pH and thiosulfate dependent microbial sulfur oxidation strategies across diverse environments.

Sulfur oxidizing bacteria (SOB) play a key role in sulfur cycling in mine tailings impoundment (TI) waters, where sulfur concentrations are typically high. However, our understanding of SOB sulfur cycling via potential S oxidation pathways (sox, rdsr, and S4I) in these globally ubiquitous contexts, remains limited. Here, we identified TI water column SOB community composition, metagenomics derived metabolic repertoires, physicochemistry, and aqueous sulfur concentration and speciation in four Canadian base metal mine, circumneutral-alkaline TIs over four years (2016 - 2019). Identification and examination of genomes from nine SOB genera occurring in these TI waters revealed two pH partitioned, metabolically distinct groups, which differentially influenced acid generation and sulfur speciation. Complete sox (csox) dominant SOB (e.g., Halothiobacillus spp., Thiomonas spp.) drove acidity generation and S2O3 2- consumption via the csox pathway at lower pH (pH ~5 to ~6.5). At circumneutral pH conditions (pH ~6.5 to ~8.5), the presence of non-csox dominant SOB (hosting the incomplete sox, rdsr, and/or other S oxidation reactions; e.g. Thiobacillus spp., Sulfuriferula spp.) were associated with higher [S2O3 2-] and limited acidity generation. The S4I pathway part 1 (tsdA; S2O3 2- to S4O6 2-), was not constrained by pH, while S4I pathway part 2 (S4O6 2- disproportionation via tetH) was limited to Thiobacillus spp. and thus circumneutral pH values. Comparative analysis of low, natural (e.g., hydrothermal vents and sulfur hot springs) and high (e.g., Zn, Cu, Pb/Zn, and Ni tailings) sulfur systems literature data with these TI results, reveals a distinct TI SOB mining microbiome, characterized by elevated abundances of csox dominant SOB, likely sustained by continuous replenishment of sulfur species through tailings or mining impacted water additions. Our results indicate that under the primarily oxic conditions in these systems, S2O3 2- availability plays a key role in determining the dominant sulfur oxidation pathways and associated geochemical and physicochemical outcomes, highlighting the potential for biological management of mining impacted waters via pH and [S2O3 2-] manipulation.

The Influence of Micronutrient Trace Metals on Microcystis aeruginosa Growth and Toxin Production.

Microcystis aeruginosa is a widespread cyanobacteria capable of producing hepatotoxic microcystins. Understanding the environmental factors that influence its growth and toxin production is essential to managing the negative effects on freshwater systems. Some micronutrients are important cofactors in cyanobacterial proteins and can influence cyanobacterial growth when availability is limited. However, micronutrient requirements are often species specific, and can be influenced by substitution between metals or by luxury uptake. In this study, M. aeruginosa was grown in modified growth media that individually excluded some micronutrients (cobalt, copper, iron, manganese, molybdenum) to assess the effect on growth, toxin production, cell morphology and iron accumulation. M. aeruginosa growth was limited when iron, cobalt and manganese were excluded from the growth media, whereas the exclusion of copper and molybdenum had no effect on growth. Intracellular microcystin-LR concentrations were variable and were at times elevated in treatments undergoing growth limitation by cobalt. Intracellular iron was notably higher in treatments grown in cobalt-deplete media compared to other treatments possibly due to inhibition or competition for transporters, or due to irons role in detoxifying reactive oxygen species (ROS).

Severe cyanobacterial blooms in an Australian lake; causes and factors controlling succession patterns.

