Dissertations / Theses on the topic 'Syntrophin'

Syntrophins are scaffolding proteins that link signaling molecules to the dystrophin protein complex at the plasma membrane. To further understand the roles of syntrophins a yeast two-hybrid screen of a human brain cDNA library was done using the PDZ domain of the recently identified brain-specific gamma 1-syntrophin, an isoform for which no signaling ligands had yet been identified. This screen yielded ten overlapping clones coding for the C-terminal portion of diacylglycerol kinase-zeta (DGK-zeta), a kinase that phosphorylates the membrane lipid diacylglycerol (DAG) to phosphatidic acid (PA). Biochemical experiments and binding assays confirmed that this kinase's C-terminus, containing the consensus PDZ-binding motif Q-E-T-A-V-COOH, was both necessary and sufficient for the interaction. This complex can be immunoprecipitated from co-transfected Hela cells, and the C-terminal-PDZ interaction appears to be required for the distribution of this complex between both the cytosol and nucleus. In the brain, DGK-zeta and gamma1-syntrophin colocalize in cerebellar Purkinje fibres and pyramidal neurons of the hippocampus and cortex, areas where dystrophin is expressed, and DGK-zeta can be detected in dystrophin immunoprecipitates from mouse brain extracts. Furthermore, biochemical experiments also show that DGK-zeta binds to the PDZ domain of alpha- and beta-syntrophins, and, endogenous syntrophin can be detected in DGK-zeta immunoprecipitates from cultured C2C12 skeletal muscle cell extracts. In normal skeletal muscle, DGK-zeta and syntrophin co-localizes at the NMJ, and the sarcolemma of oxidative fibres. In mdx mouse skeletal muscle, DGK-zeta's localization at the NMJ appears unaffected, while sarcolemmal levels are decreased in degenerating fibres but not in regenerating or regenerated ones. Together, our data suggest that syntrophin binds to DGK-zeta and links it to dystrophin-associated protein complexes in neurons and muscle cells, thereby playing a role in regulating the subcellular localization and function of this lipid kinase.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

La dystrophie musculaire de Duchenne (DMD) est la conséquence de la perte de la dystrophine, une protéine cytosquelettique indispensable au maintien mécanique et fonctionnel du sarcolemme. Notre équipe a largement étudié les entrées cationiques dans les lignées murines et a montré : 1- une augmentation anormale des influx dépendant des stocks calciques (SOCE) dans les myotubes (MT) déficients en dystrophine (dys-), 2- que les influx SOCE sont médiés par les canaux TRPC1 et TRPC4, 3- que la dérégulation des SOCE dans les MT dys- est corrigée grâce à la surexpression de l'α1-syntrophine. Au jour d'aujourd’hui, il existe peu d'éléments dans la littérature quant à la caractérisation des entrées SOCEs dans les cellules musculaires humaines et dans la DMD. Ce travail de thèse s'articule autour de deux parties : Le modèle murin, dans lequel nous avons montré un rôle indispensable de STIM1 et Orai1 dans la mise en place des entrées SOCEs et l'implication de la voie Ca2+/PLC/PKC dans l'augmentation anormale de ces entrées dans les MT murins dys-. Le modèle humain primaire, dans lequel nous avons mis en évidence : 1- une augmentation anormale des influx SOCEs dans les MT DMD et établit le profil d'expression des différentes protéines nécessaires à la mise en place de ces entrées ; 2- l'implication de la voie Ca2+/PLC/PKC dans la dérégulation des SOCEs dans les MT humains DMD et le rôle de l'α1-syntrophine dans la régulation de ces entrées dans les MT humains ; 3- la dérégulation de l'homéostasie calcique dans la DMD qui se produit par l'intermédiaire des entrées cationiques dépendantes de TRPV2 dans les cellules musculaires dystrophiques
Duchenne muscular dystrophy (DMD) is the consequence of the loss of dystrophin, a cytoskeletal protein essential for the mechanical and functional maintenance of the sarcolemma. Our group has extensively studied store-operated cation influx (SOCE) in mouse cell lines and highlighted: 1- an abnormal increase in SOCE in dystrophin-deficient (dys-) mouse myotubes (MT), 2- That SOCE are mediated by TRPC1 and TRPC4, 3- that SOCE deregulation in dys- MT is corrected by overexpression of α1-syntrophin. As of today, there is little evidence in the literature regarding the characterization of SOCE in human muscle cells and in human DMD. This thesis work is divided in two parts : In the murine model, we demonstrated an essential role of STIM1 and Orai1 in the establishment of SOCE and highlighted the involvement of Ca2+/PLC/PKC pathway in the abnormal increase of cation entry in dystrophin-deficient mouse myotubes.In primary human model, we showed: 1- an abnormal increase of SOCE in DMD MT and established the expression profile of various proteins necessary for the implementation of this influx; 2- the involvement of Ca2+/PLC/PKC in SOCE deregulation in human DMD MT and the role of α1-syntrophin in the regulation of cation entry in human MT; 3- the deregulation of calcium homeostasis in DMD that occurs through TRPV2. This work proposes a new regulatory pathway, Ca2+/PLC/PKC, for SOCE in skeletal muscle cells and provides the first elements of the disruption of calcium homeostasis in DMD human myotubes due to the absence of SOCE's regulation by the α1-syntrophin and to the overactivation of TRPV2 channels

Syntrophic acetate oxidizing bacteria convert acetate into hydrogen and carbon dioxide and through the mutualistic syntrophic partnership with methanogens the products are further converted to methane in biogas processes operating at high ammonia concentrations. There is very little known about SAOBs, only five have been characterized and had their genome analyzed. The aim of this project was to gain further knowledge about the methane producing pathway of SAOBs with a proteomic approach. Proteins were extracted from biogas sludge with a phenol-based approach and trypsin digestion and peptide recovery were performed using the Suspension Trapping method. Measurement of the peptide content was made with LC-MS/MS. The peptide profiles obtained were screened for the proteins expressed of the mesophilic SAOB Syntrophaceticus schinkii. The data supports earlier suggestions that it utilizes the Wood-Ljungdahl pathway for hydrogen production. Furthermore, the peptide profile revealed that enzymes for the glycine reductase complex and the glycine cleavage system were expressed during high ammonia concentration, indicating a potential role of these enzymes in the methane producing pathway. However, due to partial failure of the sample preparation for mass spectrometry measurements no quantification conclusions could be made. A discussion on how to further improve sample preparation methods as well as how to access the proteome to a large extent is presented.

