6)), and symbols represent calculated recovery from individual samples. Se was not recovered in the biomass or gas phases. Bioaccumulation accounted for small fractions of As recovery in the As-only and the combined metal(loid) tests, but the difference was not statistically significant (p = 0.276) (Figure 4). Arsenic was primarily recovered in the liquid phase, and recovery from the HNO3 rinses of flasks did not differ significantly (p > 0.05). Analysis of the HNO3 traps in the As-only and the combined metal(loid) tests indicated that As was not volatilized at a detectable level from any of the flasks. Arsenate accumulation in microalgae has been reported in the literature to be inhibited in phosphate-enriched media, thus, the absence of detectable As bioaccumulation in the present study may be due to the phosphate concentration of the growth medium (275 mg L-1). Figure 4: Arsenic phase partitioning in As-only (individual) and combined metal(loid) tests in the presence of growing S. maxima. Recoveries are reported as percentages of the initial As mass. Bars represent average recovery of replicate (n = α (1), β (2), δ (4), or ε (6)), and symbols represent calculated recovery from individual samples. Arsenic was recovered in the biomass in trace amounts, and no As was recovered in the HNO3 traps. In addition to the work efforts with S. maxima, the C. reinhardtii microcosm experiments have also been initiated. Unlike the Spirulina media, which is buffered at an elevated pH, the Chlamydomonas media is neutral, and as a result more prone to bacterial and/or yeast contamination during the microcosm experiments. After several C. reinhardtii tests resulted in contaminated cultures, the experimental setup and sampling protocols were altered. All phases of the experimental setup were moved to a clean bench (recently acquired as part of a separate project), microcosm sampling frequency was reduced, and ultimately a new mother culture of C. reinhardtii was obtained in an effort to prevent contamination in the microcosm experiments. Gram staining and visual observation under a light microscope of the final C. reinhardtii cultures are used to confirm that contamination has been minimized for each microcosm experiment using the improved setup/sampling protocol.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2022
Citation:
Vernaz, Joshua. "Fate and Transport of Mercury, Cadmium, Selenium, and Arsenic in the Presence of Growing Spirulina maxima". MS Thesis. Chemical Engineering. University of Nevada, Reno. May, 2022.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Lown L, Vernaz J, Dunham-Cheatham S, Hiibel SR, Gustin M. "Fate of Mercury, Cadmium, Selenium, and Arsenic in a Simulated Industrial Aquaculture Setting". International Conference of Mercury as a Global Pollutant. July 24-29, 2022.
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Progress 05/15/20 to 05/14/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Two minor changes to our experimental approach will be implemented, stemming directly from our work/results to date. Many analytical challenges were experienced and resolved with the S. maxima microcosms, and the majority of these challenges were a direct result of high levels of total dissolved solids present in the growth media due to the high pH requirements for growth of S. maxima. Because of these challenges, we plan to use the freshwater algae species Chlamydomonas reinhardtii, which does not require significant pH buffering, for the next set of microcosm testing. In addition, we plan to utilize a single combination of all 4 metal(loids) rather than a series of binary metal(loid) combinations to evaluate the impacts of the mixed metal(loids) on the algae. Toxicity tests with S. maxima demonstrated similar growth rates to the combined metals as to those observed with individual metals at equivalent levels, and similar tests will be performed with C. reinhardtii. What opportunities for training and professional development has the project provided?The project has supported the training of one post-doctoral researcher, two graduate students, and two undergraduate students on various analytical methods for mercury and other metal quantification, algae cultivation and harvesting, data analysis, and data dissemination via preparation of professional publications. How have the results been disseminated to communities of interest?No results have been disseminated to communities of interest to date, however two peer-reviewed manuscripts are in preparation, along with plans to present the project results at a professional conference later this year. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, microcosm experiments for the Se and a combination of all four metal(loid)s being considered (Se, As, Cd, and Hg) will be conducted with S. maxima, along with microcosm experiments for the individual and combined metalloids for the second microalgae species, Chlamydomonas reinhardtii. The final microcosm and sampling system developed for S. maxima will be used with C. reinhardtii, with equivalent metal(loid) concentrations initially utilized provided the toxicity testing with the new algae species support those levels. We expect that the primary MS student working on the project will complete his thesis and graduate in December 2021, with the second graduate student completing the project as she continues her PhD efforts. The results of the Cd, Se, and As microcosm experiments for both algae species will be the primary focus of the MS student's thesis, and a peer-reviewed manuscript is expected from that work. The Hg and combined metal(loid) microcosm results will be a focus of the PhD student's dissertation, and a second peer-reviewed manuscript is expected from those efforts. In addition, we believe that a "methods" paper detailing the successful microcosm setup, sampling, and analysis will be of value to the scientific community, and as such we hope to publish that as a third manuscript from the project. As part of the graduate students' training, we also hope to send each of them to a professional conference where they can present their research results; however due to the ongoing COVID situation they may only be able to participate in a virtual conference.
