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Chapter 1 reviews the literature concerning the synthesis and reactivity of oxodimethyl- enemethane metal complexes and their applications in organic synthesis. The preparation of metal-ureylene complexes and amino acid complexes of platinum are also discussed. Chapter 2 describes the synthesis of metallaphosphetane-3-oxide complexes of platinum and palladium, [M {CH(R)P(O)(Ph)CHR} L2]. Crystallographic and n.m.r. evidence for benzoyl substituted complexes, indicates that the four-membered rings are puckered, with the phosphoryl oxygen adopting an equatorial environment. The ligand substitution reactions of platinum analogues are also discussed. The preparation of new platina-2,4,3-diazaphosphetidine-3-oxide complexes, [Pt{N(R)P(O)(Ph)NR}L2] is presented in Chapter 3. An X-ray crystal structure determination on an N,N -diphenyl derivative indicates that the four-membered metalla-cyclic ring is almost planar, the nitrogen atoms of which show sp2 hybrid character. An investigation into the insertion reactions of the N,N'-dihydrogen derivatives with dimethyl acetylenedicarboxylate resulted in the isolation of six-membered platinacyclic products, characterised by n.m.r. and i.r. spectroscopy. Attempts to prepare a similar insertion product using hexafluorobut-2-yne, led only to the formation of an organic polymeric material. The formation of a novel four-membered ring species containing a phosphin-imine functionality [Pt{SP(=NPh)(Ph)NPh}L2] is also described in Chapter 3. The phos-phinimine group was shown to have Lewis base capabilities and also underwent Wittig-type reactions with C=O and C=S containing molecules to form [Pt{SP(O)(Ph)NPh}L2] and [Pt{SP(S)(Ph)S)L2] ring systems respectively. The formation and characterisation of phosphonato, phosphato and arsonato complexes of platinum(II) are described in Chapter 4. The molecular structure of a bis(phosphine)phos- phonato complex establishes the presence of a slightly puckered four-membered ring with the phosphoryl oxygen adopting an equatorial position. The decomposition in solution of a bis(benzyldiphenylphosphine) analogue led to the formation of the di-orthometallated product cis-[Pt{C6H4CH2PPh2}2], as shown by X-ray crystallography. Chapter 5 describes the synthesis of five-membered platinacycles containing amino acid derivatives, [Pt{N(R2)-CH(R1)-C(O)O}L2]. An X-ray crystal structure determination on an [N-acetylglycinato(2-)-N,O] complex, indicated the presence of an almost planar five- membered ring that contains substantial electron delocalisation within the carboxylate and amide functionalities. The reaction of these complexes in ethanol with carbon monoxide or sulphur dioxide led to the formation of bis(ethoxycarbonyl) or bis(ethylsulphonato) metal complexes respectively. The final Chapter investigates the syntheses of several new five-membered metallacyclic species which contain metal bonds to carbon, nitrogen, oxygen or sulphur. The isolation of the metallaoxacyclopentenone ring system [Pt{CH=C(NHCOMe)C(O)O}L2], formed via the rearrangement of an initially produced [Pt{N(COMe)C(=CH2)C(O)O}L2] species, was confirmed by a single crystal X-ray diffraction study. The natures of the other complexes were examined using n.m.r. and i.r. spectroscopy.

The recalcitrance of biomass represents a major bottleneck for the efficient production of fermentable sugars from biomass. Cellulase cocktails are often only able to release 75-80% of the potential sugars from biomass and this adds to the overall costs of lignocellulosic processing. The high amounts of fresh water used in biomass processing also adds to the overall costs and environmental footprint of this process. A more sustainable approach could be the use of seawater during the process, saving the valuable fresh water for human consumption and agriculture. For such replacement to be viable, there is a need to identify salt tolerant lignocellulose-degrading enzymes. We have been prospecting for enzymes from the marine environment that attack the more recalcitrant components of lignocellulosic biomass. To achieve these ends, we have carried out selective culture enrichments using highly degraded biomass and inoculum taken from a saltmarsh. Saltmarshes are highly productive ecosystems, where most of the biomass is provided by land plants and is therefore rich in lignocellulose. Lignocellulose forms the major source of biomass to feed the large communities of heterotrophic organisms living in saltmarshes, which are likely to contain a range of microbial species specialised for the degradation of lignocellulosic biomass. We took biomass from the saltmarsh grass Spartina anglica that had been previously degraded by microbes over a 10-week period, losing 70% of its content in the process. This recalcitrant biomass was then used as the sole carbon source in a shake-flask culture inoculated with saltmarsh sediment. Cultures were grown for 8 weeks and then analysed using meta-omic approaches. Meta-genomics were used to investigate the microbial community present in the final recalcitrant biomass, while combined meta-proteomics and meta-transcriptomics were used to identify putative CAZymes (Carbohydrate active enzymes). Candidate enzymes have been cloned, heterologous expressed in E. coli and characterized according to their salt tolerance.

