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Membrane tension profiles revealed cell edges uncoupling during Glioblastoma linear motility
No full textGlioblastoma cells exploit blood vessels and neuronal axons to invade the brain. At the microscale level, the geometry and confinement of these physiological tracks impact cell mechanics, particularly the plasma membrane tension and, in turn, cell migration. In the past, several studies widely explored the participation of plasma membrane tension in cellular physiological processes, such as cell spreading, polarisation, phagocytosis, and membrane trafficking, emphasising tension contribution in counteracting actin cytoskeleton remodelling. However, the role of membrane tension as a mechanical regulator of cell motility is controversial, and the scientific community still discusses whether it can regulate cell behaviour locally or globally. Discrepancies may arise from the use of different cell types and tools for probing membrane tension. Various membrane-pulling assays have been used to investigate membrane tension, and variation has been recorded during cell front protrusion. Unfortunately, current approaches are unsuitable for studying membrane tension evolution at the whole cell level. Moreover, despite the impact of cholesterol on plasma membrane tension being well consolidated, a significant number of studies focused on lipid metabolism to curb Glioblastoma progression, disregarding the mechanical effect on Glioblastoma cell invasion. This PhD project proposed a novel in-vitro vessel-mimicking approach for studying membrane tension's spatiotemporal evolution in invasive Glioblastoma cells. A fluorescent membrane dye (FLIPPER-TR) has been used to record membrane tension's evolution by analysing the fluorescence decay along the whole cell. Deep-UV micropatterning has been used as a vessel-mimicking approach to create linear cues with micro-scaled dimensionalities, similar to brain capillaries, and resemble brain vasculature's geometry and constrain. Cell-instructive patterned surfaces were coated with laminin, the most representative matrix protein of the abluminal surface of brain vasculature, to mimic the perivascular niche composition. Combining the micropatterning technique with the Fluorescence-Lifetime Imaging Microscopy (FLIM) of FLIPPER-TR probe, we have integrated subcellular tension variations with global cell motility. Results showed different membrane tension patterns at the cell front and rear, separated by a low-tension zone, which buffers tension transmission and sustains the independent behaviour of the two edges. Furthermore, the localisation and redistribution of known mechano-sensors (CAV-1 and Class I-Myosins) during linear motility confirmed differences in tension profiles. We found that the membrane tension profiles of the two cell edges are not synchronised during Glioblastoma linear migration, reflecting their uncoupled dynamics. Interestingly, cholesterol content inversely correlated with the motile behaviour of Glioblastoma cell lines; fast-motile cell lines showed higher cholesterol content than non-motile ones. Moreover, cholesterol clusters distributed differently between the cell front and rear of migrating Glioblastoma cells, matching their cell edges' tension profiles. This result highlighted a putative role of cholesterol clusters in creating tension differentials as insulators of membrane tension propagation between different cell regions. These findings pave the way to reconsidering years of literature around actin-based mechanical processes at the cell leading edge, underestimating cholesterol-rich membrane domains' contribution to tension propagation to the cell trailing edge. This PhD thesis has added evidence on membrane tension's role in the mechanical control of cell motility and cholesterol participation in Glioblastoma invasion
Hyperthermia-induced injury and adaptation in adrenocortical carcinoma cells
No full textAdrenocortical carcinoma (ACC) is a rare and aggressive malignancy that presents with limited therapeutic options, particularly for the 50% of patients who are diagnosed at late stages and are inoperable. While hyperthermia-based approaches (e.g. thermal ablation, heated intraperitoneal chemotherapy) have demonstrated promise against various solid tumours, the precise mechanisms by which hyperthermia induces ACC cell death, the effects on steroidogenesis, and whether cells develop thermal resistance, remain unclear.
This thesis investigates the impact of hyperthermia on ACC cells using in vitro models (H295R, HAC15) alongside the non-tumorigenic endothelial cells (HUVEC). Lethal exposures (≥48°C) were found to markedly reduce ACC cell viability, primarily through caspase-independent pathways consistent with necrosis and necroptosis, rather than classical apoptosis. In contrast, sublethal exposures (42–45°C) caused minimal cell death where most ACC cells survived, regained typical morphology, and eventually resumed proliferation. These surviving cells did not manifest robust thermotolerance upon rechallenge, indicating a limited capacity for long-term hyperthermia resistance.
