Les archées hyperthermophiles ont une température optimale de croissance supérieure à 80°C.Les cellules exposées à un stress thermique subissent une augmentation de la sensibilité aux agents induisant des cassures double brin de l’ADN. Les études sur les eucaryotes et bactéries ont démontré que la recombinaison homologue joue un rôle essentiel non seulement dans la réparation de l’ADN, mais aussi dans le redémarrage des arrêts de la fourche de réplication. Les enzymes associées aux étapes initiales de la recombinaison homologue chez les archées sont homologues à celles des eucaryotes, et différentes des analogues bactériens. De plus, plusieurs études ont démontré que les protéines impliquées dans la recombinaison homologue sont essentielles chez les archées hyperthermophiles, soulignant l’importance biologique de cette voie de réparation chez ces organismes particuliers. Le rôle de la recombinaison homologue pour la stabilité génomique a été bien étudié chez les eucaryotes et les bactéries, cependant, peu de ses propriétés fonctionnelles ont été étudiées chez les archées hyperthermophiles. Pour mieux comprendre le mécanisme de recombinaison homologue impliquée au niveau de la maintenance génomique chez les archées, un réseau d’interactions protéine-protéines a été révélé précédemment au laboratoire à partir des protéines de Pyrococcus abyssi. Ces travaux ont démontré de nouvelles interactions où interviennent les protéines de la réplication et les protéines de la recombinaison de l’ADN. L’objet de cette étude de thèse est de présenter deux interactions : PCNA/Mre11-rad50 (MR) complexe et Primase/RadA. Pour la première fois chez P. furiosus, une interaction physique et fonctionnelle a été démontrée entre le PCNA et le complexe MR (l’initiateur de HR). Un motif, situé en position Cterminale de Mre11, permet l’interaction avec PCNA.PCNA stimule l’activité endonucléase du complexe MR à distance proche de l’extrémité 5’ d’une cassure double brin. Cette propriété est en accord avec l’intervention ultérieure des enzymes assurant la suite du mécanisme de réparation par recombinaison homologue. Par ailleurs, les protéines RadA, Primase et P41 ont été produites et purifiées. Leurs fonctions enzymatiques ont été confirmées. Cependant, nous n’avons pas pu caractériser la fonction de l’association de RadA/Primase. / Hyperthermophilic archaea (HA) are found in high-temperature environments and grow optimally above 80°C. Usually, cells exposed to heat stress display an increased sensitivity to agents inducing double-stranded DNA breaks (DSBs). Studies in Eukaryotes and Bacteria have revealed that homologous recombination (HR) plays a crucial role not only in DNA DSBs repair, but also in the collapsed/stalled DNA replication fork restart.Recombinase and various HR-associated enzymes in archaea specifically resemble the eukaryotic homologues, rather than bacterial homologues.Furthermore, several studies have demonstrated the necessity of HR proteins in HA, suggesting that, HR is an important mechanism in HA. HR influencing genome stability has been well studied in Eukaryotes andBacteria, however, few of its functional properties have been studied in HA.To better understand how HR mechanism is involved in the archaeal genome maintenance process, a previous work proposed a protein-protein interaction network based on Pyrococcus abyssi proteins. Through the network, new interactions involving proteins from DNA replication and DNA recombination were highlighted. The targets of the study presented here for two protein interaction are: PCNA/Mre11-rad50 complex (MR complex) and Primase/RadA. For the first time in P. furiosus, we showed both physical and functional interactions between PCNA (Maestro in DNA replication) and MR complex (initiator of HR). We have identified a PCNA-interaction motif (PIP) located in the C-terminal of Mre11, and shown that PCNA stimulated MR complex endonuclease cleavage proximal to the s’ strand of DNA DSBs at physiological ionic strength. For the second interaction, we have purified the proteins PabRadA/PfuRadA, PabPrimase and PabP41, and confirmed its enzymatic functions. However, we were not able to characterize the function of Primase/RadA association.

