The importance of social behaviour is evident in human society, but there are both costs and benefits associated with cooperation and sociality throughout the animal kingdom. At what point do the benefits outweigh the costs, and when do selective pressures favour sociality and colonization over solitude and independence? To investigate these questions, we have focused on an anomalous species of spider, Anelosimus studiosus, also known now as the northern social spider. Throughout its broad range, A. studiosus is solitary and aggressive, but recently, colonies of cooperative and social individuals have been observed at northern latitudes. This leads to two research questions: 1) what characteristics differentiate the two variants behaviourally, and, 2) how are they different physiologically? Colonies and individuals were collected from multiple populations throughout the Tennessee River watershed area and maintained in a laboratory environment for quantitative and qualitative assessment of behavioural traits as well as specific neurochemical analysis by high performance liquid chromatography with electrochemical detection. After classifying individuals as social or aggressive, I looked at the influence of factors such as age, reproductive state, nutritional state, and time of day on behaviour and neurophysiology. I found correlations between social behaviours and serotonin, aggressive behaviours and octopamine (invertebrate counterpart of norepinephrine), and several other compounds associated with an increase or decrease in aggression. These studies combine techniques from multiple disciplines to contribute to the greater understanding of the proximate control of social and aggressive behaviours as well as factors influencing the evolution of sociality.

Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Microorganisms have played a critical role in geological processes and in the formation of soils throughout geological time. It is hypothesized that biological activity can also affect soil properties in short engineering time-scales. Bioactivity in sediments is determined by the classical limiting factors (i.e., nutrients, water, C for biomass, temperature and pH) as well as by pore-size geometrical limits and mechanical interactions between bacterial cells and soil particles. These constraints restrict the range of grain size and burial depth where biomediated geochemical processes can be expected in sediments, affect the interpretation of geological processes and the development of engineering solutions such as bioremediation. When biological, geometrical and mechanical limiting factors are satisfied, bioactivity can be designed to alter the mechanical properties of a soil mass, including lowering the bulk stiffness of the pore fluid through controlled gas bio-generation, increasing the shear stiffness of the soil skeleton by biomineralization, and reducing hydraulic conduction through biofilm formation and clogging. Each of these processes can be analyzed to capture the bio-chemo-hydro-mechanical coupling effects, in order to identify the governing equations that can be used for process design. Design must recognize the implications of spatial variability, reversibility and environmental impacts.

No description available.

Complex biogenic habitats created by large canopy-forming macroalgae on intertidal and shallow subtidal rocky reefs worldwide are increasingly affected by degraded environmental conditions at local scales and global climate-driven changes. A better understanding of the mechanisms underlying the impacts of complex suites of anthropogenic stressors on algal forests is essential for the conservation and restoration of these habitats and of their ecological, economic and social values. This thesis tests physical and biological mechanisms underlying the impacts of different forms of natural and human-related disturbance on macroalgal assemblages dominated by fucoid canopies along the east coast of the South Island of New Zealand. A field removal experiment was initially set up to test assemblage responses to mechanical perturbations of increasing severity, simulating the impacts of disturbance agents affecting intertidal habitats such as storms and human trampling. Different combinations of assemblage components (i.e., canopy, mid-canopy and basal layer) were selectively removed, from the thinning of the canopy to the destruction of the entire assemblage. The recovery of the canopy-forming fucoids Hormosira banksii and Cystophora torulosa was affected by the intensity of the disturbance. For both species, even a 50% thinning had impacts lasting at least eighteen months, and recovery trajectories were longer following more intense perturbations. Independently of assemblage diversity and composition at different sites and shore heights, the recovery of the canopy relied entirely on the increase in abundance of these dominant fucoids in response to disturbance, indicating that functional redundancy is limited in this system. Minor understory fucoids, which could have provided functional replacement for the dominant habitat formers, had reduced rates of growth or recruitment when the overlying canopy was disturbed. I then used a combination of field and laboratory experiments to test the impacts of physical and biotic stress sources on the dominant fucoids H. banksii and C. torulosa. The large fucoid Durvillaea antarctica was also included in one of the laboratory investigations. I assessed how altered physical and biotic conditions affect these important habitat formers, both separately and in combination. Physical stressors included increased sedimentation, nutrient enrichment and warmer water temperatures. Biotic stress originated from interspecific competition with turfs of articulated coralline algae and ephemeral, fast-growing green and brown algae. Sediment deposition severely reduced the survival and growth of recently settled H. banksii, C. torulosa and D. antarctica germlings in laboratory experiments. In the field, the recruitment of H. banksii on unoccupied substrates was significantly higher than in treatments in which sediments or mats of turf-forming coralline algae covered the substrate. This shows that sediment deposition and space pre-emption by algal turfs can synergistically affect the development of fucoid beds. Further impacts of sediment accumulation in the benthic environment were investigated using in situ and laboratory photorespirometry techniques to assess the contribution of coralline algae to assemblage net primary productivity (NPP), both in the presence and absence of sediment. The NPP of articulated corallines was reduced by sediment. Sediment accumulation among the thalli limited the access of the corallines to the light and induced photoinhibitive mechanisms. In the absence of sediment, however, coralline algae enhanced the NPP of assemblages with a fucoid canopy, showing the importance of synergistic interactions among the components of multi-layered assemblages in optimizing light use. Nutrient enrichment had a less pervasive influence on the dominant fucoids H. banksii and C. torulosa than sedimentation. In laboratory experiments, nutrients stimulated the growth of H. banksii and C. torulosa germlings. However, negative impacts of high nutrient levels were observed for the early life stages of D. antarctica. The abundance of opportunistic, fast-growing algae rapidly increased in response to nutrient enrichment both in the laboratory and in the field. Impacts of ephemeral species on fucoid early life stages were only evident in laboratory contexts, where green algae of the genus Ulva impaired both the settlement of H. banksii zygotes and the growth of its germlings. Fucoid recruitment in the field was not affected by increased covers of ephemeral algae caused by enhanced nutrient regimes, indicating that H. banksii and C. torulosa may be resistant to short-term (one year) nutrient pollution. In the laboratory, increased temperatures within the range predicted for the end of the 21st century caused increased mortality in the H. banksii, C. torulosa and D. antarctica germlings. In a separate experiment, a combination of warmer water temperatures and nutrient enrichment enhanced the growth of ephemeral green algae. These results suggest that opposite responses to altered climate conditions may contribute to shifts from complex biogenic habitats dominated by macroalgal canopies to simplified systems monopolized by a limited number of stress-tolerant species. This research contributes to a clearer mechanistic understanding of biotic and physical mechanisms shaping the structure of coastal marine hard bottom communities under increasingly stressful conditions worldwide. These findings may provide insights for other studies investigating the complex mosaic of challenges facing marine coastal ecosystems.

Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird. / Biogenic amines are small organic compounds that act as neurotransmitters, neuromodulators and/or neurohormones in vertebrates and in invertebrates. They form an important group of messenger substances and mediate their diverse effects by binding to membrane receptors that primarily belong to the large gene-family of G protein-coupled receptors. In insects, the group of biogenic amine messengers consists of five members: dopamine, tyramine, octopamine, serotonin, and histamine. Besides many other effects, some of these biogenic amines were shown, for example, to modulate gustatory sensitivity to sucrose stimuli in the honeybee (Apis mellifera). I have investigated various aspects of the aminergic signal transduction in the “model organisms” honeybee and American cockroach (Periplaneta americana). So far, I have characterized two dopamine receptors, a tyramine receptor, an octopamine receptor and a serotonin receptor of the honeybee, which is well-known for its learning and memory capacities. The receptors where expressed in cultivated mammalian cells in order to analyze their pharmacological and functional (i.e., second messenger coupling) properties. The spatiotemporal expression patterns of the respective receptor mRNA were investigated in the honeybee brain by using different techniques (RT PCR, Northern blotting, in situ-hybridization). The salivary glands of the American cockroach were used as a model object in order to investigate the cellular effects of biogenic amines. Both dopamine and serotonin trigger salivary secretion in isolated salivary glands. The quality of the secreted saliva is, however, different. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Serotonin-induced protein secretion is mediated by an increase in the intracellular concentration of cAMP. The pharmacological properties of dopamine receptors associated with cockroach salivary glands were investigated and the molecular characterization of putative aminergic receptors of the cockroach was initiated. Furthermore, I have characterized the ebony gene of the cockroach. This gene encodes an enzyme that is probably involved in the inactivation of biogenic amines in the cockroach (as in other insects). The ebony gene is expressed in the brain and in the salivary glands of the cockroach.

L’ocratossina e le ammine biogene sono due metaboliti biologici che possono essere ritrovati nei vini. Il primo di questi contaminanti è stato studiato recentemente per la sua elevata tossicità sebbene non sia rintracciabile frequentemente nei vini. Le ammine biogene sono presenti in ogni tipo di vino in differenti concentrazioni, oltretutto, alcune di loro, ad alte concentrazioni, possono causare reazioni allergiche. Gli obiettivi di questo PhD riguardano tre differenti aspetti. Indagare metodi biologici di per la decontaminazione di ocratossina A durante la vinificazione; in particolare lo studio si è focalizzato sull’uso di un ceppo Lactobacillus plantarum utilizzato come starter malolattico. Valutare la presenza di Lactobacillus spp., isolati da mosto e vino, in grado di produrre ammine biogene, usando tecniche molecolari come la reazione a catena della polimerasi (PCR) per rilevare i geni codificanti gli enzimi responsabili della sintesi di questi composti. Testare la capacità di un L. plantarum di effettuare la malolattica effettuando l’inoculo in differenti fasi della vinificazione e valutare il trend delle ammine biogene già presenti nel mosto. / Two of the major biological metabolites present in wine are the ochratoxin and the biogenic amines. The first of these contaminants was studied in recent decades because of its toxicity in humans, although its presence is not frequent in wines. The biogenic amines are present in every types of wine in different concentration, and some of them, in high concentrations, can cause allergenic reactions in humans. The objectives of this PhD regard three different aspects. Investigate the biological methods to reduce ochratoxin A in wine during winemaking; in particular the study is focused to use a Lactobacillus plantarum strain as malolactic starter. Investigate the presence of Lactobacillus spp., isolated from must and wine, able to produce the amines, using molecular techniques as polymerase chain reaction (PCR) to detect the genes that encode for the enzymes responsible of the synthesis of these compounds. Test the ability of a L. plantarum to perform MLF in relationship with inoculation time and assess the trend of biogenic amines already present in must.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.