Sublethal hyperthermia also transiently suppressed steroidogenesis, specifically Cortisol and Aldosterone biosynthesis, immediately following hyperthermia exposure; however, cells that recovered from sublethal hyperthermia regained their functional ability to synthesise steroids. Western blotting, live-cell imaging, and flow cytometry showed the involvement of necroptosis-related proteins like mixed lineage kinase domain-like protein (MLKL), and increased intracellular calcium flux upon hyperthermia exposure. Furthermore, scanning electron microscopy revealed classic morphological changes indicative of necrotic damage at lethal temperatures, alongside profound structural alterations that subsequently normalised in sub-lethally heated cells.
Collectively, these findings provide a comprehensive evaluation of hyperthermia- induced cytotoxicity in ACC cells. By delineating the thermal thresholds that govern cell death, clarifying a dominant cell-death pathway, and assessing the functional resilience of surviving cells, this thesis sets the groundwork for refining hyperthermia- based interventions.Science Foundation Ireland under Grant number [20/US/ 3676], the National Institutes of Health Grant number [R01EB028848
Writing water justice in the twenty-first century: Environmental novels, neoliberalism, and water politics
No full textThis thesis poses drought as a literary problem. It analyses seven different twenty first-century novels which respond to drought events as a way of articulating culturally and historically specific anxieties surrounding human-created climate change and global ecological destruction. These novels are: Fabienne Bayet-Charlton’s Watershed (2005); Karen Jayes’s For the Mercy of Water (2012); Dominique Botha’s False River (2013); Claire Vaye Watkins’s Gold Fame Citrus (2015); Paolo Bacigalupi’s The Water Knife (2015); Mike McCormack’s Solar Bones (2016); and Mary Costello’s The River Capture (2019). Using ecocritical and ecofeminist approaches, I argue that these novels both require and provide a means of engaging critically with water and environmental politics. They show an emphasis on freshwater’s value to the human body, which can be connected to ecofeminist reckonings with the feminisation and subordination of the body, emotions, care work, the material world, and nature in Western culture. Moreover, these texts connect the body to larger scales of economy, ecology, and society; their representations of infrastructure failure, climate change, privatisation, dispossession, over-extraction, among a host of other water-related problems, prompt thinking about how water crises are manufactured by the historical and current disregard and exploitation of nature and people under global capitalism. Embracing the biological, sensory, political, and ecological value of freshwater, I also investigate how water-inspired formal experimentation in these novels challenges (or reinforces) the gendered human/nature and mind/body dualisms that so often dominates the novel form. The novel form has a history of privileging individual psychological development against inert environments, yet some of these texts construct narrative forms that convey human embodiment and subjectivity as collective, more-than-human, political, and contingent on environmental conditions. My contention is that freshwater itself pushes the novel form in more ecologically- and socially-just directions.2026-03-2
Software as a medical device: A scoping review of challenges in the European regulatory context and how they can be addressed with the biodesign innovation process
No full textA Research Master's Thesis submitted to the University of Galway for the award of a Master's of Science in BioInnovation.Abstract
Background: Software as a medical device (SaMD) and embedded software with SaMD-like functionality are increasingly being used in healthcare settings. This is facilitated by the rapid advancements in technology over the last decade, with innovations such as Artificial Intelligence and cloud computing driving the agenda. SaMD operates independently on general-purpose computing platforms and is used for diagnosing, treating and monitoring medical conditions, or as a decision support tool by clinicians. However, the integration of SaMD devices into healthcare settings often faces challenges, especially within the European Union’s regulatory frameworks, such as the Medical Device Regulation (MDR) and the In Vitro Diagnostic Regulation (IVDR).
Methods: This study employed two complementary review methodologies to explore the challenges associated with the development, regulation, and integration of SaMD in Europe. A narrative literature review provided a synthesis of existing knowledge on SaMD, focusing on its definition, evolution, and the overarching challenges it faces. It also aimed at outlining key themes under which these challenges would be classified in the subsequent scoping review. A scoping review was then conducted, guided by the Arksey and O’Malley’s framework. A systematic search was conducted in July 2022 for the period January 2013 to July 2024. I systematically examined peer-reviewed literature published over the last decade, to identify specific challenges related to SaMD development, deployment and adoption. Challenges were categorised into regulatory, technical, clinical, market, and ethical-legal domains.
Results: The search yielded 169 papers, with 28 meeting the inclusion criteria. The findings highlighted significant challenges across all the domains. Regulatory barriers arose from stringent regulatory requirements that may not align with the evolving nature of software innovations. Technical challenges faced by SaMD developers included challenges of ensuring data quality, model validation, and achieving interoperability with existing healthcare systems. Clinically, the major challenges lay in integrating SaMD innovations without disrupting existing workflows and patient care. There was also a need for a high degree of accuracy and reliability of these algorithms, to justify their adoption. Market challenges were a result of the high costs and complexities of navigating regulatory approval and the market entry process. Ethical and legal challenges focussed on issues such as data privacy, algorithmic bias and accountability for AI decisions.
