A systematic study of in-reactor granule yield data was set up in the laboratory. Methanogenic granular sludge growth in fed batch shake flasks for different substrates appeared to decrease with the energy content of the substrate and required the presence of a mixing force. In UASB reactors, granular cell yields were found to be quite variable suggesting the involvement of complex microbiological interactions. The factor foremost in influencing the build-up in the reactor of granular sludge was the presence of high-energy carbohydrates; pre-acidified influents affected in-reactor granular sludge yield very negatively. The influence of sulphate reduction and sulphide concentrations was found not to be of primary importance. The ionic strength of the medium also had no strong influence. Granule formation proceeded somewhat better at low than at nigh strength buffer capacity. High levels of protein, however, reduced the in-reactor granular sludge build-up strongly. Measurement of the acid production by granular and fluffy sludges revealed that the former rapidly produce volatile acids, particularly propionate, while the latter do not. Measurement of the pH in the granule indicated that the outside layer of the granule is rich in acidogens. Calculations of proton and hydrogen fluxes in the granule support the concept that acidogens, able to ferment high energy carbohydrates efficiently to cells and exo-cellular binding materials, might be of primary importance for in-reactor granular growth.

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