The anaerobic digestion (AD) consists of a cascade of syntrophic interactions between several microbial groups that result in the breakdown of biodegradable organic matter in the absence of oxygen, resulting in the production of biogas, a CH4 and CO2-rich mixture that can be valorised as renewable energy. The anaerobic digestion process has been engineered for a wide range of applications and current technology developments are aimed towards the so-called co-digestion, the treatment of complex mixtures of organic materials (e.g. slaughterhouse wastes) for an increased biogas yield. Nevertheless, the AD process might be hampered by the inhibitory effect of long chain fatty acids, ammonia and sulphate arising from the hydrolysis of proteins. The main objective of this thesis is to develop innovative techniques and methodologies to degrade protein. As described throughout the following sections, these compounds are potentially inhibitors of AD process, and consequently the production of CH4 and other value-added compounds. These wastes with high concentration of proteins emit high concentrations of NH3 when are degraded. It is reported that in this situation, the main population of methanogenic archaea are inhibited. However, the populations with the ability to carry out the hydrogenotrophic methanogenesis are active in this kind of environments. Therefore, increasing acetate oxidising bacteria populations (SAOB) is possible to achieve a syntrophy relation with some hydrogenotrophic archaea (HMA) populations, in this way is possible to avoid the inhibition caused by the NH3 concentration. Furthermore, sometimes these kind of wastes are associated with rich SO42- compounds that could be easily reduced to sulphide (H2S), molecule that is toxic, corrosive and odorous, producing several problems in the AD process and industrial facilities. The biological reaction that produced these compounds is performed by sulphate-reducing bacteria (SRB). These microbial communities, SRB, have the ability to couple the oxidation of organic matter to the reduction of SO42- outcompeting directly with methane producing archaea (MPA) and homoacetogenic bacteria for common substrates, and inhibiting the CH4 production. In order to carry out this doctoral thesis, two blocks (engineering and microbiological) have been developed that have been integrated as the work progressed. Firstly, different batch-scale studies were carried out in order to monitor the physical-chemical parameters and also the evolution of the microbial communities. In microbiology field, the main objective was to explore the evidence of acetate oxidation via syntrophic interaction between SAOB-HMA. To this end, the abundances of majorities groups were quantified using qPCR analysis based on the 16S rRNA and mcrA genes combined with isotopic compounds. Furthermore, the rapid advancement of high-throughput molecular techniques, as shotgun metagenomics sequencing and binning of scaffolds, have been allowed the implementation of genome-centric approaches that generate comprehensive databases from complex environmental samples, which includes complete or nearly-complete microbial genomes. Secondly, it was necessary that the reactors operated with the appropriate parameters to stablish this syntrophic interaction. As well as, it was also to establish protocols and different parameters concerning the configuration of the reactor based on literature and results obtained from the first set of experiments. Within this block, the hydraulic retention time (HRT), temperature ranges, organic loading rate, reactor configuration was treated. Finally, within this block, the most important point was to found the optimal reactor configuration. As it is known, the structure and the various units that make up a reactor, affect directly the efficiency and optimization of the same. Along this thesis different configurations was tested.
La digestió anaeròbia (DA) consisteix en una cascada d’interaccions sintròfiques entre diferents grups de microorganismes que tenen com a principal finalitat la degradació de la matèria orgànica per obtenir biogàs, una barreja de CH4 i CO2 pot ser de gran valor en el camp de les energies renovables. El procés de la DA ha sigut dissenyat per una ampli espectre de residus, (per exemple, restes d’escorxadors) per obtenir un major rendiment de biogàs. No obstant, el procés de la DA es podria veure obstaculitzat per l’efecte inhibitori dels àcids grassos de cadena llarga, la hidròlisi de les proteïnes o els compostos sulfurats. L’objectiu principal d’aquesta tesis es desenvolupar tècniques i mètodes innovadors per la degradació de les proteïnes, principalment. Com es descriu al llarg de les pròximes secciones, aquests compostos son potencialment inhibidores del procés de la DAa, i en conseqüències, de la producció de CH4. Està descrit a la literatura que sota aquestes condicions desfavorables el principal grup de microorganismes productors de CH4 (arquees metanogèniques) es troba total o parcialment inhibit. No obstant, dins d’aquesta gran comunitat de microorganismes existeixen reductes que son capaços de mantenir la activitat metanogènica tot i trobar-se sota aquestes condicions d’inhibició, mitjançant la ruta metanogènica hidrogenotròfica. Aquesta població doncs, ha obert noves possibles vies per la obtenció de biogàs partint d’aquests productes altament inhibitoris. Una d’aquestes opcions, consisteix en incrementar aquelles poblacions que son capaces d’oxidat l’acetat per aconseguir establir una sintròfia amb les poblacions hidrogenotrofiques, evitant així, la inhibició causada per NH3. A més, en moltes ocasions aquest tipus de residus porten associats altes càrregues de compostos sulfurats, el que podria portar posteriorment a una reducció d’aquests compostos a la formació d’àcid sulfhídric (H2S). Aquesta molècula, és tòxica, corrosiva i olorosa, que a més provoca problemes dins del propi procés i és altament perjudicial per les instal·lacions. Aquesta molècula es forma principalment de manera biològica, portada a terme pels bacteris coneguts com sulfato reductors (SRB). Aquestes comunitats, SRB, tenen la capacitat d’acoblar l’oxidació de la matèria orgànica amb la reducció dels compostos sulfurats, competint directament pels substrats comuns amb els arquees hidrogenotrofics i els bacteris homoacetogenics, inhibint així la producció de CH4. Amb la finalitat de desenvolupar aquesta tesis doctoral, s’han portat a terme dos grans blocs experimentals (enginyeria i microbiologia) que a mesura que el treball avançava s’han integrat progressivament l’un amb l’altre. Dins del bloc de la microbiologia, el primer objectiu va ser l’estudi i l’anàlisi de les evidencies de la ruta sintròfica oxidadora d’acetat acoblada a la metanogenesis hidrogenotrofica, estudiant les interaccions entre aquestes dues grans comunitats. Es van quantificar mitjançant anàlisis de qPCR els dos grups majoritaris bastant-nos en els gens de referencia, 16SrRNA per bacteris i mcrA per arquees metanogèniques combinat amb un estudi de marcatge isotòpic. Complementàriament, el ràpid avenç de les tecnologies moleculars d’alt impacte com la seqüenciació shotgun i les eines bioinformàtiques, han permès desenvolupar estudis centrats en el genoma i poder generar bases de dades més accessibles i amplies relacionades amb la complexitat del microbioma ambiental. En segon lloc, un dels principals objectius del segon bloc era mitjançant els correctes paràmetres d’operació dels reactors aconseguir l’aparició i estabilitat de les sintròfies comentades anteriorment. Va ser fonamental també, establir protocols i marcar els paràmetres relacionals amb la configuració dels reactors, per aquest motiu, es va basar el disseny d’aquests digestors en els resultats obtinguts dels experiments discontinus previs. Seguint en aquesta línia, es van analitzar minuciosament paràmetres com el temps de retenció hidràulic, velocitat de càrrega orgànica, rangs de temperatura i configuració del reactor. Finalment, un dels punts crítics pel bon funcionament del projecte va ser l’elecció i disseny dels reactors. Es coneix, que el disseny i l’operació dels digestors es de vital importància per un desenvolupament òptim dels procés, per això es van provar diferents configuracions.
La digestion anaerobia (DA) consiste en una cascada de interacciones sintróficas entre diferentes grupos de microganismos que tiene como principal finalidad la degradación de la materia orgánica con el fin de obtener biogás, una mezcla de CH4 y CO2 que puede ser de gran valor en el campo de las energíaas renovables. El proceso de DA ha sido diseñado para una amplia gama de aplicaciones y para obtener un mayor rendimiento de biogás. Sin embargo, el proceso de DA podría verse obstaculizado por el efecto inhibidor de los ácidos grasos de cadena larga, el amoníaco de la hidrólisis de proteínas o los compuestos sulfurados. El objetivo principal de esta tesis es desarrollar técnicas y métodos innovadores para degradar las proteínas. Como se describe a lo largo de las siguientes secciones, estos compuestos son potecialmente inhibidores del proceso de DA y, en consecuencia, de la producción de CH4. Este tipo de residuo con alta carga de proteínas emite altas concentraciones de NH3 cuando éstas son degradadas. Está reportado que bajo estas condiciones desfavorables el principal grupo de microorganismos productores de CH4 (arqueas metanogénicas) se encuentra total o parcialmente inhibido. Sin embargo, dentro de esta gran comunidad de arqueas capaces de producir CH4, existen algunos reductos capaces de mantenerse activas bajo concentraciones de elevado nitrógeno amoniacal mediante la ruta de la metanogenesis hidrogenotrófica. Esta población abre nuevas posibilidades para obtención de biogás de este tipo de residuos. Una de estas opciones consiste en incrementar las poblaciones oxidadoras de acetato para lograr una relación sintrófica estable con estas poblacions hidrogenotrófcas, evitando de este la inhibición causada por NH3. Además, en muchas ocasiones este tipo de residuos con alta carga proteíca van asociados a una elevada concentración de compuestos sulfurados, lo que podría conllevar una reducción de éstos a ácido sulfhídrico (H2S). Esta molécula es tóxica, corrosiva y olorosa, que provoca varios problemas en DA y en las instalaciones asociadas. La reacción biológica que conlleva a la formación de este compuesto está mediada por el grupo de bacterias conocidas como bacterias sulfato reductoras (SRB). Estas comunidades microbianas, SRB, tienen la capacidad de acoplar la oxidación de la materia orgánica a la reducción del SO42- compitiendo directamente por los sustratos comunes con las arqueas productoras de CH4 y bacterias homoacetogénicas inhibiendo de este modo, la producción de biogás. Se han llevado a cabo dos grandes bloques (ingeniería y microbiología) que a medida que se avanzaba con el trabajo han ido integrándose. En primer lugar, se llevaron a cabo diferentes estudios en régimen discontinuo a escala laboratorio, monitorizando los parámetros físicoquimicos y la evolución de las comunidades microbianas. El primer objetivo fue el estudio y análisis de las evidencias de la ruta sintrófica oxidadora de acetato acoplada a una metanogenesis hidrogenotrófica, estudiando las interacciones entre estas dos comunidades. Para ello, se cuantificaron mediante qPCR los dos grupos mayoritarios basándonos en los genes de referencia, 16SrRNA para la monitorización de las bacterias, y mcrA en el caso de las arqueas metanogénicas combinando esta cuantificación con el marcaje de compuestos isotópicos. Además, se ha podido desarrollar estudios centrados en el estudio del genoma gracias a la secuanciación shotgun y las herramientas bionformáticas. En segundo lugar, uno de los principales era mediante los correctos parámetros de operación de los reactores conseguir la aparición y estabilidad de las sintrofías comentadas anteriormente. Fue fundamental establecer protocolos y marcar los parámetros relacionados con la configuración de los reactores, y para ello se basó el diseño de estos en los resultados obtenidos de los diferentes ensayos discontinuos previos. Siguiendo en esta línea, se analizaron meticulosamente parámetros como tiempo de retención hidráulico, velocidad de carga orgánica, rangos de temperatura y configuración del reactor. Finalmente, uno de los puntos críticos fue la elección y diseño de los reactores. Es bien sabido, que el diseño y operación del digestor es crucial para un desarrollo óptimo del proceso, por ello se probaron diferentes configuraciones

Tidal freshwater wetland soils contain large amounts of organic carbon, some of which is mineralized to carbon dioxide (CO2) and methane (CH4) by a diverse consortium of anaerobic microorganisms that includes fermenters, syntrophs, and methanogens (MG). These microbial groups are tightly linked and often rely on cooperative interspecies metabolisms (i.e., syntrophy) to survive. Environmental perturbations can disrupt these interactions and thus alter the rates and pathways of carbon cycling. One environmental change of particular concern in coastal wetlands is sea level rise, which can result in increased episodic saltwater intrusion events into these ecosystems. These events cause an influx of sulfate (SO4-2) to the soils and may stimulate sulfate-reducing bacteria (SRB), which can directly compete with syntrophs for energy sources (e.g., fermentation products such as butyrate). Since syntroph metabolism generates byproducts that serve as the energy source for many MG, this competition can have indirect negative effects on methanogenesis. In addition, SRB can directly compete with MG for these byproducts, particularly formate, H2, and/or acetate. The goal of this study was to understand how both MG and syntroph-MG consortia respond to and recover from SRB competition during an episodic saltwater intrusion event. To achieve this, microcosms containing soil slurry from a freshwater wetland were subjected to simulated saltwater intrusion, and metabolic inhibitors were used to isolate the activity of the various functional groups. This study focused on the breakdown of butyrate, which is a key energy source in syntroph‑MG consortia metabolisms. The observed changes in butyrate breakdown rates and byproduct accumulation during butyrate degradation assays confirmed that butyrate breakdown was mediated through syntroph-MG consortia, and that formate, rather than H2, was likely used as an electron carrier during syntrophic activity. Additions of SO4‑2 (as Na2SO4) to the freshwater microcosms stimulated SRB activity and shifted the MG community to favor acetoclastic members. These changes were accompanied by a 24% increase in CO2 production and an 80% decrease in CH4 production. Interestingly, when NaCl was added to achieve similar ionic strength, CH4 production decreased by ~32%, suggesting SRB competition is not the only factor affecting methanogenesis. Butyrate degradation rates demonstrated that while SRB were strong competitors for butyrate, concurrent syntrophic metabolism was possible. Further, data show that SRB were poor competitors for acetate, which could explain the increase in acetoclastic MG. Following removal of SRB competition, CH4 production recovered but only by ~50% after 28 days, which suggests that some MG communities in tidal freshwater wetlands may not be resilient to saltwater intrusion events. Over this same time, rates of syntrophic butyrate breakdown largely recovered, but butyrate breakdown resulted in the production of less CH4 and acetate and more CO2 and formate, indicating saltwater intrusion events may lead to persistent changes in the byproducts and pathways of carbon breakdown in tidal freshwater wetlands.

Diacylglycerol kinase-zeta (DGK-zeta) attenuates diacylglycerol (DAG) signaling by converting it into phosphatidic acid (PA). DGK-zeta binds via its C-terminus, which contains a PSD-95/ Discs-Large/ZO-1 (PDZ)-binding motif, to a family of PDZ domain-containing scaffold proteins called syntrophins. Previous studies showed that some PDZ-mediated interactions are regulated by phosphorylation of the C-terminal PDZ-binding motif, however, to my knowledge, there are no published reports which demonstrate that a phosphorylation outside of this motif regulates PDZ interactions. Here, I provide evidence that protein kinase C-mediated phosphorylation of the MARCKS domain of DGK-zeta increases its association with syntrophins by a PDZ-dependent mechanism. Compared to wild-type (wt) DGK-zeta, a mutant mimicking phosphorylation of the MARCKS domain (DGK-zeta M1) bound more to recombinant syntrophin PDZ domains in in vitro binding assays. Moreover, more endogenous syntrophin co-immunoprecipitated from lysates of COS cells infected with HA-tagged DGK-zetaM1 than with wt HA-DGK-zeta. Protein kinase C (PKC) activation by phorbol myristate acetate enhanced the interaction of wt DGK-zeta and syntrophin PDZ domains, an effect that was blocked by a specific PKC inhibitor. Consistent with the idea that the MARCKS domain regulates binding, deletion of this domain decreased binding to syntrophin PDZ domains. Surprisingly, phosphorylation-mimicking mutants of extracellular signal-regulated kinase phosphorylation sites closer to the C-terminus had no detectable effect on syntrophin binding. Collectively, my findings suggest PKC-mediated phosphorylation of the MARCKS domain regulates the PDZ-dependent interaction between DGK-zeta and syntrophins.

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With the evolution of the genus Homo, both intellectually and physically, an important phase of history began: the Neolithic farming revolution. The planet came to know an activity that so far hadn’t been part of the relations between living beings: farming. Over the centuries the landscape has changed, along with the evolution of techniques to carry on such activity. The most affected environments were forest ecosystems, as the introduction of fire and cutting tools allowed the forest to be put down and burned for later agricultural planting. The advance of this management, along with the continuous increase of domesticated species, has provided the expansion of framing for great part of the world. In modern times, this activity has reached great proportions, influenced mainly by the green revolution, genetics, chemistry and biotechnology, that became widely present in the farming environment .Therefore, it is evident the degradation that this practice brings to life. As an attempt to confront this situation agroecology has been trying to rescue and improve practices that are well-adjusted to the dynamics of nature. When dealing with a science that involves many areas of knowledge, agroecology is based on working with the flow of life and natural processes. An activity that is related to agro ecological science is agroforestry systems, which basically has the purpose of forest restoration, food production and products for use in human daily life. However, in a more intense sense of the agroforestry system, there is an activity developed by the Swiss farmer / researcher Ernst Götsch, with his theory and practice has been developing for almost forty years the work of forest recovery and food production, called Syntropic Farming. So, the work aims to perform an analysis of the Syntropic Farming, considering its foundations and empirical applications. For the analysis of the empirical experiments, three areas were selected that follow the management proposed and developed by Götsch, located in the states of Bahia, Rio Grande do Sul and in the Federal District. In order to fulfill this objective, the research had different stages. Being carried out research and readings of bibliographical references; interviews; visitation to the selected areas, to acquire knowledge of the history and composition of the agroforestry systems in question and direct observation; implantation and evaluation of an agroforestry system in a town, called Sananduva - RS, in the property of the own author. The results indicate that the Syntropic Farming shows great potential for forest recovery, together with the restoration of the micro fauna due to the constant presence of organic matter in the soil. It is noteworthy the great production of food, both in quantity and in quality. The potential of replicability of the Syntropic Farming in the three analyzed experiences was also verified, considering some selected central aspects. Since the cultivation is based on the processes of the forest, an important effect is in the autonomy that the system provides to the farmer.
Com a evolução do gênero Homo, tanto intelectualmente quanto fisicamente, viu-se iniciar uma importante fase da história: a revolução agrícola neolítica. O planeta passou a conhecer uma atividade que até então não havia feito parte das relações entre os seres vivos: a agricultura. Com o passar dos séculos a paisagem foi mudando, juntamente com a evolução de técnicas para exercer tal atividade. Os ambientes mais atingidos foram os ecossistemas florestais, pois a introdução do fogo e ferramentas de corte permitiram que a floresta fosse posta abaixo e queimada, para posterior plantio agrícola. O avanço deste manejo, juntamente com o acréscimo contínuo de espécies domesticadas proporcionaram a expansão da agricultura por boa parte do mundo. Em tempos modernos, esta atividade atingiu grandes proporções, influenciada principalmente pela revolução verde, a genética, a química e a biotecnologia, que tornam-se onipresentes nos meios agrícolas. Para tanto, fica evidente a degradação que tal prática ocasiona à vida. Na tentativa de contrapor esta situação a agroecologia tem procurado resgatar e aperfeiçoar práticas mais equilibradas com a dinâmica da natureza. Tratando de uma ciência que envolve muitas áreas do conhecimento, a agroecologia tem por base trabalhar no fluxo da vida e dos processos naturais. Uma atividade que adentra a ciência agroecológica são os sistemas agroflorestais, que em grande parte tem a finalidade de recomposição florestal, produção de alimentos e produtos para uso no cotidiano humano. Porém, num sentido mais profundo de sistema agroflorestal, está a atividade desenvolvida pelo suíço agricultor/pesquisador Ernst Götsch, que com sua teoria e prática vem desenvolvendo, há quase quarenta anos, o trabalho de recuperação florestal e produção de alimentos, chamado de Agricultura Sintrópica. Para tanto, o trabalho objetiva realizar uma análise da Agricultura Sintrópica, considerando seus fundamentos e aplicações empíricas. Para a análise das experiências empíricas foram selecionadas três áreas que seguem o manejo proposto e desenvolvido por Götsch, localizadas no estado da Bahia, Rio Grande do Sul e no Distrito Federal. Para o cumprimento do referido objetivo, a pesquisa teve diferentes etapas, sendo realizadas pesquisa e leituras de referenciais bibliográficos; entrevistas; visitação às áreas selecionadas, para conhecimento da história e da composição dos sistemas agroflorestais em questão e observação direta; implantação e avaliação de um sistema agroflorestal no município de Sananduva – RS, em propriedade do próprio autor. Os resultados indicam que a Agricultura Sintrópica mostra grande potencial para recuperação florestal, juntamente com a restauração da microfauna, devido à presença constante de matéria orgânica no solo. Destacase a grande produção de alimentos, tanto em quantidade quanto em qualidade. Também, foi constatado o potencial de replicabilidade da Agricultura Sintrópica nas três experiências analisadas, considerando alguns aspectos centrais selecionados. Por se tratar de cultivo baseado nos processos da floresta, um efeito importante está na autonomia que o sistema proporciona ao agricultor.