Archaea is one of the three domains of life and studies of archaeal biology are important for understanding of life in extreme environments, fundamental biogeochemical processes, the origin of life, the eukaryotic cell and their own, unique biology. This thesis presents four studies of the archaeal cell, using the extremophilic Sulfolobus and ocean living Nitrosopumilus as model systems. Cell division in crenarchaea is shown to be carried out by a previously unknown system named Cdv (cell division). The system shares homology with the eukaryotic ESCRT-III system which is used for membrane reorganization during vesicle formation, viral release and cytokinesis. Organisms of the phylum Thaumarchaeota also use the Cdv system, despite also carrying genes for the euryarchaeal and bacterial cell division system FtsZ. The thaumarchaeal cell cycle is demonstrated to be dominated by the prereplicative and replicative stage, in contrasts to the crenarchaeal cell cycle where the cell at the majority of the time resides in the postreplicative stage. The replication rate is remarkably low and closer to what is measured for eukaryotes than other archaea. The gene organization of Sulfolobus is significantly associated with the three origins of replication. The surrounding regions are dense with genes of high importance for the organisms such as highly transcribed genes, genes with known function in fundamental cellular processes and conserved archaeal genes. The overall gene density is elevated and transposons are underrepresented. The archaeal virus SIRV2 displays a lytic life style where the host cell at the final stage of infection is disrupted for release of new virus particles. The remarkable pyramid-like structure VAP (virus associated pyramids), that is formed independently of the virus particle, is used for cell lysis. The research presented in this thesis describes unique features of the archaeal cell and influences our understanding of the entire tree of life.

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.

Le méthane, un des principaux gaz à effet de serre, est majoritairement produit et consommé par l'activité métabolique de microorganismes affiliés aux domaines des Archaea et des Bacteria. Afin d'appréhender le cycle biogéochimique du méthane, il est essentiel d'identifier l'ensemble des acteurs impliqués dans ce dernier ainsi que les facteurs environnementaux modulant leurs activités. Les lacs d'eau douce constituent une source importante de méthane, car, dans ces écosystèmes, les conditions environnementales favorisent la méthanogenèse au détriment d'autres processus terminaux de la dégradation anaérobie de la matière organique. Au cours de cette thèse, les études sur les communautés impliquées dans le cycle biogéochimique du méthane ont été conduites dans la colonne d'eau et les sédiments anoxiques du Lac Pavin (Auvergne), unique lac méromictique de France. Cet écosystème a été choisi comme site d'étude en raison des fortes concentrations en méthane présentes dans sa couche d'eau profonde qui contrastent avec les faibles émissions de ce gaz vers l'atmosphère. Ces observations géochimiques suggèrent une intense activité de production et de consommation du méthane, offrant un cadre pertinent pour l'étude des communautés ciblées. Les approches moléculaires visant à caractériser la structure spatiale, la composition, les zones d'activité et les facteurs (ascendants et descendants) potentiellement impliqués dans la régulation des communautés de méthanogènes et de méthanotrophes ont été, au cours de ce travail, systématiquement associées à des approches culturales et microcalorimétriques afin d'acquérir des données sur la physiologie des microorganismes impliqués dans le cycle du méthane. Les résultats obtenus mettent en évidence que les communautés de méthanogènes sont distribuées sur l'ensemble de la colonne d'eau anoxique et dans la strate superficielle des sédiments profonds. Ce groupe métabolique, essentiellement représenté par des espèces affiliées aux Methanosaetaceae et aux Methanoregulaceae, est particulièrement actif dans la zone benthique qui constituerait la source principale de méthane dans cet écosystème. Une nouvelle espèce méthanogène, Methanobacterium lacus, a été isolée de ces sédiments et décrite, et vient enrichir le faible nombre d'espèces méthanogènes isolées à ce jour à partir des lacs d'eau douce. L'étude écophysiologique de cette souche suggère que la température pourrait en partie expliquer la faible représentativité des Methanobacteriales dans cet écosystème. Une partie du méthane semble être directement consommée dans la zone anoxique (pélagique et benthique). L'existence de ce processus d'oxydation anaérobie, soutenu par les approches microcalorimétriques, pourrait être, dans les sédiments profonds, sous la dépendance de lignées candidates archéennes dont la physiologie reste encore énigmatique. Le remplacement progressif des méthanogènes par 2 lignées candidates d'archaea (MBG-D et MCG) le long du profil sédimentaire suggère qu'elle se développe dans des niche contrastées. La régulation putative des communautés archéennes par les virus a été analysée. Cette étude est la première à rapporter la présence de particules virales de type "archaeovirus" dans un environnement non-extrême (en termes de température, pH et salinité) ainsi que des particules virales pouvant représentées de nouvelles familles de virus. Une activité virale intense est suggérée dans ces sédiments par le nombre important de cellules infectées, comparativement à d'autres sédiments, et par le changement concomitant de la structure de la communauté virale et procaryotique avec la profondeur. Bien qu'une partie du méthane soit probablement oxydée en anaérobiose, la consommation de ce métabolite est principalement dépendante de l'activité de méthanotrophes aérobies dominées par des espèces affiliées au genre Methylobacter, un des principaux genres de méthanotrophes rencontré en milieu d'eau douce. (...)

