The feasibility of using brown seaweed, Laminaria digitata, as feedstock for generating bioenergy and biomaterials

PhD ThesisThe societal need to develop sustainable renewable energy sources has seen a recent increase in the amount of research on anaerobic digestion technologies. Biofuels from algae, known as third generation biofuels, are taking a lead interest in this regard. The characteristics of the biopolymer components of seaweed, particularly brown algae, make it suitable for methanogenic digestion, and brings advantages over other biofuel feedstocks which displace terrestrial food crops from agricultural production. This thesis investigates the feasibility of using brown seaweed, Laminaria digitata (LD), as a viable feedstock for continuous generation of bioenergy (methane) via the anaerobic digestion process, and biomaterial production from thermochemical processes. Results of methane yield from an initial bio-methane potential (BMP) assessment, using a modified BMP method, on pre-treated and dried samples gave yields of between 141 ± 5.77 mL CH4 gVS-1 and 207 ± 0.07 mL CH4 gVS-1. Analysis of the thermochemical properties of the seaweed by pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) identified sixty-four compounds present in all samples, twenty which have been previously reported as major pyrolysis products of Laminaria digitata. Proton Nuclear Magnetic Resonance (1H NMR) analysis of extracted sodium alginate (biomaterial) fraction, gave results in agreement with reported literature on mono and diad frequencies, homopolymeric mannuronic FM (0.36 - 0.46) (FMM = 0.33 - 0.47 ), guluronic (0.54 - 0.64) (FGG =0.19 - 0.25) blocks with alternating block fractions of (FGM =0.17 - 0.21) and (FMG = 0.17 - 0.21). The M/G ratio obtained (1.18 - 1.79) is an indication that the alginate extracted from L. digitata can be used to produce soft and elastic gels rather than brittle ones. Alginate is a major polysaccharide component of brown seaweeds which degrades to glyceraldehyde-3-phosphate and pyruvate as final products during anaerobic digestion. The triad frequencies (FGGG = 0.14 - 0.17, FMGM = 0.11 - 0.126, FGGM = FMGG = 0.05 - 0.09) and the average block lengths are (NG = 2.15 - 2.22 and NM = 2.61 - 3.85) were also evaluated. BMP studies on the effect of temperature on biogas production from L. digitata feedstock showed the trend 35 °C > 25 °C > 45 °C > 55 °C, similar results being found in continuous fermentations, with mesophilic (35 °C) reactors giving better 4 cumulative methane yield than thermophilic (55 °C) reactors. Optimisation of the process using a multivariate technique, fit and multiple regression model, showed the interaction terms for mesophilic and thermophilic reactors were the best indicators of optimal methane production compared to other terms. Research into the potential of mixed co-digestion of the L. digitata feedstock is important as it helps to overcome the limitations of using a mono-digestion feedstock of L. digitata, such as high hydrogen sulphide production, limited availability of L. digitata biomass, and seasonal variation in algal composition. Mono- and co-digestion of L. digitata (LD) with a stimulated food waste (SFW) were assessed using various mix ratios LD100:0%, LD90:10%, LD75:25%, LD50:50%. BMP results showed the co-digested mix ratios exhibited both antagonist (LD90:10%) and synergetic (LD75:25%) effects. In the continuous study, the mono-digestion of LD100:0% was characterized by an accumulation of high total volatile fatty acids (tVFA) concentrations, reduced pH, and an increased FOS: TAC ratio, when the organic loading rate (OLR) was increased, leading to reactor failure. It was proposed that co-digestion brought about the dilution of inhibitory compounds, faster acclimatization of microorganisms to high salinity (chloride) levels in the presence of low ammonia concentrations at high loading rate. Trace element supplementation (TES) during anaerobic digestion of the macroalgae feedstock in various mix ratios: control (TES 0), TES 1 (0.1 mg/l Se, 0.1 mg/l W), TES 2 (0.1 mg/l Se, 0.1 mg/l W, 0.5 mg/l Co, 0.1 mg/l Mo), TES 3 (0.1 mg/l Se, 0.1 mg/l W, 0.5 mg/l Co, 0.1 mg/l Mo, 0.5 mg/l Ni, 0.05 mg/l Cu) and TES 4 (0.1 mg/l Se, 0.1 mg/l W, 0.5 mg/l Co, 0.1 mg/l Mo, 0.5 mg/l Ni, 0.05 mg/l Cu, 0.5 mg/l Fe, 0.1 mg/l Zn) in batch reactors improved methane yield by 17% - 50%, and stimulated a steady digestion process in a continuous reactor when added weekly with increase in OLR compared to a reactor without trace element which led to reactor instability, and eventually failure.Tertiary Education Trust Fund (TEFUND) Nigeria and the University of Port Harcourt, Choba, Nigeri

