Microbial Physiology and Stress Adaptation of Anaerobic Methanotrophs Maider Junkal Echeveste Medrano
Microbial Physiology and Stress Adaptation of Anaerobic Methanotrophs Maider Junkal Echeveste Medrano
Echeveste Medrano, M.J. (2025). Microbial Physiology and Stress Adaptation of Anaerobic Methanotrophs [PhD Thesis, Radboud University]. Nijmegen, The Netherlands. Online PDF + flipbook ISBN 978-94-6506-810-7 Provided by thesis specialist Ridderprint, ridderprint.nl Printing Ridderprint Layout and design Richard Guenne, persoonlijkproefschrift.nl This research was financially supported by the Netherlands Ministry of Education, Culture and Science together with the Netherlands Organization for Scientific Research (NWO) through SIAM gravitation grant 024.002.002. The financial support provided by Radboud University for the printing of this thesis is acknowledged. All rights reserved. A copy of this thesis has been supplied on the condition that anyone who consults it recognizes that copyright rests with the author. No part of this thesis may be copied or used in any form without the explicit consent of the author. © Maider Junkal Echeveste Medrano, 2025
Microbial Physiology and Stress Adaptation of Anaerobic Methanotrophs Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.M. Sanders, volgens besluit van het college voor promoties in het openbaar te verdedigen op donderdag 10 april 2025 om 10:30 uur precies door Maider Junkal Echeveste Medrano geboren op 12 september 1996 te Irun (Spanje)
Promotoren Prof. dr. C.U. Welte Prof. dr. ir. M.S.M. Jetten Copromotor Dr. I. Sánchez-Andrea (IE University Segovia, Spanje) Manuscriptcommissie Prof. dr. H.J.M. op den Camp Prof. dr. A. Probst (Universität Duisburg-Essen, Duitsland) Dr. M.C. Schölmerich (Eidgenössische Technische Hochschule Zürich, Zwitserland)
Microbial Physiology and Stress Adaptation of Anaerobic Methanotrophs Dissertation to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. J.M. Sanders, according to the decision of the Doctorate Board to be defended in public on Thursday, April 10, 2025 at 10:30 AM by Maider Junkal Echeveste Medrano born on September 12, 1996 in Irun (Spain)
PhD supervisors Prof. dr. C.U. Welte Prof. dr. ir. M.S.M. Jetten PhD co-supervisor Dr. I. Sánchez-Andrea (IE University, Segovia, Spain) Manuscript Committee Prof. dr. H.J.M. op den Camp Prof. dr. A. Probst (University of Duisburg-Essen, Germany) Dr. M.C. Schölmerich (ETH Zürich, Switzerland)
Para la (R)Amona, Ibai y Jon E (x2)
TABLE OF CONTENTS Summary 11 Samenvatting 16 Resumen 20 Laburpena 24 Chapter 1 Introduction and thesis outline 29 Chapter 2 Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments 43 Chapter 3 Contrasting methane, sulfide, and nitrogen loading regimes in bioreactors shape microbial communities originating from methane-rich coastal sediment of the Stockholm Archipelago 75 Chapter 4 Unraveling nitrogen, sulfur, and carbon metabolic pathways and microbial community transcriptional responses to substrate deprivation and toxicity stresses in a bioreactor mimicking anoxic brackish coastal sediment conditions 103 Chapter 5 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea 137 Chapter 6 Osmoregulation in freshwater anaerobic methaneoxidizing archaea under salt stress 165 Chapter 7 Methanotrophic flexibility of “Ca. Methanoperedens” and its interactions with sulfate-reducing bacteria in the sediment of meromictic Lake Cadagno 203 Chapter 8 Synthesis and discussion 231 References 259 Appendices 291 Research data management 292 Acknowledgments | Agradecimientos | Eskerrak 294 Curriculum vitae 300 List of publications 301 Outreach 302
S (Summary) Summary Samenvatting Resumen Laburpena
12 Summary SUMMARY Coastal ecosystems are dynamic hotspots for methane. They are highly sensitive to anthropogenic impacts and climate-change derived challenges. This PhD dissertation investigated the mechanisms by which anaerobic methanotrophs, important microorganisms in counteracting methane emissions, responds to coastal ecosystem stressors such as sulfide, nitric oxide, and salinity-induced osmotic pressure. Additionally, sulfide was studied in the context of sulfatedependent anaerobic oxidation of methane (S-AOM) in meromictic Lake Cadagno. To conduct this investigation, we used a complementary approach, combining co-cultures of anaerobic methanotrophs in bioreactors, enrichments started with anoxic coastal sediments, along with biogeochemical measurements, meta-omics analysis, stable isotope activity tests, molecular assays, and physiological studies (Figure 1). In Chapter 1, we introduce the environmental relevance of methane oxidation in coastal ecosystems and present the potential of employing a culture of anaerobic methane oxidizing archaea (ANME) from the genus “Candidatus (Ca.) Methanoperedens” as a study model. Ultimately, we identify current knowledge gaps and outline the research questions that guided this thesis. In Chapter 2, we concluded that a new genus of ANME archaea might be able to act as a methane biofilter in the coastal Stockholm Archipelago under hypoxic but not euxinic conditions due to sulfide toxicity. ANME-2 Metagenome Assembled Genome (MAG) 011 was first classified as ANME-2b on the original paper and later updated with the latest Genome Taxonomy Database (GTBD) classification for this thesis (September 2024) as genus QBUR01 closely related to “Ca. Methanocomedens” (canonical ANME-2a). For this investigation, we defined sulfide inhibition for sulfate S-AOM with half inhibitory thresholds at ~ 1 mM sulfide. We also hypothesized that the sulfite reductase from the Fsr Group II belonging to the identified ANME MAG could aid in sulfite detoxification. In Chapter 3, we monitored two coastal anoxic sediment microbiomes for over a year under distinct methane, nitrogen (nitrate and ammonium), and sulfide
13 Summary concentrations. Here, we defined the Gammaproteobacteria as the most dominant and fluctuating group. The nitrogen reduction processes differed between the two systems: the oligotrophic bioreactor stimulated Dissimilatory Nitrate Reduction to Ammonium (DNRA) whereas denitrification dominated the eutrophic bioreactor. Methanotrophs in the oligotrophic systems (including biofilm growth) consisted of a diverse yet poorly described Methylomonadaceae genus Methylovulum and subgroup KS41 that competed for nitrate with the main driver of DNRA, “Ca. Methanoperedens BLZ2” spp. Moreover, the oligotrophic system selected for the novel MAGs Pseudomonadales IMCC2047 and Rugosibacter with divergent Membranebound MonoOxygenase (CuMMOs) with potential for short alkane degradation. The eutrophic systems selected for better described Methylomonadaceae Methylomonas and Methylobacter species. This investigation highlighted the metabolic versatility of microaerophilic methanotrophs from the family Methylomonadaceae to cope with sulfide, low oxygen, and with potential for denitrification. In Chapter 4, we investigated the flexibility of a stable anoxic co-culture of sulfide oxidizing denitrifiers, anaerobic ammonium-oxidizing (anammox) bacteria, and nitrate/nitrite-dependent anaerobic oxidation of methane (N-DAMO) partners to cope with nitrogen deprivation, and sulfide and nitric oxide stress, mimicking dynamic changes that occur in brackish coastal sediment conditions as described in Chapter 3. Ammonium removal resulted in the disappearance of “Ca. Nitrobium versatile” and anammox bacteria from the initial co-culture. DNRA activity, mainly promoted by “Ca. Methanoperedens”, was unable to sustain anammox activity. Upon ammonium re-introduction, “Ca. Methylomirabilis lanthanidiphila” was replaced by “Ca. Methylomirabilis tolerans” and anammox “Ca. Scalindua rubra” with “Ca. Kuenenia stuttgartienses”. Sulfide and nitric oxide stressors enriched for Thiohalobacteraceae as sulfide oxidizing denitrifiers, and sulfide:quinone oxidoreductase expression was elevated. This research helps to gain insights on the complex microbial community dynamics that can occur coastal ecosystems or engineered ecosystems like wastewater treatment plants. In Chapter 5, we investigated the stress response to sulfide in freshwater anaerobic methanotrophs. We observed a decrease in nitrate-dependent methane oxidation activity after 0.5 mM sulfide exposure to “Ca. Methanoperedens BLZ2” both after
14 Summary short-term and particularly after long-term (6.5 weeks) exposure. During the shortterm sulfide exposure, sulfide detoxification seemed to proceed via sulfide-oxidizing bacteria. Long-term exposure at 0.25 mM sulfide/day stimulated the expression of Group III Dsr-LP sulfite reductases belonging to “Ca. Methanoperedens” spp. Furthermore, “Ca. Methanoperedens” storage polymers were used during the longterm sulfide exposure probably linked to the stress response. In Chapter 6, we reported the long-term bioreactor acclimation of freshwateradapted “Ca. Methanoperedens Vercelli” to marine salinities. We described N(ε)-acetyl-β-L-lysine as a key osmolyte that is exclusively produced by “Ca. Methanoperedens” in the enrichment via the gene pair kamA and ablB. We expanded the production potential of N(ε)-acetyl-β-L-lysine across a universal evolutionary tree focused on archaea and demonstrated that horizontal gene transfer (HGT) from Firmicutes gave rise to the production potential of this osmolyte in ANME archaea. We additionally hypothesize that N(ε)-acetyl-β-L-lysine production potential might have been acquired from “Ca. Methanoperedens spp.” into Borgs also via HGT. Both polyhydroxyalkanoates and sialic acids or negatively charged monosaccharides belonging to the extracellular polysaccharide layer, were linked to “Ca. Methanoperedens” and indicated to be employed as coping mechanisms for salinity stress. In Chapter 7, we expanded on the electron acceptor flexibility of “Ca. Methanoperedens” and addressed the possibility for S-AOM in the sediment of meromictic Lake Cadagno. We described the interaction of “Ca. Methanoperedens” spp. with an undescribed Desulfobacterota class QYQD01. In this chapter, we additionally recovered five different “Ca. Methanoperedens” MAGs from sediment and incubations with sulfate and manganese oxide. We described the potential of these MAGs to engage in Extracellular Electron Transfer (EET) via distinct Multi-heme-Cytochrome (MHCs) or OmcZ nanowires. Furthermore, we explored the genomic traits of Desulfobacterota class QYQD01 as a facultative sulfate reducing bacteria (SRB) syntroph. We also investigated the widespread co-abundance of Desulfobacterota class QYQD01 with “Ca. Methanoperedens” species mainly in marine and groundwater systems.
15 Summary In Chapter 8, we synthesized our research outcomes, reflected on the methodologies used and integrated our findings into the broader environmental context of coastal and engineered ecosystems. Figure 1 | Figuur 1 | Figura 1 | 1. Irudia. Graphical abstract of presented PhD dissertation. Chapters are separated following a “Top down” approach: Environmental, Dynamics or Physiology. The main findings of each Chapter are hereby highlighted.
16 Samenvatting SAMENVATTING Kustecosystemen zijn dynamische hotspots voor methaan, maar ze zijn ook zeer gevoelig voor de effecten van menselijk handelen en de uitdagende gevolgen van klimaatverandering. In dit proefschrift onderzochten we de adaptatiemechanismen die anaerobe methaanoxideerders, belangrijke microorganismen in de bestrijding van methaanemissies, gebruiken om zich aan te passen aan stressfactoren in kustecosystemen, zoals sulfide, stikstofmonoxide en osmotische druk veroorzaakt door zoutgehalte. Bovendien hebben we het gebruik van sulfide bestudeerd in de context van sulfaatafhankelijke anaerobe oxidatie van methaan (S-AOM) in het meromictische Zwitserse meer Cadagno. Om dit onderzoek uit te voeren, combineerden we het kweken van anaerobe methaan-oxideerders in bioreactoren en werden bioreactoren gestart met anoxische kustsedimenten voor het verrijken van anaerobe methanotrofen. Daarnaast maakten we gebruik van biogeochemische metingen, meta-omics analyses, stabiele isotoopactiviteitstesten, moleculaire assays en fysiologische studies (Figuur 1). In Hoofdstuk 1 introduceren we de milieurelevantie van methaanoxidatie in kustecosystemen en de mogelijkheden van het gebruik van een cultuur van anaerobe methaan-oxiderende archaea (ANME) van het geslacht “Ca. Methanoperedens” als studiemodel. We identificeren de huidige kennishiaten en schetsen de onderzoeksvragen die als onderwerp voor deze thesis hebben gediend. In Hoofdstuk 2 kwamen we tot de conclusie dat een nieuw geslacht van ANME archaea mogelijk kan fungeren als een methaanbiofilter in de kustwateren van de Stockholmse scherenkust onder hypoxische, maar vanwege sulfidetoxiciteit niet onder euxinische omstandigheden. ANME-2 MAG 011 werd in de oorspronkelijke publicatie eerst geclassificeerd als ANME-2b en in dit proefschrift (september 2024) bijgewerkt met de nieuwste “Genome Taxonomy Database” (“GTBD”)-classificatie als geslacht QBUR01, en is daarmee nauw verwant aan “Ca. Methanocomedens” (ANME-2a). Voor dit onderzoek hebben we de 50% sulfideremming voor sulfaat S-AOM vastgesteld met ~ 1 mM sulfide. We veronderstellen dat de sulfietreductase van de “Fsr Groep II” gecodeerd in de ANME MAG zou kunnen helpen bij sulfidedetoxificatie.
