Microscale structure and function of anaerobic-aerobic granules containing glycogen accumulating organisms

The spatial arrangement and metabolic activity of 'Candidatus Competibacter phosphatis' was investigated in granular sludge from an anaerobic-aerobic sequencing batch reactor enriched for glycogen-accumulating organisms. In this process, the electron donor (acetate) and the electron acceptor (oxygen) were supplied sequentially in each phase. The organism, identified by fluorescence in situ hybridisation, was present throughout the granules; however, metabolic activity was limited to a 100-mum-thick layer immediately below the surface of the granules. To investigate the cause of this, oxygen microsensors and a novel microscale biosensor for volatile fatty acids were used in conjunction with chemical staining for intracellular storage polymers. It was found that the limited distribution of activity was caused by mass transport limitation of oxygen into the granules during the aerobic phase. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.