Impacts
What was accomplished under these goals?
A reliable and reproducible experimental design for quantifying Hg, As, Cd and Se in liquid, biomass and gas phases following the growth of microalgae has been established (Figs. 1 and 2). Briefly, a microcosm experiment consists of five flasks dosed with either a single element (Hg, Cd, Se, As) or a combination of all four elements. Of these flasks, three are biological replicates and two are abiotic replicates. Two additional flasks containing algae but no elemental spikes and one flask with media only serve to control for algae growth and contamination monitoring. Flasks are inoculated, dosed, and sealed in a light- and temperature-controlled growth chamber and bubbled continuously with carbon-scrubbed ambient air for 14 days. The effluent from each flask enters two sequential 30-mL bubblers containing concentrated nitric acid to capture the gas phase metals. These gas phase samples are collected from the bubbler traps daily. Flasks are manually agitated and rotated within the growth chamber daily to ensure uniform exposure to light. To calculate the mass balance, initial concentrations of elements are determined directly from the microcosm after dosing. Following 14 days of growth, percent element recovery is calculated from the sum of the element recovered in media, biomass, glassware, and nitric traps. To date, an informative mass balance has been performed for Hg, As, and Cd with Spirulina maxima. A microcosm study with Se and replicate Cd are currently underway and analogous mass balance experiments in Chlorella vulgaris or Chlamydomonas reinhardtii are planned for completion later this year. Two manuscripts presenting findings and method development are in preparation. Considerable time was spent on method development. A number of liquid trap solutions containing potassium permanganate and sulfuric acid were initially evaluated to capture Hg with poor results. Concentrated nitric acid was established as an effective Hg trap solution and was tested for Hg retention over time with a satisfactory outcome. Capture of both elemental and oxidized Hg forms was confirmed in nitric acid. Additional experimental variables were refined to great effect, including manifold construction, spike concentration, ambient air flow rate, bubbler volume, and bubbler configuration. A successful mass balance was performed with Hg. In the gas phase, approximately 6% of the total Hg was captured in nitric traps under biotic conditions (Fig. 3, representative microcosm) and 4.5% under abiotic conditions. Cation exchange membranes (CEMs) were evaluated to distinguish reactive oxidized Hg (ROM) and elemental Hg, however the total mass captured over 14 days was negligible (<1%), thus the use of CEMs will not continued. The majority of Hg was associated with the biomass phase under biotic conditions (57%) or sorbed to the microcosm under abiotic conditions (77%). Hg contained in the media represented the second largest fraction in both biotic and abiotic flasks (Fig. 3). Figure 1: Schematic of the flask portion of the microcosm experimental apparatus. (A) secondary container (9"x13"x4" plastic bins used), (B) manifold tubing (1/4"ID PVC tubing), (C) PTFE tubing, (D) rotameter, (E) silicone connector, (F) glass straw (inlet straw reaches bottom of flask, outlet only reaches headspace), (G) stopper, (H) zip ties (two to form a collar around flask, three to form a chain that connects the collar between straws), (I) 2L flask, (J) 600mL media with or without algae. Figure 2: Cation exchange membrane (CEM) and nitric trap configuration. CEMs are excluded in single element experiments with As, Cd, and Se. (A) Flask, (B) Silicone stopper, (C) Inlet and outlet glass straws, (D) Silicone connector, (E) PTFE tubing, (F) Nitric traps, (G) Soda lime trap, (H) Outlet manifold