Ewen Silvester

Proton and Iron Binding by the Cyanobacterial Toxin Microcystin-LR

Microcystins (MCs) are a group of hepatotoxins produced by cyanobacteria that have not had their functional role or the environmental factors that trigger production clearly determined. One suggestion is that microcystins are siderophores (i.e., ligands with an extremely high affinity with iron, typically with stability constants substantially greater than 10(25)). In this work, we explore proton and iron binding with microcystin-LR (MC-LR). Using UV-visible spectroscopy and a HPLC peak retention time-based method, the two acid dissociation constants associated with the carboxylic groups of MC-LR were determined to be: pKa₁ = 2.17 and pKa₂ = 3.96. Cyclic voltammetry provides evidence for the formation of at least two Fe(III)-MC-LR complexes, with the Fe(III) reduction peak significantly shifted to more reducing potentials in the presence of MC-LR. These complexes have been interpreted as a rapidly formed initial complex (Complex 1) and a more stable, and slower forming, Complex 2. The stability constant for Fe(III)-MC-LR (Complex 2) was estimated to be approximately 10(13) in 60% v/v MeOH/water at 0.1 M ionic strength. The electrochemical experiments provide no evidence for the formation of a complex between Fe(2+) and MC-LR. Given that most MC-LR is released only upon cell lysis, and coupled with the moderate strength of the stability constant with Fe(III) determined in this study, it appears unlikely that that MC-LR is an extracellular siderophore. If MC-LR is involved in iron regulation in cyanobacteria, it is more likely as a shuttle for iron across the cell membrane or in intracellular processes.

Acidification and buffering mechanisms in acid sulfate soil wetlands of the Murray-Darling Basin, Australia.

The acid generation mechanisms and neutralizing capacities of sulfidic sediments from two inland wetlands have been studied in order to understand the response of these types of systems to drying events. The two systems show vastly different responses to oxidation, with one (Bottle Bend (BB) lagoon) having virtually no acid neutralizing capacity (ANC) and the other (Psyche Bend (PB) lagoon) an ANC that is an order of magnitude greater than the acid generation potential. While BB strongly acidifies during oxidation the free acid generation is less than that expected from the measured proton production and consumption processes, with additional proton consumption attributed to the formation of an acid-anion (chloride) FeIII (oxyhydr)oxide product, similar to akaganéite (Fe(OH)2.7Cl0.3). While such products can partially attenuate the acidification of these systems, resilience to acidification is primarily imparted by sediment ANC.

High Spatial Resolution Infrared Micro-Spectroscopy Reveals the Mechanism of Leaf Lignin Decomposition by Aquatic Fungi

Organic carbon is a critical component of aquatic systems, providing energy storage and transfer between organisms. Fungi are a major decomposer group in the aquatic carbon cycle, and are one of few groups thought to be capable of breaking down woody (lignified) tissue. In this work we have used high spatial resolution (synchrotron light source) infrared micro-spectroscopy to study the interaction between aquatic fungi and lignified leaf vein material (xylem) from River Redgum trees (E. camaldulensis) endemic to the lowland rivers of South-Eastern Australia. The work provides spatially explicit evidence that fungal colonisation of leaf litter involves the oxidative breakdown of lignin immediately adjacent to the fungal tissue and depletion of the lignin-bound cellulose. Cellulose depletion occurs over relatively short length scales (5–15 µm) and highlights the likely importance of mechanical breakdown in accessing the carbohydrate content of this resource. Low bioavailability compounds (oxidized lignin and polyphenols of plant origin) remain in colonised leaves, even after fungal activity diminishes, and suggests a possible pathway for the sequestration of carbon in wetlands. The work shows that fungi likely have a critical role in the partitioning of lignified material into a biodegradable fraction that can re-enter the aquatic carbon cycle, and a recalcitrant fraction that enters long-term storage in sediments or contribute to the formation of dissolved organic carbon in the water column.

Rehabilitation options for inland waterways impacted by sulfidic sediments – Field trials in a south-eastern Australian wetland

