Role of Extracellular Polymeric Substances EPS in Aerobic Granulation

Extracellular polymeric substances (EPS) are sticky materials secreted by cells, and may play an important role in cell adhesion phenomena, formation of matrix structure, microbial physiology, and improvement of long-term stability of granules (Schmidt and Ahring, 1994; Tay et al., 2001c; Liu et al., 2004b; McSwain et al., 2005). High polysaccharide content could facilitate cell-to-cell interaction and further strengthen microbial structure through the formation of a polymeric matrix. The accumulation of EPS as capsular material and peripheral slime has been correlated with biological adhesion and aggregation processes (Costerton et al., 1981; Tay et al., 2001c; Liu et al., 2002). The metabolic blocking of exopolysaccha-ride synthesis was found to prevent microbial aggregation (Cammarota and Sant'Anna, 1998; Yang et al., 2004). EPS in granules were hypothesized to bridge two neighboring bacterial cells physically to each other as well as with other inert particulate matter, and settle out as aggregates (Liu et al., 2004b).

Individual bacterium

Individual bacterium

Extracellular Bacteria Images

Polymeric chain of EPS

Fig. 4.3. Schematic representation of extracellular polymeric substance-enhanced biogranulation (Liu et al., 2004b).

Polymeric chain of EPS

Fig. 4.3. Schematic representation of extracellular polymeric substance-enhanced biogranulation (Liu et al., 2004b).

EPS has been observed in different types of biogranules by scanning electron microscopy and transmission electron microscopy. In the biogranulation process, EPS could provide an extensive surface area for bacterial binding (Fig. 4.3). Furthermore, extracellular polysaccharide matrices surrounding aggregated bacteria can provide sites available for attraction of organic and inorganic materials (Yu et al., 2001; Sponza, 2002; Liu et al., 2004b). Evidence shows that the formation of biogranules is a micro-bial evolution instead of a random aggregation of suspended microbes (El-Mamouni et al., 1995; Fang, 2000; Tay et al., 2001c).

The spatial distribution of EPS in biogranules should be correlated to microbial evolution and distribution during the formation of biogranules. Investigation into the spatial distribution of EPS with depth in hetero-trophic biofilms showed that EPS production yields tended to decrease with biofilm depth (Zhang and Bishop, 2001). This is probably due to the fact that viable biomass loses its ability to produce EPS in the deeper sections of biofilms because of the lower microbial activity resulting from lower nutrient availability. More recently, Wang et al. (2005b) found that the outer shell of aerobic granule was composed of poorly soluble and non-easily biodegradable EPS, whereas its core part was filled with readily soluble and biodegradable EPS. Figure 4.4 shows that the fluorescent

Eps Extracellular Polymeric Substances

200 400 600 800 Distance to surface (|m)

Fig. 4.4. Cross section view of aerobic granules; (a) fresh granule; (b) granule stained by calcofluor white. Bar: 100 ^m; (c) profile of the dye fluorescence intensity distribution along the granule radius from the surface to the center (arrow) (Wang et al., 2005b). (See Color Plate Section before the Index.)

200 400 600 800 Distance to surface (|m)

1000

Fig. 4.4. Cross section view of aerobic granules; (a) fresh granule; (b) granule stained by calcofluor white. Bar: 100 ^m; (c) profile of the dye fluorescence intensity distribution along the granule radius from the surface to the center (arrow) (Wang et al., 2005b). (See Color Plate Section before the Index.)

dye (calcofluor white) was mainly attached to the outer shell of the granule, while the fluorescence was very weak in the center of the granule. In addition, the EPS produced by bacteria could be utilized as a secondary substrate in the deeper layers or zones of aerobic granules, where readily degradable substrates were either not available or limiting (Chi, 2005; Wang et al., 2005b). It appears that the spatial distribution and properties of EPS rather than its absolute quantity in aerobic granules play an essential role in stabilizing the structure and maintaining the strength of microbial aggregates (Wang et al., 2005b).

It appears from Fig. 4.4 that the fluorescence intensity profile in the direction of granule radius indicates that most calcofluor white-stained

EPS is situated in the outer shell of the granule with a depth of 400 |xm below the granule surface. Therefore, ^-linked EPS would be mainly located in the outer shell of the granule. It is believed that the insoluble EPS present in the granule shell would play a protective role with respect to the structure stability and integrity of aerobic granules, i.e. insoluble EPS may serve as the backbone of aggregated structure, while the easily biodegradable EPS located at the core of granules would play a less important role.

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