Local Conditions Structure Unique Archaeal Communities in the Anoxic Sediments of Meromictic Lake Kivu

Meromictic Lake Kivu is renowned for its enormous quantity of methane dissolved in the hypolimnion. The methane is primarily of biological origin, and its concentration has been increasing in the past half-century. Insight into the origin of methane production in Lake Kivu has become relevant with the recent commercial extraction of methane from the hypolimnion. This study provides the first culture-independent approach to identifying the archaeal communities present in Lake Kivu sediments at the sediment-water interface. Terminal restriction fragment length polymorphism analysis suggests considerable heterogeneity in the archaeal community composition at varying sample locations. This diversity reflects changes in the geochemical conditions in the sediment and the overlying water, which are an effect of local groundwater inflows. A more in-depth look at the archaeal community composition by clone library analysis revealed diverse phylogenies of Euryarchaeota and Crenarachaeota. Many of the sequences in the clone libraries belonged to globally distributed archaeal clades such as the rice cluster V and Lake Dagow sediment environmental clusters. Several of the determined clades were previously thought to be rare among freshwater sediment Archaea (e.g., sequences related to the SAGMEG-1 clade). Surprisingly, there was no observed relation of clones to known hydrogentrophic methanogens and less than 2% of clones were related to acetoclastic methanogens. The local variability, diversity, and novelty of the archaeal community structure in Lake Kivu should be considered when making assumptions on the biogeochemical functioning of its sediment

Enrichissement d'une communauté microbienne anaérobie oxydante du méthane à partir de sédiments marins : évaluation des performances en bioréacteurs