Cyanobacterial blooms have major impacts on the ecological integrity and anthropogenic value of freshwater systems. Chrysosporum ovalisporum, a potentially toxic cyanobacteria has been rare in Australian waters until recently when is has bloomed in a number of lake and river systems. The aim of this study was to determine drivers of its growth and growing dominance. We performed regular monitoring of Mannus Lake, a small freshwater reservoir in South-Eastern Australia that has recently undergone extremely dense bloom events. Blooms of the diazotrophic Chrysosporum ovalisporum occurred in both summers of the 19 month study during periods of persistent thermal stratification. Following the C. ovalisporum blooms, non-diazotrophic taxa (Microcystis aeruginosa and Woronichinia sp.) dominated the phytoplankton community under less stratified conditions. Thermal stratification and nitrogen availability appeared to be the primary drivers of changes in cyanobacterial community structure. We propose that the observed transition from C. ovalisporum to M. aeruginosa and/or Woronichinia sp. may be a result of nitrogen limitation in early summer, which combined with persistent thermal stratification led to an ecological advantage for the nitrogen-fixing C. ovalisporum. Mixing events caused the senescence of the C. ovalisporum bloom, likely supplementing the nutrient budget of the lake with atmospherically derived N and alleviating N limitation to non-diazotrophic taxa. Non-diazotrophic cyanobacterial growth then increased, albeit at much lower biovolumes compared to the initial bloom. Overall, the results demonstrate the role of thermal stratification and nutrient cycling in structuring the cyanobacterial community and provide insights into the environmental factors driving the proliferation of the relatively new, potentially toxic cyanobacterium C. ovalisporum in Australian waters.

Sorption, degradation and microbial toxicity of chemicals associated with hydraulic fracturing fluid and produced water in soils.

Spills of hydraulic fracturing (HF) fluids and of produced water during unconventional gas extraction operations may cause soil contamination. We studied the degradation and microbial toxicity of selected HF chemical components including two biocides (methylisothiozolinone- MIT, chloromethylisothiozolinone- CMIT), a gel-breaker aid (triethanolamine -TEA), and three geogenic chemicals (phenol, m-cresol and p-cresol) in ultrapure water, HF fluid and produced water in five different soil types (surface and subsurface soils). The degradation of the two biocides (in soils treated with HF fluid or ultrapure water) and of the three geogenic chemicals (in soils treated with produced water) was rapid (in all cases DT50 values < 2 days in surface soils). In contrast, the loss of TEA was much slower in soils, especially in those treated with HF fluid (DT50 > 30 days). Sorption coefficients (Koc in L/Kg) in these soils ranged from 71 to 733 for TEA, 64-408 for MIT and 11-72 for CMIT. In terms of soil microbial toxicity, exposure to HF fluid and produced water reduced microbial respiration, albeit temporarily. The overall microbial activities in surface soils contaminated with produced water had fully recovered in most soils. In contrast, the HF fluid addition to soils completely inhibited the nitrification in all soils, with little recovery over the 60 day experimental period. In the case of produced water exposure, three out of five surface soils showed complete recovery in nitrification during the study period. The functional genes for nitrogen fixation (nifH) and carbon cycling (GA1) and microbial community composition (16 S rRNA) were significantly affected by HF fluid in some soils. Overall, the study shows that the HF fluid can have significant detrimental impact on soil microbial functions, especially on nitrogen cycling. More work is needed to identify the exact cause of microbial toxicity in soils contaminated with HF fluid.

Assessing the importance of cobalt as a micronutrient for freshwater cyanobacteria.

Micronutrients play key roles in numerous metabolic processes in cyanobacteria. However, our understanding of whether the micronutrient cobalt influences the productivity of freshwater systems or the occurrence of cyanobacterial blooms is limited. This study aimed to quantify the concentration of Co necessary for optimal cyanobacterial growth by exposing Microcystis aeruginosa to a range of Co concentrations under culture conditions. Extended exposure to concentrations below ˜0.06 µg · L-1 resulted in notable inhibition of M. aeruginosa growth. A clear negative relationship was observed between Co concentration in solution and intracellular Fe quota of M. aeruginosa, possibly due to decreased transport of Fe at higher Co concentrations. Cyanocobalamin and any Co within the structure of cyanocobalamin appears to be non-bioavailable to M. aeruginosa, instead they likely rely on the synthesis of a structural variant - pseudocobalamin, which may have implications for the wider algal community as the variants of cobalamin are not necessarily functionally exchangeable. To evaluate the likelihood of Co limitation of cyanobacterial growth under field conditions, a survey of 10 freshwater reservoirs in South-Eastern Australia was conducted. Four of the ten sites had dissolved Co concentrations below the 0.06 µg · L-1 threshold value. All four of these sites rarely undergo cyanobacterial blooms, strengthening evidence of the potential for Co to limit growth, perhaps either alone or in combination with phosphorus.