Characterisation of biocatalyst production within an integrated biorefinery context

With the emerging interest in integrated biorefinery concepts, there is a need to identify and develop profitable product streams and ensure the utilisation of as many waste streams as possible. Early stage bioprocess development for these processes can be facilitated by the use of high throughput bioreactor platforms that enables rapid, quantitative and scalable data acquisition. This thesis aims to establish high throughput methodologies for the production and characterisation of industrial biocatalysts within an integrated biorefinery context. Specifically, the work focuses on the production of the CV2025 ω-Transaminase (CV2025 ω-TAm) in Escherichia coli BL21 (DE3) using sugar beet vinasse, a bioethanol waste stream, as a fermentation feedstock. The high throughput platform to be explored is a 24-well, controlled microbioreactor (MBR) that provides individual monitoring and control of process parameters at the well level. Initially, batch E. coli BL21 (DE3) fermentations expressing CV2025 ω-TAm were established in the controlled MBR using a synthetic medium to provide benchmark data on cell growth and enzyme expression. These cultures indicated a good degree of monitoring and control with respect to process parameters as well as culture reproducibility across the wells. Significant enhancements in relation to maximum biomass concentration (Xmax), yield of biomass on substrate (YX/S) and CV2025 ω-TAm specific activity of 3.7, 1.9 and 2.2-fold, respectively, were shown in the MBR compared to conventional shake flask system, also representing a 31-fold volumetric reduction. Optimisation of CV2025 ω-TAm production in the MBR showed that the best cell growth and enzyme titre was achieved with an early induction (6 h), 0.1 mM IPTG and 0.024 mmol IPTG gdcw-1, yielding enhancements in Xmax, YX/S and CV2025 ω-TAm specific activity of 1.04, 1.2 and 1.4-fold, respectively over the non-optimised cultures. Control of dissolved oxygen (DO) levels between 30 - 50% oxygen saturation had no significant impact on cell growth and CV2025 ω-TAm titre. Evaluation of vinasse as a fermentation feedstock for CV2025 ω-TAm production has led to several novel findings. Characterisation of vinasse showed that the feedstock comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 (DE3) cell growth and CV2025 ω-TAm production were feasible in cultures using 17 to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The D-galactose in vinasse was shown to facilitate auto-induction of the pQR801 plasmid leading to comparable CV2025 ω-TAm expression as obtained in IPTG-induced cultures. Assessment of different vinasse pre-processing options confirmed the relevance of the dilution step in reducing polyphenol concentrations to below inhibitory levels. Moreover, the use of pasteurised vinasse was found to be promising for large scale applications. Further medium optimisation studies in the MBR showed the benefit of supplementing vinasse with specific media components. Supplementation of diluted vinasse medium with 10 g L-1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, Xmax, CV2025 ω-TAm volumetric and specific activity, respectively, over those achieved in non-supplemented cultures. Additionally, the CV2025 ω-TAm titre attained here represented 81% of that obtained using an optimised synthetic medium. Investigation into the metabolic preferences of E. coli BL21 (DE3) when grown on a complex carbon source like vinasse showed the sequential metabolism of D-mannitol before glycerol utilisation, which was followed by the simultaneous metabolism of glycerol, D-xylitol, D-dulcitol and acetate thereafter. Finally, scale-up of the optimal conditions for CV2025 ω-TAm production using both synthetic and vinasse-based media, from the controlled MBR to a 7.5 L stirred tank reactor (STR) was shown based on matched kLa values and specific aeration rates. Results showed a good reproducibility with respect to cell growth, substrate consumption and CV2025 ω-TAm production between the scales, representing a 769-fold volumetric scale translation. The feasibility of further intensification of CV2025 ω-TAm production in STR at higher kLa values using both synthetic and vinasse-based media was also demonstrated leading to enhancements of 1.4 and 1.9-fold in enzyme titre, respectively. Overall, this work has established high throughput methodologies for the characterisation, optimisation and scale-up of industrial biocatalyst production. The approach was demonstrated within the context of an integrated sugar beet biorefinery. However, the utility of the high throughput approach is considered generally applicable across the industrial biotechnology sector