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.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-09Bitstream added on 2014-06-13T19:43:44Z : No. of bitstreams: 1 santana_am_dr_jabo.pdf: 5909203 bytes, checksum: 3d660be8aee8bbcfabdb656e3b158b48 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste trabalho avaliou-se o desempenho de sistema de tratamento combinado anaeróbio-aeróbio constituído por dois reatores anaeróbios de fluxo ascendente com manta de lodo (UASB), em série, em escala piloto (volumes de 510 e 209 L, respectivamente) seguidos de um reator em batelada seqüencial (RBS - volume de trabalho 210 L) aeróbio, tratando águas residuárias de suinocultura com concentrações médias de sólidos suspensos totais (SST) variando de 5 a 11 g L-1 e submetidos a tempos de detenção hidráulica (TDH) de 28 e 14 h no primeiro reator (R1), 11 e 6 h no segundo reator (R2) e de 58 e 26 h no RBS. As eficiências médias de remoção de DQO total e SST variaram de 54 a 90% e de 54 a 96%, respectivamente, no conjunto de reatores UASB em dois estágios (R1+R2), com carga orgânica volumétrica (COV) de 11 a 26 g DQO (L d)-1 no R1. A produção volumétrica máxima de metano de 1,613 m3 CH4 (m3 reator d)-1 ocorreu no R1, com COV de 19 g DQO (L d)-1 e TDH de 14 h. No RBS aeróbio, como pós-tratamento do efluente gerado nos reatores UASB, as eficiências médias de remoção foram 89%, 93%, 61%, 89% e 71% para a DQO total, SST, P-total, NTK e NT, respectivamente, com COV variando de 0,4 a 3,6 g DQO (L d)-1. Assim, no sistema de tratamento combinado anaeróbio-aeróbio (R1+R2+RBS), as eficiências médias de remoção da DQO total, SST, P-total, NTK e NT atingiram valores de 96 a 99%, 96 a 99%, 77 a 85%, 76 a 97% e 68 a 89%, respectivamente, e dos micronutrientes de 77 a 98%, 94 a 99%, 83 a 97% e de 62 a 99% para Fe, Zn, Cu e Mn, respectivamente, Para os coliformes termotolerantes, as eficiências de remoção médias foram de 93,80 a 99,99%, obtendo-se valores mínimos de 2,3 x 103 NMP/100 mL. A atividade metanogênica específica do lodo dos reatores UASB foi mais elevada quando se utilizou o propionato + butirato como fonte de substrato, e quando os reatores foram operados... / In this work a combination between aerobic and anaerobic treatment systems constituted by two Upflow Anaerobic Sludge Reactor (UASB) in series, in a pilot scale (510 and 209 L volume each respectively) followed by a sequential batch reactor (SBR – 210L net volume) aerobic, treating swine residual water having from 5 to 11 g L-1 of Total Suspended Solids (TSS) and under a hydraulic detention timing (HDT) of 28 and 14 hours in the first reactor (R1), 11 and 6 hours in the second reactor (R2) and 58 and 26 hours in the Sequential Batch Reactor (RBS). The COD and TSS average efficiency varied from 54 to 90% and from 54 to 96% respectively in the UASB two stages (R1 and R2), in a volumetric organic load (VOL) varying from 11 to 26g COD (L d)-1 in the R1. The maximum methane volumetric production was 1.613 m3 CH4 (m3 reactor d)-1 in the R1, having a 19g COD (L d)-1 and an HDT of 14h. Using an aerobic SBR as an effluent pretreater that was generate in the UASB reactors, it was reached an average of removing efficiency of 89%, 93%, 61%, 89% and 71% to total COD, TSS, total P, TNK and TN, respectively, COD varying from 0.4 to 3.6 g COD (L d)-1. This way, in the anaerobic-aerobic combined system (R1 + R2 + RBS) the total COD, TSS, total P, TNK and TN, average removing reached values from 96 to 99%, 96 to 99%, 77 to 85%, 76 to 97% and 68 to 89%, respectively. To Fe, Zn, Cu and Mn the average efficiency was from 77 to 98%, 94 to 99%, 83 to 97% and 62 to 99%, respectively. To the thermotolerant coliforms the removal efficiency average was from 93.80 to 99.99% reaching the lower value of 2.3 x 103 MPN/100 mL. The sludge specific methanogenic activity in the UASB reactors was more elevated when was used the propionate + butyrate as substrate in conjunction with the operation of 28 HDT, 11h and TSS affluent around 5 g L-1, reaching 0.443 and 0.206 mmol CH4 (g SV h)-1 in the reactors R1 and R2, respectively.

