For bacteria in an anaerobic culture to form granules, a number of conditions have to be fulfilled. The contributions of physical, chemical, and biological factors to granulation process could not be considered separately. So far, no model seems able to depict the entire anaerobic granulation process reasonably. Based on the existing mechanisms for formation of anaerobic granules, a general four-step model to better describe anaerobic granulation is proposed as follows.
Step 1: Physical movement to initiate bacterium-to-bacterium contact or bacterial attachment onto nuclei. The forces involved in this step include:
• Hydrodynamic force.
• Diffusion force.
• Thermodynamic forces, e.g. Brownian movement.
• Cell mobility. Cells can move by means of flagella, cilia, and pseu-dopods, while cell movement may also be directed by a signaling mechanism.
Step 2: Initial attractive physical, chemical, and biochemical forces to keep stable multicellular contacts. These attractive forces are:
• Opposite charge attraction.
• Thermodynamic forces including free energy of surface; surface tension.
• Filamentous bacteria that can serve as a bridge to link or grasp individual cells together.
It should be emphasized that the hydrophobicity of bacterial surface plays a crucial role in the initiation of biofilms and anaerobic granules (Mahoney et al., 1987; van Loosdrecht et al., 1987; Teo et al., 2000; Tay et al., 2000a). According to the thermodynamics theory, increasing the hydrophobicity of cellular surfaces would cause a corresponding decrease in the excess Gibbs energy of the surface, which in turn promotes cell-to-cell interaction and further serves as a driving force for bacteria to self-aggregate out of liquid phase (hydrophilic phase).
• Hydrogen liaison.
• Formation of ionic pairs.
• Formation of ionic triplet.
• Interparticulate bridge and so on.
• Cellular surface dehydration.
• Cellular membrane fusion.
• Signaling and collective action in bacterial community.
As described by the proton translocation-dehydration theory (Teo et al., 2000; Tay et al., 2000a), cellular surface dehydration and membrane fusion could lead to initiation of anaerobic granulation, while cooperative self-organization of bacteria will assist to form an organized spatial structure (Shapiro, 1998; Ben-Jacob et al., 2000).
Step 3: Microbial forces to make cell aggregation mature:
• Production of extracellular polymer by bacteria, such as exopolysac-charides.
• Growth of cellular cluster.
• Metabolic change and genetic competence induced by environment, which facilitate the cell-cell interaction and result in a highly organized microbial structure.
Step 4: Steady-state three-dimensional structure of microbial aggregate shaped by hydrodynamic shear forces. The microbial aggregates are finally shaped by hydrodynamic shear force to form a certain structured community. The shape and size of microbial aggregates are predominantly determined by the interactive strength/pattern between aggregates and hydrodynamic shear force, microbial species, and substrate loading rate.
The present general four-step model for anaerobic granulation attempts to broadly cover the current understanding of the entire granulation process as much as possible. It should be realized that identification of gross engineering events in relation to anaerobic granulation is relatively easy. But to identify the events at molecular or genetic level, a more profound understanding of the mechanisms responsible for anaerobic granulation is required. As Tolker-Nielsen and Molin (2000) noted, "it probably does not make sense to make firm decisions about one or the other explanation as the rule for community development".
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