Microbiological aspects of granular methanogenic sludge

The settling characteristics of anaerobic sludge are enhanced by the formation of microbial conglomerates. Various types of conglomerates having different structures, were distinguished in the present study, viz. granules, pellets and flocs (chapter 1). Granular methanogenic sludge, often developing in upflow anaerobic sludge blanket reactors, is optimal for an economic application of the system. One of the hypotheses to explain the formation of these granules is that bacteria would excrete extracellular polymers, responsible for the formation of stable, well settling aggregates. Scanning electron micrographs showed that indeed extracellular material can be detected in granular methanogenic sludge grown on waste water of a liquid sugar plant (chapter 3). Various methods were used to quantify the fraction of this material and to elucidate its chemical composition: 1 - 2% of the dry weight of the granules appeared to consist of extracellular sugars. The relative proportions of these sugars varied somewhat with the extraction methods used and the batch of granules analysed.Granular methanogenic sludge grown on waste water of a sugar refinery consisted of a diverse microflora composed of various types of bacteria (chapter 3). As these granules had been formed on a complex waste water the growth of granular methanogenic sludge was studied under better defined conditions with respect to substrate supply. In chapter 2 it was shown that growth of methanogenic sludge granules is possible in bench scale UASB reactors with a volume of one liter. The first series of experiments was carried out with waste water of the sugar factory on which granular sludge had been found to develop in a full scale UASB reactor. Development and growth of granular methanogenic sludge was possible when a UASB reactor with a volume of one liter had been seeded with disintegrated granular sludge. However, when the reactor had been seeded with digested sewage sludge more fluffy pellets developed. Subsequent experiments showed that various compounds could be omitted from artificially prepared waste waters that originally reflected the composition of the waste water of the sugar refinery. Cultivation of methanogenic sludge granules proved possible on ethanol or propionate as most simple substrates. Feeding granular methanogenic sludge for prolonged periods with acetate as sole energy source resulted in weaker, more fluffy pellets.Incubation of granular methanogenic sludge in batch culture under growth- supporting conditions generally resulted in the overgrowth of the original seed granules by dispersed growing bacteria and development of fluffy pellets. Cultivation of granular methanogenic sludge in batch culture was only possible in experiments where propionate was stoichiometrically converted to methane and acetate (and probably carbon dioxide).By studying the potential methanogenic activities of granular methanogenic sludges (chapter 4) it was shown that the biomass consisted for a significant part of acetoclastic methanogens. These potential specific activities showed that obligately acetoclastic methanogens were quantitatively more important than hydrogenotrophic methanogens. These conclusions were drawn from a comparison between the potential methanogenic activities measured in the present experiments, and literature data on the potential activities of various groups of anaerobic bacteria. This approach provided a reliable tool for the estimation of the microbial composition of methanogenic consortia and was faster and more accurate than MPN counts. The resulting estimates of the acetoclastic biomass with respect to the occurrence of obligately and facultatively acetoclastic methanogens were corroborated by microscopic observations and indicated that Methanothrixsoehngenii -like organisms are the most numerous acetoclastic methanogens in granular sludge.An indirect method for the determination of the potential methanogenic activity of anaerobic biomass as suggested by others, viz. the estimation of the amount of F 420 present in methanogenic consortia was tested with granular sludge in the present study. It was shown (chapter 5) that a good correlation existed between the F 420 content of different granular consortia and their potential methanogenic activity on formate. No correlation, however, was observed between the F 420 content and the potential acetoclastic methanogenic activity of such consortia. This indicates that F 420 content is not a reliable parameter for assessing the overall potential methanogenic activity of anaerobic biomass.In densely packed biolayers mass transfer limitation will influence the conversion capacity of the biomass at low substrate concentrations. The results reported in chapter 6 show that the effect of mass transfer limitation in granular methanogenic sludge is dependent on the composition of the biolayer, viz. its potential specific activity, the thickness, and on the type of substrate. To detect significant effects methanogenic granules had to be cultivated on specific substrates which resulted in the development of uniform biolayers. Even with this material mass transfer resistance was of no significance unless biolayer thickness reached values above 1 mm. These results indicate that mass transfer resistance will generally not be effective in industrial reactors. They furthermore show that not only the outer parts, but also the inner parts of the granules consisted of actively catabolizing bacteria.