The accumulation of significant pools of sulfidic sediments in inland wetlands and creeks is an emerging risk for the management of inland waterways. We used replicated plot trials to appraise the viability of various strategies for neutralizing oxidized, acidified sulfidic sediments in a highly degraded wetland. Of the twenty different treatments trialed only addition of calcium hydroxide or calcium carbonate, burning of wood, and planting of Phragmites australis, Typha domingensis and Atriplex nummularia into beds prepared with CaCO3 or P. australis and T. domingensis into beds of sediment and mulch, decreased total actual acidity (TAA) in the top 5 cm of sediment in the first two weeks following treatment. Only the calcium hydroxide treatments and planting of P. australis, T. domingensis and A. nummularia into beds prepared with CaCO3 decreased TAA for a longer period of time (6 months). None of the treatments, except the planting of P. australis into beds prepared with lime, decreased TAA in the 5-30 cm layer of sediments. Therefore, the only effective treatment appears to be the application of highly alkaline ameliorants which need to be transported to the site. A survey of the wetland was undertaken to estimate the total amount of actual and potential acidity stored in the wetlands sediment and overlying water and showed that up to 1200 tonnes of calcium carbonate would be required to neutralise all of the actual and potential acidity in the 10 ha wetland. However, neutralisation of the remaining water in the wetland (about 12.5 ML) would produce approximately 2750 m3 of metal rich sludge (approximately 100 tonnes dry weight) that would require separate disposal.

Salinity-induced acidification in a wetland sediment through the displacement of clay-bound iron(II)

Environmental context Acidification of inland waterways is an emerging issue worldwide, mostly because it disturbs the balance of reduced sulfur species in soils, sediments and mine tailings. We describe a pathway for wetland acidification through salt displacement and oxidation of Fe2+ from clay minerals. This alternative pathway for acidification raises environmental concerns because an increasing number of inland waterways are affected by increasing salinity. Abstract A wetland near the Murray River (south-eastern Australia) was found to have significant levels of exchangeable reduced iron (Fe2+) in the sediment clay-zone, and the potential for acidification under high salinity and oxidising conditions. Cation exchange experiments using purified clay from this site show relative affinities consistent with the lyotrophic series: Fe2+>Ca2+>Mg2+>H+>K+>Na+. This relative affinity is confirmed in Fe2+ displacement experiments using natural sediment clay. Proton production during oxidation of salt-treated sediments corresponds to that expected for the oxidation and hydrolysis of Fe2+ displaced from clay interlayers, taking into account the buffering properties of the sediment matrix. This work shows that wetland acidification can occur in low sulfur-containing wetlands and is not exclusively a problem associated with sulfidic sediments.

Decomposition of native leaf litter by aquatic hyphomycetes in an alpine stream

Despite the recognised significance of hyphomycetes in the degradation of leaf litter in streams, few studies have been carried out in alpine environments and none in Australian alpine streams. We hypothesised that the fungal communities responsible for leaf decomposition would change over immersion time, and would respond differently at different sites and on different types of vegetation. Leaf bags containing Epacris glacialis (F. Muell.), Eucalyptus pauciflora (Sieber ex. Spreng) and Eucalyptus delegatensis (R.T. Baker) were deployed at different sites in a stream in the Victorian Alpine National Park, south-eastern Australia. Leaf colonisation was delayed for 2 weeks and decay constants for E. pauciflora and E. delegatensis were 0.004–0.005 and 0.006 respectively. Maximum fungal biomass on leaves was similar to that in previous published studies, whereas sporulation rates were two or three orders of magnitude lower, indicating a reduced reproductive effort. Sporulation and DNA-based studies combined showed that fungal communities on the decomposing leaf material changed over time and exhibited significant preferences for leaf type and study site. We have shown that aquatic hyphomycetes can degrade physically tough leaves of Australian alpine plant species, potentially contributing to pathways for particulate carbon to enter alpine-stream food webs.

Ionic regulation in an alpine peatland in the Bogong High Plains, Victoria, Australia

Environmental context. Australian alpine peatlands are thought to have an important role in maintaining water quality in the associated headwater streams. This study has confirmed that these peatlands can significantly modify stream water through a range of mechanisms, including: nutrient uptake, salt sequestering, and the export of organic carbon. While the significance of this chemical regulation to down stream processes is yet to be fully understood, it is clear that these systems have considerable potential to modify water composition. Abstract. Heathy Spur 1 (HS-1) is an intact alpine peatland in the Bogong High Plains, Victoria, Australia, that serves as a reference system for understanding the impacts of historical land use practices (cattle grazing, water diversion) and wildfire. The major ion chemistry in the groundwater feed and drainage water at HS-1 was studied over seasonal timescales during ‘dry weather’ periods; conditions that allow a simple hydrological model to be used, where the groundwater is assumed to partition between evapotranspiration and stream discharge. With this model the acid neutralising capacity (ANC) of stream discharge can be understood in terms of evapotranspiration and proton uptake associated with nitrate and sulfate removal. Stream discharge ANC is strongly partitioned towards exported dissolved organic carbon, shifting the buffering intensity to lower pH compared to the groundwater. Given the extremely low alkalinity of the regional groundwater, these alpine peatlands likely have a critical role in increasing headwater stream buffering capacity.

