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.

The influence of salinity on the chronic toxicity of shale gas flowback wastewater to freshwater organisms.

The potential environmental risk associated with flowback waters generated during hydraulic fracturing of target shale gas formations needs to be assessed to enable management decisions and actions that prevent adverse impacts on aquatic ecosystems. Using direct toxicity assessment (DTA), we determined that the shale gas flowback wastewater (FWW) from two exploration wells (Tanumbirini-1 and Kyalla 117 N2) in the Beetaloo Sub-basin, Northern Territory, Australia were chronically toxic to eight freshwater biota. Salinity in the respective FWWs contributed 16% and 55% of the chronic toxicity at the 50% effect level. The remaining toxicity was attributed to unidentified chemicals and interactive effects from the mixture of identified organics, inorganics and radionuclides. The most sensitive chronic endpoints were the snail (Physa acuta) embryo development (0.08-1.1% EC10), microalga (Chlorella sp. 12) growth rate inhibition (0.23-3.7% EC10) and water flea (Ceriodaphnia cf. dubia) reproduction (0.38-4.9% EC10). No effect and 10% effect concentrations from the DTA were used in a species sensitivity distribution to derive "safe" dilutions of 1 in 300 and 1 in 1140 for the two FWWs. These dilutions would provide site-specific long-term protection to 95% of aquatic biota in the unlikely event of an accidental spill or seepage.

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.

Probabilistic risk assessment of mine-derived copper in the Ok Tedi/Fly River, Papua New Guinea.

The Ok Tedi mine discharges waste rock and tailings into the Ok Tedi River in Papua New Guinea. This has resulted in elevated copper concentrations throughout the Ok Tedi/Fly River system, which can potentially impact aquatic biota. Ten years of measured copper and toxicity monitoring data were used to assess the risk of chronic effects from the mine-derived copper. Cumulative probability plots of dissolved and labile copper were compared to a species sensitivity distribution (SSD) of published copper toxicity data for four regions of the river. The Cu-SSD was used to estimate the risk of chronic effects to aquatic organisms in the Ok Tedi/Fly River at a range of potential copper exposure scenarios. The risk to species at the median labile copper concentration for each region showed a gradient effect with distance downstream from the mine and only the most sensitive (0.2-11%) species were at risk. There were copper exceedances of the region-specific guideline values (GV) and default guideline value (DGV) 88% and 74% of the time, respectively, in the Ok Tedi region (closest to the mine) and this is considered a high risk of chronic effects. Measured copper concentrations in the middle Fly River, lower Fly River (farthest downstream of the mine) and the river at Kiunga (reference site) exceeded the region-specific GVs and DGVs less frequently to rarely and present a lower risk of chronic effects from copper. The risk was supported using toxicity tests with the local microalgal species Chlorella sp. Comparison of recent (2010-2020) and historical (1996-2004) copper monitoring data from the Ok Tedi/Fly River indicates a decrease in the labile copper concentrations (30-76%) at key sites from impacted regions and a subsequent decrease in risk. This coincides with improved mining practices aimed at reducing the copper load into the Ok Tedi/Fly River.

Improved prediction of sediment toxicity using a combination of sediment and overlying water contaminant exposures.

The choice of sediment quality assessment methodologies can strongly influence assessment outcomes and management decisions for contaminated sites. While in situ (field) methods may potentially provide greater realism, high costs and/or complex logistics often prevent their use and assessment must rely on laboratory-based methods. In this study, we utilised static-renewal and flow-through ecotoxicology tests in parallel on sediments with a wide range of properties and varying types and concentrations of contaminants. The prediction of chronic effects to amphipod reproduction was explored using multiple linear regression (MLR). The study confirmed the considerable over-estimation of the risk of toxicity of contaminated sediments in field locations when assessments rely on the results of laboratory-based static and static-renewal tests. Improved prediction of toxicity risks was achieved using a combination of contaminant exposure measures from sediment and overlying water. Existing sediment and water quality guideline values (GVs) were effective for predicting risks posed by sediments containing mixtures of common metal and organic contaminants. For 17 sediments with paired data sets from static-renewal and flow-through tests, the best prediction of toxicity to reproduction was achieved using a 2-parameter MLR that included hazard quotients for sediment contaminants and toxic units for dissolved metals (r2 = 0.892). The inclusion of particle size, organic carbon and acid-volatile sulfide did not improve toxicity predictions, despite these parameters being recognised as modifying contaminant bioavailability. The use of dilute-acid-extractable metal concentrations in place total recoverable metal concentrations did not improve the predictions. The study also confirmed that sediments existing within the estuarine and marine bays of Sydney Harbour pose significant risks of adverse effects to benthic organisms.

Diffusive Milli-Gels (DMG) for in situ assessment of metal bioavailability: A comparison with labile metal measurement using Chelex columns and acute toxicity to Ceriodaphnia dubia for copper in freshwaters.

Fluctuations in concentrations of bioavailable metals occur in most natural waters. In situ measurements are desirable to predict risks of adverse effects to aquatic organisms. We evaluated Diffusive Milli-Gels (DMG), a new in situ passive sampler, for assessing the bioavailability and toxicity of copper in waters exhibiting a wide range of characteristics. The performance was compared to an established Chelex-column method that measures labile copper concentrations by discrete sampling, and the ability to predict acute toxicity to the cladoceran, Ceriodaphnia dubia. The labile copper concentrations measured by the DMG and Chelex-column methods decreased with increasing dissolved organic carbon (DOC) (1.9-15 mg L-1) and hardness (21-270 mg CaCO3 L-1 hardness), with 20-70% of total dissolved copper being present as labile copper. Toxicity decreased with increasing DOC and hardness. Strong linear relationships existed between the EC50 for C. dubia and DOC, and when the EC50 was related to either the labile copper concentrations measured by DMG (r2 = 0.874) or the Chelex column (0.956) methods. The study demonstrates that the DMG passive sampler is a relevant tool for the in situ assessment of environmental risks posed by metals whose toxicity is strongly influenced by speciation.

Global Sensitivity Analysis for the determination of parameter importance in bio-manufacturing processes.

The present paper describes the application of GSA (Global Sensitivity Analysis) techniques to mathematical models of bioprocesses in order to rank inputs such as feed titres, flow rates and matrix capacities for the relative influence that each exerts upon outputs such as yield or throughput. GSA enables quantification of both the impact of individual variables on process outputs, as well as their interactions. These data highlight those attributes of a bioprocess which offer the greatest potential for achieving manufacturing improvements. Whereas previous GSA studies have been limited to individual unit operations, this paper extends the treatment to an entire downstream process and illustrates its utility by application to the production of a Fab-based rattlesnake antivenom called CroFab [(Crotalidae Polyvalent Immune Fab (Ovine); Protherics U.K. Limited]. Initially, hyperimmunized ovine serum containing rattlesnake antivenom IgG (product), other antibodies and albumin is applied to a synthetic affinity ligand adsorbent column to separate the antibodies from the albumin. The antibodies are papain-digested into Fab and Fc fragments, before concentration by ultrafiltration. Fc, residual IgG and albumin are eliminated by an ion-exchanger and then CroFab-specific affinity chromatography is used to produce purified antivenom. Application of GSA to the model of this process showed that product yield was controlled by IgG feed concentration and the synthetic-material affinity column's capacity and flow rate, whereas product throughput was predominantly influenced by the synthetic material's capacity, the ultrafiltration concentration factor and the CroFab affinity flow rate. Such information provides a rational basis for identifying the most promising strategies for delivering improvements to commercial-scale biomanufacturing processes.

A methodology for centrifuge selection for the separation of high solids density cell broths by visualisation of performance using windows of operation.

Expression systems capable of growing to high cell densities are now readily available and are popular due to the benefits of increased product concentration. However, such high solids density cultures pose a major challenge for bioprocess engineers as choosing the right separation equipment and operating it at optimal conditions is crucial for efficient recovery. This study proposes a methodology for the rapid determination of suitable operating conditions for the centrifugal recovery of high cell density fermentation broths. An ultra scale-down (USD) approach for the prediction of clarification and dewatering levels achieved in a range of typical high-speed centrifuges is presented. Together with a visualisation tool, a Window of Operation, this provides for the rapid analysis of separation performance and evaluation of the available operating conditions, as an aid in the selection of the centrifuge equipment most appropriate for a given process duty. A case study examining centrifuge selection for the processing of a high cell density Pichia pastoris culture demonstrates the method. The study examines semi-continuous disc-stack centrifuges and batch-operated machines such as multi-chamber bowls and Carr Powerfuges. Performance is assessed based on the variables of clarification, dewatering and product yield. Inclusion of limits imposed by the centrifuge type and design, and operation itself, serve to constrain the process and to define the Windows of Operation. The insight gained from the case study provides a useful indication of the utility of the methodology presented and illustrates the challenges of centrifuge selection for the demanding case of high solids concentration feed streams.