La dystrophine est une protéine du cytosquelette normalement exprimée sous la membrane des cellules musculaires squelettiques. L'absence de cette protéine dans la Dystrophie Musculaire de Duchenne (DMD) entraîne la nécrose des fibres musculaires, résultant entre autres d'une dérégulation des mouvements calciques à travers le sarcolemme et par conséquent d'une augmentation du calcium libre dans le myoplasme. A l'heure actuelle, le lien entre l'absence de dystrophine et l'altération calcique n'est toujours pas établi et l'objectif de ce travail a été de le mettre en évidence. Les expériences ont été principalement réalisées sur les lignées cellulaires SolC1 déficientes en dystrophine et SolD6 exprimant la mini-dystrophine ainsi que sur des cultures primaires de souris normales et de souris mdx, modèle animal de la DMD. Notre étude démontre que, dans les cellules déficientes en dystrophine, les entrées calciques activées par la déplétion en calcium du réticulum sarcoplasmique, sont considérablement augmentées. Par la technique de siRNA, nous avons pu identifier les canaux TRPC1 et TRPC4 par où transitent les influx cationiques dans les myotubes SolD6. Nous avons également décrit pour la première fois un lien moléculaire entre TRPC1/TRPC4 et la dystrophine, l’α1-syntrophine et le domaine PDZ de cette dernière. Ce complexe α1-syntrophine/TRPCs est réduit dans les cellules déficientes en dystrophine car l'expression de l'α1-syntrophine au sarcolemme est diminuée. Nous suggérons qu'une régulation normale des entrées de calcium à travers TRPC1/TRPC4 dépend de l'association entre ces canaux non dépendants du potentiel et l'α1-syntrophine. En effet, la surexpression de l'α1-syntrophine dans les cellules déficientes en dystrophine rétablit l'entrée de calcium. Inversement, des expériences avec des siRNAs dirigés contre l'α1-syntrophine entrainent une augmentation des influx cationiques dans les cellules exprimant la mini-dystrophine à des niveaux proches des influx mesurés dans les cellules déficientes en dystrophine. En plus de son rôle de protéine d'échafaudage, l'α1-syntrophine serait donc cruciale pour réguler l'activité des canaux TRPC1/TRPC4 dans le muscle squelettique. D'autre part, nous avons pu mettre en évidence par des traitements pharmacologiques que l'influx cationique exacerbé des cellules SolC1 déficientes en dystrophine est dépendant de la voie PLC/PKC. Dans ces myotubes, l’absence de la dystrophine et/ou de l'α1-syntrophine entrainent à travers TRPC1 des entrées accrues de calcium, potentialisées par la voie PLC/PKC. Ce travail de thèse a mis clairement en évidence un influx cationique dépendant des canaux TRPCs et régulé par l'α1-syntrophine dans la cellule musculaire squelettique. L'absence de cette dernière au sarcolemme pourrait conférer une nouvelle sensibilité au canal TRPC1 entrainant alors sa suractivation et une entrée incontrôlée de calcium dans le cytoplasme des cellules déficientes en dystrophine
The dystrophin is a cytoskeleton protein normally expressed underneath the sarcolemma of skeletal muscle. The lack of this protein in Duchenne Muscular Dystrophy leads to muscle necrosis and to increased intracellular free calcium in the cytoplasm. Actually, the link between calcium mishandling and the absence of dystrophin is not well established and the aim of my study is to demonstrate it. Our works showed that cationic influx activated by calcium depletion of sarcoplasmic reticulum is strongly increased. We identified TRPC1 and TRPC4 channels supporting cationic influx in myotubes expressing mini-dystrophin. We also described for the first time a molecular link between dystrophin and TRPC1/TRPC4 channels, the alpha1-syntrophin. We suggested that normal regulation of syntrophin overexpression leads to reduction of abnormal cationic influx in dystrophin-deficient myotubes. Conversely, alpha1-syntrophin repression leads to increased cationic entry in myotubes expressing mini-dystrophin. The presence at normal level of this protein appears to be crucial for normal regulation of TRPC1/TRPC4 channels in skeletal muscle. On the other hand, we demonstrated an increased cationic influx supported by TRPC in dystrophin-deficient myotubes, which seems to be potentiated by PLC/PKC pathway

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass
Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass.
Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt.

This thesis evaluates a novel technique to increase the active biomass inside continuously stirred tank biogas reactors with possible benefits of shorter retention times, higher degree of degradation, higher methane yield and tolerance of higher organic loading rates. The technique includes addition of magnetic biomass carriers to the process which, after adhesion of active microorganisms, can be magnetically separated at reactor outflow and reintroduced to the process. The evaluation of magnetic biomass carriers included methods such as batch experiments, quantitative real-time polymerase chain reaction and continuous reactor experiments with different organic loading rates and addition of volatile fatty acids. The results show that reintroduction of magnetic biomass carriers does indeed work: an accumulated biomass of microorganisms is achieved inside the reactor during a continuous process. Magnetite was selected as the most promising biomass carrier, microbiological studies of the particles show that microbiological colonization of magnetite is present with preferential adhesion of hydrogenotrophic methanogens, important for the methanogenesis. The anaerobic digestion with magnetite as biomass carrier present increased process stability and elevated degrading potential of volatile fatty acids, as well as leading to higher methane content when subjected to increased organic load. Thus, the total gas production is increased in certain situations when using magnetic biomass carriers, why further studies of appropriate hydraulic retention times, organic loading rates and substrates are warranted.

Biogas är benämningen för metangas (CH₄) som är producerad via anaerob (syrefri) rötning av biologiskt material. I Linköping finns en av Sveriges största biogasanläggningar. Anläggningen drivs av Svensk Biogas AB som ägs av Tekniska Verken i Linköping AB (publ.). I anläggningen rötas stora mängder proteinrikt substrat vilket leder till höga halter av ammoniak (NH₃) och ammonium (NH₄⁺) i rötkammaren. Ammoniak (NH₃) är toxiskt för de metanbildare som i en välmående process står för den största delen av metangasproduktionen. Höga halter av ammoniak (NH₃) och ammonium (NH₄⁺) kan inhibera dessa metanbildare vilket leder till minskad gasproduktion.

Detta examensarbete syftar främst till att genom ett kontinuerligt rötkammarexperiment utreda om zeoliter är ett lämpligt hjälpmedel för att reducera ammonium (NH₄⁺) i en anaerob process. Vid sänkta halter ammonium (NH₄⁺) är hypotesen att de mikroorganismer som är aktiva i den mest effektiva metanbildningsvägen återetableras. Arbetet syftar även till att experimentellt utreda vilka effekter zeoliter i sin helhet har på den anaeroba processen. Zeoliters effekt vid låga zeolitkoncentrationer utreds i en serie utrötningsexperiment i batch. Dessutom har en materialstudie kring zeoliternas kapacitet i olika miljöer genomförts.