Physical and chemical drivers of vegetation in groundwater-source pools on the Bogong High Plains, Victoria

The wetland communities intimately associated with groundwater sources in the Australian alps are poorly documented compared with the broader (and more obvious) alpine peatlands. In the present work, we report on the vegetation observed immediately around such sources and the characteristics of the supplying groundwater so as to understand the likely factors controlling the observed vegetation assemblage. Thirty-two groundwater sources were identified across three catchments on the Bogong High Plains, Victoria (Australia), and the vegetation associated with these sources surveyed. Groundwater sources occurred across a range of altitudes (1667–1854 m), independent of aspect, and were hydrologically connected to (upstream of) peatlands. Localised mounding adjacent to the groundwater sources resulted in the formation of pools (‘groundwater source pools’). The vegetation within the pools was dominated by bryophytes, with the aquatic bryophyte Blindia robusta the most common species. The groundwater was deficient in major ions, and similar to rainwater apart from elevated concentrations of CO2 accumulated in the groundwater-recharge process. The high CO2 concentrations, combined with the near-constant temperature conditions provided by sustained groundwater flow, are thought to be likely drivers for the high abundance of B. robusta. Although the relative contributions of rain and snow to aquifer recharge are not fully understood for the Australian Alps, these ecosystems are likely to be vulnerable to the changes in precipitation regime that are predicted under climate-change scenarios.

Proteins are a major component of dissolved organic nitrogen (DON) leached from terrestrially aged Eucalyptus camaldulensis leaves

Environmental context Dissolved organic nitrogen often constitutes the largest portion of the dissolved nitrogen pool yet is a commonly overlooked nutrient source in aquatic systems. Terrestrially aged Eucalyptus camaldulensis, a common lowland leaf litter species, rapidly released proteinaceous dissolved organic nitrogen during the first 24h of leaching. The results indicate that terrestrial leaf litter may play an important role in satisfying nutrient demand within aquatic systems through both direct deposition and floodplain interactions. Abstract Understanding sources and forms of dissolved nitrogen is of critical importance to the management of aquatic systems worldwide. Dissolved organic nitrogen (DON) often constitutes the largest portion of the dissolved nitrogen pool, yet is commonly overlooked as a nutrient source to aquatic food webs, likely owing to its bound nature within organic material and the non-specific methods by which it is measured. In this study, we determined the protein and peptide (dissolved combined amino acid (DCAA)) contribution to DON leached from Eucalyptus camaldulensis leaves over 24h. The distribution of proteinaceous material in unleached and leached leaves was characterised using Fourier-transform infrared (FTIR) microspectroscopy to determine the likely source of DCAA within the leaf tissue. DCAAs were found to be a significant component (38.5%) of the leached DON; however, >90% of the leaf protein remained in the leaves after 24h. FTIR microspectroscopy shows that proteinaceous material is strongly partitioned to fungal colonised palisade cells in the leaf mesophyll, with evidence for depletion of this material after leaching. Comparison of leaching kinetics in the presence and absence of a microbial inhibitor (sodium azide) suggests that microbial uptake or adsorption commences within the timescales of these leaching experiments. The work shows that DON in the form of peptides and proteins leached from leaf litter is a likely source of bioavailable nutrients to in-stream and floodplain systems.

A predicted change in the amino acid landscapes available to freshwater carnivores

Anthropogenic effects lead to nonrandom alterations to macroinvertebrate communities, which may lead to alteration of the ‘micronutrient landscapes’ experienced by higher consumers. Understanding how amino acid composition varies among taxa and guilds is an essential step toward predicting how micronutrient landscape alteration will affect carnivores. We assessed whether: 1) wild macroinvertebrate prey varied in their amino acid compositions, 2) variation in amino acid composition was correlated with economical selection pressures or functional traits that may separate the compositions of functional feeding groups (FFGs) or is simply a result of phylogeny, and 3) simulated anthropogenic change in the composition of macroinvertebrate communities affects the amino acid composition of the nutrient landscape. Amino acid composition varied significantly among taxa and was strongly correlated with phylogeny but not FFGs. The amino acid compositions of holometabolous insects (those that undergo complete metamorphosis; Trichoptera and Diptera) differed from those of hemimetabolous insects (those that undergo incomplete metamorphosis; Ephemeroptera and Hemiptera). This separation can be explained by morphological and physiological traits involved in pupation or nymphal development. Simulated deterioration of macroinvertebrate communities changed the amino acid landscape, resulting in lower availability of threonine, phenylalanine, proline, and tyrosine to carnivores. Amino acid availability to carnivores is likely to respond more to the loss of taxonomic lineages than to loss of prey FFGs. Our study provides a critical first step toward understanding how changes to macroinvertebrate communities might affect the availability of amino acids to higher consumers.