Recommendations and Conclusion: A wide variety of challenges are faced by researchers and companies developing and marketing SaMD devices. The Biodesign Innovation Framework provides a structured needs-driven approach to addressing some of the challenges faced in developing and integrating software-based innovations into healthcare. It promotes interdisciplinary collaboration, iterative development and early stakeholder engagement. By engaging with developers within this framework, regulators can better align rules and guidelines with technological advancements. Through the same process developers can improve clinical integration strategies and stakeholders can work together to comprehensively address ethico-legal concerns through a holistic approach. Future research should focus on repeated cross-sectional studies to evaluate the impact of evolving regulations. This will help to expand stakeholder engagement to keep pace with the rapidly changing landscape of digital healthcare solutions. This approach will help to bridge gaps in the medical device innovation lifecycle, ensuring that SaMD technologies are safe and widely accepted by all stakeholders
Evaluation of the environmental performance of novel anaerobic digestion biorefineries for the development of a sustainable circular bioeconomy in Ireland
No full textThe circular bioeconomy (CBE) is a novel paradigm that seeks the integration of circularity principles into the life cycle of renewable biological resources, to help keep humanity within safe operating conditions, respecting planetary boundaries. This doctoral thesis advances state-of-the-art environmental evaluation of anaerobic digestion (AD) biorefineries as core technological platforms for the development of a CBE. The work comprises a series of distinct yet related studies. Initially, a critical evaluation of 76 peer-reviewed studies on environmental life cycle assessment (LCA) of biorefineries was conducted to establish the state-of-the-art. Inconsistent approaches to model biogenic emissions, digestate management, and product substitution (i.e., avoiding production of equivalent products in other systems) were major methodological challenges.
To overcome these limitations, a novel, comprehensive, and open-access LCI model, LCAD 2.0, was developed and applied. First, a systematic evaluation of 150 prospective AD-biorefinery configurations using cattle manure, food waste, and grass elucidated critical factors defining environmental performance and nutrient circularity, inter alia: type of feedstock, energy substitution potential, fugitive methane emissions, emissions during digestate application, and the application of carbon capture technologies. AD-biorefinery systems using organic waste as feedstocks, in particular those with high methane yield, such as food waste - offer the strongest potential for environmental mitigation, especially when considering avoided impacts from counterfactual waste management.
Then, an integrated techno-economic and environmental assessment of a biomethane plant processing food waste at industrial scale in Ireland was undertaken. This revealed significant economic challenges linked to high volatility and market fluctuations after disruptive events such as the war in Ukraine and COVID-19. The calculated levelised cost of energy was 2.9 times higher than values previously defined in 2019 and 1.6 times the benchmark established in the National Biomethane Strategy, making policy incentives crucial for financial viability. On the other hand, although environmental mitigation potential is possible, with climate mitigation up to 222 kgCO2-eq/tonne of food waste digested, this mitigation potential is contingent upon the type of energy used in the plant, proper control of biogenic emissions, and efficient digestate application according to the calendar defined by the Nitrates Directive.
Finally, six novel biorefinery configurations producing biofuel, biofertilisers, platform chemicals, and elemental carbon were assessed for environmental performance. All six evaluated configurations achieved environmental mitigation potential, with maximum climate mitigation between 340 and 542 kgCO2-eq/tonne food waste, though simpler configurations generally outperformed more complex ones due to energy-intensive purification and chemical consumption. This research contributes to the body of knowledge on AD-biorefinery sustainability, by applying comprehensive expanded boundary LCA to novel AD-biorefinery configurations. It presents LCA practitioners and researchers with a new tool to overcome methodological challenges linked to
data collection, advancing robust environmental evaluation of prospective biorefineries needed to power the CBE
Maigheotides A-C, Peptides from the Deep-Sea Black Coral Phanopathes sp.