Materialstudien visade att den valda zeoliten som studerades, clinoptilolite, hade en maximal katjonbytarkapacitet på ≈ 19 mg NH₄⁺/g zeolit. Vidare konstaterades att zeoliter med mindre diameter än 1 mm har avsevärt bättre kapacitet än zeoliter med större diameter. I det kontinuerliga rötkammarexperimentet konstaterades att clinoptilolite kan användas i en rötkammare för att reducera ammoniumhalten (NH₄⁺). Detta utan att några allvarliga processtörningar uppstår. Cirka 175 g zeolit/l krävdes för att reducera ammoniumhalten (NH₄⁺) från 5300 mg NH₄⁺/l till 3200 mg NH₄⁺/l. Det är inte realistisktatt använda så stora mängder zeoliter i en fullskalig anläggning. Mikrobiologiskt sett observerades ingen förändring av de metanbildarna som dominerar den effektivaste metanbildningsvägen.

Resultaten från utrötningsförsöket i batch visade att zeolittillsatser av 5 g/l respektive 10 g/l hade en klart positiv effekt på metanbildningen jämfört utan zeolittillsats. I de batcher med 1-10 g zeolit/l startade metangasproduktionen ≈ 14 dagar tidigare än batcherna med 0 g zeolit/l. 16 dagar efter att experimentet startade hade batcherna med 5-10 g zeolit/l producerat ≈ 500 ml metangas (CH₄) jämfört med serien utan zeoliter som vid samma tidpunkt producerat ≈ 75 ml metangas (CH₄). Utrötningsgraden ökade i samtliga serier med zeolittillsats jämfört med serien utan zeoliter. Tillsatsen av 5 g zeolit/l ökade den specifika metangasproduktionen med ≈ 19 % jämfört med utan zeolittillsatser. Slutsatsen är att clinoptilolite i små koncentrationer, mellan 5-10 g/l, påverkar så väl kinetiken som utrötningsgraden för den anaeroba processen på ett positivt sett. Den optimala koncentrationen av clinoptilolite i en mesofil anaerob process bör ligga mellan 5-10 g zeolit/l.

Methane (CH₄) is formed by anaerobic (oxygen-free) digestion of biological materials. One of Sweden's largest biogas plants is placed in Linköping. The plant is operated by Svensk Biogas AB, which is owned by Tekniska Verken i Linköping AB (publ.). In their biogas plant a large amount of protein rich material is handled. High amounts of protein leads to high levels of ammonia (NH₃) and ammonium (NH₄⁺) in the digestion chamber. High levels of ammonia (NH₃) are toxic to the most dominant methane forming microorganism. High concentrations of ammonia (NH₃) and ammonium (NH₄⁺) can inhibit these methane forming microorganisms which may lead to a reduction in gas production.

This aim with this master thesis was to reduce high ammonium levels by adding zeolites to a lab scale continuous digestion chamber. The hypothesis is that at reduced levels of ammonium (NH₄⁺) the most effective methane forming microorganism will reestablish. This thesis also aims to experimentally investigate all types of effects that zeolites may have in an anaerobic digestion process. Which effect zeolites at low concentration have in a digestion chamber will be investigated by using lab scale batch digestion chambers. In addition, a material study on the capacity of the zeolites in different environments will be investigated.

The material study showed that the selected zeolite, clinoptilolite, had a cat ion exchange capacity around 19 mg NH₄⁺/g zeolite. It was also found that the zeolites with a diameter less than 1 mm had significantly better capacity than zeolites with larger diameter. In the continuous digestion experiment it was found that clinoptilolite can be used in a digestion chamber to reduce high levels of ammonium (NH₄⁺). This without any serious disorder on the process. Around 175 g zeolite/l was needed to reduce ammonium levels (NH₄⁺) from 5300 mg NH₄⁺/l to 3200 mg NH₄⁺/l. However, it is not realistic to use such large amounts of zeolites in a full-scale digestion chamber. No changes in the culture of methane forming microorganisms were found.

The results of the batch experiment showed that concentrations of 5 g zeolite/l and 10 g zeolite/l had a positive effect on the methanogenesis compared to batches without additives. In the batches with 1-10 g zeolite/l the forming of methane began about 14 days earlier than in the batches without any zeolites. After 16 days, batches with 5-10 g zeolite/l had produced about 500 ml of methane (CH₄), compared with series without additives, which at the same time had produced about 75 ml of methane (CH₄). The methane yield increased in all series which included zeolites compared to the batches without zeolites. Addition of 5 g zeolite/l increased the specific methane production by approximately 19 % compared to no additives. The conclusion is that clinoptilolite in small concentrations; 5-10 g/l have a positive effect on as well the kinetics as on the methane yield for the anaerobic process. The best concentration of zeolites in a mesophile anaerobic digestion chamber appears to be between 5-10 g zeolite/l.

Les procédés anaérobies de traitement de déchets apparaissent clairement pouvoir répondre à l'enjeu socio-économique actuel que représente la valorisation énergétique de la fraction organique contenue dans les déchets ménagers. En effet, les processus de dégradation anaérobies font intervenir en cascade, différentes réactions et populations de micro-organismes permettant de transformer la matière organique en biogaz riche en méthane. Une bonne connaissance des effets des paramètres opérationnels sur l'orientation des métabolismes s'avère ainsi nécessaire à l'émergence de solutions permettant d'optimiser ces procédés. Ceci est notamment le cas pour la dernière étape, appelée méthanogenèse. Dans ce contexte, l'approche isotopique reposant sur la mesure de la composition isotopique (13C/12C) du méthane et du dioxyde de carbone, devrait pouvoir répondre à cet objectif en permettant l'identification des métabolismes à l'origine de la production du méthane. La transposabilité à l'étude de la digestion anaérobie des déchets de cette approche isotopique déjà utilisée dans les écosystèmes naturels, a tout d'abord été vérifiée expérimentalement. Les effets de certains paramètres opérationnels connus pour avoir un impact fort sur le processus de digestion anaérobie, tels que la température et la concentration en azote ammoniacal, ont ensuite été étudiés. Il a été mis en évidence qu'en condition thermophile, la méthanogenèse acétoclaste observée en condition mésophile, était remplacée par une oxydation syntrophique de l'acétate lors de la digestion anaérobie des déchets ménagers. Des expériences sur acétate ont montré que cet effet sur les voies métaboliques n'était toutefois pas systématique et pourrait ne pas être dû à un effet direct d'une augmentation de la température, mais plutôt à l'accroissement de la concentration en ammoniaque qui en résulte. D'autres expériences ont clairement établi qu'une augmentation de la concentration en azote ammoniacal conduisait également à la mise en place de l'oxydation syntrophique de l'acétate. Le couplage de l'approche isotopique avec des analyses microbiologiques a révélé que cette réaction d'oxydation syntrophique de l'acétate, à haute concentration en azote ammoniacal, pouvait s'établir telle que déjà décrite, par la mise en place d'une relation symbiotique bactéries/archées hydrogénotrophes strictes, mais également de manière différente en impliquant des membres de la famille Methanosarcinaceae qui pourraient réaliser seuls les deux étapes de la réaction (oxydation et méthanogenèse hydrogénotrophe). L'application de l'approche isotopique a également permis de mettre en évidence, lors d'une expérience visant à simuler la recirculation de différents effluents au sein d'une installation de stockage de déchets bioactive, l'influence de la nature de l'effluent sur l'orientation des métabolismes méthanogènes. Enfin, l'influence de la proportion de déchets verts, lors de la co-digestion biodéchets / déchets verts, sur la concentration en ions ammonium libérés ainsi que sur l'orientation du métabolisme en résultant, a été étudiée. Les potentialités d'une utilisation de l'approche isotopique sur site ont également été investiguées au travers d'une campagne de mesures sur une installation de stockage de déchets non dangereux
Anaerobic waste treatment processes are clearly part of the answer to a current important socio-economic issue in waste management: energy production from the organic fraction of municipal solid waste. The anaerobic digestion of municipal solid waste is a complex process involving numerous reactions and microorganism communities. At the end of the degradation process, some biogas with a particularly high methane content is produced. A detailed knowledge on how operational parameters affect metabolism orientations is required to optimize these treatment processes. This is in particular the case for the last degradation reaction called methanogenesis. In this context, an isotopic approach based on isotopic composition measurements (13C/12C) for methane and carbon dioxide can provide some clues with regard to this objective. Indeed, this methodology enables the determination of the methanogenic pathways by which methane is produced.Transferability of the isotopic approach used for natural ecosystems to the field of anaerobic digestion of municipal solid waste was first experimentally verified. In a second time, the effects of some operational parameters known to strongly impact the anaerobic digestion process, such as temperature and ammonia concentration, were studied. During anaerobic digestion of reconstituted municipal solid waste in thermophilic conditions, it was shown that aceticlastic methanogenesis (occurring in mesophilic conditions) was replaced by a syntrophic acetate oxidation reaction. Additional experiments using acetate as sole substrate were performed and showed that this effect on the metabolic pathways was not systematic. Consequently, it cannot be due to a direct effect of the temperature increase. It could rather be explained by the induced and indirect increase in ammonia concentration. Additional experiments clearly demonstrated that an increase in ammonia concentration led to the establishment of a syntrophic acetate oxidation reaction. The isotopic approach was combined with microbiological analyses and showed that the syntrophic acetate oxidation reaction occurring at high ammonia concentration during acetate incubations could have been performed through a syntrophic relationship between bacteria and strict hydrogenotrophic archaea, as previously described in the literature. Interestingly, the syntrophic acetate oxidation could also have occurred using a different pathway relying on members of the Methanosarcinaceae family putatively able to perform the two steps of the reaction (oxidation and hydrogenotrophic methanogenesis). In addition, the implementation of the isotopic approach during an experiment designed to simulate a landfill bioreactor evidenced the influence of the effluent's nature on the methanogenesis metabolism orientation. The influence of green waste proportion during the co-digestion of biowaste / green waste mixtures on resulting ammonia concentrations and methanogenesis pathways was also studied through dedicated experiments. Finally, the potential of the isotopic approach for landfill-scale application was investigated through a measurement campaign on a landfill site