17 Samenvatting In Hoofdstuk 3 hebben we gedurende een jaar twee microbiële gemeenschappen in anoxische kustsedimenten gemonitord onder verschillende methaan-, stikstof- (nitraat en ammonium) en sulfideconcentraties. Hierbij hebben we vastgesteld dat de Gammaproteobacteriën de meest dominante groep vormden. De stikstofverwijderingsprocessen verschilden tussen de twee systemen: de oligotrofe bioreactor stimuleerde dissimilatoire nitraatreductie tot ammonium (DNRA), terwijl denitrificatie domineerde in de eutrofe bioreactor. Methanotrofen in de oligotrofe systemen (inclusief biofilmgroei) bestonden uit een diverse, maar nog niet goed beschreven, geslacht van Methylomonadaceae, namelijk Methylovulum, en subgroep KS41. Deze twee aerobe methanotrofen concurreerden om nitraat met de belangrijkste aanjager van DNRA, “Ca. Methanoperedens BLZ2” spp. Bovendien selecteerde het oligotrofe systeem voor twee nieuwe MAGs Pseudomonadales IMCC2047 en Rugosibacter. Beide MAGs codeerden een divergent membraangebonden mono-oxygenase (CuMMOs), die mogelijk verantwoordelijk is voor de afbraak van korte alkanen. De eutrofe systemen selecteerden voor beter beschreven Methylomonadaceae-soorten, Methylomonas en Methylobacter. Het onderzoek uit dit hoofdstuk benadrukte de metabole veelzijdigheid van microaerofiele methanotrofen uit de familie Methylomonadaceae en hun vermogen om om te gaan met sulfide, lage zuurstofconcentraties en met de mogelijkheid tot denitrificatie. In Hoofdstuk 4 onderzochten we de flexibiliteit van een stabiele anoxische co-cultuur van sulfide-oxiderende denitrificeerders, anammox en nitraat-/ nitrietafhankelijke anaerobe oxidatie van methaan (N-DAMO) partners om om te gaan met stikstofgebrek, en sulfide- en stikstofmonoxide-stress. Dit onderzoek bootste de dynamische veranderingen na die mogelijk optreden in kustsedimentcondities, zoals beschreven in Hoofdstuk 3. Het omzetten en verwijderen van ammonium resulteerde in het verdwijnen van “Ca. Nitrobium versatile” en anammox-bacteriën uit de initiële co-cultuur. De DNRA-activiteit, voornamelijk uitgevoerd door “Ca. Methanoperedens”, was niet in staat om de anammox-activiteit te ondersteunen. Na herintroductie van ammonium werd “Ca. Methylomirabilis lanthanidiphila” vervangen door “Ca. Methylomirabilis tolerans” en anammox “Ca. Kuenenia stuttgartienses” werd vervangen door “Ca. Scalindua rubra”. De toegevoegde sulfide en stikstofmonoxide verrijkten
18 Samenvatting de cultuur met Thiohalobacteraceae als sulfide-oxiderende denitrificeerders, en verhoogden de expressie van sulfide:quinone oxidoreductase. Het onderzoek uit dit hoofdstuk heeft geholpen om inzicht te krijgen in de complexe microbiële gemeenschapsdynamiek die kan optreden in kustecosystemen of door de mens gemaakte ecosystemen, zoals afvalwaterzuiveringsinstallaties. In Hoofdstuk 5 bestudeerden we de stressreactie op sulfide van zoetwater anaerobe methanotrofen. We zagen een afname in nitraatafhankelijke methaanoxidatieactiviteit na blootstelling aan 0,5 mM sulfide in “Ca. Methanoperedens BLZ2”, zowel na kortetermijn- als langetermijnblootstelling (6,5 weken). Tijdens de kortetermijnblootstelling aan sulfide leek sulfidedetoxificatie plaats te vinden via sulfide-oxiderende bacteriën. Langetermijnblootstelling aan 0,25 mM sulfide per dag stimuleerde de expressie van Groep “III Dsr-LP” sulfietreductases behorend tot “Ca. Methanoperedens” spp. Bovendien werden “Ca. Methanoperedens” opslagpolymeren gebruikt tijdens de langetermijnblootstelling aan sulfide, waarschijnlijk gekoppeld aan de stressreactie. In Hoofdstuk 6 beschrijven we de langetermijnbioreactoracclimatisatie van zoetwater-aangepaste “Ca. Methanoperedens Vercelli” aan mariene zoutgehaltes. We toonden aan dat N(ε)-acetyl-β-lysine een belangrijke osmoliet is die in de cultuur wordt geproduceerd door “Ca. Methanoperedens” via het genenpaar kamA en ablB. We lieten zien dat het potentieel voor de productie van N(ε)-acetylβ-lysine aanwezig is in veel micro-organismen, en we berekenden een universele evolutionaire stamboom van de kamA- en ablB-genen en toonden aan dat horizontale genoverdracht (HGT) vanuit Firmicutes hoogstwaarschijnlijk heeft geleid tot de productie van deze osmoliet in ANME-archaea. We veronderstelden dat de N(ε)- acetyl-β-lysine genen mogelijk ook verworven zijn door “Ca. Methanoperedens spp.” via zogenaamde Borgs. Zowel polyhydroxyalkanoaten als extracellulaire siaalzuren werden door “Ca. Methanoperedens” gebruikt als aanpassingsmechanismen voor zoutstress. In Hoofdstuk 7 onderzochten we de elektronacceptorflexibiliteit van “Ca. Methanoperedens” en de mogelijkheid voor “S-AOM” in het sediment van het Zwitserse meromictische meer Cadagno. We beschrijven de interactie van “Ca.
19 Samenvatting Methanoperedens” spp. met een tot nu toe onbeschreven Desulfobacterota klasse QYQD01. Bovendien hebben we vijf verschillende “Ca. Methanoperedens” MAGs kunnen assembleren uit het originele sediment en de incubaties met sulfaat en mangaanoxide. Verder beschrijven we de genen in deze MAGs met betrekking tot het vermogen om Extracellulaire Electron Transfer (EET) te doen via Multi-hemeCytochromen (MHCs) of OmcZ nanodraadjes. We onderzochten ook het genoom van de Desulfobacterota klasse QYQD01 als een facultatieve SRB-syntroof. Deze Desulfobacterota klasse QYQD01 komt vaak samen voor met “Ca. Methanoperedens” in mariene en grondwater systemen. Het afsluitende Hoofdstuk 8 bevat een discussie van de belangrijkste bevindingen van dit proefschrift, inclusief de beschrijving van nieuwe ANME-groepen en hun fysiologische en genomische kenmerken, en aanbevelingen voor toekomstig onderzoek.
