Mechanical and cell-to-cell adhesive properties of aggregated Methanosarcina.

The mechanical and adhesive properties as well as the turgor pressure of microbes play an important role in cell growth and aggregation. By applying AFM together with finite element modelling, one can determine the cell wall structural homogeneity, mechanical and cell-to-cell adhesive properties for aggregated Methanosarcina barkeri cells. This also allows a novel approach to determine in-aggregate turgor pressure determination. Analyzing the AFM force-indentation response of the aggregates under loads less than 10 nN, our study reveals structural inhomogeneity of the polymeric part of the cell wall material and suggests that the cell wall consists of two layers of methanochondroitin (external: with a thickness of 3 ± 1 nm and internal: with a thickness of 169 ± 30 nm). On average, the hyperelastic finite element model showed that the internal layer is more rigid (µ = 14 ± 4 MPa) than the external layer (µ = 2.8 ± 0.9 MPa). To determine the turgor pressure and adhesiveness of the cells, a specific mode of indentation (under a load of 45 nN), aimed towards the centre of the individual aggregate, was performed. By modelling the AFM induced decohesion of the aggregate, the turgor pressure and the cell-to-cell adhesive interface properties could be determined. On average, the turgor pressure is estimated to be 59 ± 22 kPa, the interface strength is 78 ± 12 kPa and the polymer network extensibility is 2.8 ± 0.9 nm. We predict that internal cell wall comprised highly compressed methanochondroitin chains and we are able to identify a conceptual model for stress dependent inner cell wall growth.

Anaerobic biodegradation of pentachlorophenol in a fixed-film reactor inoculated with polluted sediment from Santos-São Vicente Estuary, Brazil.

This paper discusses the results of pentachlorophenol (PCP) anaerobic biodegradation in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor operated under methanogenic and halophylic conditions. The system was inoculated with autochthonous microorganisms taken from a site in the Santos-São Vicente Estuary (state of São Paulo, Brazil) severely contaminated with PCP, phenolic compounds, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and heavy metals. The inoculum was previously enriched for methanogenesis activity by changing glucose concentrations and under halophylic condition. PCP was added to the HAIB reactor as sodium salt (NaPCP) at an initial concentration of 5 mg l(-1) and increased to 13, 15, and 21 mg l(-1). Organic matter removal efficiency ranged from 77 to 100%. PCP removal efficiency was 100%. Denaturing gradient gel electrophoresis profile showed changes in the structure of Bacteria domain, which was associated with NaPCP and glucose amendments. The diversity of Archaea remained unaltered during the different phases. Scanning electron microscope examinations showed that cells morphologically resembling Methanosarcina and Methanosaeta predominated in the biofilm. These cells were detected by fluorescence in situ hybridization with the Methanosarcinales (MSMX860) specific probe. The results are of great importance in planning the estuary's restoration by using anaerobic technology and autochthonous microorganisms for bioremediation.

Development of methanogenic consortia in fluidized-bed batches using sepiolite of different particle size.

The addition of support materials, such as sepiolite, to fluidized-bed anaerobic digesters enhances the methane production by increasing the colonization by syntrophic microbiota. However, the efficiency in the methanogenesis depends on the particle size of the support material, the highest level of methane production being obtained by the smaller particle size sepiolite. Because of the porosity and physico-chemical characteristics of these support materials, the anaerobic microbial consortia formed quickly (after one week of incubation). The predominant methanogenic bacteria present in the active granules, detected both by immunofluorescence using specific antibodies and by scanning electron microscopy, were acetoclastic methanogens, mainly Methanosarcina and Methanosaeta.

Transfer of Methanolobus siciliae to the genus Methanosarcina, naming it Methanosarcina siciliae, and emendation of the genus Methanosarcina.

A sequence analysis of the 16S rRNA of Methanolobus siciliae T4/M(T) (T = type strain) showed that this strain is closely related to members of the genus Methanosarcina, especially Methanosarcina acetivorans C2A(T). Methanolobus siciliae T4/M(T) and HI350 were morphologically more similar to members of the genus Methanosarcina than to members of the genus Methanolobus in that they both formed massive cell aggregates with pseudosarcinae. Thus, we propose that Methanolobus siciliae should be transferred to the genus Methanosarcina as Methanosarcina siciliae.

Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.

Immunochemistry and localization of the enzyme disaggregatase in Methanosarcina mazei.

The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains.