Arsenic cycling in marine phytoplankton and microbiota

Abstract

The transfer of arsenic in marine food-webs has been widely researched, largely to assess whether the ingestion of arsenic-containing seafood poses significant health risks to humans. Studies investigating arsenic cycling in marine food-webs have, however, generally overlooked single-celled organisms such as phytoplankton, bacteria and other microbiota such as fungi, yeasts etc. These organisms fulfil various ecological roles in marine systems and also form the base of most food-webs. In addition phytoplankton and bacteria/microbiota have also been hypothesised to play key roles in marine arsenic cycling via both the transfer of incorporated arsenic in food-webs and also regulating the arsenic species that are present in the environment i.e. in the water-column and sediments. Phytoplankton, bacteria and other microbiota have often been overlooked in research investigating marine arsenic cycling in marine food-webs due to a lack of understanding as to their ecological importance in marine systems and also as a result of the inability to reliably study arsenic cycling in these organisms in situ. Consequently, to investigate arsenic cycling in marine phytoplankton, bacteria and other associated microbiota laboratory-based experimentation is a necessity; however, the ecological validity of different experimental approaches used to study arsenic cycling in these organisms has never been assessed. It is therefore unknown as to whether different experimental approaches used to investigate arsenic cycling in these organisms actually influence the arsenic cycling processes observed. This thesis has two specific aims, which firstly were to increase the understanding of the roles that marine phytoplankton, bacteria and associated microbiota play in marine arsenic cycling. In addition this thesis also assessed the merits of various experimental approaches used to investigate arsenic cycling in marine phytoplankton, bacteria and associated microbiota to illustrate if different experimental approaches influenced the arsenic cycling processes observed. For marine phytoplankton, the influence of culture regime, arsenic and nutrient exposure and the presence of bacteria/microbiota were tested to illustrate if these variables could illicit changes to arsenic cycling processes. From an arsenic cycling perspective, it was observed from cultures of the marine phytoplankton Dunaliella tertiolecta and Thalassiosira pseudonana that nutrient availability particularly that of phosphate (PO43-) drives arsenic uptake probably as a result of structural similarities between arsenate – As(V) and PO43-. In addition D. tertiolecta and T. pseudonana were observed to accumulate arsenic in lipid-soluble cell fractions, possibly as a replacement for phosphorus. Arsenoribosides were observed to be major arsenic components in both phytoplankton species, particularly in hydrolysed lipid extracts which suggests that arsenoribosides may be formed via the degradation of arsenolipids rather than via an extension to the Challenger [1] pathway as had been previously assumed. In addition, arsenobetaine (AB) was not present in any phytoplankton tissue samples analysed here, which provides further evidence that the formation of AB occurs in marine animals in situ. Experimental variables such as culture regime and arsenic/nutrient exposures were observed to directly alter the arsenic cycling processes performed by both D. tertiolecta and T. pseudonana. The use of batch cultures was deemed not suitable for the observation of environmentally valid arsenic cycling processes as these cultures encompassed a closed system and as a result extreme cell densities, rapid nutrient depletion and accumulation of dead cell material resulted in the movement of arsenic in the culture occurring as a series of fluxes from water → live cells → dead cells rather than mimicking how arsenic cycles in the environment. These issues were minimised in continuous culture systems as the constant replenishment of arsenic and nutrient resources coupled with the removal of cell culture resulted in more stable cell densities, stable nutrient concentrations and less dead cell tissue accumulation, which resulted in arsenic cycling being more analogous to nature. Arsenic and nutrient exposures were also illustrated to directly influence arsenic uptake, arsenic accumulation and arsenic species formation in D. tertiolecta. Arsenic exposure at concentrations exceeding ambient concentrations (≈ 2 μg L-1) resulted in inorganic arsenic, primarily As(V) accumulating in the lipids, whist nutrient exposure, particularly that of PO43- facilitated arsenic uptake and also possibly the accumulation of arsenic in the lipids. The influence of bacteria and associated microbiota on the arsenic cycling processes performed by D. tertiolecta were minimal as it was observed that sterile D. tertiolecta cultures contained bacteria and thus experiments of this nature are compromised as differences in arsenic cycling processes between sterile and non-sterile organisms/cultures cannot be tested. In addition, the physical/chemical properties of the culture media combined with the likely release of anti-microbial secondary metabolites from D. tertiolecta cells also limited the presence of foreign bacteria/microbiota, thus also making the effects of antagonistic bacteria/microbiota on phytoplankton arsenic cycling untestable. This thesis also aimed to illustrate the role of bacteria and associated microbiota in the degradation of arsenoribosides. Arsenoribosides, despite being the major arsenic species in marine macro-algae have never been detected in seawater, with bacteria/microbiota hypothesised as the means behind the degradation of arsenoribosides. When macro-algal tissue from the marine species Ecklonia radiata was decomposed under laboratory conditions, arsenoribosides were lost from tissues, yet were rarely detected in seawater. Dimethylarsenoethanol (DMAE) was found both in decomposing E. radiata tissue and in seawater/sand porewaters of experimental microcosms and was shown to persist in microcosms containing autoclaved sand and seawater. Consequently, it was proposed that microbial communities from multiple niches in marine ecosystems plus the inherent complexity of these microbial communities are responsible for the degradation of arsenoribosides, with the initial degradation of arsenoribosides → DMAE occurring in decomposing macro-algal tissues, as all experimental microcosms used contained microbial communities from E. radiata tissues and in all cases arsenoribosides were degraded to DMAE. DMAE was subsequently released into the surrounding environment and persisted in seawater and sand porewaters of microbially altered (reduced microbial complexity) microcosms but was degraded to As(V) in natural microcosms, suggesting that bacteria/microbiota inhabiting sand and seawater environments are responsible for the removal of DMAE, which also explains why this arsenic species has also never been found in seawater. The degradation of arsenoribosides by marine bacteria and associated microbiota was investigated using three experimental approaches, namely culture media incubation experiments, macro-algal tissue decomposition studies and field studies. It was observed that the degradation of arsenoribosides was far more efficient in macro-algal tissue decomposition studies than in culture media incubation experiments in which arsenoribosides persisted, which is unlike what occurs in nature. It was observed that the high niche availability in macro-algal tissue decomposition studies resulted in the degradation of arsenoribosides being a good representation of how the arsenoriboside degradation occurs environmentally. Niche availability is limited in culture media incubation experiments and the physical/chemical properties of culture media are highly selective, which results in considerably decreased microbial diversity, which disrupts networks and interactions between species which are likely to be essential in determining if bacteria/microbiota can perform complex arsenic cycling processes such as the degradation of arsenoriboside

Date of Award	2013
Original language	English
Supervisor	Bill Maher (Supervisor) & Simon FOSTER (Supervisor)