20 Resumen RESUMEN Los ecosistemas costeros son “hotspots” dinámicos para el metano. Estos son altamente sensibles a los impactos antropogénicos y a los desafíos derivados del cambio climático. Esta tesis doctoral investigó los mecanismos por los cuales los metanótrofos anaerobios, microorganismos importantes para contrarrestar las emisiones de metano, responden a los factores de estrés en los ecosistemas costeros, tales como el sulfuro de hidrógeno, el óxido nítrico y la presión osmótica inducida por la salinidad. Además, se estudió el papel del sulfuro de hidrógeno en el contexto de la oxidación anaerobia de metano acoplada a la reducción del sulfato (S-OAM) en el lago meromíctico Cadagno. Para llevar a cabo esta investigación, se utilizó un enfoque complementario que combinó cocultivos de metanótrofos anaerobios en biorreactores, enriquecimientos iniciados con sedimentos costeros anóxicos, junto con mediciones biogeoquímicas, análisis multiómico, pruebas de actividad con isótopos estables, ensayos moleculares y estudios fisiológicos (Figura 1). En el Capítulo 1, introducimos la relevancia ambiental de la oxidación de metano en ecosistemas costeros y, presentamos el potencial de emplear un cultivo de arqueas oxidantes de metano anaerobias (“ANME”) del género “Ca. Methanoperedens” como modelo de estudio. Finalmente, se identifican las brechas actuales de conocimiento y se delinean las preguntas de investigación que guiaron esta tesis. En el Capítulo 2, concluimos que un nuevo género de arqueas “ANME” podría actuar como biofiltro de metano en el archipiélago costero de Estocolmo bajo condiciones hipóxicas, pero no en condiciones de euxinia, debido a la toxicidad del sulfuro de hidrógeno. El genoma ANME-2 011 fue inicialmente clasificado como ANME-2b en el artículo original y más tarde lo actualizamos con la última clasificación de la “Genome Taxonomy Database” (“GTBD”) para esta tesis (Septiembre 2024), como el género QBUR01, estrechamente relacionado con “Ca. Methanocomedens” (ANME-2a). Para esta investigación, definimos la inhibición por sulfuro de hidrógeno para S-OAM con umbrales de inhibición media de ~ 1 mM de sulfuro de hidrógeno. Asimismo, hipotetizamos que la reductasa de sulfito del
21 Resumen grupo “Fsr II” perteneciente al genoma ANME-2 011 identificada, podría contribuir a la detoxificación del sulfito. En el Capítulo 3, monitoreamos durante más de un año dos microbiomas de sedimentos costeros anóxicos bajo distintas concentraciones de metano, nitrógeno (nitrato y amonio) e sulfuro de hidrógeno. Para esta investigación, definimos a los Gammaproteobacteria como el grupo más dominante y variable. Los procesos de reducción de nitrógeno difirieron entre los dos sistemas: el biorreactor oligotrófico estimuló la reducción disimilatoria de nitrato a amonio (“DNRA”), mientras que la desnitrificación dominó en el biorreactor eutrófico. Los metanótrofos en los sistemas oligotróficos (incluido el crecimiento en biopelículas) consistieron en un género diverso pero poco descrito de Methylomonadaceae, Methylovulum, y el subgrupo KS41, que competían por nitrato con el principal contribuidor a la “DNRA”, “Ca. Methanoperedens BLZ2”. Además, el sistema oligotrófico seleccionó nuevos genomas clasificados como Pseudomonadales IMCC2047 y Rugosibacter con monooxigenasas de membrana divergentes (“CuMMOs”) con potencial para degradación de alcanos de cadena corta. Los sistemas eutróficos seleccionaron especies mejor descritas de Methylomonadaceae, como Methylomonas y Methylobacter. Esta investigación destacó la versatilidad metabólica de los metanótrofos microaerófilos de la familia Methylomonadaceae para contrarrestar el sulfuro de hidrógeno, la limitación de oxígeno y con potencial para la desnitrificación. En el Capítulo 4, se investigó la flexibilidad de un cocultivo anóxico estable de desnitrificadores oxidantes de sulfuro de hidrógeno, anammox y socios de la oxidación anaerobia del metano acoplada a la reducción del nitrato/nitrito (“NDAMO”) para enfrentarse a la privación de nitrógeno, y el estrés por sulfuro de hidrógeno y óxido nítrico, simulando cambios dinámicos que ocurren en sedimentos costeros salobres como se describió en el Capítulo 3. La eliminación de amonio resultó en la desaparición de “Ca. Nitrobium versatile” y bacterias anammox del cocultivo inicial. La actividad “DNRA”, promovida principalmente por “Ca. Methanoperedens”, no pudo sostener la actividad anammox. Tras la reintroducción de amonio, “Ca. Methylomirabilis lanthanidiphila” fue reemplazada por “Ca. Methylomirabilis tolerans” y anammox “Ca. Scalindua rubra” por “Ca. Kuenenia stuttgartienses”. Los factores de estrés por sulfuro de hidrógeno y óxido
22 Resumen nítrico enriquecieron a la familia Thiohalobacteraceae como desnitrificadores oxidantes de sulfuro de hidrógeno, y se observó una elevada expresión de la oxidoreductasa de sulfuro:quinona. Esta investigación proporciona conocimientos sobre las dinámicas complejas de comunidades microbianas que pueden ocurrir en ecosistemas costeros o artificiales como plantas de tratamiento de aguas residuales. En el Capítulo 5, se investigó la respuesta al estrés por sulfuro de hidrógeno en metanótrofos anaerobios de agua dulce. Observamos una disminución en la actividad de oxidación anaerobia del metano acoplada a la reducción del nitrato tras la exposición a 0,5 mM de sulfuro de hidrógeno en “Ca. Methanoperedens BLZ2” tanto a corto como, especialmente, a largo plazo (6,5 semanas). Durante la exposición a corto plazo, la detoxificación del sulfuro de hidrógeno pareció proceder a través de bacterias oxidantes del sulfuro de hidrógeno. La exposición prolongada a 0,25 mM de sulfuro de hidrógeno/día estimuló la expresión de reductasas de sulfito del grupo “Dsr-LP III” pertenecientes a “Ca. Methanoperedens”. Además, los polímeros de almacenamiento de “Ca. Methanoperedens” se utilizaron durante la exposición prolongada al sulfuro de hidrógeno, probablemente relacionados con la respuesta al estrés. En el Capítulo 6, reportamos la aclimatación a largo plazo en un biorreactor de “Ca. Methanoperedens Vercelli”, adaptado a agua dulce, a condiciones de salinidad marina. Describimos a la N(ε)-acetil-β-L-lisina como osmolito clave producido exclusivamente por “Ca. Methanoperedens” en el enriquecimiento, mediante los genes kamA y ablB. Ampliamos el potencial de producción de N(ε)-acetil-β-Llisina en un árbol evolutivo universal centrado en arqueas, y demostramos que la transferencia horizontal de genes (THG) desde Firmicutes permitió la capacidad de producir este osmolito en arqueas “ANME”. Además, planteamos la hipótesis de que la capacidad de producción de N(ε)-acetil-β-L-lisina podría haber sido adquirida desde especies de “Ca. Methanoperedens” hacia Borgs también a través de THG. Tanto los polihidroxialcanoatos como los ácidos siálicos o monosacáridos con carga negativa, pertenecientes a la sustancias poliméricas extracelulares se vincularon con “Ca. Methanoperedens” y se indicaron como mecanismos de adaptación al estrés por salinidad.
23 Resumen En el Capítulo 7, ampliamos la flexibilidad de los aceptores de electrones de “Ca. Methanoperedens” y abordamos la posibilidad de S-OAM en los sedimentos del lago meromíctico Cadagno. Describimos la interacción de especies de “Ca. Methanoperedens” con una clase de Desulfobacterota no descrita, denominada QYQD01. En este capítulo, también recuperamos cinco diferentes genomas de “Ca. Methanoperedens” a partir de sedimentos e incubaciones con sulfato y óxido de manganeso. Describimos el potencial de estos genomas para participar en la transferencia de electrones extracelular (“EET”) a través de diferentes citocromos “multi-heme” (“MHCs”) o nanocables “OmcZ”. Además, exploramos los rasgos genómicos de la clase QYQD01 de Desulfobacterota como un organismo sintrófico facultativo sulfato reductor. Investigamos, asimismo, la correlación de abundancia generalizada de la clase QYQD01 de Desulfobacterota con especies de “Ca. Methanoperedens”, principalmente en sistemas marinos y de aguas subterráneas. En el Capítulo 8, sintetizamos los resultados de nuestra investigación, reflexionamos sobre las metodologías empleadas e integramos nuestros hallazgos en el contexto ambiental más amplio de ecosistemas costeros y ecosistemas artificiales
24 Laburpena LABURPENA Itsasertzeko ekosistemak metanoarentzako “hotspot” dinamikoak dira. Giza jarduerek eragindako inpaktuekiko zein klima-aldaketak sortutako erronken aurrean oso sentikorrak dira. Tesi honetan, metanoaren isuriak arintzeko garrantzitsuak diren mikroorganismoak, metanotrofo anaerobikoak, itsasertzeko ekosistemetan hidrogeno sulfuroa, nitrogeno monoxidoa eta gazitasunak sortutako presio osmotikoa eragindako estresarekiko nola erantzuten duten aztertu zen. Gainera, hidrogeno sulfuroa, sulfatoaren-mendeko metanoaren oxidazio anaerobikoa (S-MOA) ikertzeko erabili zen Cadagno aintzira meromiktikoaren testuinguruan. Ikerketa burutzeko, ikuspegi osagarria erabili zen, metanotrofo anaerobikoen aberaste-kulturak bioerreaktoreetan erabiliz, itsasertzeko sedimentu anoxikoekin hasitako aberaste-kulturekin, neurketa biogeokimikoekin, metaomika analisiekin, isotopo egonkorren aktibitate-probekin, saiakera molekularrekin eta ikerketa fisiologikoekin batera (1. Irudia). Lehenengo kapituluan, itsasertzeko ekosistemetan metanoaren oxidazioak duen garrantzia aurkezten dugu eta “Candidatus (Ca.) Methanoperedens” generoko metanoaren oxidazio anaerobia (“ANME”) aurrera eramaten duten arkeoen aberaste-kultura ikerketa-eredu gisa erabiltzeko potentziala azaltzen dugu. Azkenik, egungo ezagutza-hutsuneak identifikatzen ditugu, tesia gidatu duten ikerketa-galderak zehaztuz. Bigarren kapituluan, “ANME” arkeo genero berri batek metanoaren bioiragazki gisa Stockholm Artxipelagoaren itsasertzeko baldintza hipoxikoetan jardun dezakeela ondorioztatu genuen, baina ez baldintza euxinikoetan, hidrogeno sulfuroaren toxikotasuna dela eta hain zuzen ere. Hasieran, ANME-2 011 genoma ANME-2b bezala sailkatu zen eta, ondoren, “Genome Taxonomy Database” (“GTDB”) sailkapenarekin (2024ko Irailean), “Ca. Methanocomedens” (ANME-2a) QBUR01 generora lotuta eguneratu zen. Ikerketa honetarako, hidrogeno sulfuroaren inhibizio-mugak S-MOA-n definitu genituen, hidrogeno sulfuroaren erdi-ataria ~ 1 mM-koa izanik. Gainera, identifikatutako ANME MAG-aren “Fsr II” taldeko sulfito-erreduktasak sulfitoaren desintoxikazioan lagundu dezakeela hipotesi modura proposatu genuen.
25 Laburpena Hirugarren kapituluan, bi sedimentu anoxikoen mikrobiomak urtebete baino gehiagoz aztertu genituen, metano, nitratoa, amonioa eta hidrogeno sulfuroaren kontzentrazio desberdinekin. Gammaproteobacteria taldea nagusiena eta aldakorrena bezala identifikatu genuen. Nitrogenoaren erredukzio-prozesuak desberdinak izan ziren bi sistemen artean: bioerreaktore oligotrofikoak nitratutik amoniora disimilazio bidezko erredukzioa (“DNRA”) sustatu zuen, desnitrifikazioa nagusi zen bitartean bioerreaktore eutrofikoan. Sistema oligotrofikoko metanotrofoek (biogeruzaren hazkuntza barne) Methylovulum eta KS41 taldeko Methylomonadaceae generoen dibertsitate handia erakutsi zuten, eta nitratoaren erredukzioagatik lehiatu ziren “DNRA”-ren eragile nagusia zen “Ca. Methanoperedens BLZ2” espeziearekin. Gainera, sistema oligotrofikoak Pseudomonadales IMCC2047 eta Rugosibacter genoma berriak aberastu zituen, “Membrane-bound MonoOxygenase” (“CuMMOs”) entzima dibergenteak izanik, alkano laburren degradazio potentzialarekin. Sistema eutrofikoak ezagunagoak diren generoen aberaste-kulturak, Methylomonas eta Methylobacter, faboratu zituen. Ikerketa honek Methylomonadaceae familiako metanotrofo mikroaerofilikoen moldagarritasun metabolikoa nabarmendu zuen, hidrogeno sulfuroa zein oxigeno kontzentrazio baxuei moldatzeko eta desnitrifikaziorako potentzialarekin. Laugarren kapituluan, hidrogeno sulfuroaren oxidatzaile desnitrifikatzaileak, anammox bakterioak eta nitrato/nitritoaren mendeko metanoaren oxidazio anaerobioa (“N-DAMO”) bideratzen zuten aberaste-kultura anoxiko eta egonkor baten moldagarritasuna aztertu genuen, nitrogeno gabeziari eta hidrogeno sulfuroaren zein nitrogeno monoxidoaren estresari aurre egiteko. Modu honetan, hirugarren kapituluan aurkeztutako itsasertzeko sedimentu gazi-gezetako aldaketa dinamiko naturalak islatuz. Amonio gabetza “Ca. Nitrobium versatile” eta anammox bakterioen desagerpena eragin zuen hasierako aberaste-kulturatik. Gehienbat “Ca. Methanoperedens” sustatutako “DNRA”, ez zen anammox jarduera mantentzeko gai izan. Amonioa berriro gehitzerakoan, “Ca. Methylomirabilis lanthanidiphila”, “Ca. Methylomirabilis tolerans”-ekin ordezkatu zen eta anammox “Ca. Scalindua rubra”, “Ca. Kuenenia stuttgartienses”-ekin. Hidrogeno sulfuroa eta nitrogeno monoxidoaren estresen eraginez, desnitrifikatzaile oxidatzaileak ziren Thiohalobacteraceae familiako bakterioek aberastu egin ziren, eta sulfuro:kinona oxidoerreduktasen adierazpena areagotu zen. Ikerketa honek