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (AOM-SR) is a biological process mediated by anaerobic methanotrophs (ANME) and sulfate reducing bacteria. Due to its relevance in regulating the global carbon cycle and potential biotechnological application for treating sulfate-rich wastewater, AOM-SR has drawn attention from the scientific community. However, the detailed knowledge on ANME community, its physiology and metabolic pathway are scarcely available, presumably due to the lack of either pure cultures or the difficulty to enrich the biomass. To enhance the recent knowledge on ANME distribution and enrichment conditions, this research investigated AOM-SR with the following objectives: (i) characterize the microbial communities responsible for AOM in marine sediment, (ii) enrich ANME in different bioreactor configurations, i.e. membrane bioreactor (MBR), biotrickling filter (BTF) and high pressure bioreactor (HPB), and (iii) assess the AOM-SR activity under different pressure and temperature conditions.The microbes inhabiting coastal sediments from Marine Lake Grevelingen (the Netherlands) was characterized and the ability of the microorganisms to carry out AOM-SR was assessed. By performing batch activity tests for over 250 days, AOM-SR was evidenced by sulfide production and the concomitant consumption of sulfate and methane at approximately equimolar ratios and a sulfate reduction rate of 5 µmol sulfate per gram dry weight per day was attained. Sequence analysis of 16S rRNA genes showed the presence of ANME-3 in the Marine Lake Grevelingen sediment.Two bioreactor configurations, i.e. MBR and BTF were operated under ambient conditions for 726 days and 380 days, respectively, to enrich the microorganisms from Ginsburg Mud Volcano performing AOM. The reactors were operated in fed-batch mode for the liquid phase with a continuous supply of gaseous methane. In the MBR, an external ultra-filtration membrane was used to retain the biomass, whereas, in the BTF, biomass retention was achieved via biomass attachment to the packing material. AOM-SR was recorded only after ~ 200 days in both bioreactor configurations. The BTF operation showed the enrichment of ANME in the biofilm by Illumina Miseq method, especially ANME-1 (40%) and ANME-2 (10%). Interestingly, in the MBR, aggregates of ANME-2 and Desulfosarcina were visualized by CARD-FISH. Acetate production was observed in the MBR, indicating that acetate was a possible intermediate of AOM. Although both bioreactor configurations showed good performance and resilience capacities for AOM enrichment, the sulfate reduction rate was slightly higher and faster in the BTF (1.3 mM day-1 at day 280) than the MBR (0.5 mM day-1 at day 380).In order to simulate cold seep conditions and differentiate the impact of environmental conditions on AOM activities, sediment highly enriched with the ANME-2a clade was incubated in HPB at different temperature (4, 15 and 25 oC at 100 bar) and pressure (20, 100, 200 and 300 bar at 15 oC) conditions. The incubation at 100 bar pressure and 15 oC was observed to be the most suitable condition for the ANME-2a phylotype, which is similar to in-situ conditions where the biomass was sampled, i.e. Captain Aryutinov Mud Volcano, Gulf of Cadiz. The incubations at 200 and 300 bar pressures showed the depletion in activities after 30 days of incubation. Incubation of AOM hosting sediment at in-situ condition could be a preferred option for achieving high AOM activities and sulfate reduction rates.In this thesis, it has been experimentally demonstrated that biomass retention and the continuous supply of methane can favor the growth of the slow growing anaerobic methane oxidizing community in bioreactors even under ambient conditions. Therefore, locating ANME habitats in shallow environments and enriching them at ambient conditions can be advantageous for future environmental biotechnology applicationsL'oxydation anaérobie du méthane (AOM) couplé à la réduction du sulfate (AOM-SR) est un processus biologique médié par méthanotrophes anaérobie (ANME) et de bactéries sulfato-réductrices. La communauté scientifique s'inquiète de AOM, en raison de sa pertinence dans la régulation du cycle global du carbone et de la potentielle application biotechnologique pour le traitement de sulfate riches eaux usées.Pour améliorer les connaissances récentes sur les conditions de distribution et d'enrichissement ANME, cette recherche a étudié AOM-SR avec les objectifs suivants: (i) caractériser les communautés microbiennes responsables de AOM dans les sédiments marins, (ii) de les enrichir dans les bioréacteurs avec différentes configurations, à savoir bioréacteur à membrane (MBR), filtre biotrickling (BTF) et bioréacteur à haute pression (HPB), et (iii) d'évaluer l'activité de l'ANME et le processus AOM dans différentes conditions de pression et de température.Les microbes habitant peu profonde dans les sédiments de Marine lac Grevelingen (Pays-Bas) ont été caractérisés et leur capacité de faire AOM-SR a été évaluée. Un test d'activité a été réalisée en discontinu pour 250 jours, AOM-SR est mise en évidence par la production de sulfure et de la prise concomitante de sulfate et de méthane dans des rapports équimolaires et il a été atteint 5 µmoles par gramme de poids par jour de taux de réduction du sulfate. L'analyse des séquences de gènes 16SrRNA a montré la présence de méthanotrophes anaérobie ANME-3 dans les sédiments marins du lac Grevelingen.Deux configurations de bioréacteurs, à savoir MBR et BTF ont été opérés dans des conditions ambiantes pendant 726 jours et 380 jours, respectivement, pour enrichir les micro-organismes de Ginsburg Mud Volcano performantes AOM. Les réacteurs sont exploités en mode fed-batch pour la phase liquide avec un apport continu de méthane. Dans le MBR, une membrane d'ultrafiltration externe a été utilisée pour retenir la biomasse, alors que, dans la BTF, la rétention de biomasse a été accomplie par la fixation de la biomasse sur le matériau d'emballage. AOM-SR a été enregistrée seulement après ~ 200 jours dans les deux configurations de bioréacteurs. L'opération du BTF a montré l'enrichissement de l'ANME dans le biofilm par la méthode Illumina Miseq, en particulier ANME-1 (40%) et ANME-2 (10%). Dans le MBR, les agrégats d'ANME-2 et Desulfosarcina ont été visualisées par CARD-FISH. La production d'acétate a été observée dans le MBR, ce qui indique que l'acétate était un possible intermédiaire d'AOM. Bien que les deux configurations de bioréacteurs ont montré de bonnes performances, le taux de réduction du sulfate était légèrement plus élevée et plus rapide dans la BTF (1,3 mM par jour âpres 280 jours) que le MBR (0,5 mM par jour jour âpres 380 jours).Afin de simuler les conditions de suintement froid et de différencier l'impact des conditions environnementales sur AOM, les sédiments fortement enrichi avec le clade ANME-2a ont été incubées dans HPB à différentes températures (4, 15 et 25 °C à 100 bars) et pressions (20, 100, 200 et 300 bar à 15 °C). L'incubation à une pression de 100 bar et 15 ° C a été observé comme la condition la plus appropriée pour la phylotype ANME-2a, qui est similaire aux conditions in situ (Capitaine Aryutinov Mud Volcano, Golfe de Cadix). L'incubation de ce sédiment aux conditions in situ pourrait être une option privilégiée pour obtenir une activité AOM-SR plus élevée.Dans cette thèse, il a été démontré expérimentalement que la rétention de la biomasse et l'approvisionnement continu de méthane peuvent favoriser la croissance de la lente communauté microbienne qui oxyde le méthane en anaérobiose dans des bioréacteurs, même dans des conditions ambiantes. Par conséquent, la localisation des habitats de ANME dans des environnements peu profonds et l'enrichissant dans des conditions ambiantes peut être avantageuse pour les futures applications de la biotechnologie environnemental