No full textBlack corals are poorly studied with respect to their secondary metabolite chemistry. With the aim of discovering new bioactive metabolites, we collected deep-sea black corals from the Irish continental margin. Our analysis of the black coral Phanopathes sp. yielded three new peptides (maigheotides A-C). Through a combination of 2D NMR, mass spectrometry, and Marfey’s analysis, the absolute configuration of all three structures was elucidated. The maigheotides are characterized by a non-canonical amino acid ΔZ -Trp, the cyclized N-terminal Glu (pyroGlu), and C-terminal amide (maigheotide B). Similar characteristics are critical features found in neuropeptides. Screening in a number of assays has identified only weak activity, although the breadth of assays screened was limited by the small mass of peptides obtained.This work was supported by Science Foundation Ireland (SFI) and the Marine Institute under the Investigators Programme Grant No. SFI/15/1A/3100, cofunded under the European Regional Development Fund 2014- 2020, to A.L.A. along with the project NMBLI Grant-Aid Agreement PBA/MB/16/01, and the U.S. National Institutes of Health grants R56 AI154922 and R21 AT010939 to B.J.B. Subsea photographs taken by University of Galway, copyright Marine Institute, during cruise CE18012 funded under SFI/ 15/1A/3100. Alexa Parimbelli provided DNA sequences under Irish Research Council project GOIPG/2022/765. A portion of this work was performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory’s Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility, which is supported by National Science Foundation Cooperative Agreement DMR-2128556 and the State of Florida
Macromolecular crowding optimisation and application in co-culture setting for tendon regeneration
No full textTendon is a connective tissue that links bone to muscle, allowing for maintenance of skeleton posture, joint movement, energy storage and transmission of muscle force to bone. Tendon is a hypocellular and hypovascular tissue of poor self-regeneration capacity. Current surgical treatments are of limited success, frequently resulting in reinjury. Upcoming cell therapies are primarily based on tenocytes, a cell population of limited self-renewal capacity in vitro or mesenchymal stromal cells, a cell population prone to ectopic bone formation in vivo. Over the years mono- or multi- factorial cell culture technologies have failed to effectively maintain tenocyte phenotype in culture during expansion or to prime mesenchymal stromal cells towards tenogenic lineage prior to implantation. Upon these limitations the concept of co-culture was conceived. The utilisation of co-culture techniques to induce mesenchymal stromal cell lineage-specific differentiation is a compelling area of research within the field of tissue engineering. Through co-culture, functional tissue-specific cell populations can be developed for tissues of low cellularity and cells of reduced function and / or limited expansion capacity ex vivo, such as tendon.
Providing cells with relevant stimuli to support their phenotype and function through in vitro recapitulation of the in situ microenvironment with biophysical, biological and biochemical cues is of paramount importance. A strategy that has yet to be investigated in co-culture setting is macromolecular crowding, the addition of macromolecules in cell culture media that by decreasing diffusion via restriction of molecular motion, they favour protein-substrate interactions and result in enhanced and accelerated extracellular matrix deposition. In native tissue context, a continuous dynamic reciprocity between cells and their surrounding extracellular matrix maintains tissue-specific cell phenotype and/or differentiates mesenchymal stromal cells towards a tissue-specific lineage, via a finely-tuned spatiotemporal sequence of biophysical, biochemical and biological signalling cascades. This work puts forward the notion that co-culture can be significantly improved in a macromolecular crowding milieu due to the enhanced extracellular matrix present. To validate this, first an optimal (with respect to highest extracellular matrix deposition in the shortest period of time) macromolecular crowding agent that enhances and accelerates extracellular matrix deposition should be identified (objective 1, chapter 2) and then its potential in direct co-culture of mesenchymal stromal cells / tenocytes should be assessed (objective 2, chapter 3).
For objective 1, the potential of gum Arabic, gum gellan, gum karaya and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and a ready-to-use powder consisting in the combination of kappa and lambda carrageenan isoforms were used as control) were assessed. WS1 skin fibroblasts were chosen for simplicity purposes and as there was limited supply of tenocytes and mesenchymal stromal cells. Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/ml, 75 µg/ml and 100 µg/ml gum gellan; and the 500 µg/ml and 1,000 µg/ml gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrated the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential. Considering that carrageenan outperformed the assessed gums in extracellular matrix deposition, this macromolecular crowding agent, along with a Ficoll cocktail that has been used in other macromolecular crowding studies, were progressed to the second objective.
In objective 2, carrageenan and Ficoll were assessed in bone marrow mesenchymal stromal cell alone, tenocyte alone and bone marrow mesenchymal stromal cell / tenocyte direct co-cultures (no macromolecular crowding cultures were used as control). These two macromolecular crowding agents were selected to also assess whether the chemistry of the macromolecular crowding agents can affect cellular phenotype. Carrageenan increased extracellular matrix deposition without altering cell metabolic activity, proliferation and morphology. Ficoll, on the other hand, did not notably enhance extracellular matrix deposition. Subsequent biological analysis via Western blotting and lectin microarray revealed chondrogenic and osteogenic lineage commitment, but not enhanced tenogenic lineage commitment and no cell-type-specific carbohydrate, respectively.
Collectively, these data demonstrate that there are more than the physical properties of a macromolecular crowding agent to determine the rate of extracellular matrix deposition (e.g. negatively charged and polydispersed macromolecules enhance and accelerate extracellular matrix deposition but other factors intervene as size and shape of the crowder), whilst the chemical properties of a macromolecular crowding agent determine cell fate (e.g. sulphated and neutral polysaccharides induce osteogenic and/or chondrogenic lineage commitment)
Ticks, hosts, habitats, and humans: A One Health approach for investigating Lyme borreliosis risk in Ireland
No full textLyme borreliosis (or Lyme disease) is a zoonotic infection, transmitted to humans via the bite of a tick which is carrying a pathogenic genospecies of the Borrelia burgdorferi sensu lato bacterial complex. As a zoonotic disease, a One Health approach is necessary to fully understand Lyme disease risk. Furthermore, while the geographic range of both pathogen and vector is broad, differences in pathogen genospecies, vector species, habitats, and host assemblages between regions means that the drivers of Lyme borreliosis risk vary geographically. While Lyme borreliosis epidemiology, ecology, and risk may be better understood in some regions of Europe, significant knowledge gaps remain in relation to Lyme borreliosis in Ireland. These include the geographic and demographic variability in the epidemiology of Lyme borreliosis in Ireland; the effect of various host, habitat, and abiotic factors on ecological markers of disease risk; and the disease awareness and perceptions of at-risk groups.
To address these knowledge gaps, a One Health, trans-disciplinary approach to examining Lyme disease in Ireland, incorporating methods from the disciplines of epidemiology, ecology, and social science have been adopted in this study. Using meta-analytic and field study techniques, the ecological markers of disease risk (nymphal tick abundance, nymphal tick infection prevalence, density of infected nymphs) have been defined for Ireland, and discussed in the context of data obtained from an epidemiological observational analysis. This work also assessed the drivers of the above markers of disease risk. Results suggest that woodland habitat factors (such as size and type) can affect nymphal infection prevalence. We have also found that nymphal abundance (and therefore bite risk) peaks in April, has an inverse and seasonal relationship with deer activity, and an inverse relationship with robin activity. Finally, a study of individuals whose occupation or
recreational activities increase their exposure to Lyme borreliosis has generated information on risk perceptions, awareness, disease knowledge, and training levels amongst this cohort in Ireland. We have identified a lack of geographical variability in perceived risk, a lack of training amongst at-risk groups, and knowledge gaps in relation to risk habitats and disease symptoms.
The abovementioned findings of this work have implications for the future study of human populations, ticks, wildlife hosts, and the Lyme borreliosis pathogen in Ireland. The findings also have implications for the design, timing, and implementation of future awareness campaigns aiming to decrease Lyme borreliosis risk in Ireland
Celticism and the Volk: Tracing the ideas and networks that shaped Irish archaeology
No full textThe scholarly networks that shaped the discipline of archaeology were often highly politicized. Antiquarianism in Ireland was largely a colonial endeavor. The majority Gaelic-Catholic population were excluded from the social and professional circles from which antiquaries were drawn. By denigrating Ireland’s past, colonial antiquaries made the case for the imposition of Britain’s cultural values in Ireland. During the later-eighteenth century, romantic nationalism and the Ossian vogue generated international scholarly interest in Ireland’s ‘Celtic’ past. The Celtic Revival movement was initiated in the early nineteenth century by Anglo-Irish intellectuals. As the movement developed it took root among the expanding Gaelic Irish middle class. ‘Celtic’ antiquities, art, and literature became the iconography of a new ethnic nationalism. During the interwar years, Nazi Germany ‘weaponized’ ethnicity. The ‘Celtic’ regions of northern Europe were seen to preserve the lifeways, traditions, and values of the imagined golden age of Nordic Aryan supremacy in Europe. This was the past the Nazis wanted to recreate in the present. Archaeologists, folklorists, and linguists were deployed by the Nazis to infiltrate Celtic movements. Ireland’s leading archaeologist during the interwar years was an Austrian Nazi. The legacy of this period remains deeply embedded in archaeological practice in Ireland to this day.peer-reviewe
r/K Selection-based strategies for enriching acetoclastic methanogens in anaerobic digestion systems
No full textAnaerobic digestion (AD) is a widely used biotechnology for the treatment of solid waste or wastewater and the generation of renewable energy in the form of methane. Methanogenesis is the final and often rate-limiting step in AD, directly determining overall system performance and energy recovery efficiency. Among the methanogenic pathways, acetoclastic methanogenesis, converting acetate to methane, contributes up to 60% of total methane production. Remarkably, this essential step is performed exclusively by two archaeal genera: Methanosarcina and Methanothrix. Although both utilize acetate as their substrate, they exhibit fundamentally different ecological strategies and physiological traits. Methanosarcina is an r-strategist with high growth rates and broad metabolic flexibility, while Methanothrix is a K-strategist characterized by strong substrate affinity and long-term persistence under nutrient-limited conditions. Despite their central roles in methane production, the competitive dynamics, enriching mechanisms, and syntrophic interactions of these methanogens under different operational conditions remain insufficiently understood. A deeper understanding of these mechanisms is crucial for improving AD stability, enabling targeted enrichment of functional microorganisms, and guiding the rational design of next-generation anaerobic bioreactors.