tubing, (I) Acrylic support, (J) CEM filter housing (A) (B) Figure 3: Hg partitioning in biotic (A) and abiotic (B) microcosm studies following 14 days of growth with S. maxima. A successful mass balance was performed with As. The experiment was configured similarly to the successful Hg mass balance, although without CEMs preceding the gas traps. In biotic treatments, 0.4% ± 0.2% (avg ± S.D.) of the recovered As was associated with biomass, 98.9% ± 0.4% of the recovered As was associated with liquid, and 0.7% ± 0.2% of the recovered As was sorbed to the microcosm. In abiotic treatments, 99.2% was recovered in the liquid phase and 0.8% was sorbed to the microcosm. Among As-dosed microcosms, no As was detected in select gas trap samples. For most flasks, the initial dose of arsenic was recovered, ± 10%. A successful mass balance was performed with Cd, though with a different gas collection configuration. In this configuration, the microcosm effluent entered a single bubbler containing 100 mL concentrated nitric acid. The contents of the bubbler were not sampled daily, and there was no bubbler downstream to capture breakthrough Cd. The bubbler was sampled after 14 days. In the biotic treatments, the liquid phase contained 79.4% ± 2.2% (avg ± S.D.) of the recovered Cd and the biomass contained 20.6% ± 2.2% (avg ± S.D.) of the recovered Cd. In the abiotic treatment, the liquid phase contained the entirety of the recovered Cd. In biotic and abiotic treatments, the gas phase analysis did not recover any Cd. This experiment is being replicated with the gas collection configuration described for As. Two additional toxicity tests with higher doses of each element were conducted. In the first test, 125mL flasks were filled with a 50mL suspension of S. maxima and dosed with 1 ppb, 10 ppb, 100 ppb, 300 ppb, 500 ppb, or 1000 ppb of each element. Concurrently, 2L flasks were monitored for growth in the growth chamber to compare growth between experimental settings. Absorbance at 600 nm and 680 nm were recorded daily. Maximum growth rate was determined by calculating the greatest slope from four consecutive days for each flask. Each treatment was conducted in triplicate. Flasks dosed to 100 ppb and greater died. The data from this toxicity test is reflected in Figure 4. Growth inhibition was not observed at 10 ppb compared to control at 600 nm (p=0.52) and 680 nm (p=0.44) absorbances. Maximum growth rates measured from were not significantly different between 2-L flasks placed in the growth chamber and 125mL flasks in the toxicity test at 600 nm (p=0.19) and 680 nm (p=0.23) absorbances. Figure 4: Maximum growth rates observed in toxicity tests of S. maxima dosed with equal concentrations of Cd, As, Se, and Hg. In the second toxicity test, 125mL flasks were filled with a 50 mL suspension of S. maxima and dosed with varied concentrations of each element. These concentrations were determined by estimating the dose of each element required to receive a signal above the ICP-MS detection limit after dilution. The "standard" treatment comprised 30 ppb Cd, 60 ppb As, 560 ppb Se, and 0.11 ppb Hg. Absorbance was measured t 600 nm and 680 nm daily. Toxicity was studied at 0.5X standard, 1X standard, and 2X standard concentrations, each with and without dosed Hg. Each treatment was conducted in triplicate. The data from this toxicity test is reflected in Figure 5. At standard concentration, maximum growth rates were not significantly different from control at 600 nm (p=0.74 Hg-treated, p=0.93 no Hg) and 680 nm (p=0.80 Hg-treated, p=0.93 no Hg) absorbances. P-values were slightly lower for double standard concentrations. Figure 5: Maximum growth rates observed in toxicity tests of S. maxima dosed with inequal concentrations Cd, As, Se, and Hg. The standard concentration was 30 ppb Cd, 60 ppb As, 560 ppb Se, and 0.11 ppb Hg.