博士
國立成功大學
環境工程學系
89
Chemical production of Purified Terephthalic Acid (PTA) as the raw material was one of the important petrochemical and related industry in Taiwan. In addition to acetate, wastewater generated from the PTA manufacturing process mainly contained aromatic compounds including terephthalate (isomers), 4-methylbenzoate and benzoate. In the preliminary studies of biodegradability assays, the results revealed that the aromatic compounds were relatively slowly mineralized by the methanogenic consortia with the acclimation periods of more than 3 to 16 months. The thesis was thus aimed for providing the ecological insight into the microbial diversity associated with the degradation of terephthalate, 4-methylbenzoate and benzoate by the methanogenic consortia. Energetics analysis suggested that the carboxylated aromatic compounds had to be degraded syntrophically by fermenting bacteria at the conditions with the low levels of acetate and hydrogen which were manipulated by the methanogens in the methanogenic consortia. The syntrophic degradation was evidently supported by the observation that the primary intermediates such as acetate and hydrogen were identified in the degradation of aromatic compounds. With built-up of the acetate and/or hydrogen by adding the selective inhibitors (2-bromoethanesulfonic acid or sodium molybdate), the anaerobic degradation of terephthalate and 4-methylbenzoate reached the thresholds with the actual Gibbs free energy change in the range from —72 to —82 kJ/mol, equivalent to the energy quantum needed for the synthesis of one ATP unit, approximately. The energy conserved in the bacteria responsible for degrading terephthalate and 4-methylbenzoate was substantially higher than that for degrading benzoate and fatty acids. It was likely that the higher energy was conserved from the decarboxylation and demethylation processes. In addition, anaerobic degradation of terephthalate and 4-methylbenzoate was shown to be rate-limited in the fermentation step. Morphological characterization of the mature biofilm and the granulated sludge individually obtained from the anaerobic fluidized bed (AnFB) reactor and the upflow anaerobic sludge bed (UASB) reactors treating PTA wastewater was conducted by using the scanning and the transmission electron microcopies. The micrographs clearly demonstrated the architectures of the biofilm and the granule with the distribution of microbial clusters that were composed of the unidentified bacteria in syntrophic association with the hydrogenotrophic methanogen-like (Methanobrevibacter-like) and/or the acetoclastic methanogen-like (Methanosaeta-like and Methanosarcina-like) bacteria. The comparative results further revealed the morphotypic similarity of the overall microbial composition but the distribution discrepancy of the Methanosaeta-like and Methanosarcina-like populations between the biofilm and the granule degrading the mixed substrates of PTA wastewater. In the methanogenic consortia individually enriched with terephthalate, 4-methylbenaoate and benzoate, the methanogen-like populations were quite similar but the predominant bacteria with the rod morphotypes were obviously different. The rod bacteria with the distinctive traits were also frequently observed in the biofilm and granule from the AnFB and UASB reactors treating PTA wastewater, suggesting the role in the anaerobic degradation of aromatic compounds. Using 16S rDNA-based molecular techniques, the phylogenetic diversity of the members within the methanogenic consortia degrading terephthalate, 4-methylbenzoate and benzoate was investigated. 16S rDNA sequence fragments were amplified by polymerase chain reaction (PCR) with genomic DNA directly extracted from the methanogenic consortia and retrieved by the cloning and sequencing approach. Most of the retrieved 16S rDNA sequences were related to the populations without the known isolates, suggesting the unique community structures. Comparative analysis of the identified clonal sequences and closely related references from the public database indicated that the bacterial members, which might be involved in the aromatic degradation were phylogenetically affiliated with the prevalent divisions including Proteobacteria (delta subdivision), green non-sulfur bacteria (subdivision I), candidate novel MBA1 and many others, specific to the individual community. It was suggested that the members represented by the delta-proteobacterial sequences were the predominant populations in the three communities. More importantly, that the identified delta-proteobacterial sequences were individually classified into three clusters in the phylogenetic tree seemed to depend on the aromatic substrates, suggesting that the populations degrading terephthalate, 4-methylbenzoate and benzoate were probably different. Furthermore, the Archaea members within the terephthalate-enriched methanogenic consortium were found to closely affiliate with the Methanosaeta- and the Methanospirillum-related populations. Fluorescence in situ hybridization (FISH) with the group-specific, 16S rRNA-targeted oligonucleotide probes was used to localize the members in the granules. The fluorescent signals correctly hybridized with the targets revealed that the random or mixed distribution of the microbial populations was elucidated for the non-layered topology of the granule. The FISH technique with the 16S rRNA-targeted probe detected the presence of the Methanobacteriaceae, which the corresponding sequences were not retrieved in the clone library, further suggesting that the microbial diversity of the community could not completely unveiled by only one method. Based on the phenotypic and phylotypic database established in this thesis, it can provide the microbial indicators or information for the further application in the anaerobic processes treating PTA wastewater and in the isolation of the novel anaerobic microorganisms.