This study aims to: (i) develop a novel modeling framework that integrates r/K selection theory to simulate the competitive dynamics between Methanosarcina and Methanothrix under varying operational conditions; (ii) investigate the effects of reactor configuration (continuous-flow reactors (CFRs) and sequencing batch reactors (SBRs)), solids retention time (SRT), and substrate type (acetate and ethanol) on the enrichment and energy metabolism of acetoclastic methanogens; and (iii) elucidate the mechanisms of microbial cooperation, including potential amino acid cross-feeding and interspecies electron transfer, mediated by acetoclastic methanogens in acetate- and ethanol-fed anaerobic systems.
A novel AD modeling framework was developed by integrating r/K selection theory into conventional kinetic models to simulate the competitive exclusion dynamics between Methanosarcina and Methanothrix. Thermodynamic energy dissipation principles enabled the successful derivation of the kinetic parameters for Methanosarcina and Methanothrix. Sensitivity analysis identified acetate concentration and SRT as key factors influencing methanogen dominance, with low-substrate conditions and long SRTs favoring Methanothrix, and high-substrate, short-SRT environments favoring Methanosarcina. CFRs offered more stable conditions that promoted the gradual dominance of Methanothrix, especially under low acetate concentrations.
Subsequent reactor-scale experiments were conducted in acetate-fed systems to explored how reactor configuration (SBR vs. CFR) and SRT influence the enrichment and energy metabolism of acetoclastic methanogens. The results showed that short SRTs (10 and 15 days) and SBRs favored the dominance of Methanosarcina, reflecting its rapid-growth, resource-responsive characteristics. In contrast, Methanothrix achieved greater relative abundance under prolonged SRTs (25 and 50 days) in CFRs, consistent with its adaptation to stable, low-substrate environments. Interestingly, SBRs supported the co-existence of both genera, possibly due to the substrate concentration gradients established during cyclic feeding. Beyond methanogens, operational parameters also impacted acetate-oxidizing bacterial communities, with genera such as Pseudomonas, Thauera, and Desulfocurvus. Furthermore, genes associated with energy conservation, such as ATPase complexes and electron transport genes (such as Ech, Vho/Vht, Fpo, Mtr), exhibited mode-dependent patterns, suggesting that methanogenesis in each system followed different metabolic metabolism. Auxotrophy of amino acid observed in dominant microbial taxa indicated potential amino acid cross-feeding interactions, which may support metabolic complementarity.
In addition, operational mode significantly influenced methanogen and syntroph distribution in ethanol-fed systems. CFRs achieved full ethanol and volatile fatty acid degradation, while SBRs showed acetate and butyrate accumulation. Methanothrix displayed a higher relative abundance in CFR (6.1%) compared to SBR (1.7%), whereas Methanosarcina was more prevalent in SBR (1.2%) than in CFR (0.06%). Additionally, acetoclastic methanogenesis was not detected in SBR. Genes associated with hydrogen and electron transfer were more abundant in CFRs, supporting enhanced syntrophic cooperation and the potential occurrence of direct interspecies electron transfer (DIET) between Geobacter and Methanothrix.
Overall, this research reveals how life-history strategies and environmental factors shape methanogen selection and microbial network dynamics in anaerobic digesters. It provides both theoretical insights and practical tools to guide the design and operation of AD systems toward functionally optimized and ecologically stable microbial communities