Celebrating 40 years anaerobic sludge bed reactors for industrial wastewater treatment

The key for modern high-rate biotechnology, whatever system is considered, is immobilization of proper bacteria. In fact, the required high sludge retention in anaerobic sludge bed systems is based on immobilization, which generally leads to the formation of well-balanced microbial consortia. The presence of these consortia is considered a prerequisite for proper anaerobic process operation, particularly considering the occurrence of various syntrophic conversion reactions in the anaerobic degradation of most organic compounds, the detrimental effect of higher concentrations of specific intermediates, and the strong effect of environmental factors like pH and redox potential. Significant progress in the knowledge of the fundamentals of the immobilization process has been made since the development and successful implementation of high-rate anaerobic treatment systems in the seventies (Hulshoff Pol et al. 2004). In the absence of fixed or free floating inert support material, a so-called “auto-immobilization” will occur, which is understood as the immobilization of bacteria on themselves or on very fine inert or organic particles present in the wastewater, forming dense bacterial conglomerates. The bacterial conglomerates will mature on due time and form round shaped granular sludge.

The phenomenon of sludge granulation has puzzled many researchers from very different disciplines. Granulation, in fact, is a completely natural process and proceeds in all systems where the basic conditions for its occurrence are met, i.e. on mainly soluble substrates applying HRTs lower than the bacterial doubling times. Owing to the very low growth rate of the crucial aceticlastic methanogenic bacteria, particularly under sub-optimal conditions, the latter conditions are easily met. Anaerobic granule formation is mostly observed in anaerobic bioreactors that are operated in upflow mode (Hulshoff Pol et al. 2004). However, successful granulation was also observed in anaerobic sequencing batch reactors (Sung and Dague 1995; Wirtz and Dague 1996). Maybe for the first time, sludge granulation was found to occur in the up-flow Dorr Oliver Clarigesters applied in South Africa since the 1950s. However, this only became apparent by observation of sludge samples taken from such a digester in 1979 (Lettinga 2014). Surprisingly enough, no attention was given to the characteristics of the Clarigester sludge such as size, form and the mechanical strength, density and porosity of sludge flocs/aggregates. Despite all the efforts made to develop systems with a high sludge retention, nobody apparently noticed that the major part of the sludge consisted of a granular type of sludge. While studying the start-up and feasibility of anaerobic upflow filters, Young and McCarty (1969) already recognized the ability of anaerobic sludge to form very well settleable aggregates. These granules were as large as 3.1 mm in diameter and settle readily. In AF experiments with potato starch wastewater and methanol solutions conducted in the Netherlands, similar observations were made (Lettinga et al. 1976, 1979). Whereas the interest in anaerobic wastewater treatment in the USA and South Africa diminished, large emphasis on developing industrial scale systems was put in the Netherlands, where instalment of new surface water protection acts coincided with the world energy crisis of the seventies as outline above. As a result, increasing emphasis could be afforded on applied and fundamental research in this field, particularly also on the phenomenon of sludge granulation (Lettinga et al. 1987). A worldwide growing interest occurred from both the engineering and the microbiological field. As a result, the insight in the mechanism of the sludge granulation process for anaerobic treatment has been elucidated sufficiently, at least for practical application (e.g. De Zeeuw 1982, 1987; Hulshoff Pol and Lettinga 1986; Wiegant and de Man 1986; Hulshoff Pol et al. 1987, 2004; Dolfing 1987; Wu et al. 1991; van Lier et al. 1994; Fang et al. 1994; Liu et al. 2003; Song et al. 2010; Habeeb et al. 2011; Abbasi and Abbasi 2012; Subramanyam 2013). Granulation can proceed under mesophilic, thermophilic and psychrophilic conditions. It is considered of big practical importance to further unravel the fundamentals concerning the growth of mixed balanced granular aggregates, not only from the microbial but also from the process engineering point of view.

A variety of process operational and external factors are effective on granule stability, e.g. HRT, VLR, temperature, pH, upflow velocity, presence of divalent cations and heavy metals, salinity, and nutrient availability (Habeeb et al. 2011; Abbasi and Abbasi 2012; Calderon et al. 2013; Ismail et al. 2008). The seed sludge and the chemical composition of the industrial wastewater have significant impact on the chemical composition of the granular sludge (Batstone et al. 2004). In addition, Macro- and micronutrients, e.g. iron, copper, calcium, magnesium, cobalt and aluminum are vital for the aggregation of the cells (Subramanyam 2013).