Publications
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Progress 05/15/19 to 05/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Major changes/probelms that have occurred during this reporting period include a change in graduate student personnel and the COVID-19 disruption. Neither issue have changed the research approach at this point, however both resulted in a delay and significant slow down in research activity during the spring/summer of 2020. One MS student who was not supported by the project (supported by a graduate teachoing position) but was responsible for analytical testing and some method development decided in late December to leave the graduate program. As a result, the other MS student working on and supported by the project, as well as a post doc in the lab have had to expand their responsibilities and analytical capabilities. We have also hired a new graduate student that will start in August to help fill the role of the student who has left. Regarding the COVID-19 disruption, we are working closely with our campus officials to ensure the health and safety of all project personnel. Bench experiments were halted for a significant period of time, but have now resumed on a limited basis, per our campus and state guidelines. All team personnel are maximizing the physical time allowed in the labs, and are working remotely for all data analysis and other non-lab bench work. We continue to meet virtually on a regular basis to ensure that the project remains on task and schedule. What opportunities for training and professional development has the project provided?The project has supported the training of one post doctoral researcher, two graduate students, and three undergraduate students on various analytical methods for mercury and other metal quantification, algae cultivation and harvesting, and data analysis. How have the results been disseminated to communities of interest?No results have been disseminated to communities of interest to date, however one peer-reviewed manuscript is in preparation. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, microcosm experiments for individual metal(loids) and combinations of metal(loid)s will be conducted with S. maxima. Traps for methylated selenium species will also be developed; calcium sulfate is being explored as a potential trap material currently. A manuscript will be prepared for the results of S. maxima experiments. Initial experiments, such as metal(loid) stress tests, will be conducted for the next microalgae species. It is anticipated that the methods developed for the S. maxima microcosm experiments will not require operational modifications and will be able to be directly employed with the saltwater microalgae species planned later in the project.
Impacts
What was accomplished under these goals?
The freshwater microalgae Spirulina maxima have been grown successfully in controlled microcosms representative of natural and industrial settings. S. maxima were grown and maintained in UTEX Spirulina medium made with ACS-grade reagents to allow for careful control of mercury, cadmium, selenium, and arsenic concentrations. Metal(loid) concentrations were adjusted by addition of solutions of mercuric chloride, cadmium chloride, selenium selenate, and sodium arsenate dibasic heptahydrate in ultrapure water. Natural and industrial settings were replicated by the adjustment of these metal(loid) concentrations. Metal(loid) stress tests were conducted to determine the effect of these elements on the growth of S. maxima. Inhibition on maximum growth rate was not seen at the maximum concentration tested, 1000 ng metal(loid) / L. This concentration served as the maximum metal(loid) concentration for all experiments during this reporting period. Troubleshooting for S. maxima microcosm experiments is ongoing, specifically the metal(loid) capture in the gaseous effluent of the microcosms. The proposed methods for capture and quantification have been adapted prevent pressure drop associated with downstream traps from negatively impacting the microcosms. For mercury species, Tenax traps were initially proposed to speciate the mercury volatilized from microcosms. S. maxima was found to produce methylmercury in insignificant amounts and elemental mercury was the most prevalent mercury species sorbed to Tenax traps, thus the Tenax was not required as a mercury sorbent material. Gold-coated quartz traps, the replacement for Tenax traps intended to sorb all mercury species, were excluded from experiments due to inconsistencies in thermal desorption. Iodated activated carbon was found to be a suitable mercury sorbent material. Mercury analysis of these traps will be conducted in a direct mercury analyzer (Milestone DMA-80). Breakthrough curve experiments have been conducted to determine trap deployment times for iodated activated carbon traps.
Publications
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