Structure and stability of methanogenic granular sludge

Immobilization of anaerobic bacteria was essential for the development of high rate anaerobic systems for the treatment of waste waters. The most widely applied anaerobic reactor type in which solids retention time is uncoupled from the hydraulic retention time is the Upflow Anaerobic Sludge Blanket (UASB) reactor. In this reactor type methanogenic granular sludge is formed by self-immobilization of methanogenic consortia. The aim of the work presented in this thesis was to study microbiological aspects of the immobilization of methanogenic consortia. To identify factors which may be of significant importance for the immobilization of anaerobic bacteria into methanogenic granular sludge a brief overview is given on the adhesion of bacteria in general as well as the microbial degradation processes that occur in anaerobic methanogenic environments (Chapter 1). Since anaerobic degradation is carried out by a series of specialized bacteria a complex biomass will result if particulate organic material or a complex medium is used as influent of an UASB reactor. To reduce the complexity of the microbial population in methanogenic granular sludge two laboratory-scale UASB reactors were fed with ethanol or propionate as substrate under defined conditions as described in Chapter 2. The chemical and bacteriological composition of methanogenic granular sludge grown under defined conditions was studied and compared with that of methanogenic granular sludge grown on complex media (Chapter 3, 4, 5). In Chapter 3 the role of extracellular polymers in the stability of methanogenic granules was investigated. The presence of extracellular polymers with different densities and structures in methanogenic aggregates was demonstrated by electron microscopy. A three step physical disintegration procedure was used to extract water-soluble extracellular polymers from three granular sludge types. DNA was used as an intracellular marker to correct for cell lysis. Upto 3.5 mg polysaccharides/g volatile suspended solids and 5.5 mg protein/g volatile suspended solids were found to be extracellular. The amounts of extracellular polymers were much lower than reported before. However, the extracellular polymer concentration in the intermicrobial space was still high enough to form gels of sufficient strength to stabilize granules to a certain extend. Inorganic precipitates were regularly observed by electron microscopy in methanogenic granular sludge. They may play an important role in the stability of methanogenic aggregates. Chemical analysis showed high concentrations of calcium phosphates in propionate-grown granular sludge and in granular sludge from an UASB reactor at a paper- mill. The two sludge types were used to study the effect of calcium removal by a calcium specific chelant (EGTA) on granule stability (Chapter 4). A remarkable reduction of the granule strength of paper-mill granular sludge was found after EGTA treatment. Propionate-grown granules disintegrated completely when high EGTA concentrations were applied. The bacteriological composition and ultrastructure of mesophilic granular sludge from a sugar refinery, ethanol-grown and propionate-grown granular sludge was studied with complementary methods (Chapter 5). The bacteria] composition of the three types of granules showed that Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol grown granules, whereas all types of methanogens were abundantly present in granules from a full scale reactor operated on a waste stream of a liquid sugar plant. The changes in bacterial population of the granular sludge after cultivation on ethanol or propionate could be explained by the physiological properties of the bacteria involved. With propionate as substrate a remarkable structure of two types of clusters of bacteria was observed by electron microscopical analysis. In one of these clusters, consisting of two morphological types of bacteria, one bacterial species labeled with antiserurn against Methanobrevibacter arboriphilus AZ, whereas the other bacterial species most likely was the propionate oxidizing bacterium. The other type of cluster consisted of bundles of Methanothrix, which was confirmed by labeling with antiserum against Methanothrix soehngenii. The finding of the microbial cluster consisting of Methanobrevibacter arboriphilus AZ and the probable propionate oxidizing bacterium corresponded well with thermodynamic and kinetic calculations discussed in Chapter 1. The adhesion properties of several isolates from granular methanogenic sludge and anaerobic culture collection strains were determined by measuring their hydrophobicity and electrophoretic mobility (Chapter 6). All the newly isolated bacteria were highly hydrophobic, indicating that bacteria which are immobilized in UASB reactors happen to have hydrophobic surface characteristics. The same was true for Methanothrix soehngenii. The abundant presence of Methanothrix soehngenii in methanogenic granular sludge is not only due to its high hydrophobicity but also to its high affinity for acetate and its rod shaped morphology causing that other bacteria or inorganic particles may be entangled. An ethanol degrading homoacetogenic bacterium was isolated from ethanol adapted granular sludge. The isolated strain which differed from known homoacetogenic bacteria was named Clostridium granularum The characterization and immobilization of this newly isolated homoacetogenic bacterium is described in Chapter 7. The initial immobilization of this strain from suspension into aggregates was studied in a special designed recycle UASB system. Aggregates of Clostridium granularum of upto 0.1 mm were formed in this system during batch operation. Aggregate formation was enhanced when the strain was grown in the presence of a hydrogenotrophic methane bacterium. To determine the overall effect of environmental changes in UASB reactors on the growth of granular methanogenic sludge, particle size distribution measurements were made with two independent methods namely sedimentation velocities and image analysis. With the image analysis method the presence of granules with a diameter as low as 0.05 mm could be determined, whereas the sedimentation velocity method allowed only the detection of particles larger than 0.5 mm. In Chapter 8 the mean granule diameter was used as a characteristic parameter to determine the effect of substrate concentration on granular sludge. Larger granules were obtained by using high substrate concentrations (6900 mg COD/l), whereas low substrate concentrations led to small granules through granule disintegration (2050 mg COD/l). Conclusions The use of single substrates for the cultivation of methanogenic granular sludge in UASB reactors allows to elucidate structure-function relationships in methanogenic granules. The spatial orientation of bacteria in propionate grown granular sludge is a major evidence for the ecological impact of interspecies hydrogen transfer in methanogenic bacterial consortia. The presence of such spatial orientation is dependent upon the composition of the medium. The stability of methanogenic granular sludge is dependent on the presence of extracellular polymers and inorganic precipitates. The granule size is dependent on the influent substrate concentration. All bacteria isolated from methanogenic granular sludge were highly hydrophobic, indicating that the UASB reactor concept selects for bacteria with good adherence properties. Since the median particle size can be considered as an overall parameter for different physiological and technological conditions, measurement of the size distribution may be used for judging the quality and stability of granular sludge in full scale reactors.