Municipal solid waste (MSW) is any non-industrial waste produced in households and public or commercial institutions. 3.4 billion tonnes of MSW will be produced annually by 2050, but unsustainable practices like landfilling and incineration currently dominate MSW management. The organic fraction of MSW (OMSW) typically comprises ~50% lignocellulose-rich material but is underexplored as a biomanufacturing feedstock. This thesis investigated OMSW as a feedstock for producing renewable biofuels and chemicals. Uniquely, the OMSW-derived fibre used in this project was produced via a commercial autoclave pre-treatment from a realistic and reproducible MSW mixture. The OMSW fibre was subjected to comprehensive compositional analysis and hydrolysis, and OMSW hydrolysate was analysed for sugars, metals and marker inhibitors to evaluate fermentability. Waste residues were investigated as a feedstock for biogas production. Next, the growth and productivity of eight diverse and biotechnologically useful microbial species was characterised on OMSW fibre hydrolysate supplemented with 1% yeast extract and the best candidate was further characterised and improved for industrial applications. The OMSW fibre contained a large polysaccharide fraction, comprising 38% cellulose and 4% hemicellulose. Hydrolysate of OMSW fibre was high in D-glucose and D-xylose, low in inhibitors, deficient in nitrogen and phosphate and abundant in potentially toxic metals. Hydrolysis residues contained a six-fold greater metal concentration but generated 33.4% more biomethane in anaerobic digestion compared to unhydrolysed fibre. Microbial screening identified three species that robustly and efficiently fermented OMSW fibre hydrolysate: Saccharomyces cerevisiae, Zymomonas mobilis and Rhodococcus opacus. These species could theoretically produce 139 Kg and 136 Kg of ethanol and 91 Kg of triacylglycerol (TAG) per tonne of OMSW, respectively. R. opacus had the highest fermentation productivity, concurrently using D-glucose and D-xylose and producing TAG to 72% of maximum theoretical yield. Expression of a heterologous thioesterase in R. opacus to augment lauric acid production proved unsuccessful and requires further work. Overall, this study showed that OMSW is a promising renewable feedstock for biomanufacturing. The microorganisms identified through this work grew robustly and efficiently on OMSW fibre hydrolysate and are promising candidates for developing an OMSW biorefining platform.

Plant lignocellulose is the most abundant raw material on the planet and a promising substrate for biofuel production. While this complex polymer is efficiently degraded by a range of naturally occurring microbial communities, cost- and energy-efficient industrial use is hampered by its recalcitrance to degradation. By gaining a better understanding of how microbial lignocellulose degrading communities function we may be able to improve industrial processes. In this thesis, I used a combination of ecological and evolutionary approaches to uncover the species and functional traits that drive lignocellulolytic microbial community productivity. I found that the presence of key highly active cellulolytic bacteria increased the productivity of microbial consortia. Specifically, we identified two species, Cellulomonas sp. D13 and Paenibacillus sp. A8, with a range of cellulase and hemicellulase enzymes that have potential for application in industrial processes. Experimental evolution revealed that the rate of phenotypic adaptation of a focal bacterial species, Stenotrophomonas sp. D12, to growth on wheat straw was accelerated by the presence of other competing species. The trajectory of focal species evolution was determined by both the identity and the ecological and evolutionary responses of the competing species. Genome sequencing of evolved clones suggested that genetic adaptation by the focal species to degrade wheat straw involved mutations targeting regulatory genes involved in catabolite repression and carbon storage, two systems that may represent promising targets for the improvement of industrial strains. Overall these results suggest the ecological and evolutionary approaches can be used to design and improve microbial consortia for lignocellulose bioconversion.