Tese de doutoramento em Engenharia Química e Biológica
Anaerobic degradation of long-chain fatty acids (LCFA), is not yet completely understood. Previous studies suggest that different microorganisms might be involved in the degradation of saturated and unsaturated LCFA and that these compounds inhibit severely the microbial activity, especially the methanogenic activity. In this study, the toxic effect of saturated- (palmitate (C16:0) and stearate (C18:0)) and unsaturated-LCFA (oleate (C18:1)), towards pure cultures of hydrogenotrophic methanogens was evaluated by measuring cells viability and methanogenic activity of Methanospirillum hungatei and Methanobacterium formicicum. The presence of hydrogenotrophic (M. formicicum and M. hungatei) and acetoclastic (Methanosaeta concilii and Methanosarcina. mazei) methanogens in oleate and palmitate enrichment cultures was detected by PCR-DGGE fingerprinting techniques. Acetoclastic methanogens and M. formicicum grew in oleate and palmitate enrichment cultures but M. hungatei only grew in the palmitate’s enrichment. M. hungatei was more sensitive than M. formicicum particularly to unsaturated LCFA. The later was also the most abundant hydrogenotroph detected during the continuous treatment of a synthetic wastewater composed mainly by oleate, as determined by PCR-DGGE. In the same study, M. concilii was identified as the most representative acetoclast. In order to investigate differences between the proteins expressed during the degradation of saturated and unsaturated LCFA, a metaproteomics experiment was designed, in which an anaerobic sludge was incubated with palmitate, stearate and oleate. The same COGs functional categories were identified in the different conditions. The majority of the proteins were assigned to functional categories, energy production and conversion, posttranslational modification and lipid metabolism. Most of the proteins identified belong to Methanosaeta concilli, Syntrophobacter fumaroxidans, Pelobacter propionicus and Pelotomaculum thermopropionicum. Methanosaeta concilii was indeed the most abundant archaea detected by pyrosequencing analysis of the 16S rRNA gene, but the other microorganisms were not even detected by pyrosequencing. Studying metaproteomes of complex microbial communities is still a big challenge especially because most of their genomes are not sequenced which difficult proteins identification. Likewise, when analyzing the proteome of the co-culture, Syntrophomonas zehnderi and M. formicicum, specialized on the degradation of LCFA, M. formicicum’s proteome could be much better characterized compared to S. zehnderi’s, since the genome of a very close related strain of the former is available in public databases and the genome of S. zehnderi is not. S. zehnderi was a dominant microorganism in oleate degrading enrichment cultures under methanogenic and non-methanogenic conditions, stablishing close relationships with hydrogenotrophic methanogens and homoacetogenic bacteria, respectively.
Os ácidos gordos de cadeia longa (AGCL) podem ser convertidos a metano por digestão anaeróbia. Contudo, aspectos relacionados com a microbiologia desta conversão ainda não estão completamente compreendidos. Estudos anteriores sugerem que diferentes microrganismos possam estar envolvidos na degradação de AGCL saturados e insaturados e ainda que os AGCL inibem a actividade microbiológica e de forma mais severa a actividade metanogénica. Neste trabalho foi avaliado o efeito toxico dos ACGL saturados (palmitato (C16:0) e estearato (C18:0)) e insaturados (oleato (C18:1)) sobre culturas puras de Methanobacterium formicicum e Methanospirillum hungatei monitorizando a sua viabilidade celular e actividade metanogénica. A presença destes organismos hidrogenotróficos bem como de dois organismos acetoclásticos, Methanosaeta concilii e Methanosarcina mazei, em culturas mistas enriquecidas em degradadores de palmitato e de oleato, foi determinada por PCR-DGGE. Todos permaneceram nos dois enriquecimentos com excepção do M. hungatei que não foi capaz de crescer no enriquecimento com oleato. Este microrganismo mostrou ser mais sensível do que o M. formicicum aos AGCL insaturados, segundo os resultados de viabilidade celular e actividade metanogénica. Em reactores anaeróbios alimentados com um efluente sintético, composto maioritariamente por ácido oleico, M. formicicum e M. concilii foram identificados como os metanogénicos predominantes. Foi efectuado um estudo de metaproteómica com o objectivo de detectar diferenças na expressão de proteínas por parte de uma cultura mista a crescer em AGCL saturados e insaturados. A maioria das proteínas identificadas nos vários ensaios estava relacionada com processos metabólicos de produção de energia, incluindo o metabolismo dos lípidos, e modificação pós-traducional. A maioria das proteínas identificadas corresponde a proteínas dos seguintes microrganismos: Methanosaeta concilli, Syntrophobacter fumaroxidans, Pelobacter propionicus e Pelotomaculum thermopropionicum. Estas amostras foram paralelamente classificadas taxonomicamente com base nos resultados de pirosequenciação do gene que codifica para a subunidade 16S do rRNA. Segundo esta análise a Methanosaeta concilii foi identificada como o organismo metanogénico mais dominante, contudo, não foram detectadas sequências correspondentes a S. fumaroxidans, P. propionicus ou P. thermopropionicum. A falta de informação genética/proteica sobre os microrganismos envolvidos na degradação de AGCL dificulta a identificação de proteínas que poderão ser relevantes neste processo. Analisou-se também a expressão proteica de dois organismos sintróficos, Syntrophomonas zehnderi and M. formicicum, que convertem os AGCL a metano. Neste caso particular foi possível obter uma melhor caracterização do proteoma de M. formicicum do que de S. zehnderi, consequência do facto de apenas o genoma de uma estirpe próxima do primeiro se encontrar sequenciado. A S. zehnderi foi identificada como uma bactéria dominante em culturas especializadas na degradação de oleato quer em condições metanogénicas, estabelecendo relações de sintrofia com microorganismos metanogénicos hidrogenotróficos, quer em condições não metanogénicas onde a interacção como organismos homoacetogénicos terá sido favorecida.
Research grant (SFRH/BD/48960/2008) from the Portuguese Foundation for Science and Technology (FCT) and European Social Fund (POPHQREN)
Project FCOMP-01-0124-FEDER-014784, financed by the FEDER funds through the Operational Competitiveness Programme (COMPETE) and by national funds through the Portuguese Foundation for Science and Technology (FCT)

Dissertação de mestrado em Bioinformatics
Microbial communities have gained particular interest and have been used for practical applications such as biorefineries, and bioremediation. However, studying these communities has proven to be difficult due to the absence of experimental protocols and computational tools like the ones available for single organisms. In this work, we present Genome-Scale Metabolic models both for Methanospirillum hungatei strain JF1 and Syntrophobacter fumaroxidans strain MPOBT, together with a model that combines both into one community model. The genome-scale metabolic model reconstruction of S. fumaroxidans was performed in merlin whereas, the methane-producing archaeon M. hungatei was reconstructed in KBase’s environment and the model curation was performed in merlin. OptFlux and BioCoISO, a tool implemented over COBRApy developed specifically for debugging model pathways, were used for curating and validating both models. The metabolism of each individual organism was assessed through its model reconstruction. In silico simulations demonstrated the production of various compounds of interest such as formate in M. hungatei and acetate in S. fumaroxidans. The meta-model representing the community composed by both organisms was assembled using FRAMED, and it was able to describe the metabolic exchanges between the formate scavenger M. hungatei and the syntrophic partner S. fumaroxidans. The reconstructed models can be used to study further the metabolic interactions between these bacteria.
As comunidades microbianas são de especial interesse e têm sido usadas para aplicações práticas como em biorrefinarias e biorremediação. No entanto, o estudo destas comunidades tem sido difícil devido há falta de protocolos experimentais e ferramentas computacionais, como os que existem para cada organismo individualmente. Neste trabalho são apresentados os modelos metabólicos à escala genómica para estirpe JF1 de Methanospirillum hungatei e a estirpe MPOBT de Syntrophobacter fumaroxidans, juntamente com um modelo que combina ambos os modelos criados num modelo de comunidade. A reconstrução do modelo metabólico à escala genómica de S. fumaroxidans foi realizada no merlin, enquanto que o modelo da bactéria produtora de metano M. hungatei foi reconstruído na KBase e a curação manual efetuada no merlin. OptFlux e BioColSO, uma ferramenta implementada sobre o COBRApy, desenvolvida especificamente para a correção de vias do modelo, foram usadas para a curação e validação de ambos os modelos. O metabolismo de cada organismo foi acedido através das respetivas reconstruções realizadas para cada um. Simulações in silico demonstraram a produção de vários compostos de interesse como o formato no caso de M. hungatei e acetato no caso de S. fumaroxidans. O meta-modelo criado que representa a comunidade formada por ambos os organismos foi criado a partir de uma ferramenta presente no FRAMED, e este é capaz de descrever as trocas metabólicas entre M. hungatei e S. fumaroxidans. Os modelos reconstruídos podem ser usados para estudar no futuro as interações metabólicas entre estas duas bactérias.

abstract: Peatlands represent 3% of the earth’s surface but have been estimated to contain up to 30% of all terrestrial soil organic carbon and release an estimated 40% of global atmospheric CH4 emissions. Contributors to the production of CH4 are methanogenic Archaea through a coupled metabolic dependency of end products released by heterotrophic bacteria within the soil in the absence of O2. To better understand how neighboring bacterial communities can influence methanogenesis, the isolation and physiological characterization of two novel isolates, one Methanoarchaeal isolate and one Acidobacterium isolate identified as QU12MR and R28S, respectively, were targeted in this present study. Co-culture growth in varying temperatures of the QU12MR isolate paired with an isolated Clostridium species labeled R32Q and the R28S isolate were also investigated for possible influences in CH4 production. Phylogenetic analysis of strain QU12MR was observed as a member of genus Methanobacterium sharing 98% identity similar to M. arcticum strain M2 and 99% identity similar to M. uliginosum strain P2St. Phylogenetic analysis of strain R28S was associated with genus Acidicapsa from the phylum Acidobacteria, sharing 97% identity to A. acidisoli strain SK-11 and 96% identity similarity to Occallatibacter savannae strain A2-1c. Bacterial co-culture growth and archaeal CH4 production was present in the five temperature ranges tested. However, bacterial growth and archaeal CH4 production was less than what was observed in pure culture analysis after 21 days of incubation.
Dissertation/Thesis
Masters Thesis Biology 2018