New Insights Into Acidithiobacillus thiooxidans Sulfur Metabolism Through Coupled Gene Expression, Solution Chemistry, Microscopy, and Spectroscopy Analyses.

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S0 and S 2 ⁢ O 3 2 - metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidans S 2 ⁢ O 3 2 - metabolism model involves the conversion of S 2 ⁢ O 3 2 - to SO 4 2 - , S0 and S 4 ⁢ O 6 2 - , mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO 3 2 - , S 2 ⁢ O 3 2 - and polythionates in the A. thiooxidans S0 metabolism model. Our combined results shed light onto the important role specifically of TetH in S 2 ⁢ O 3 2 - metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S0 oxidation. Finally, our data suggest that formation of intracellular S 2 ⁢ O 3 2 - is a critical step in S0 metabolism, and that recycling of internally generated SO 3 2 - occurs, through comproportionating reactions that result in S 2 ⁢ O 3 2 - . Electron microscopy and spectroscopy confirmed intracellular production and storage of S0 during growth on both S0 and S 2 ⁢ O 3 2 - substrates.

A Review of the Effect of Trace Metals on Freshwater Cyanobacterial Growth and Toxin Production.

Cyanobacterial blooms are becoming more common in freshwater systems, causing ecological degradation and human health risks through exposure to cyanotoxins. The role of phosphorus and nitrogen in cyanobacterial bloom formation is well documented and these are regularly the focus of management plans. There is also strong evidence that trace metals are required for a wide range of cellular processes, however their importance as a limiting factor of cyanobacterial growth in ecological systems is unclear. Furthermore, some studies have suggested a direct link between cyanotoxin production and some trace metals. This review synthesises current knowledge on the following: (1) the biochemical role of trace metals (particularly iron, cobalt, copper, manganese, molybdenum and zinc), (2) the growth limitation of cyanobacteria by trace metals, (3) the trace metal regulation of the phytoplankton community structure and (4) the role of trace metals in cyanotoxin production. Iron dominated the literature and regularly influenced bloom formation, with 15 of 18 studies indicating limitation or colimitation of cyanobacterial growth. A range of other trace metals were found to have a demonstrated capacity to limit cyanobacterial growth, and these metals require further study. The effect of trace metals on cyanotoxin production is equivocal and highly variable. Better understanding the role of trace metals in cyanobacterial growth and bloom formation is an essential component of freshwater management and a direction for future research.

Toxicity of dissolved and precipitated forms of barium to a freshwater alga (Chlorella sp. 12) and water flea (Ceriodaphnia dubia).

Barium is present at elevated concentrations in oil and gas produced waters, and there is no international water quality guideline value to assess the potential risk of adverse effects to aquatic biota. Sulfate concentration largely controls the solubility of barium in aquatic systems, with insoluble barium sulfate (barite) assumed to be less bioavailable and less toxic than dissolved barium. We exposed aquatic biota to dissolved barium only and to a mixture of dissolved and precipitated barium. The chronic dissolved barium 48-h growth rate inhibition effect concentrations, (EC10 and EC50) for the tropical freshwater alga Chlorella sp. 12 were 40 mg/L (27-54 mg/L 95% confidence limits [CL]), and 240 mg/L (200-280 mg/L 95% CL), respectively. The acute EC10 and EC50 values for 48-h immobilization of the water flea (Ceriodaphnia dubia) by dissolved barium were 14 mg/L (13-15 mg/L 95% CL) and 17 mg/L (16-18 mg/L 95% CL), respectively. Chlorella sp. 12 was significantly more sensitive to precipitated barium than to dissolved barium, whereas the opposite seemed likely for C. dubia. Ceriodaphnia dubia was predicted to be chronically sensitive to dissolved barium at concentrations measured in produced waters and receiving waters, based on a predicted chronic EC10 of 1.7 mg/L derived from the acute EC50/10. Further chronic toxicity data that account for barium toxicity in dissolved and precipitated forms are required to derive a barium guideline for freshwater biota. Environ Toxicol Chem 2018;37:1632-1642. © 2018 SETAC.

On the mechanism of nanoparticulate CeO2 toxicity to freshwater algae.

The factors affecting the chronic (72-h) toxicity of three nanoparticulate (10-34nm) and one micron-sized form of CeO2 to the green alga, Pseudokirchneriella subcapitata were investigated. To characterise transformations in solution, hydrodynamic diameters (HDD) were measured by dynamic light scatter, zeta potential values by electrophoretic mobility, and dissolution by equilibrium dialysis. The protective effects of humic and fulvic dissolved organic carbon (DOC) on toxicity were also assessed. To investigate the mechanisms of algal toxicity, the CytoViva hyperspectral imaging system was used to visualise algal-CeO2 interactions in the presence and absence of DOC, and the role of reactive oxygen species (ROS) was investigated by 'switching off' ROS production using UV-filtered lighting conditions. The nanoparticulate CeO2 immediately aggregated in solution to HDDs measured in the range 113-193nm, whereas the HDD and zeta potential values were significantly lower in the presence of DOC. Negligible CeO2 dissolution over the time course of the bioassay ruled out potential toxicity from dissolved cerium. The nanoparticulate CeO2 concentration that caused 50% inhibition of algal growth rate (IC50) was in the range 7.6-28mg/L compared with 59mg/L for micron-sized ceria, indicating that smaller particles were more toxic. The presence of DOC mitigated toxicity, with IC50s increasing to greater than 100mg/L. Significant ROS were generated in the nanoparticulate CeO2 bioassays under normal light conditions. However, 'switching off' ROS under UV-filtered light conditions resulted in a similar IC50, indicating that ROS generation was not the toxic mechanism. The CytoViva imaging showed negligible sorption of nanoparticulate CeO2 to algal cells in the presence of DOC, and strong sorption in its absence, suggesting that this was the toxic mechanism. The results suggest that DOC in natural waters will coat CeO2 particles and mitigate toxicity to algal cells.

Derivation of a water quality guideline for aluminium in marine waters.

Metal risk assessment of industrialized harbors and coastal marine waters requires the application of robust water quality guidelines to determine the likelihood of biological impacts. Currently there is no such guideline available for aluminium in marine waters. A water quality guideline of 24 µg total Al/L has been developed for aluminium in marine waters based on chronic 10% inhibition or effect concentrations (IC10 or EC10) and no-observed-effect concentrations (NOECs) from 11 species (2 literature values and 9 species tested including temperate and tropical species) representing 6 taxonomic groups. The 3 most sensitive species tested were a diatom Ceratoneis closterium (formerly Nitzschia closterium; IC10 = 18 µg Al/L, 72-h growth rate inhibition) < mussel Mytilus edulis plannulatus (EC10 = 250 µg Al/L, 72-h embryo development) < oyster Saccostrea echinata (EC10 = 410 µg Al/L, 48-h embryo development). Toxicity to these species was the result of the dissolved aluminium forms of aluminate (Al(OH4 (-) ) and aluminium hydroxide (Al(OH)3 (0) ) although both dissolved, and particulate aluminium contributed to toxicity in the diatom Minutocellus polymorphus and green alga Dunaliella tertiolecta. In contrast, aluminium toxicity to the green flagellate alga Tetraselmis sp. was the result of particulate aluminium only. Four species, a brown macroalga (Hormosira banksii), sea urchin embryo (Heliocidaris tuberculata), and 2 juvenile fish species (Lates calcarifer and Acanthochromis polyacanthus), were not adversely affected at the highest test concentration used.

Transformations of nanomaterials in the environment.

Increasing use of engineered nanomaterials with novel properties relative to their bulk counterparts has generated a need to define their behaviors and impacts in the environment. The high surface area to volume ratio of nanoparticles results in highly reactive and physiochemically dynamic materials in environmental media. Many transformations, e.g. reactions with biomacromolecules, redox reactions, aggregation, and dissolution, may occur in both environmental and biological systems. These transformations and others will alter the fate, transport, and toxicity of nanomaterials. The nature and extent of these transformations must be understood before significant progress can be made toward understanding the environmental risks posed by these materials.

Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility.

Metal oxide nanoparticles are finding increasing application in various commercial products, leading to concerns for their environmental fate and potential toxicity. It is generally assumed that nanoparticles will persist as small particles in aquatic systems and that their bioavailability could be significantly greater than that of larger particles. The current study using nanoparticulate ZnO (ca. 30 nm) has shown that this is not always so. Particle characterization using transmission electron microscopy and dynamic light scattering techniques showed that particle aggregation is significant in a freshwater system, resulting in flocs ranging from several hundred nanometers to several microns. Chemical investigations using equilibrium dialysis demonstrated rapid dissolution of ZnO nanoparticles in a freshwater medium (pH 7.6), with a saturation solubility in the milligram per liter range, similar to that of bulk ZnO. Toxicity experiments using the freshwater alga Pseudokirchneriella subcapitata revealed comparable toxicity for nanoparticulate ZnO, bulk ZnO, and ZnCl2, with a 72-h IC50 value near 60 microg Zn/ L, attributable solely to dissolved zinc. Care therefore needs to be taken in toxicity testing in ascribing toxicity to nanoparticles per se when the effects may be related, at least in part, to simple solubility.

Azo dye method for mapping relative sediment enzyme activity in situ at precise spatial locations.

Existing methodology for measuring microbial enzyme activity in aquatic sediments involves horizontal sectioning of sediment cores into centimeter slices, followed by determination of the enzyme activity of each homogenized sediment slice. At best, this approach provides only one-dimensional information on the distribution of microbial activity. This paper describes the development of a novel technique to map sediment enzyme activity in situ at millimeter spatial resolution. Naphthol AS enzyme substrates were loaded onto filter membranes by evaporation from an organic solvent. The membranes were attached to plastic cards to form rigid probes, which were deployed vertically in sediments for a fixed time period. The exposed membranes were developed in a diazonium salt solution, resulting in the formation of a colored precipitate where substrate hydrolysis had occurred. The chromogenic reaction was calibrated and quantified by immersing substrate-loaded membranes in a series of solutions of known enzyme activity. A flatbed scanner and image analysis software were used to produce digitized images and to generate two-dimensional maps of enzyme activity. The technique was used to map the spatial features of esterase activity in aquatic sediment samples from wetland areas and enabled the precise locations of microbial activity "hotspots" to be identified.

Effect of initial cell density on the bioavailability and toxicity of copper in microalgal bioassays.

Algal toxicity tests based on growth inhibition over 72 h have been extensively used to assess the toxicity of contaminants in natural waters. However, these laboratory tests use high cell densities compared to those found in aquatic systems in order to obtain a measurable algal response. The high cell densities and test duration can result in changes in chemical speciation, bioavailability, and toxicity of contaminants throughout the test. With the recent application of flow cytometry to ecotoxicology, it is now possible to use lower initial cell densities to minimize chemical speciation changes. The speciation and toxicity of copper in static bioassays with the tropical freshwater alga Chlorella sp. and the temperate species Selenastrum capricornutum (Pseudokirchneriella subcapitata) were investigated at a range of initial cell densities (10(2)-10(5) cells/ml). Copper toxicity decreased with increasing initial cell density. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h median effective concentration [EC50]) increased from 4.6 to 16 microg/L for Chlorella sp. and from 6.6 to 17 microg/L for S. capricornutum as the initial cell density increased from 10(2) to 10(5) cells/ml. Measurements of anodic stripping voltammetry-labile, extracellular, and intracellular copper confirmed that at higher initial cell densities, less copper was bound to the cells, resulting in less copper uptake and lower toxicity. Chemical measurements indicated that reduced copper toxicity was due primarily to depletion of dissolved copper in solution, with solution speciation changes due to algal exudates and pH playing a minor role. These findings suggest that standard static laboratory bioassays using 10(4) to 10(5) algal cells/ml may seriously underestimate metal toxicity in natural waters.