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Our understanding of microbial communities associated with anaerobic digestion (AD) currently relies strongly on metagenomic sequencing, which can reveal phylogenetic diversity, but does not provide information concerning microbial activity or the close associations that may form between syntrophic organisms. Approaches that facilitate charting of process-targeted variation in microbial community activities are important for understanding how the microbiology of AD functions as a single biological machine. Here I identify subsets of metabolically specialised microbes as they respond to substrate availability in AD systems using bioorthogonal non-canonical amino acid tagging (BONCAT) and affinity-based cell separation (ABCS). The results demonstrate the specific labelling, visualisation and separation of microbes that actively participate in volatile fatty acid (VFA) degradation and suggest a differential response to octanoic acid by specialists within the microbial community. Analysis of metagenomic sequences from a time series of BONCAT-ABCS samples reveals that this method enriched a distinct microbial community with genetically-derived metabolisms consistent with and changing according to the observed metabolic outcomes. Proteomic data generated from BONCAT-ABCS help to resolve the functional landscape of enriched microbial community and give better resolution of identified translationally/metabolically active taxa. This enrichment approach allows us to determine the temporal response of those microbes most likely to engage in octanoic acid degradation in AD sludge. This method can be applied to the identification of specialist microbes with a role in degradation of a range of other compounds in AD, enhancing our understanding of microbial community interactions and facilitating the development of strategies for process optimisation.

Plant-virus-vector interactions mediate important ecological processes and can significantly reduce crop production. Almost all plant viruses require vectors for transmission and use sophisticated mechanisms to achieve this: they manipulate host plants to release volatile chemical that attract their vectors. This chemoattraction has been demonstrated in above-ground interactions but has never been studied in viruses transmitted by soil-dwelling nematodes. Tobacco rattle virus (TRV) is an important pest of potato, transmitted by trichodorid nematodes. This work uses a model plant system to investigate the effects of TRV infection on host root architecture, root volatile release and chemoattraction of its nematode vectors. TRV infection alters root structure, producing a smaller and more compact root system. It modifies root volatile profiles compared to uninfected plants, which leads to trichodorids preferentially moving towards infected plants. The TRV genome contains genes known as 2b and 2c; implicated for nematode transmission. Mutations in these genes reduce the severity of root architecture symptoms, remove differences in the profile of volatiles released from infected roots compared with uninfected ones and, in the case of 2b, make plants less attractive to nematodes than plants infected with viruses without mutations. The release of the volatile 2-ethyl-1-hexanol significantly increased in roots infected by TRV. When added to uninfected plants, it made them more attractive to nematodes than untreated counterparts. This work demonstrates TRV manipulates the production of host root volatiles, leading to increased attraction of trichodorid vectors. 2b and 2c are important in this interaction and 2-ethyl-1-hexanol is a strong component of the attractant signal. This new knowledge shows nematode transmitted viruses use similar mechanisms to attract their vectors as their better-studied aboveground counterparts and contributes to the study of volatile- mediated rhizosphere interactions with implications for agricultural pest control.

Over recent decades salt marshes have been restored as a cost-effective response to coastal biodiversity loss and flood management. Previous research on established sites has demonstrated that restored marshes have significantly different sediment properties than natural marshes which appears to broadly impact ecosystem function. A study was conducted to examine the differences in natural and realigned salt marshes in terms of plant biodiversity, sediment characteristics (bulk density, water content, pH and nutrients) and microbial communities in Colchester Essex, UK. We studied three pairs of natural and realigned salt marshes of different ages, 13, 62, 118 years since breaching of the sea wall. Furthermore, we studied monthly changes of sediment characteristics of a newly realigned site from breach to 14 months of tidal inundation. In addition to monthly sediment changes, we placed invertebrate exclusion chambers in the newly realigned marsh to examine the effect of bioturbation in changing sediment characteristics and microbial communities. Sediment characteristics of our realigned marshes were significantly different than those of the natural marshes. In addition, natural marshes displayed higher variability and heterogeneity in nutrient and water content than our realigned marshes. Within our newly realigned salt marsh we observed that despite the different starting sediment characteristics, 14 months after inundation our realigned site was broadly similar to the natural marsh but only on the top 5cm of sediment, indicating the presence of a relic agricultural layer which can affect the hydrology and development of the system. Macro-invertebrate colonization has shown that it can influence the geochemical characteristic and microbial communities of sediment in a newly realigned salt marsh. Microbial communities' composition and abundances within a newly realigned marsh are significantly different from natural marshes 14 months post inundation.

Arsenic is a toxic metalloid contaminating soil and water supplies in many regions worldwide, and its entry into the food chain poses a serious health risk to millions of people. Arsenic is toxic to plants, lowering the rate of photosynthesis and inhibiting growth, resulting in a reduction in crop yields. Therefore, there is a pressing need to improve arsenic tolerance in plants and reduce accumulation of arsenic in crops. To this end, an arsenite efflux transporter from yeast was heterologously expressed in rice plants under the control of tissue specific promoters. Expression in different tissues led to altered arsenic tolerance and tissue distribution, indicating that this is a promising strategy for the future development of arsenic tolerant varieties. The role of NIP aquaporins in arsenic transport and tolerance in the model plant Arabidopsis was explored in Chapter 3. Group II NIPs were identified as relevant targets for engineering arsenic tolerance and reducing seed/grain arsenic accumulation in crops. Further targets for engineering arsenic tolerance were identified from a collection of T- DNA insertion mutants of Arabidopsis using forward and reverse genetic screening approaches. Two of these were further characterised for their role in arsenic tolerance and accumulation.

Anaerobic Digestion (AD) is a biologically mediated technology that is used as a method for managing and obtaining energy from organic waste materials. Through the biological action of Bacteria, Fungi and Archaea, in the absence of oxygen, the organic waste is converted to biogas, mainly methane, which can be used as a fuel source. This gas can be burned to generate electricity, heat, injected into the grid or used to fuel vehicles. I have developed a single stage, lab scale anaerobic digester that is a reflection of full-scale process systems. This model reactor facilitates the collection of samples for metagenomic sequencing, along with process data, providing an insight to the AD process. Three experiments were carried out (using the lab model) to determine (i) the dynamic changes that occur in microbial AD communities, (ii) the rate at which these communities change and (iii) if the observed changes are comparable between numerous systems run under the same conditions. The use of amplicon sequencing appears to be a common method used to study the composition of microbial communities, especially in AD, but this method is prone to inaccuracies and so alternative methods were developed, as described in this thesis. By applying the use of shotgun metagenomic sequencing, combined with various contig assemblers and a custom clustering method, more detail on the microbes present and their functions in AD is obtained compared to targeted sequencing. Pipelines to interpret large datasets generated through Next Generation Sequencing (NGS) have been developed and utilised in this project. We have identified microbes that are present within the AD system, and the time-scale of the dynamic changes. This method has also revealed novel methanogens that are important in the AD process.