Metabolic cross-feeding is an important process that can broadly shape microbial communities. Comparative genomic analysis of >6000 sequenced bacteria from diverse environments provides evidence to suggesting that amino acid biosynthesis has been broadly optimized to reduce individual metabolic burden in favor of enhanced cross-feeding to support synergistic growth across the biosphere. Still, little is known about specific cross-feeding principles that drive the formation and maintenance of individuals within a mixed population. Here, we devised a series of synthetic syntrophic communities to probe the complex interactions underlying metabolic exchange of amino acids. We experimentally analyzed multi-member, multi-dimensional communities of Escherichia coli of increasing sophistication to assess the outcomes of synergistic cross-feeding. We find that biosynthetically costly amino acids including methionine, lysine, isoleucine, arginine and aromatics, tend to promote stronger cooperative interactions than amino acids that are cheaper to produce. Furthermore, cells that share common intermediates along branching pathways yielded more synergistic growth, but exhibited many instances of both positive and negative epistasis when these interactions scaled to higher-dimensions. This system enabled the identification of synergistic pairings and optimal expression levels of amino acid exporters of arginine, threonine and aromatics towards drastic improvements of ecosystem productivity. Tradeoffs identified in these mutualistic systems between secretion, relative abundance and absolute community productivity have implication in the evolution of cooperative behaviors. Long-term evolution of these synthetic communities highlight transporter over-expression, amino acid pool redistribution, and perturbations to nitrogen regulation as strategies to circumvent imposed metabolic dependencies. To address this potentially problematic genomic plasticity, a genetically reassigned organism is leveraged to investigate synthetic metabolic dependencies showing improved biocontainment and potential for microbial consortia control. These results improve our basic understanding of microbial syntrophy while also highlighting the utility and limitations of current approaches to modeling and controlling the dynamic complexities of microbial ecosystems. This work sets a foundation for future endeavors in microbial ecology and evolution, and presents a platform to develop better and more robust engineered synthetic communities for industrial biotechnology.

Increasing demand for freshwater and energy is pushing towards the development of alternative technologies that are sustainable. One of the realistic solutions to address this is utilization of the renewable resources like wastewater. Conventional wastewater treatment processes can be highly energy demanding and can fails to recover the full potential of useful resources such as energy in the wastewater. As a consequence, there is an urgent necessity for sustainable wastewater treatment technologies that could harness such resources present in wastewaters. Advanced treatment process based on microbial electrochemical technologies (METs) such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) have a great potential for the resources recovery through a sustainable wastewater treatment process. METs rely on the abilities of microorganisms that are capable of transferring electrons extracellularly by oxidizing the organic matter in the wastewater and producing electrical current for electricity generation (MFC) or H2 and CH4 production (MEC). Propionate is an important volatile fatty acid (VFA) (24-70%) in some wastewaters and accumulation of this VFA can cause a process failure in a conventional anaerobic digestion (AD) system. To address this issue, MECs were explored as a novel, alternative wastewater treatment technology, with a focus on a better understanding of propionate oxidation in the anode of MECs. Having such knowledge could help in the development of more robust and efficient wastewater treatment systems to recover energy and produce high quality effluents. Several studies were conducted to: 1) determine the paths of electron flow in the anode of propionate fed MECs low (4.5 mM) and high (36 mM) propionate concentrations; 2) examine the effect of different set anode potentials on the electrochemical performance, propionate degradation, electron fluxes, and microbial community structure in MECs fed propionate; and 3) examine the temporal dynamics of microbial communities in MECs fed with low or high concentration of acetate or propionate relating to the reactor performance. Overall, the findings from these studies provides new knowledge on propionate oxidation in MECs. The discovery of such findings may shed light on the development of an energy positive wastewater treatment process capable of producing a high quality effluent.

Dissertação de mestrado em Bioengenharia
Biogas is an alternative to substitute fossil fuels and is mostly composed by methane, a gas component that is produced by microorganisms called methanogens that use compounds such as acetate, hydrogen and carbon dioxide as substrates. In syntrophic communities, methanogens and bacteria exchange electrons for energetic purposes, normally through the use of soluble small chemical compounds that act as shuttles, such as hydrogen or formate. However, it has been recently suggested that in some cases this electron exchange can be performed directly or with the aid of conductive materials, which can potentially be a more energy conserving approach, thus improving the efficiency of methanogenesis. This project addressed the study of the effect of carbon nanotubes (CNT) in methane production by methanogenic communities, in two distinct studies. In the first study, assays were performed in batch with pure cultures of hydrogenotrophic, Methanobacterium formicicum and Methanospirillum hungatei, and acetoclastic methanogens, Methanosaeta concilii and Methanosarcina mazei, in the presence of CNT. Also, growth of pure cultures of M. formicicum was tested in the absence of a reducing agent. The results showed that CNT presence in pure cultures of the hydrogenotrophic methanogens resulted in an increased methane production and reduced lag phases. Although CNT also accelerated methane production by the acetoclastic pure cultures at 1 g/L CNT, a higher concentration of 5 g/L CNT, inhibited the methane production and induced longer lag phases. In the second study, co-cultures of butyrate-degrading bacteria Syntrophomonas wolfei and M. hungatei were grown with and without CNT, in order to determine the effect of CNT in butyrate-oxidizing syntrophic communities. The presence of CNT accelerated methane production by this co-culture, with methane production rate reaching a value of 1.03±0.03 mM/d for co-cultures incubated with 5 g/L CNT, while co-cultures in the absence of CNT only registered a rate of 0.72±0.01 mM/d. Adaptation of sludge in anaerobic bioreactors was also performed with the long-term objective of adding CNT, in order to assess its effect in complex microbial communities. By analysing the results obtained in this work, it was concluded that CNT improved hydrogen and acetate conversion to methane by pure cultures of methanogens and by syntrophic co-cultures of S. wolfei and M. hungatei. However, future studies with CNT are encouraged in order to understand the mechanisms by which CNT influences the methanogenic activity and in which cases it can be involved in interspecies electron transfer, since with the results obtained it remains unclear if they have a role in electron exchange between different species.
O biogás, que é composto maioritariamente por metano, é um componente gasoso produzido por microorganismos que usam como substrato moléculas simples, como o acetato, o hidrogénio ou o dióxido carbono, é uma conhecida alternativa aos combustíveis fósseis como fonte de energia. Em comunidades sintróficas, os organismos metanogénicos e as bactérias presentes trocam eletrões entre si com o objetivo de obtenção de energia para o seu crescimento, através de pequenos compostos químicos solúveis que servem de transporte, como o hidrogénio e o formato. Estudos recentes indicam que em alguns casos, estas trocas de eletrões podem ser realizadas diretamente ou por meio de materiais condutores, sendo esta uma abordagem que poderá ser mais favorável de um ponto de visto energético, aumentando assim a eficiência da metanogénese. Este projeto visou o estudo do efeito de nanotubos de carvão (NTC) nos mecanismos que envolvem a produção de metano em comunidades metanogénicas, através de dois diferentes estudos. No primeiro estudo, foram realizados ensaios em batch com culturas puras dos organismos metanogénicos hidrogenotróficos, Methanobacterium formicicum e Methanospirillum hungatei, e acetoclásticos, Methanosaeta concilii e Methanosarcina mazei, na presença de NTC. Além disso, também foram testadas culturas puras de M. formicicum na presença de NTC, mas sem adição de um agente redutor. Pelos resultados obtidos, verificou-se que os NTC provocaram um aumento na produção de metano e redução da fase de latência nas culturas puras dos metanogénicos hidrogenotróficos. Apesar de a presença dos NTC também ter acelerado a produção de metano nas culturas dos acetoclásticos com 1 g/L de NTC, numa maior concentração de 5 g/L de NTC, foi registada uma inibição na produção de metano e um aumento na duração das fases de latência. Num segundo estudo foram inoculadas culturas mistas contendo Syntrophomonas wolfei, uma bactéria que metaboliza butirato, e M. hungatei. Os resultados obtidos mostraram que a produção de metano também foi acelerada nas culturas mistas inoculadas com NTC, atingindo uma taxa de produção de metano de 1.03 ± 0.03 mM/d para as culturas incubadas com 5 g/L NTC, enquanto que na ausência de NTC, a taxa obtida foi de apenas 0.72 ± 0.01 mM/d. No âmbito deste trabalho, também foi realizada uma adaptação de biomassa em reatores anaeróbios, com o objetivo a longo prazo de adicionar os NTC para estudar o seu efeito em comunidades microbianas complexas. Por último, a análise dos resultados obtidos neste trabalho permitiu concluir que os NTC têm um efeito benéfico na conversão dos substratos a metano pelos microorganismos metanogénicos e pelas co-culturas sintróficas de S. wolfei e M. hungatei. Contudo, serão necessários mais estudos com os NTC para compreender melhor de que forma estes poderão estar envolvidos nos mecanismos de transferência de eletrões entre espécies, pois a sua influência neste processo continua por determinar.