Performance assessment and enrichment of anaerobic methane oxidizing microbial communities from marine sediments in bioreactors

L'oxydation anaérobie du méthane (AOM) couplé à la réduction du sulfate (AOM-SR) est un processus biologique médié par méthanotrophes anaérobie (ANME) et de bactéries sulfato-réductrices. La communauté scientifique s'inquiète de AOM, en raison de sa pertinence dans la régulation du cycle global du carbone et de la potentielle application biotechnologique pour le traitement de sulfate riches eaux usées.Pour améliorer les connaissances récentes sur les conditions de distribution et d'enrichissement ANME, cette recherche a étudié AOM-SR avec les objectifs suivants: (i) caractériser les communautés microbiennes responsables de AOM dans les sédiments marins, (ii) de les enrichir dans les bioréacteurs avec différentes configurations, à savoir bioréacteur à membrane (MBR), filtre biotrickling (BTF) et bioréacteur à haute pression (HPB), et (iii) d'évaluer l'activité de l'ANME et le processus AOM dans différentes conditions de pression et de température.Les microbes habitant peu profonde dans les sédiments de Marine lac Grevelingen (Pays-Bas) ont été caractérisés et leur capacité de faire AOM-SR a été évaluée. Un test d'activité a été réalisée en discontinu pour 250 jours, AOM-SR est mise en évidence par la production de sulfure et de la prise concomitante de sulfate et de méthane dans des rapports équimolaires et il a été atteint 5 µmoles par gramme de poids par jour de taux de réduction du sulfate. L'analyse des séquences de gènes 16SrRNA a montré la présence de méthanotrophes anaérobie ANME-3 dans les sédiments marins du lac Grevelingen.Deux configurations de bioréacteurs, à savoir MBR et BTF ont été opérés dans des conditions ambiantes pendant 726 jours et 380 jours, respectivement, pour enrichir les micro-organismes de Ginsburg Mud Volcano performantes AOM. Les réacteurs sont exploités en mode fed-batch pour la phase liquide avec un apport continu de méthane. Dans le MBR, une membrane d'ultrafiltration externe a été utilisée pour retenir la biomasse, alors que, dans la BTF, la rétention de biomasse a été accomplie par la fixation de la biomasse sur le matériau d'emballage. AOM-SR a été enregistrée seulement après ~ 200 jours dans les deux configurations de bioréacteurs. L'opération du BTF a montré l'enrichissement de l'ANME dans le biofilm par la méthode Illumina Miseq, en particulier ANME-1 (40%) et ANME-2 (10%). Dans le MBR, les agrégats d'ANME-2 et Desulfosarcina ont été visualisées par CARD-FISH. La production d'acétate a été observée dans le MBR, ce qui indique que l'acétate était un possible intermédiaire d'AOM. Bien que les deux configurations de bioréacteurs ont montré de bonnes performances, le taux de réduction du sulfate était légèrement plus élevée et plus rapide dans la BTF (1,3 mM par jour âpres 280 jours) que le MBR (0,5 mM par jour jour âpres 380 jours).Afin de simuler les conditions de suintement froid et de différencier l'impact des conditions environnementales sur AOM, les sédiments fortement enrichi avec le clade ANME-2a ont été incubées dans HPB à différentes températures (4, 15 et 25 °C à 100 bars) et pressions (20, 100, 200 et 300 bar à 15 °C). L'incubation à une pression de 100 bar et 15 ° C a été observé comme la condition la plus appropriée pour la phylotype ANME-2a, qui est similaire aux conditions in situ (Capitaine Aryutinov Mud Volcano, Golfe de Cadix). L'incubation de ce sédiment aux conditions in situ pourrait être une option privilégiée pour obtenir une activité AOM-SR plus élevée.Dans cette thèse, il a été démontré expérimentalement que la rétention de la biomasse et l'approvisionnement continu de méthane peuvent favoriser la croissance de la lente communauté microbienne qui oxyde le méthane en anaérobiose dans des bioréacteurs, même dans des conditions ambiantes. Par conséquent, la localisation des habitats de ANME dans des environnements peu profonds et l'enrichissant dans des conditions ambiantes peut être avantageuse pour les futures applications de la biotechnologie environnementaleAnaerobic oxidation of methane (AOM) coupled to sulfate reduction (AOM-SR) is a biological process mediated by anaerobic methanotrophs (ANME) and sulfate reducing bacteria. Due to its relevance in regulating the global carbon cycle and potential biotechnological application for treating sulfate-rich wastewater, AOM-SR has drawn attention from the scientific community. However, the detailed knowledge on ANME community, its physiology and metabolic pathway are scarcely available, presumably due to the lack of either pure cultures or the difficulty to enrich the biomass. To enhance the recent knowledge on ANME distribution and enrichment conditions, this research investigated AOM-SR with the following objectives: (i) characterize the microbial communities responsible for AOM in marine sediment, (ii) enrich ANME in different bioreactor configurations, i.e. membrane bioreactor (MBR), biotrickling filter (BTF) and high pressure bioreactor (HPB), and (iii) assess the AOM-SR activity under different pressure and temperature conditions.The microbes inhabiting coastal sediments from Marine Lake Grevelingen (the Netherlands) was characterized and the ability of the microorganisms to carry out AOM-SR was assessed. By performing batch activity tests for over 250 days, AOM-SR was evidenced by sulfide production and the concomitant consumption of sulfate and methane at approximately equimolar ratios and a sulfate reduction rate of 5 µmol sulfate per gram dry weight per day was attained. Sequence analysis of 16S rRNA genes showed the presence of ANME-3 in the Marine Lake Grevelingen sediment.Two bioreactor configurations, i.e. MBR and BTF were operated under ambient conditions for 726 days and 380 days, respectively, to enrich the microorganisms from Ginsburg Mud Volcano performing AOM. The reactors were operated in fed-batch mode for the liquid phase with a continuous supply of gaseous methane. In the MBR, an external ultra-filtration membrane was used to retain the biomass, whereas, in the BTF, biomass retention was achieved via biomass attachment to the packing material. AOM-SR was recorded only after ~ 200 days in both bioreactor configurations. The BTF operation showed the enrichment of ANME in the biofilm by Illumina Miseq method, especially ANME-1 (40%) and ANME-2 (10%). Interestingly, in the MBR, aggregates of ANME-2 and Desulfosarcina were visualized by CARD-FISH. Acetate production was observed in the MBR, indicating that acetate was a possible intermediate of AOM. Although both bioreactor configurations showed good performance and resilience capacities for AOM enrichment, the sulfate reduction rate was slightly higher and faster in the BTF (1.3 mM day-1 at day 280) than the MBR (0.5 mM day-1 at day 380).In order to simulate cold seep conditions and differentiate the impact of environmental conditions on AOM activities, sediment highly enriched with the ANME-2a clade was incubated in HPB at different temperature (4, 15 and 25 oC at 100 bar) and pressure (20, 100, 200 and 300 bar at 15 oC) conditions. The incubation at 100 bar pressure and 15 oC was observed to be the most suitable condition for the ANME-2a phylotype, which is similar to in-situ conditions where the biomass was sampled, i.e. Captain Aryutinov Mud Volcano, Gulf of Cadiz. The incubations at 200 and 300 bar pressures showed the depletion in activities after 30 days of incubation. Incubation of AOM hosting sediment at in-situ condition could be a preferred option for achieving high AOM activities and sulfate reduction rates.In this thesis, it has been experimentally demonstrated that biomass retention and the continuous supply of methane can favor the growth of the slow growing anaerobic methane oxidizing community in bioreactors even under ambient conditions. Therefore, locating ANME habitats in shallow environments and enriching them at ambient conditions can be advantageous for future environmental biotechnology application