La saline de Sfax est un milieu hypersalin localisé dans la zone centrale de la côte est de Tunisie. Dans ce travail, nous avons isolé, identifié, et caractérisé des souches halophiles produisant des peptides antimicrobiens (halocines), afin de mieux comprendre leur rôle dans les interactions microbiennes au sein des milieux hypersalins. Deux étangs salins (TS18, 390 g.L-1 NaCl et M1, 200 g.L-1 NaCl) ont été choisis pour l’échantillonnage. Trente-cinq souches de procaryotes halophiles ont été isolées et caractérisées, dont 11 ont présenté une activité antimicrobienne. Parmi ces souches, 3 produisent des substances antimicrobiennes de nature protéique. A l’aide de PCR et RT-PCR nous avons montré que les souches Halobacterium salinarum ETD5 et ETD8 exprimaient le gène de l’halocine S8, un peptide de 3,6 kDa préalablement purifié d’une souche S8a non identifiée. Le peptide a été purifié à partir de cultures de la souche Hbt. salinarum ETD5. Après purification bioguidée, les fractions actives révèlent deux bandes de 8 et 14 kDa présentant une activité antimicrobienne. L’analyse par séquençage Nterminal et spectrométrie de masse a permis d’identifier ces deux halocines. La bande de 8 kDa correspond à une halocine S8 de 81 acides aminés qui subirait une maturation post-traductionnelle protéolytique différente de celle initialement décrite dans la littérature. Le clonage et le séquençage du gène codant le précurseur de l’halocine S8 démontrent que la séquence est identique chez les deux souches ETD5 et S8a. La bande de 14 kDa correspond à une nouvelle halocine, l’halocine S14. L’halocine S14 correspond à une forme tronquée en partie N-terminale de la Mn-superoxyde dismutase (SOD) d’Hbt. salinarum. Il pourrait s’agir d’évolution divergente d’un gène codant deux protéines distinctes, ou d’une maturation différente de la SOD. Ce travail permettra de mieux connaître les molécules intervenant dans les interactions microbiennes dans les milieux hypersalins, des milieux extrêmes susceptibles de révéler des structures et des modes d’action originaux. / The solar saltern of Sfax is a hypersaline located in the central area of the eastern coast of Tunisia. In this study, we isolated, identified, and characterized halophilic strains producing antimicrobial peptides (halocins), aiming to understand their role in microbial interactions in hypersaline environments. Two ponds (TS18, 390 g.L-1 NaCl and M1, 200 g.L-1 NaCl) were selected for sampling. Thirty-five halophilic strains have been isolated and characterized, among which 11 displayed antimicrobial activity. Three of them produced antimicrobial substances of proteinaceous nature. Using PCR and RT-PCR, we have demonstrated that Halobacterium salinarum ETD5 and ETD8 express the gene encoding halocin S8, a 3.6 kDa peptide previously isolated from a strain S8 unidentified. The peptide was purified from cultures of strain Hbt. salinarum ETD5. Following bioguided purification, the active fractions revealed two protein bands of 8 and 14 kDa exhibiting antimicrobial activity. N-terminal sequencing and mass spectrometry analyses allowed identification of these two halocins. The 8 kDa band corresponds to halocin S8, undergoing a different proteolytic post-translational processing from that originallydescribed. Cloning and sequencing of the gene encoding the precursor of halocin S8 showed that the sequence is identical for both strains ETD5 and S8a. The 14 kDa band is a new halocin termed halocin S14. Halocin S14 corresponds to an N-terminally truncated portion of the archaeal Mnsuperoxide dismutase (SOD). This could result from divergent evolution of a gene encoding two distinct proteins, or a different post-translational processing of SOD. Our study helps to better understand which molecules are involved in microbial interactions within hypersaline environments and and how they contribute to the competitions in such extreme environments, which are susceptible to give rise to original structures and modes of action.