An investigation of the isolation, characterisation and application of hydantoinases for the industrial production of amino acids

This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids

Biomethane production from macroalgae

Irish brown seaweeds have been identified as a potential bio-resource with potentially high specific methane yields. Anaerobic digestion is deemed the most feasible technology due to its commercial viability for handling such wet feedstock. However, the biomethane potential of seaweed is highly dependent on its chemical composition which can vary by species type, cultivation method, and time of harvest. This study aims to investigate and optimize the process for the production of biomethane from Irish brown seaweeds focusing on the key technology bottlenecks including for seaweed characterization, biomethane potential assessment, optimization of long-term anaerobic digestion and suitable pre-treatment technologies to enhance potential gas yields. Laminaria digitata and Ascophyllum nodosum were tested for seasonal variation. From the characterization and batch digestion of L. digitata, August was found to be the optimal month for harvest due to high organic matter content, low level of ash and ultimately highest biomethane yield. The specific methane yield of 53 m3 CH4 t-1 wwt in August was 4.5 times higher than the yield in December (12 m3 CH4 t-1 wwt), with ash content the key factor in seasonal variation. For A. nodosum, the optimal harvest month was October with polyphenol content found to be a more influential factor than ash. The gross energy yields from both species were evaluated in the range of 116-200 GJ ha-1 yr-1. Continuous digestion trials were subsequently designed for S. latissima and L. digitata to optimize the key digestion parameters. Results from mono-digestion and co-digestion with dairy slurry revealed that both seaweeds could be digested at maximum biomethane efficiency to a loading rate of 4 kg VS m-3 d-1. Accumulation of salt in the digesters was a concern for long term digestion and it was reasoned that suitable pretreatment may be required prior to digestion. Various pre-treatments were subsequently tested on L. digitata to enhance the gas yield. It was found that maceration after hot water washing yielded 25% more specific methane and up to 54% salt removal as compared to untreated L. digitata. The experiments undertaken aim to assist in providing a basic guideline for feasible design and operation of seaweed digesters in Ireland