Valorization of Bombyx mori pupae and Moringa oleifera leaf as sources of alternative protein: Structural, functional and antioxidant properties

This study compared protein concentrates extracted from silkworm pupae (SPP) and moringa leaf (MLP) using alkaline extraction followed by isoelectric precipitation with commercial whey protein concentrate (WPC). SPP demonstrated higher protein extraction yield of 5.99 % and protein recovery of 66.02 % compared to MLP (4.39% and 55.27% of extraction yield and protein recovery, respectively). Proximate composition analysis revealed that SPP contained high protein of 65.40 % with ∼13 % fat content, while MLP had lower protein (48.70%) and fat content (0.33%). Particle size analysis showed small mean diameters of ∼123 nm for WPC, ∼192 nm for SPP, and ∼1485 nm for MLP with low polydispersity index (0.35 to 0.51). Secondary structure analysis highlighted β-sheet as predominant in WPC and MLP, while SPP exhibited a major β-turns structure. Solubility studies indicated that SPP had lowest solubility compared to others. In terms of functional properties, MLP exhibited the highest water and oil holding capacities, and foaming capacity, while SPP showed the greatest emulsifying capacity and stability. After passing simulated intestinal digestion, SPP exhibited the highest recovery of TPC, ABTS, DPPH, and FRAP. This study emphasizes the feasibility and advantages of utilizing SPP and MLP as food ingredients for various food applications