Les structures des communautés procaryotes (diversité β) ont été caractérisées au sein de deux compartiments du benthos en milieu littoral: les sédiments et les bivalves fouisseurs.Dans les sédiments de surface, les communautés bactériennes et archéennes ont été ciblées par ARISA (Automated Ribosomal Intergenic Spacer Analysis) et T-RFLP (Terminal-Restriction Fragment Length Polymorphism) sur le gène codant pour l’ARN 16S respectivement. Sur 29 sites des zones intertidale et subtidale du Bassin d’Arcachon, les communautés procaryotes présentaient des patrons de distribution spatiale corrélés à ceux de la macrofaune benthique mais représentaient une faible diversité cumulée par habitat. L’extension à des écosystèmes proches (Vasière Ouest Gironde, Estuaire de la Gironde) a mis en évidence une forte proportion d’OTU (Operational Taxonomic Unit) rares et une diminution de la similarité entre les communautés avec l’éloignement géographique des écosystèmes. Chez les bivalves, les communautés bactériennes ont été caractérisées successivement à l’échelle de l’individu, de l’organe isolé et de l’espèce. Le suivi d’une cohorte de coques n’a pas montré d’influence de la présence du parasite Bucephalus minimus sur la structure de ces communautés à l’échelle de l’individu. En revanche, prises à l’échelle de l’organe en comparant la coque à la palourde, les communautés bactériennes associées aux bivalves se sont différenciées en fonction des organes (e.g branchies, tube digestif, reste des tissus), de l’habitat et/ou de l’espèce. / The structures of prokaryotic communities (β diversity) were characterized in sediments and burrowing bivalves.In the sediments, the bacterial and archaeal communities were analyzed by ARISA (Automated Ribosomal Intergenic Spacer Analysis) and 16S RNA gene T-RFLP (Terminal-Restriction Fragment Length Polymorphism) respectively. In 29 subtidal and intertidal sites of the Arcachon Bay, the spatial patterns of prokaryotic and benthic macrofaunal communities were correlated whereas prokaryotic community species exhibited a low cumulated diversity per habitat. Extending to neighboring ecosystems (West-Gironde mud patch, Gironde estuary) revealed the occurrence of a high proportion of rare OTU (Operational Taxonomic Unit) and a decrease of community similarities with geographic distance among ecosystems.For the bivalves, the whole individual, the organ-scale and the species-scale were successively considered. A cockle cohort monitoring indicated no influence of the parasite Bucephalus minimus occurrence on bacterial community structure at the individual-scale. Comparing cockle and clam at the organ level demonstrated that bivalve associated bacterial communities could differ among organs (e.g. gills, digestive gut, others tissues), among habitats and/or between species.

Foram estudados três procedimentos para o tratamento prévio de amostras provenientes de três sistemas de biodigestão anaeróbia operados com resíduos sólidos urbanos padronizados (RSUDp), com o fim de promover o desprendimento de células microbianas de fibras e outros materiais, e com isso obter um inóculo mais homogêneo para contagens celulares. Os tratamentos foram os seguintes: (a) suspensão de amostras em tampão fosfato e processamento em liqüidificador; (b) homogeneização mecânica e manual de amostras em solução mineral anaeróbia; (c) amostras submetidas a banho de ultra-som. O método do Número Mais Provável (NMP) foi utilizado para avaliar o número de bactérias celulolíticas e arqueas metanogênicas, em meio mineral contendo celulose em pó. Não foram constatadas diferenças entre os três tratamentos empregados, considerando-se os valores encontrados para ambos os grupos, da ordem de 102 células/mL. O tratamento com ultra-som foi escolhido para outras determinações em função da simplicidade quanto à manipulação. Assim, foram operados dois biorreatores anaeróbios com RSUDp para avaliação da reprodutibilidade dos valores das amostras após tratamento com ultra-som, para a determinação do número de microrganismos (celulolíticos, acetogênicos e metanogênicos) e avaliação da atividade microbiana anaeróbia na degradação do RSUDp. Os resultados das contagens de bactérias celulolíticas e arqueas metanogênicas pelo método do NMP em meio contendo celulose não foram superiores a 103 células/mL, e as réplicas foram bastante semelhantes. O aumento do número do grupo metanogênico pôde ser correlacionado com o aumento dos teores de metano no biorreator amostrado. As contagens de organismos acetogênicos e metanogênicos em meio mineral com fontes específicas revelou valores até 103 células/mL. Os tipos morfológicos predominantes nos exames microscópicos dos sistemas de biodigestão foram bacilos curvos, retos, sarcinas e cistos de sarcinas e as amostras dos tubos de contagem mostraram para celulolíticas, bacilos, cocos e sarcinas, para acetogênicas bacilos, bacilos espessos, cocos e cistos de sarcinas e para as arqueas metanogênicas bacilos fluorescentes e sarcinas. A evolução do processo de biodigestão anaeróbia nos dois últimos sistemas operados mostrou-se bastante próxima a proposta por BARLAZ et al. (1989b), em que o número de bactérias celulolíticas e de arqueas metanogênicas aumenta na fase considerada metânica acelerada. O conteúdo de sólidos totais diminuiu em 50% ao longo do processo e os teores de ácidos orgânicos voláteis diminuíram de 27,5 para 8, 79 g de ác. acético/L. / Three different treatments were applied to samples from three anaerobic biodigestion systems operated with standardized municipal solid waste to promote the release of microbial cells adhered to fibers and other materials, resulting in a more homogeneous inoculum to cellular counting. The following treatments were applied to the samples: a) suspension in phosphate buffer followed by blending; b) mechanical and hand homogenization in anaerobic mineral solution; c) sonication in ultrasound bath. The Most Probable Number (MPN) technique was employed to evaluate the populations of cellulolytic bacteria and methanogenic Archaea, in mineral medium containing powdered cellulose. The populations of both groups of microorganisms were close to 102 cells/mL independently of the treatment applied to release the cells and no clear distinction among them could be made concerning their efficiencies. In view of this fact the ultrasound treatment was employed in all other determinations due to its simple execution. Two anaerobic bioreactors operated with standardized municipal solid waste were monitored to evaluate the reliability of sonication as a procedure for cell release, to determine microorganism populations (cellulolytic, acetogenic bacteria and methanogenic Archaea) and to evaluate the microbial anaerobic activity concerning the biodegradation of the standardized municipal solid waste. Cellulolytic bacteria and methanogenic Archaea had similar populations (lower than 103 cells/mL) as well as acetogenic and methanogenic microorganisms (up to 103 cells/mL). The increase in the methanogenic population could be directly related to the increase of methane production in the studied reactor. The morphological types which predominate in the microscopic examinations of the biodigestion systems were curved and straight rods and sarcina. The tubes for MPN countings showed the presence of rods, coccus and sarcina for cellulolytic bacteria; rods, tick rods, coccus and sarcina for acetogenic bacteria and fluorescent rods and sarcina for methanogenic Archaea. The evolution of the anaerobic biodigestion process in the latter two monitored systems was very similar to the one proposed by BARLAZ et al. ( 1989b) where the number of cellulolytic bacteria and methanogenic Archaea increase during the accelerated methane phase. The total content of solids decreased 50% during the process and the volatile acidity decreased from 27,5 g acetic acid/L to 8,79 g acetic acid/L.