26 Laburpena itsasertzeko ekosistemetan edo hondakin-uren tratamendu-instalazio bezalako teknologizaturiko ekosistemetan gertatzen diren mikroorganismo-komunitateen dinamika konplexuak ulertzen laguntzen du. Bosgarren kapituluan, hidrogeno sulfuroaren eragina aztertu genuen ur gezako metanotrofoetan. Hidrogeno sulfuroaren (0,5 mM) esposizio laburraren eta, bereziki, luzearen ondoren (6,5 aste), “Ca. Methanoperedens BLZ2” nitratuaren mendeko metanoaren oxidazioa gutxitu zuela behatu genuen. Epe laburreko esposizioarekin, desintoxikazioa hidrogeno sulfuroarekin bakterio oxidatzaileen bidez gertatu zen. Epe luzeko esposizioan (0,25 mM hidrogeno sulfuro/egun), “Ca. Methanoperedens”-ri loturiko “Dsr-LP III. Taldeko” sulfito-erreduktasak adierazpena areagotu zuen. Era berean, “Ca. Methanoperedens”-en biltegiratze polimeroak erabili ziren epe luzeko hidrogeno sulfuroaren esposizioaren ostean, estresari aurre egiteko erantzun moduan. Seigarren kapituluan, ur-gezetara egokitutako “Ca. Methanoperedens Vercelli”-k itsas gazitasunera egokitzeko bioerreaktore aklimatazio luzea ikertu genuen. “Ca. Methanoperedens”-ek soilik ekoizturiko N(ε)-azetil-β-L-lysina osmolito klabea deskribatu genuen, kamA eta ablB gene bikotearen bidez sortuta. N(ε)-azetil-β-L-lisina-ren ekoizpen potentziala arkeoetan oinarritutako eboluzio zuhaitz unibertsal batean zabaldu genuen eta gene transferentzia horizontala (GTH) Firmicutes-etik izan zela behatu genuen, osmolitoaren ekoizpen potentziala “ANME” arkeoetan ahalbidetuz. Horrez gain, N(ε)-azetil-β-L-lisinaren ekoizpen potentziala “Ca. Methanoperedens spp.”-tik Borg-etara GTH-ren bidez lortu zitekeela hipotetizatu genuen. Gainera, “Ca. Methanoperedens”-ekin loturiko polihidroxialkanoatoak eta azido sialikoak edo zelula kanpoko polisakarido geruzako karga negatiboko monosakaridoak, gazitasunak sortutako presioari aurre egiteko mekanismo gisa erabiltzen zirela iradoki genuen. Zazpigarren kapitulu honetan, “Ca. Methanoperedens”-en elektroi hartzaileen malgutasuna aztertu genuen eta Cadagno aintzira meromiktikoko sedimentuan S-MOA-aren posibilitatea aztertu genuen. “Ca. Methanoperedens” espezieen eta deskribatu gabeko Desulfobacterota klaseko QYQD01-en arteko elkarrekintza
27 Laburpena deskribatu genuen. Gainera, kapitulu honetan bost “Ca. Methanoperedens” genoma berreskuratu genituen bai sedimentutik zein sulfatoarekin eta manganeso oxidoarekin prestatutako inkubazioetatik. Genoma hauek “Extracellular Electron Transfer” (EET) prozesuan parte hartzeko gaitasuna dutela deskribatu genuen, “Multi-heme-Cytochrome” (MHC) edo “OmcZ” nanohari bereizien bidez. Era berean, Desulfobacterota klaseko QYQD01-en ezaugarri genomikoak aztertu genituen, fakultatiboak diren sulfato erreduzizatzaile sintrofiko gisa. Bukatzeko, Desulfobacterota klaseko QYQD01-en eta “Ca. Methanoperedens” espezieen pareko ugaritasun zabala ikertu genuen, nagusiki itsas eta lurpeko ur sistemetan. Zortzigarren kapituluan, gure ikerketaren emaitzak laburtu ditugu, baliatutako metodologien inguruan hausnartuz, eta gure aurkikuntzak itsasertzeko eta teknologizaturiko ekosistemen testuinguru orokorrean integratuz.
1 Introduction and thesis outline
30 Chapter 1 GREENHOUSE GAS METHANE IN THE CONTEXT OF COASTAL ECOSYSTEMS Methane (CH₄) is a potent greenhouse gas and plays a significant role in global warming, contributing to climate change (Saunois et al., 2016; Saunois et al., 2024). Despite a shorter atmospheric lifetime (~ 12 years) than of carbon dioxide (CO₂) (up to 200 years), methane has a global warming potential approximately 80 times over a 20-year period, making it a critical component for climate change modelling (Inman, 2008; IPCC, 2014, 2023; Pachauri et al., 2014). Coastal ecosystems, particularly wetlands, are hotspots for methane production due to high organic matter input and the reduced, anoxic conditions in sediments processes (Malone & Newton, 2020; Wallenius et al., 2021; Wells et al., 2020) (Figure 1). Furthermore, they are dynamic zones where methane emissions are prevalent due to complex carbon, nitrogen and sulfur cycling (Siefert & Plattner, 2004). Organic matter deposition and the anoxic conditions in sediments create ideal environments for methanogenesis - the biological process through for which methanogenic archaea produce methane. Methanogenesis is fueled by a variety of substrates, including acetate, methanol, hydrogen and dimethyl sulfide (Kurth et al., 2020). Agriculture, waste, and fossil fuel activities, along with aquatic ecosystems, are the primary contributors to methane emissions. The main methane sinks include atmospheric chemical reactions, soils, and (potentially) certain engineered biotechnological systems (Figure 1) (Rosentreter et al., 2021; Saunois et al., 2024). However, not all methane produced in these natural anoxic layers escapes into the atmosphere. A large portion is consumed by methane-oxidizing microorganisms in both anoxic and oxic zones, creating a natural biofilter that mitigates greenhouse gas emissions (Glodowska et al., 2022; Guerrero-Cruz et al., 2021; Kalyuzhnaya et al., 2019; Venetz et al., 2023; Welte et al., 2016) (Figure 1). The microbial methane oxidation occurs across various ecosystems, including oceans, wetlands, soils, and lakes, as well as engineered systems such as Waste Water Treatment Plants (Figure 1) (Rosentreter et al., 2021; Saunois et al., 2024; Wang et al., 2017).
31 Introduction and thesis outline Microbial communities including methanotrophs Sources Sinks Ocean Wetlands Biotechnology Atmospheric chemical reactions Potential methane sink Soil Lakes Agriculture and waste Fossil fuel production and burning Figure 1. Schematic diagram on the different sources and sinks of methane. Sizes of trees, industries and microbes are not to scale. Pink arrows show the sources of methane, agriculture and waste being the biggest anthropogenic source with about 217 teragrams of CH4 per year. Wetlands are the biggest natural sources of methane emissions with about 181 teragrams of CH4 per year. Other minor natural emissions of methane are inland waters, oceans, termites, wild animals, permafrost and vegetation. Methanotrophs and their metabolic partners can act as a methane biofilter in some environments such lakes and ocean depending on the microbial composition and the availability of electron acceptors. Orange arrows show the sinks of methane, atmospheric chemical degradation being the biggest sink with 518 teragrams of CH4 per year. Diverse methanotrophs could be cultivated and studied and further used in biotechnological applications to produce high-value natural products of interest to humans. Edited from (Cervantes et al., 2021) using Adobe Illustrator 2024 and the Integration and Application Network Symbol Library from the Center for Environmental Science of the University of Maryland (USA). Size of arrow is relative to the contribution to methane sink or source. 1
32 Chapter 1 THE INNER WORKINGS OF THE METHANE BIOFILTER The natural methane biofilter is a microbial network that operates in both oxic and anoxic conditions, playing a pivotal role in mitigating methane emissions from coastal ecosystems. The biofilter comprises two primary groups of microorganisms: methane-oxidizing bacteria (MOB) (Kalyuzhnaya et al., 2019) and anaerobic methane-oxidizing archaea (ANME) (Chadwick et al., 2022; Timmers et al., 2017) (Figure 2). Aerobic methane oxidation primarily occurs in the oxygen-rich layers of water columns and sediments, where MOB use oxygen to oxidize methane into carbon dioxide. The Alphaproteobacteria methanotrophs appear to be more pronounced in well-oxygenated environments, while the Gammaproteobacteria methanotrophs seem to tolerate better less-oxygenated or anoxic zones (Li et al., 2024). The adaptation of methanotrophs to also operate in (micro)oxic zones make the methane biofilter a flexible system that responds to oxygen fluctuations in sediments and water columns (Li et al., 2024; Reis et al., 2024). These metabolic adaptations might include potential for denitrification, fermentation or metal oxide reduction under oxygen-limited conditions (Kits et al., 2015; Li et al., 2023). Anaerobic oxidation of methane (AOM), mediated by ANME archaea, typically occurs in deeper anoxic sediments where electron acceptors such as sulfate, nitrate, iron-, and manganese-oxides are available (Figure 2) (Chadwick et al., 2022; Egger et al., 2018; Glodowska et al., 2022; Knittel & Boetius, 2009). In marine environments, ANME archaea from the groups ANME-1, ANME-2abc and ANME-3 act in concert with sulfate-reducing bacteria (SRB), coupling sulfate reduction to methane oxidation (Murali et al., 2023b) (Figure 3). Intriguingly, there is also evidence for iron reduction in ANME-2a enrichments (Slobodkin et al., 2023). The sedimentary interval commonly known as the sulfate-methane transition zone (SMTZ) has been extensively used to localize the AOM process in marine and brackish systems, as this is the place where the sedimentary methane biofiltering takes place and were described in Chapters 2-3 and including Chapter 7, with a particular sulfate-rich freshwater SMTZ.
33 Introduction and thesis outline In brackish and freshwater environments, metabolically highly flexible ANME species such as “Ca. Methanoperedens” use nitrate, metal oxides (iron, manganese), humic substances as electron acceptors, thereby playing a critical role in methane mitigation in these ecosystems (Cai et al., 2022; Haroon et al., 2013; Leu et al., 2020a; Pelsma et al., 2023a; Valenzuela et al., 2020) (Figure 2 and 3). Sulfate-AOM for “Ca. Methanoperedens” is a particular topic of interest, because of its enigmatic and unique occurrence in sulfate-rich freshwater systems like meromictic lakes or groundwater systems (Bell et al., 2022; Su et al., 2020), included also in Chapter 7. O2 CH4 SO4 2NO3 - NO2 - MnO2 Fe(OH)3 Methane-oxidizing bacteria or MOB ANME-2a / “Ca. Methanoperedens” ANME-1, ANME-2abc, ANME-3 (“Ca. Methanoperedens”) “Ca. Methanoperedens” “Ca. Methylomirabilis” (MOB) Methanogens Methane biofilter Lorem ipsum Figure 2. An example schematic illustrating chemical gradients relevant to methane oxidation in the water column and sediment, along with the microbial groups catalyzing these reactions. The dashed black gradient for methane indicates that methanogens are present only within the anoxic sediment interval, while the remaining methane diffuses upward to the surface. The parentheses highlight less common environments for the methanotrophic groups mentioned. Edited using Adobe Illustrator 2024. 1
34 Chapter 1 ON “Ca. Methanoperedens” AS A METHANOTROPHIC STUDY MODEL “Ca. Methanoperedens” has demonstrated a highly versatile metabolism, allowing it to thrive in various ecosystems, including freshwater anoxic environments (e.g. wetland) and soils (e.g. peat) and groundwater systems. It has also been reported in marine metagenomes, although with reduced coverage compared to marine ANME (Chapter 7) (Figure 3). This suggests that “Ca. Methanoperedens” serves as a widespread methane biofilter across diverse natural ecosystems, making it a favorable subject for study. Bioreactors have been successfully used to enrich this environmentally relevant genus (Figure 3). Over the past decade, distinct cultures have been successfully obtained under conditions with nitrate, manganese, and iron oxides. In nitrate bioreactor enrichments, “Ca. Methanoperedens” pairs with nitrite scavengers. During nitrate/nitrite-dependent anaerobic methane oxidation (N-DAMO), “Ca. Methanoperedens” collaborates with methanotrophic nitritescavenging partners from the genus “Ca. Methylomirabilis” (Figure 3) (Chapter 5). Furthermore, “Ca. Methanoperedens” works with anaerobic ammoniumoxidizing (anammox) bacteria as nitrite scavengers, facilitating dissimilatory nitrate reduction to ammonium and providing ammonium in exchange for nitrite removal by anammox bacteria (Chapter 4). Other nitrite scavengers may include autotrophic or heterotrophic denitrifiers (Chapter 6). The ability of “Ca. Methanoperedens” to use various electron acceptors highlights the resilience of the methane biofilter, but this system remains sensitive to environmental stressors. Lateral gene transfer was emphasized as a way of Methanoperedenaceae to expand the presence of e.g. multiheme c-type cytochrome (MHCs) (Leu et al., 2020b). Recent findings have revealed that the presence of mobile genetic elements (MGEs) associated to “Ca. Methanoperedens” could enhance its known metabolic versatility. These elements have been linked to plasmids and viruses in bioreactor systems, as well as to novel extrachromosomal elements known as Borgs found in environmental soil samples (Al-Shayeb et al., 2022; Schoelmerich et al., 2022).
35 Introduction and thesis outline Figure 3 below depicts methods that can be utilized to explore the physiology of “Ca. Methanoperedens” in bioreactor systems. Common approaches to study “Ca. Methanoperedens” include activity assays (13C-CH₄) labeling to infer methane oxidation potential, as well as metagenomics and metatranscriptomics to analyze genetic potential and transcriptional activity. ‘Ca. Methanoperedens’ “Ca. Methanoperedens” marine ANME Metagenome Metatranscriptome Metaproteome Metabolomics rt-qPCR qPCR Sialic acids FISH NO3 - MnO2 Fe(OH)3 PHA Activity Assays CH4 13 C-CH4 13C-CO 2 Figure 3. Cartoon illustrating a wetland as an exemplary environment for enriching and characterizing “Ca. Methanoperedens” in a bioreactor. The most common habitats for “Ca. Methanoperedens” include freshwater (eutrophic) organic-rich anoxic environments, such as wetlands. In contrast, other anaerobic methanotrophic archaea (ANME) and those involved in sulfate-dependent anaerobic oxidation of methane are found in marine settings. Various methods employed in this PhD dissertation for characterizing an enrichment culture of “Ca. Methanoperedens” are categorized into activity, microbial community structure analysis, spatial distribution/morphology examination, and evaluation of granules or extracellular polymeric substance (EPS) layers. Methane oxidation potential assays are represented by the monitoring of 13C-CO₂ labeled production signal derived from 13C-CH₄. Reverse Transcriptase (RT)-quantitative PCR (RT-qPCR); Polyhydroxyalkanoates (PHAs). Images were obtained from Biorender and the Integration and Application Network Symbol Library from the Center for Environmental Science of the University of Maryland (USA) and edited using Adobe Illustrator 2024. 1
36 Chapter 1 COASTAL ECOSYSTEM STRESSORS: EUTROPHICATION AND SEA-LEVEL RISE Coastal ecosystems are subject to a variety of anthropogenic stressors, with eutrophication and sea-level rise being particularly induced by climate change (Figure 4) (Howarth et al., 2011; Malone & Newton, 2020; Wallenius et al., 2021). Eutrophication in coastal ecosystems leads to an excess of organic carbon degradation coupled with sulfate reduction, resulting in the buildup of sulfide (Żygadłowska et al., 2024a). Elevated sulfide levels can disrupt microbial community functions, leading to reduced methane oxidation rates (Chapter 2 and 4). Rising sulfide concentrations can also inhibit key metabolic processes by damaging copper- and iron-containing cofactors (Jin et al., 1998) and suppressing methanogenesis (Karhadkar et al., 1987). It also poses toxicity risks to aquatic life, especially fish and invertebrates, and negatively impacts water quality for human recreational use (Riesch et al., 2015) (Figure 4, A-B). Sea-level rise, driven by climate change, poses a significant threat to coastal methane biofilters by altering methane dynamics (IPCC, 2023; Kaushal et al., 2021) (Figure 4, C-D). As with sea levels rise, saltwater intrusion into freshwater and brackish systems imposes hyperosmotic stress on freshwater microbial communities and can reduce methane oxidation rates (Ho et al., 2018; Osudar et al., 2017). While some freshwater “Ca. Methanoperedens” have been observed in marine environments (Chapter 7), the long-term effects of salinity on their methaneoxidizing capacity and physiological adaptations remain poorly understood. A common microbial strategy for coping with osmotic stress involves the accumulation of intracellular osmolytes. Osmolytes are water-soluble organic compounds that accumulate within cells to balance hyperosmotic pressure. These compounds include sugars, amino acids, polyols, and their derivatives (BebloVranesevic et al., 2017; Gregory & Boyd, 2021; Guan et al., 2017).
37 Introduction and thesis outline Eutrophication Sea level rise A C B D Figure 4. Panels A and B depict the process of eutrophication, with Panel A presenting a cartoon image and Panel B illustrating a real-case scenario of severe eutrophication featuring a scuba diver in the marine lake Mar Menor in Spain (“Mar Menor, cuando el desprecio al medio ambiente se vuelve contra nosotros,” El País, February 23rd 2020). Panels C and D represent sea-level rise, with Panel C showing a cartoon illustration and Panel D depicting a real scenario on the Pacific Ocean island of Tuvalu, which is highly impacted by rising sea levels. Here, a couple on a motorbike pass through the narrowest point of Fongafale Island in the Funafuti atoll, part of the island nation Tuvalu. The Pacific Ocean is on the left, and a lagoon is in the right. The coral island atoll nation has been identified as one of the world’s most vulnerable islands to climate change. (“This Pacific island country is disappearing. What happens next?” National Geographic, July 8th 2024). Panel A and C images were obtained from the Integration and Application Network Symbol Library from the Center for Environmental Science of the University of Maryland (USA). 1
38 Chapter 1 KNOWLEDGE GAPS ON METHANOTROPHIC RESPONSE TO COASTAL-STRESSORS Despite significant progress in understanding methane cycling in coastal ecosystems, several key knowledge gaps remain. First, while the roles of aerobic and anaerobic methanotrophs are well-established, the physiological limits of these microorganisms under stress conditions such as sulfide toxicity and salinity stress are poorly understood. For example, the mechanisms by which sulfide inhibits methanotrophic activity and the potential for microbial adaptation to sulfidic environments remain largely unexplored and are therefore addressed in Chapters 2, 4, and 5. In this regard, the putative coupling of S-AOM with “Ca. Methanoperedens” in the sulfate-containing sediments of Lake Cadagno is relevant and, this coupling was further explored in Chapter 7. Additionally, the impact of sea-level rise on methane biofilter efficiency, particularly in coastal systems, warrants further investigation and is addressed in Chapter 3 and 6. Although some freshwater methanotrophs have been shown to withstand marine salinities, the overall resilience of these communities in the face of increasing salinity gradients is not fully understood. Finally, there is a need for more detailed studies on the interplay between eutrophication, sulfide accumulation, salinity increase and methane oxidation rates. While elevated organic matter inputs clearly enhances methanogenesis, the long-term effect on methane biofilter functioning, particularly in relation to microbial community shifts and enzyme inhibition and overall physiological adaptations are not yet well explored These knowledge gaps are critical for developing accurate and predictive models of methane emissions and for designing effective management strategies to mitigate greenhouse gas emissions from vulnerable coastal ecosystems.
www.ridderprint.nlRkJQdWJsaXNoZXIy MTk4NDMw