Shipworms are marine bivalve molluscs, known for their wood boring abilities. They use modified shells to bore into and grind wood, which is then digested. The shipworm's ability to feed on lignocellulose is dependent on the presence of endosymbiotic bacteria that live in the animal's gills inside specialized eukaryotic cells called bacteriocytes. These bacteria provide the animal with hydrolytic enzymes for wood digestion, which are translocated from the gills to the caecum, the main site of wood digestion. Unlike other lignocellulose degrading organisms, which harbour symbiotic microbes in their digestive tract, the shipworm caecum hosts only few bacteria but contains a large amount of carbohydrate active enzymes (CAZymes) of both endogenous and bacterial origin. This study investigates the anatomical, physiological and molecular basis of wood digestion in the shipworm Lyrodus pedicellatus. A combination of meta-transcriptomics, meta-proteomics and microscopic studies of the shipworm digestive organs were used, coupled with recombinant production and characterisation of some of the most expressed lignocellulolytic enzymes. This multidisciplinary analysis revealed how two structures, the food groove (a mucus stream utilised by filter feeding molluscs to transport food particles from the gills to the digestive system) and the crystalline style (a rotating structure hosted in the stomach involved in extra-cellular digestion) have been co-opted in shipworms to translocate bacteria and their enzymes from the gills to the caecum, to facilitate wood digestion. The transcriptomic and proteomic results indicate that bacterial lignocellulolytic enzymes are expressed in the gills, while the endogenous enzymes are mainly produced by the digestive glands, with complementary CAZy classes being expressed by the bacteria and the shipworms, indicating a subdivision of roles. Five bacterial CAZymes were recombinantly expressed and characterised, showing activity on cellulose, galacto-glucomannan and xylan. This study provides new insights into the mechanisms of wood digestion in shipworms, which may have biotechnological relevance.

Lignocellulose is an abundant agricultural waste product presenting an attractive renewable feedstock for ethanol production. Currently, lignocellulosic bioprocessing is economically constrained requiring expensive pretreatments, high enzyme loadings and large volumes of freshwater imposing water security concerns. Seawater is an abundant and inexpensive prospective replacement solvent. The discovery of highly active halotolerant enzymes with novel tolerances may enable seawater incorporation in bioprocessing; sustainably improving process economics and mitigating water security concerns. Salt marshes are intertidal ecosystems where lignocellulose is remineralised within the predominantly marine organic rich sediments; representing model ecosystems for targeted bioprospecting of highly active, halotolerant and inhibitor resistant enzymes. In this thesis, salt marsh carbon cycling is explored using an in situ degradation experiment. Metatranscriptomics in conjunction with metasecretome proteomics enabled the identification of the lignocellulose-associated enzymatic profile at the ecosystem level and their most likely taxonomic origin. Amplicon sequencing was conducted to reveal the abundance profile for key taxa. Investigations revealed 11,268 identifiable biomass associated proteins, 410 of which were annotated as putative carbohydrate active domains within 307 proteins. The ecosystem level profile displayed a preference for cellulose over matrix polysaccharide deconstruction. Carbohydrate esterase family 1 enzymes were highly abundant; however, lignin modifying enzymes were not, suggesting a decoupling mechanism for accessibility in lieu of oxidative modification. Fungi made no contribution to the lignocellulolytic library; the dominant lignocellulolytic producing taxa were bacteria, identified as the Proteobacteria and Bacteroidetes families Flavobacteriaceae, Alteromonadaceae, Cytophagaceae, Saccharospirillaceae, Marinilabiaceae, Flammeovirgaceae, Bacteroidaceae and Vibrionaceae and Sphingobacteriaceae. A spectrum of potential enzymes were selected for expression in a heterologous system; two enzymes were characterised in depth and both demonstrated halotolerance. This included a putative GH6; identified as an exo-ß-1,4-glucanase with significant activity on crystalline cellulose. A second protein displayed a product profile representative of a ß-1,4-glucosidase with significant transglycosylase activity on oligosaccharide substrates.

Silicon (Si) is taken up from the soil as monosilicilic acid by plant roots, transported to leaves and deposited as phytoliths, amorphous silica (SiO2) bodies, which are a key component of anti-herbivore defense in grasses. Silicon transporters have been identified in important crop species such as rice and barley, but the mechanisms behind the transport of Si remain poorly characterised in many non-crop grasses. Specifically, the extent to which Si uptake and deposition is driven by Si transporter expression remains disputed. Induction of Si defenses in response to herbivory suggests plants exhibit control over Si uptake and distribution. This thesis investigated the effects of wounding and Si addition on foliar Si concentration and deposition, and on Si transporter gene expression, in different genotypes of the economically important forage grass Festuca arundinacea, which exhibits variation in Si uptake and deposition. Following Si addition and damage, varieties differed in Si concentration, the numbers of leaf spines, and in the magnitude of the increase in Si uptake induced by damage. Some previous studies suggest trade-offs may exist between Si and carbon, but thus far such potential trade-offs have not been investigated intra-specifically, nor have there been any tests of the existence of trade-offs between different types of Si-based defenses. Trade-offs between leaf spines and phytoliths, and between Si and the key structural component, lignin, were found. This thesis presents novel findings on how Si defenses are mobilised in response to damage, how they are regulated at the level of gene expression, and how Si is deposited in different structures on the leaf surface and within cells. These findings have implications for improved understanding of plant defense and for the targeted selection of traits during breeding for sustainable crop protection.

Marginal (‘upland’) areas in the UK are enjoyed by many tourists, and provide ecosystem services of clean water, soil carbon storage and agricultural produce (breeding livestock, meat and wool). However, poor financial return from the agricultural land puts it at risk of abandonment. Hay meadows in marginal areas are prized for their biodiversity, which is maintained by their traditional, low-input management including sheep production. If their management was abandoned, their biodiversity would fall. This thesis examined if bioenergy production (by anaerobic digestion (AD)) could provide a sufficiently high income compared to sheep farming, to prevent total abandonment. In addition to encouraging biodiversity, this could mitigate climate change by reducing fertiliser input and producing renewable energy. In marginal areas in northern England, fresh vegetation from five hay fields (not receiving inorganic fertiliser) was compared to five silage fields (receiving inorganic fertiliser), because grass silage is commonly used in AD. Biodiversity; biomass yield; biomethane production by laboratory AD; greenhouse gas (GHG) emissions; and financial returns from sheep farming or using the silage/hay crop in AD were measured. Compared to silage fields, the hay fields had greater biodiversity, lower GHG emissions but similar biomethane production. And hay was more financially viable in AD than silage, partly due to lower cost. However, in order to make a similar profit per ha to that achieved by sheep production, a farmer would have to sell their hay to a nearby AD plant (such as a dairy farm AD), rather than form a co-operative AD. Bioenergy is commonly perceived as an ‘enemy’ of biodiversity, but my work shows that bioenergy and biodiversity could co-exist, through AD of hay.

The declining reserves of fossil fuel twined with an increasing concern about the environmental consequences of burning these fuels and rising carbon dioxide levels, means that a more sustainable replacement is required. Lignocellulosic biomass is an attractive candidate that has been shown to be the best sustainable alternative source to produce bioethanol for liquid transportation fuels. It has enormous availability, is renewable and cost-effective. As an agricultural residue, it does not compete with food production. However, lignocellulosic biomass of plant cell walls is composed mainly of cellulose, hemicellulose and lignin, which are extremely resistant to digestion. Converting this biomass to useful products of fermentable sugars for bioethanol production has met with little success as harsh pretreatment and costly enzyme applications are required. An arsenal of enzymes and a synergistic mechanism are required to deconstruct recalcitrant lignocellulosic biomass for an efficient production of lignocellulosic bioethanol. To achieve this goal, this study used transcriptomic and proteomic approaches with the objective of identifying new genes and enzymes involved in lignocellulose degradation. This revealed that the only one AA10 of Cellulomonas fimi was among the highest enzymes identified during the degradation of cellulose. Another other 20 hypothetical proteins co-expressed with CAZymes have been identified including a potentially exclusively new C. fimi β-glucosidase (PKDP1) that contains a PKD-domain and oxidoreductase predicted function of PQQ-domain. A naturally mutagenized C. fimi population also was screened from an adaptive evolution experiment involving exposure to a wheat straw environment. One of the strains in the adaptive population (Strain-6) showed a higher association with wheat straw biomass, which may be an indication of the strategy that being used by the adapted strain to tackle obstinate substrates to sustain growth. These results show many new enzymes would be revealed from the C. fimi repertoire in order to have a better enzymatic cocktails for lignocellulose breakdown. For the future, this encourages a deeper understanding of lignocellulose deconstruction mechanisms by an orchestra of multiple enzymes in a bacterial system.

The filamentous fungus Aspergillus niger is a common mould, and has risen to worldwide impact through industrial fermentation that is the chief source of the world’s citric acid. As well as the high market value chemical citric acid, A. niger fermentation supplies an array of enzymes of biotechnological importance. Industrial fermentation processes continue to be dependent on sucrose-based feed-stocks, and rising energy costs and tightening resources are prompting a shift to more sustainable methods. A. niger fermentation is also a promising platform for the sustainable production of many chemicals that could help solve coming world challenges. A. niger has within it the metabolic potential to achieve these goals, but these can only be realised with more efficient strain development techniques. This project built on the recent systems biology studies of A. niger to create in silico tools that guide rational engineering strategies. A dynamic metabolic model, relevant to the industrial setting of batch fermentation, of A. niger organic acid production was developed and shown to accurately capture physiological characteristics. An empirical characterisation of organic acid fermentation by the wild-type ATCC1015 strain was used to inform the model, and revealed new findings. The onset of citric acid production coincided with a shift to phosphate-limited growth, caused by a rapid phosphate uptake and storage of phosphate as polyphosphate. The role of polyphosphate in organic acid fermentation has not previously been described. The dynamic model was probed by the engineering of seven targets. To better inform further engineering, the genome-scale metabolic network of A. niger was updated and made specific to the ATCC1015 strain, the parent of citric acid producing strains. Finally, a genetic algorithm was developed for in silico evolution of A. niger organic acid production, and applied to suggest strategies for optimising the production of different organic acids.

The production of second generation biofuels from agricultural residues is an attractive alternative to the use of conventional first generation feedstocks, which are also important food resources. However, these alternative feedstocks predominately consist of lignocellulose, the main structural component of the plant cell wall, and expensive physicochemical and enzymatic pre-treatments are required before fermentation into biofuel. Therefore, the discovery of novel enzymes capable of deconstructing lignocellulose, in conditions that would be amenable to industry, is an important goal. The work, presented in this thesis, has explored the degradation of lignocellulose by a community of composting microbes, enriched for growth on wheat straw. Culturable members of the community, were isolated and assessed for their enzymatic activities towards lignin, cellulose and xylan. From these studies, a promising Ascomycota was identified, Graphium sp., which was capable of utilising both crystalline cellulose and xylan as carbon sources for growth. Transcriptomic studies were performed on Graphium sp. with and without wheat straw present, representing the first molecular information generated from an organism of this genus. From this 680 putative proteins were annotated as containing carbohydrate active domains. Proteomics added further depth to the analysis, with investigations focused on secreted proteins both located in the culture supernatant and bound to the insoluble lignocellulose substrate. Six secreted proteins were identified as targets for further analysis, and three of these were successfully isolated either from the native host, or a heterologous system. This included a lytic polysaccharide monooxygenase that appeared active on both chitin and cellulose, and a GH7 whose activity on cellulose was demonstrated. An intriguing protein, which showed low homology to a dioxygenase, was also expressed, though its role in the lignocellulose degrading environment has yet to be established.

Second generation biofuels are seen as a sustainable solution to the problem of dwindling fossil fuels stocks. However, the process of converting lignocellulosic biomass to sugars for fermentation is expensive due the recalcitrance of these materials to enzymatic digestion. The identification of quantitative trait loci (QTL) in the model grass species Brachypodium distachyon was undertaken in order to improve our understanding of genes that affect straw digestibility. Initially, the study focused on the analysis of natural accessions to determine if there was variation, in terms of digestibility and cell wall composition, within the species and to identify lines suitable for producing recombinant inbred lines (RILs). This information was successfully used to initiate the production of a RIL population that can be used in future research. I made use of a pre-existing RIL population produced previously from a bi-parental cross between Bd21 and Bd3.1 to study pathogen resistance. This RIL population was screened for straw digestibility using a semi-automated robotic platform. This data together with the genotype data was used to identify QTL linked to digestibility. A single QTL was detected on chromosome 5 together with a further QTL on chromosome 3 that acted in epistasis. A candidate gene for each of the QTLs was identified by reviewing those located within the QTL regions. The chromosome 5 candidate gene encodes a glycosyl hydrolase family 43 family protein likely involved in xylan biosynthesis and the chromosome 3 candidate gene is a cellulose synthase-like subfamily A protein that has a possible glucomannan 4-beta-mannosyltransferase function. Functionality was analysed by studying the cell wall composition of selected RILs and corresponding Arabidopsis thaliana T-DNA lines to determine any differences in the secondary cell wall structure. The results indicated that the differences in digestibility are associated with subtle differences in cell wall composition.