Microbial Production of Bio-Based Chemicals: A Biorefinery Perspective

A shift from fossil- to renewable biomass feedstock for the emerging bio-based economy requires the development and adoption of new sustainable technologies that are more suited for transformation of biomass components to chemicals, materials and energy. This thesis presents investigations on the development of processes based on industrial biotechnology as a key element for the production of chemicals from agro-/industrial by-products. The chemicals of interest are the ones that could potentially serve as building blocks, platforms, for other chemicals and polymers. Glycerol, a by-product of biodiesel production, was used as raw material for the production of propionic acid, 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP), while methacrylic acid (MA) was produced from 2-methyl-1,3-propanediol, a by-product of butanediol production. Different strategies to overcome the bottlenecks such as product inhibition existing in the bioprocesses for production of the chemicals were studied. Fermentation of glycerol to propionic acid was studied using Propionibacterium acidipropionici. High cell density cultivations were used to overcome the low production rate caused by slow microbial growth and product-mediated toxicity. Increasing the cell density by immobilization and sequential batch recycling improved the production rates by 2- and 6-fold, respectively, over that obtained using conventional batch fermentation. Potato juice, a by-product of potato starch processing, was shown to be a promising, inexpensive nitrogen/vitamin source for the growth of the organism and propionic acid production. Lactobacillus reuteri was employed as a whole cell biocatalyst for the conversion of glycerol to 3HPA and 3HP in aqueous solution. Production of 3HPA using glycerol dehydratase activity of the cells, limited by substrate inhibition and product toxicity, was performed in a fed-batch mode with in situ complexation of the hydroxyaldehyde with bisulfite, and subsequent removal through binding to an anion exchanger. This resulted in increase in production of 3HPA from 0.45 g/g biocatalyst in a batch process to 5.4 g/g. 3HP is formed as an oxidation product of 3HPA, however its accumulation as a product of glycerol metabolism in wild-type L. reuteri has not been reported earlier. The metabolic fluxes through the glycerol reductive and oxidative pathways were calculated using variable volume fed-batch operation. The glycerol feeding strategies were optimized to yield complete conversion of 3HPA into equimolar mixture of 3HP and 1,3PDO, the products that can be easily separated from each other. MA was quantitatively produced at high purity from 2-methyl-1,3-propanediol by a novel process involving integrated biological and chemical catalysis. Whole resting cells of Gluconobacter oxydans were used for selective oxidation of the substrate to the corresponding hydroxycarboxylic acid, which upon dehydration over TiO2 at 210 degree Celsius yielded MA. This process offers a potential, significantly greener alternative to the acetone-cyanohydrin process used for MA production, involving highly toxic substrates, large amounts of waste and greenhouse gas emissions

Novozym 435 : the “perfect” lipase immobilized biocatalyst?

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by polyIJmethyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the most widely used commercial biocatalyst in both academy and industry. Here, we review some of the success stories of N435 (in chemistry, energy and lipid manipulation), but we focus on some of the problems that the use of this biocatalyst may generate. Some of these problems are just based on the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. Finally, some solutions (N435 coating with silicone, enzyme physical or chemical crosslinking, and use of alternative supports) are proposed. However, the N435 history, even with these problems, may continue in the coming future due to its very good properties if some simpler alternative biocatalysts are not developed

An NADH dependent reductase for isolated enzyme and whole cell catalysis.

Isolated enzymes and whole cell biocatalysts can both be applied for the synthesis of chiral hydroxy compounds. It is hypothesized that whole cells can easily be employed for such reactions using simple technology which is robust. This is because whole cells contain all the necessary enzymes and metabolic pathways for cofactor regeneration. This also means that the enzymes and their cofactors are well-protected within their natural cell environment. In contrast, it is hypothesized that isolated enzymes require complicated and expensive purification procedures. They also require the stoichiometric addition of cofactors (or methods employed for their regeneration), and are susceptible to inactivation since they are isolated from their natural cell environment. The aim of this thesis was to systematically compare a whole cell biocatalyst (Trichosporon capitatum (MY 1890)) and an NADH dependent isolated reductase (tetralone reductase) in the synthesis of a chiral alcohol (6-bromo-P-tetralol). The comparison was carried out to ascertain which type biocatalyst is preferred, and also to establish whether the general hypotheses (as stated above) are true with respect to each biocatalyst. Comparison of the isolated enzyme and whole cell biocatalyst showed that there were significant differences with respect to each of the systems. These included differences in: the biocatalytic purity, the reaction methodology, the system efficiency, and the effects on the biocatalyst from the addition of substrate and solvent. The isolated enzyme methods were much more complicated than the whole cell methods, from the preparation of the isolated enzyme through to the bioreduction. This was because a novel protein purification process needed to be set up and a cofactor regeneration system was required. However, the isolated enzyme system showed higher substrate conversions than the whole cell system. At 1 g/L, a conversion of 86% after 420min was achieved, whereas the whole cell system exhibited a conversion of 79% after 450min. It was hypothesized that the whole cell system suffered from lower conversions due to the substrate and product accumulating inside the cell membrane and disrupting cell metabolism. In the same configuration, the whole cell system also suffered from lower reaction rates which were attributed to mass transfer limitations through the cell membrane. The addition of a solvent enhanced whole cell biocatalytic reaction rates, but only at low substrate concentrations. The isolated enzyme system was susceptible to inactivation, and increased solvent concentrations caused a detrimental affect on the reaction rates and conversions. This was most likely due to the solvent causing an irreversible change in the active site conformation. The similarities and differences of employing an NADH dependent reductase and a whole cell biocatalyst for the production of a chiral alcohol are discussed in this thesis