Anaerobic oxidation of methane coupled to thiosulfate reduction in a biotrickling filter

Microorganisms from an anaerobic methane oxidizing sediment were enriched with methane gas as the substrate in a biotrickling filter (BTF) using thiosulfate as electron acceptor for 213days. Thiosulfate disproportionation to sulfate and sulfide were the dominating sulfur conversion process in the BTF and the sulfide production rate was 0.5mmoll(-1)day(-1). A specific group of sulfate reducing bacteria (SRB), belonging to the Desulforsarcina/Desulfococcus group, was enriched in the BTF. The BTF biomass showed maximum sulfate reduction rate (0.38mmoll(-1)day(-1)) with methane as sole electron donor, measured in the absence of thiosulfate in the BTF. Therefore, a BTF fed with thiosulfate as electron acceptor can be used to enrich SRB of the DSS group and activate the inoculum for anaerobic oxidation of methane coupled to sulfate reduction

Dynamic modeling of anaerobic methane oxidation coupled to sulfate reduction: role of elemental sulfur as intermediate

The process dynamics of anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR), and the potential role of elemental sulfur as intermediate are presented in this paper. Thermodynamic screening and experimental evidence from the literature conclude that a prominent model to describe AOM-SR is based on the concept that anaerobic methane oxidation proceeds through the production of the intermediate elemental sulfur. Two microbial groups are involved in the process: (a) anaerobic methanotrophs (ANME-2) and (b) Desulfosarcina/Desulfococcus sulfur reducers cluster (DSS). In this work, a dynamic model was developed to explore the interactions between biotic and abiotic processes to simulate the microbial activity, the chemical composition and speciation of the liquid phase, and the gas phase composition in the reactor headspace. The model includes the microbial kinetics for the symbiotic growth of ANME-2 and DSS, mass transfer phenomena between the gas and liquid phase for methane, hydrogen sulfide, and carbon dioxide and acid-base reactions for bicarbonate, sulfide, and ammonium. A data set from batch experiments, running for 250 days in artificial seawater inoculated with sediment from Marine Lake Grevelingen (The Netherlands) was used to calibrate the model. The inherent characteristics of AOM-SR make the identification of the kinetic parameters difficult due to the high correlation between them. However, by meaningfully selecting a set of kinetic parameters, the model simulates successfully the experimental data for sulfate reduction and sulfide production. The model can be considered as the basic structure for simulating continuous flow three-phase engineered systems based on AOM-SR

Enrichment of sulfate reducing anaerobic methane oxidizing community dominated by ANME-1 from Ginsburg Mud Volcano (Gulf of Cadiz) sediment in a biotrickling filter

This study was performed to enrich anaerobic methane-oxidizing archaea (ANME) present in sediment from the Ginsburg Mud Volcano (Gulf of Cadiz) in a polyurethane foam packed biotrickling filter (BTF). The BTF was operated at 20 (±2) °C, ambient pressure with continuous supply of methane for 248 days. Sulfate reduction with simultaneous sulfide production (accumulating ∼7 mM) after 200 days of BTF operation evidenced anaerobic oxidation of methane (AOM) coupled to sulfate reduction. High-throughput sequence analysis of 16S rRNA genes showed that after 248 days of BTF operation, the ANME clades enriched to more than 50% of the archaeal sequences, including ANME-1b (40.3%) and ANME-2 (10.0%). Enrichment of the AOM community was beneficial to Desulfobacteraceae, which increased from 0.2% to 1.8%. Both the inoculum and the BTF enrichment contained large populations of anaerobic sulfur oxidizing bacteria, suggesting extensive sulfur cycling in the BTF

Enrichment of ANME-2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with 13C labeled methane, 13C labeled inorganic carbon was produced and the AOM rate based on 13C-inorganic carbon was 1.2 μmol/(gdw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME-2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol gVSS −1day−1 compared to lactate (32 µmol gVSS −1day−1), acetate (26 µmol gVSS −1day−1) and methane (4.7 µmol gVSS −1day−1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane