The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45؇) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45؇). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.

Adhesion of bacteria involved in anaerobic consortia was investigated in upflow anaerobic sludge bed reactors and was related to surface thermodynamics. The adhesion of hydrophilic cells appeared to be enhanced at a low liquid surface tension (␥ LV), while the adhesion of hydrophobic cells was favored at a high ␥ LV. Growth in protein-rich growth media resulted in low granular biomass yields; addition of polycations, such as poly-L-lysine and chitosan, increased the ␥ LV and the granular biomass yield. On the basis of the results of activity tests and microbial counts with wash-out cells, we identified two types of structured granules that were related to the influence of ␥ LV. In one type of granules, hydrophilic acidogens surrounded a more hydrophobic methanogenic association. These granules were selected at a low ␥ LV provided that carbohydrates were available as substrates. The other type of granules was selected at a high ␥ LV ; hydrophobic cells (i.e., methanogens) were predominant throughout these granules. The granules which had acidogens as solid-phase emulsifiers around a methanogenic association appeared to allow more stable reactor performance. Decreasing the ␥ LV in the reactor by adding trace amounts of a surfactant also increased reactor stability.

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Laboratory studies of in-reactor granular-sludge yield (Ygran) were carried out to investigate the effect of substrates rich in proteins. Both lab-scale upflow anaerobic sludge blanket (UASB) and shake-flask systems were used to monitor the behaviour of the sludge. Influent based on molasses with a COD to N ratio of 100:2.5-3.0 gave good Ygran; increasing substitution of the carbohydrate COD by protein COD resulted in a deterioration of the sludge characteristics. The negative effects appear to be related to the protein rather than to the NH4+ formed. Of the various environmental parameters examined, the surface tension of the mixed liquor was the main deviating factor. At a proper range of oxygenation i.e., by means of monitoring of both the input COD to oxygen ratio (gCOD gO2−1) and the daily oxygen loading rate (mgO2 gVSS−1.d−1), the UASB reactors treating the nitrogen-rich wastewater showed enhanced Ygran with only a minor reduction in methanogenesis. A range of working conditions in which this principle can be applied to decrease fluffy growth to the advantage of granular increase is proposed.

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