Enhancing biomethane production from anaerobic digestion of Sargassum muticum

Higher methane yields from the anaerobic digestion (AD) of Sargassum muticum could improve the process energy balance and make its use during AD more economical and energetically favourable. Previous research showed that after 28 days of AD, methane yields from S. muticum were low (17% of the theoretical yield). This thesis aims to identify the causes of the low methane yield. Biochemical methane potential tests of freshly harvested, rinsed, and freeze-dried (FD) spring and summer S. muticum sampled over three years delivered yields of 27–39% and 24–32% of the theoretical, respectively. FD samples were extracted with water or with 70% (v/v) aqueous methanol (MeOH); methane yields per gram volatile solids of the extracted samples were higher than the untreated FD samples by up to 19.1% and 26.6%, respectively. Proximate, ultimate, and biochemical analyses showed that untreated FD biomass contained ash contents of 24.2–28.1% dry weight, with total dietary fibre representing 49.8– 67.4% of the organic fraction. Water- or MeOH-extracted spring and summer biomass were higher in total dietary fibre content (75.3–82.8% of the organic fraction) and lower in soluble dietary fibre (SDF) and phenolic content than the FD samples. Indices calculated for bioconversion of the biomass to methane were negatively correlated with the SDF and phenolic contents. Water-extracted spring biomass had lower SDF content than the FD and water- extracted summer samples and produced higher methane yields. The aqueous MeOH extract of S. muticum was examined after repeated extraction (×9) with 1% polyvinylpolypyrrolidone (PVPP). The PVPP-treated extract was 93.7% lower in phenolic content (Folin-Ciocalteu assay) and 24.4% higher in protein content than the untreated extract, with no significant difference in the lipid contents. MeOH-extracted biomass combined with the PVPP-treated extract produced 85.7% higher methane yields than when combined with untreated MeOH extracts. Membrane filtration of the untreated MeOH extract yielded a high molecular weight (MW) (≥ 5 kDa) fraction, which contained 90.7% of the total phenolic content of the extract. It inhibited methane yields by 41.9% when combined with MeOH-extracted biomass; however, since the methane yields of the extracted biomass remained low (≤ 32% of the theoretical, an increase from ≤ 27%), recalcitrance of the total dietary fibre also represents a limiting factor. Improving methane conversion of the residual fibre fraction after phenolics’ extraction is required to utilise this biomass in a biorefinery approach efficiently

Optimisation of biogas generation from brown seaweed residues: compositional and geographical parameters affecting the viability of a biorefinery concept

Very recently, integrated biorefinery approaches are being developed with the aim to produce high-value products for a variety of industries in conjunction with green energy from sustainable biomass. Macroalgae (seaweed) have been regarded as more sustainable compared to terrestrial crops, since they do not occupy land for growth. Macroalgal biomass changes greatly according to species and harvest season, which affects its chemical energy potential. This study was conducted seasonally on five species of brown seaweed over a yearlong period to investigate the effects of chemical composition variations, bioproducts extraction processes and inoculum acclimatation on methane production. As a result of the bioproducts extraction, it was found the seaweed residues exhibit a great potential to produce methane. Stoichiometric methane yield and C:N ratio changed in favour of an improved digestibility with bioconversion rates greater than 70% in some instances, i.e. achieved by Laminaria species and on the West coast Fucus serratus. The two Laminaria species investigated also presented the highest CH4 production rate, with Laminaria digitata reaching 523 mL CH4 gVS-1 and L. saccharina peaking at 535 mL CH4 gVS-1 with acclimatised and non-acclimatised sludge respectively