The morphological and spatial structure of granules in a UASB reactor was examined by MacLeod et al. (1990). They found that the granular aggregates were three-layered structures. Whereas the exterior layer of the granule contained a heterogeneous microbial population, the middle layer consisted of more homogeneous biomass. Moreover, the internal core consisted of a “single species”, like Methanothrix-like cells, later renamed to Methanosaeta spec. (Patel and Sprott 1990). Similar findings have been reported in the study of Baloch et al. (2008), in which anaerobic granules were found to possess a multi-layered structure with complex microbial ecology and dominating methanogenic subpopulations. Apparently, Methanosaeta plays an important role in sludge granulation (Fang et al. 1994). The structured characteristics and layered ‘ecological zones’ of the granules were defined as a stable metabolic arrangement that creates optimal nutritional and environmental conditions for all microorganisms included in it (Guiot et al. 1992). The carbon source or substrate was considered the most important factor affecting the microstructure of the UASB granules (Grotenhuis et al. 1991; Fang et al. 1994; Batstone et al. 2004). The extent of required acidification and the acidogenesis rate of the substrate affects the concentration profiles of the substrate, metabolites in the granule and its structure. For example, granules in a UASB reactor treating sucrose and brewery wastewaters had a three-layered structure; however, the ones in a UASB reactor treating glutamate exhibited a rather uniform structure. McHugh et al. (2003) reported that, in a granule, a central core of acetoclastic methanogens is surrounded by a layer of hydrogen and/or formate producing acetogens, and hydrogen and/or formate consuming methanogens. Outside layer of this granule structure consists of microorganisms that hydrolyze and acidify the complex organic matter (Liu et al. 2003). Methanosaeta spp. populations have been found abundant in stable granules in various studies. Apparently, these organisms are necessary for the successful operation of anaerobic sludge bed reactors. Methanogens related to Methanosaeta spp. have a filamentous morphology, are more or less hydrophobic, have an electrophoretic mobility of about 0, and are considered the most important component of the granule structure, providing support for other microorganisms in the granule (Grotenhuis et al. 1992, Song et al. 2010; Calderon et al. 2013). It is hypothesized that after the formation of such methanogenic nucleus, acetogenic bacteria adhere, followed by the formation of biofilm layers consisting of hydrogenotrophic methanogens (Abbasi and Abbasi 2012). On the other hand, the bacteriophage in the granular sludge may cause the breakdown of the granules (Subramanyam 2013).

Molecular techniques are increasingly used to study the microbial community structure of environmental ecosystems like anaerobic granular sludge without cultivation (Batstone et al. 2004). By using molecular techniques, Sekiguchi et al. (1999) localized the methanogens in anaerobic granular sludge systems. They showed that a significant fraction of the granule is inactive and this probably consists of cellular fragments. The spatial information associated with a protein or pathway inside the cell can influence the end-behavior of a molecular network (Agapakis et al. 2012). Satoh et al. (2007) combined 16S rRNA gene-based molecular techniques with microsensors to provide direct information about the phylogenetic diversities, spatial distributions, and activities of bacteria and archaea in anaerobic granules. They found that acid and H2 production occurred in the outer part of the granule, below which H2 consumption and CH4 production were found.

In essence, sludge granulation finds its ground in the fact that bacterial retention is imperative when dilution rates exceed the bacterial growth rates (van Loosdrecht et al. 2002). Immobilization further requires the presence of support material and/or specific growth nuclei (Hulshoff Pol et al. 1983), as well as the presence of exopolymeric substances (EPS) acting as a kind of glue creating a microbial matrix (Vanderhaegen et al. 1992). The occurrence of granulation can be explained as follows:

• Proper growth nuclei, i.e. inert organic and inorganic bacterial carrier materials as well as bacterial aggregates, are already present in the seed sludge.

• Finely dispersed matter, including viable bacterial matter, will become decreasingly retained, once the superficial liquid and gas velocities increase, applying dilution rates higher than the bacterial growth rates under the prevailing environmental conditions. As a result, film and/or aggregate formation automatically occurs.

• The size of the aggregates and/or biofilm thickness are limited, viz. it depends on the intrinsic strength (binding forces and the degree of bacterial intertwinement) and the external forces exerted on the particles/films (shear stress). Therefore, at due time, particles/films will fall apart, evolving a next generation. The first generation(s) of aggregates, indicated by Hulshoff Pol et al. (1983) as “filamentous” granules, are quite voluminous and in fact more a flock than a granule.

• Retained secondary growth nuclei will grow in size again, but also in bacterial density. Growth is not restricted to the outskirts, but also proceeds inside the aggregates. At due time, they will fall apart again, evolving a third generation, etc.

During the above described selection process, both organic and hydraulic loading rates gradually increase, increasing the shear stress inside the system. For mainly soluble wastewaters that are partly acidified, granular sludge will be easily cultivated. Table 2 lists some common characteristics of methanogenic granular sludge.

Full size table