Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

Interspecies electron transfer is a key process in methanogenic and sulphate-reducing environments. Bacteria and archaea that live in syntrophic communities take advantage of the metabolic abilities of their syntrophic partner to overcome energy barriers and break down compounds that they cannot digest by themselves. Here, we review the transfer of hydrogen and formate between bacteria and archaea that helps to sustain growth in syntrophic methanogenic communities. We also describe the process of reverse electron transfer, which is a key requirement in obligately syntrophic interactions. Anaerobic methane oxidation coupled to sulphate reduction is also carried out by syntrophic communities of bacteria and archaea but, as we discuss, the exact mechanism of this syntrophic interaction is not yet understood.

Electron transfer in syntrophic communities of anaerobic bacteria and archaea

It has been a decade since multicellularity was proposed as a general bacterial trait. Intercellular communication and multicellular coordination are now known to be widespread among prokaryotes and to affect multiple phenotypes. Many different classes of signaling molecules have been identified in both Gram-positive and Gram-negative species. Bacteria have sophisticated signal transduction networks for integrating intercellular signals with other information to make decisions about gene expression and cellular differentiation. Coordinated multicellular behavior can be observed in a variety of situations, including development of E. coli and B. subtilis colonies, swarming by Proteus and Serratia, and spatially organized interspecific metabolic cooperation in anaerobic bioreactor granules. Bacteria benefit from multicellular cooperation by using cellular division of labor, accessing resources that cannot effectively be utilized by single cells, collectively defending against antagonists, and optimizing population survival by differentiating into distinct cell types.

Thinking about bacterial populations as multicellular organisms

Upflow Anaerobic Sludge Bed (UASB) wastewater (pre-)treatment systems represent a proven sustainable technology for a wide range of very different industrial effluents, including those containing toxic/inhibitory compounds. The process is also feasible for treatment of domestic wastewater with temperatures as low as 14-16 degrees C and likely even lower. Compared to conventional aerobic treatment systems the anaerobic treatment process merely offers advantages. This especially is true for the rate of start-up. The available insight in anaerobic sludge immobilization (i.e. granulation) and growth of granular anaerobic sludge in many respects suffices for practice. In anaerobic treatment the immobilization of balanced microbial communities is essential, because the concentration of intermediates then can be kept sufficiently low. So far ignored factors like the death and decay rate of organisms are of eminent importance for the quality of immobilized anaerobic sludge. Taking these factors into account, it can be shown that there does not exist any need for 'phase separation' when treating non- or slightly acidified wastewaters. Phase separation even is detrimental in case the acidogenic organisms are not removed from the effluent of the acidogenic reactor, because they deteriorate the settleability of granular sludge and also negatively affect the formation and growth of granular sludge. The growing insight in the role of factors like nutrients and trace elements, the effect of metabolic intermediates and end products opens excellent prospects for process control, e.g. for the anaerobic treatment of wastewaters containing mainly methanol. Anaerobic wastewater treatment can also profitably be applied in the thermophilic and psychrophilic temperature range. Moreover, thermophilic anaerobic sludge can be used under mesophilic conditions. The Expanded Granular Sludge Bed (EGSB) system particularly offers big practical potentials, e.g. for very low strength wastewaters (COD << 1 g/l) and at temperatures as low as 10 degrees C. In EGSB-systems virtually all the retained sludge is employed, while compared to UASB-systems also a substantially bigger fraction of the immobilized organisms (inside the granules) participates in the process, because an extraordinary high substrate affinity prevails in these systems. It looks necessary to reconsider theories for mass transfer in immobilized anaerobic biomass. Instead of phasing the digestion process, staging of the anaerobic reactors should be applied. In this way mixing up of the sludge can be significantly reduced and a plug flow is promoted. A staged process will provide a higher treatment efficiency and a higher process stability. This especially applies for thermophilic systems.

Anaerobic digestion and wastewater treatment systems

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.

https://aem.asm.org/content/aem/57/7/1942.full.pdf

Bacteriological composition and structure of granular sludge adapted to different substrates.

Role of substrate concentration in particle size distribution of methanogenic granular sludge in UASB reactors

Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed.

Localization and quantification of extracellular polymers in methanogenic granular sludge.

The effect of the calcium-specific chelant ethylene glycol-bis(\-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on methanogenic granular sludge from a laboratory-scale upflow anaerobic sludge-blanket (UASB) reactor fed propionate and from a full-scale reactor treating paper-mill waste-water was studied. Upon treatment with EGTA both sludge types showed a decrease in the calcium and phosphorus content and a release of protein and polysaccharides, leading to a decrease in strength of papermill granular sludge and a disintegration of propionate-grown granules. After treatment of propionate-grown granular sludge with high EGTA concentrations, the methanogenic activity with propionate and acetate as test substrates decreased by 88 and 33%, respectively. The marked reduction in propionate oxidation activity may be caused by a disruption of the special juxtapositioning of bacteria in the granules.

Effect of ethylene glycol-bis(β-aminoethyl ether)- N,N -tetraacetic acid (EGTA) on stability and activity of methanogenic granular sludge

The hydrophobicities and electrophoretic mobilities of isolates from methanogenic anaerobic granular sludge were measured and compared with those of strains from culture collections. All new isolates were highly hydrophobic, indicating that the upflow anaerobic sludge blanket reactor concept selects for hydrophobic bacteria. Methanothrix soehngenii, a methanogen often observed in methanogenic granular sludge, was highly hydrophobic and showed low electrophoretic mobility at pH 7. The role of this strain in the formation of methanogenic granular sludge is discussed.

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J.T.C. Grotenhuis

Papers

Bacteriological composition and structure of granular sludge adapted to different substrates.

Role of substrate concentration in particle size distribution of methanogenic granular sludge in UASB reactors

Localization and quantification of extracellular polymers in methanogenic granular sludge.

Effect of ethylene glycol-bis(β-aminoethyl ether)- N,N -tetraacetic acid (EGTA) on stability and activity of methanogenic granular sludge

Hydrophobicities and Electrophoretic Mobilities of Anaerobic Bacterial Isolates from Methanogenic Granular Sludge