The dwindling reserves of fossil fuels, coupled with the environmental consequences of burning these fuels, mean that a more sustainable alternative is required. Bioethanol produced from lignocellulosic biomass is an attractive candidate for the replacement of liquid transportation fuels. Lignocellulosic biomass is composed of plant cell walls, which are extremely resistant to digestion. Converting this biomass to fermentable sugars for bioethanol production therefore requires energetic pretreatment and expensive enzyme applications. To make lignocellulosic bioethanol a commercial reality, the conversion efficiency needs to be improved and one approach of doing so is to produce crops that are more susceptible to hydrolysis. To this end, this study used a forward genetic approach with the objective of identifying genes that affect the digestibility of plant biomass. A chemically mutagenised population of the model grass Brachypodium distachyon was screened for improved saccharification with industrial cellulases. This revealed 12 mutant lines with heritable increases in saccharification. Characterisation of these 12 mutants revealed a range of different alterations in cell wall composition. Interestingly, a number of the mutant lines showed no change in lignin content, which is thought to be the major contributor to cell wall recalcitrance. These results show that saccharification can be significantly improved through a number of distinct modifications of the cell wall, giving the potential for combining more than one of these modifications in biofuel crops to obtain even higher ethanol yields. Furthermore, the mutations seem to have little effect on plant growth, development or stem strength, important traits for crop field performance. Candidate causal mutations of three of the high saccharification mutants have been identified and characterisation of the mutated genes has begun. In the long term, this will enable subsequent examination of orthologous genes in the relevant cereal and grass crops used for biofuel production.

Ionic liquids as ‘green solvents’ are becoming ever more important in a wide variety of chemical applications. Certain ionic liquids, based on the imidazolium cation in particular, have been shown to be suitable for solubilising lignocellulosic materials, such as woods, rapid growing grasses and waste agricultural products, which do not interfere with food based crops. For these reasons, from waste or unneeded biomass materials, useful products can be produced, which have fewer ethical and economical issues. The major difficulty with exploiting lignocellulosic materials is the pre-treatment step, to be able to access the chemical products within biomass. Current methods of pre-treatment to produce biofuels involve concentrated corrosive acids or high temperature and pressure reactors (steam explosion), both of which are environmentally unfriendly. Ionic liquids can be designed to be environmentally friendly, as well as potential biodegradability, depending on the choice of the ions used in synthesis. This work has successfully shown the use of diethanolammonium chloride, (which is cheap and biodegradable), to pre-treat miscanthus with a 12 times improvement of sugar release compared to 4 times improvement with dilute acid. Similar improvements have been observed with poplar biomass. Lignin removal has been identified as the major benefit in this IL pre-treatment, while hemicellulose removal also played a role. Surface morphology is a factor but cellulose crystallinity was not determined to be a factor with this ionic liquid. A yield of 30% bio-ethanol was achieved with one step saccharification and fermentation but considerable improvements are required for the process to be more efficient.

Transportation accounts for a large proportion of global CO2 emissions, estimated at 22% of all anthropogenically produced CO2 in 2010, with most of this coming from the use of liquid transportation fuels. Second generation biofuels offer a sustainable opportunity to decarbonise this sector of global energy use. To realise the potential of second generation biofuels more efficient methods of deconstructing their lignocellulosic starting material must be developed. Understanding how the marine wood borer, Limnoria quadripunctata, is able to do this biochemically without the assistance of microbial symbionts may inspire new techniques to achieve this in an industrial setting. Whilst the anatomy of Limnoria has been well studied, and the transcriptome of its hepatopancreas (HP), a secretory organ of the digestive system, has been previously elucidated, little is known about how the animal is able to derive nutrients from its diet of wood without the aid of microbial symbionts. In this work a proteomic study of the HP and gut tissue of Limnoria was carried out and the data generated analysed in concert with that of the HP transcriptome to identify proteins which may be involved in lignocellulose digestion in the animal. In this way the glycosyl hydrolases, hemocyanins (HCs), ferritins and leucine rich repeat proteins were identified as having the potential to be involved in lignocellulose digestion. Work was undertaken to characterise the function of HC proteins in Limnoria. Reverse transcriptase quantitative polymerase chain reaction analysis showed all five Limnoria HC genes to be solely expressed in the HP, whilst Western blot and proteomic examination showed HC protein to be present in the HP and gut, as well as the rest of the body. This indicates that HCs are transported post translation to the gut, the site where ingested wood particles are segregated and presumably digested. In vivo experiments suggested that a phenoloxidase enzyme activity was associated with the production of peroxides in the gut of Limnoria. Using a soluble extract of Limnoria tissue a di-phenoloxidase (DPO) enzyme activity was demonstrated which it was possible to inhibit using a phenoloxidase inhibitor. Mass spectrometry analysis implicated HC protein as the species responsible for this activity. Multiple sequence alignment showed that the Limnoria HC sequences possess the conserved features associated with the ability to carry out a DPO enzyme activity described in HC proteins from other arthropod species. Attempts were made to heterologously express Limnoria HC proteins in a number of systems with limited success being achieved using the ArcticExpress strain of Escherichia coli. Partially pure heterologous protein produced in this way was able to show a DPO enzyme activity which was inhibited by a phenoloxidase inhibitor. Mass spectrometry analysis implicates HC as the protein species responsible for this activity. Based on the findings of this work it seems possible that the hemocyanin proteins of Limnoria aid the animal in digesting wood by contributing to the formation of reactive peroxide compounds in its gut which attack lignocellulose. This process may also contribute towards the sterility of the gut by creating an inhospitable environment for microbial colonisation. Further work is now required to determine the mechanisms by which it occurs.

A need to reduce anthropogenic carbon emissions has prompted a trend for industry to switch from fossil to biorenewable feedstock, but it remains unclear if this is always a ‘low carbon’ decision. The concept that biomaterials can have lower greenhouse gas (GHG) emissions than petrochemicals has been grasped enthusiastically, however one aspect in particular has been somewhat overlooked when considering life time emissions; their disposal. Low carbon waste management is not a new concept, but its application to waste streams with high bio content is not well understood. This thesis employs mixed methods to investigate the impact of end of life scenarios on the GHG emissions associated with biorenewable materials. A hybrid life cycle assessment (LCA) of a biomaterial and petrochemical product shows that end of life scenarios have a bigger impact on overall GHG emissions for waste biomaterials than those based on petrochemicals and shows that biomaterials can be lower carbon if disposal is taken into account. In order to understand how such benefits from biomaterials can be realised, fourteen interviews with biomaterial industry stakeholders were then conducted and provided insights from which policy options to promote low carbon disposal are developed. A focus group with nine experts considered these options and made recommendations to raise the profile of disposal via encouraging product purity, stimulating demand and updating collection infrastructure. One other recommendation was to provide more transparency regarding the benefits of particular disposal options on specific biomaterials and in order to help with this the final part of this thesis is devoted to the development of a low carbon decision support tool for biomaterial disposal options based on LCA and tested on two hemp biorefineries. This tool was used to rank all the disposal options according to GHG emissions as well as cost effectiveness, particularly useful in locations where preferred strategies may not be available for example where there is no district heating infrastructure to support Combined Heat and Power (CHP). Its results confirm the waste hierarchy but also shows novel technologies such as ‘ethanol from waste’ are can be both low carbon and economically competitive. This tool can both help biorefinery operators to design low carbon disposal options into their products, as well as help guide waste and biomaterial policy decisions. The tool suggests that existing UK disposal infrastructure for municipal solid waste streams is not designed with biomaterial waste in mind, and that a rethink in waste disposal and its funding may be required to ensure future bio-based economies achieve better reductions in carbon emissions.