Distribution of EPS and Cell Surface Hydrophobicity in Aerobic Granules Wang Zhiwu ([email protected]
) Liu Yu ([email protected]
) Tay Joo-Hwa ([email protected]
Civil Engineering Research
Aerobic granulation as a promising biotechnology for wastewater treatment has attracted intensive research attention. Most research on aerobic granulation has been focused on the cultivation and physical characteristics of aerobic granules. So far, the structure of anaerobic granules has been studied extensively, and extracellular polysaccharides (EPS) have been found to contribute to the build-up of the matrix structure and the stability of anaerobic granules (Liu et al., 2004). The failure of microbial aggregation has been found to be correlated to the metabolic blocking of EPS synthesis, while EPS deficiency would result in a weak structure of anaerobic granules. Compared to anaerobic granules, little is currently known about the internal structure of aerobic granules as well as the EPS and cell hydrophobicity distribution in aerobic granules. Thus, this study investigated the distribution of EPS and hydrophobicity in aerobic granules as well as the essential role of EPS in aerobic granulation.
Materials and methods Aerobic granules were cultivated in a sequencing batch reactor (SBR) and were fed with sodium acetate as the sole carbon source. EPS were extracted from the ground granules according to the cold aqueous technique, and the extracted EPS were analyzed using calorimetric method. In this study, the sliced aerobic granule was stained with 300 mg L-1 calcofluor white (Fluorescent Brightener 28, Sigma) for 1 hour in 20 mL Phosphate Buffered Saline. The samples were then rinsed with 2 mL of Milli-Q water to decrease the background fluorescence and were visualized with Epifluorescence Microscope BX-FLA-3 (Olympus, Japan). In addition, intact and sectioned aerobic granules were visualized by Image Analyzer (IA) (Olympus Imaging Analyzing System SZX9, Japan). Hydrophobicity of the aerobic granule was determined and expressed as the percentage of cells adhering to the hexadecane after 15 min partitioning.
Figure 1. Cross section view of the aerobic granule in bright field (A) and dark field (B) visualization modes, Bar: 500 µm
granules were sliced and the respective density of the outer and the inner layer was then measured. Results indicate that the density of the outer layer of granule was higher than that of the core part of the aerobic granule. Hence, the core part of granule was composed of transparent and jelly-like substances, while the outer shell of the granule mainly consisted of dense materials leading to an opaque structure. EPS and hydrophobicity distribution in aerobic granule The outer shell of the aerobic granule was separated from the core part and the corresponding EPS content as well as hydrophobicity was determined. Figure 2 shows that the EPS content in the core part of the granule is nearly 5 times higher than that in the shell part of aerobic granule. To localize the EPS distribution, the sliced aerobic granule was stained by calcofluor white, and was then visualized by epifluorescent microscopy. Using a fresh granule as the reference (Figure 3), it was found that the fluorescent dye was mainly attached to the outer shell of the granule, while the fluorescence was very weak in the center of the granule. The fluorescence intensity profile in the direction of the granule radius further shows that most calcofluor white stained EPS are situated in the outer shell of the granule with a depth of 400 (m below the granule surface. This may imply that the (-linked EPS would be mainly located in the outer shell of the granule. In
Results The heterogeneous structure of aerobic granules Figure 1 shows that the aerobic granule has an opaque outer layer with a depth of about 800 (m from the granule surface and a relatively transparent inner core. It appears that the highly transparent center part of the granule was not void, and was instead filled with jelly-like substances. Five aerobic
Figure 2. Hydrophobicity (black) and EPS (gray) distribution in the shell and core part of the aerobic granule
Figure 3. Cross section view of aerobic granules, A: fresh granule; B: granule stained by calcofluor white, Bar: 100 µm
fact, calcofluor white has been commonly used to label the (β-linked EPS (deBeer et al., 1996). In addition, Figure 2 shows the respective hydrophobicity of the shell and core of aerobic granule. It seems that the granule outer shell has a much higher hydrophobicity than the granule core.
 Aquino S.F., Stuckey D.C. (2003) Production of soluble microbial products (SMP) in anaerobic chemostats under nutrient deficiency. J Environ Eng-Asce 129: 1007-1014.  deBeer D., OFlaharty V., Thaveesri J., Lens P., Verstraete W. (1996) Distribution of extracellular polysaccharides and flotation of anaerobic sludge. Appl Microbiol Biotechnol 46: 197-201.  Laspidou C.S., Rittmann B.E. (2002) A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water. Res 36: 2711-2720.  Liu Y.Q., Liu Y., Tay J.H. (2004) The effects of extracellular polymeric substances on the formation and stability of biogranules. Appl Microbiol Biotechnol 65: 143-148.  Tay J.H., Tay S.T.L., Ivanov V., Pan S., Jiang H.L., Liu Q.S. (2003) Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment. Lett Appl Microbiol 36: 297-301.
Previous research showed that substrate could only penetrate to a depth of 800 (m below the aerobic granule surface (Tay et al., 2003). This may indicate that a nutrient deficient situation would be encountered in the core part of aerobic granules, as shown in Figure 1. There is evidence that the nutrient deficiency condition would induce the production of soluble EPS. It has been reported that compared to a balanced culture, 10 times more soluble EPS was secreted in nutrient deficiency culture (Aquino and Stuckey, 2003). This provides a plausible explanation for the enhanced
Civil Engineering Research
Figure 1 shows that aerobic granules have a heterogeneous structure consisting of a dense outer shell and a loosestructure core, while the outer shell of the aerobic granule is mainly composed of (-linked EPS which are more hydrophobic (Figures 2 and 3). According to their physicochemical properties, EPS can be classified into bound EPS and soluble EPS, while generally, only soluble EPS are considered to be biodegradable (Laspidou and Rittmann, 2002). This may imply that the EPS detected in the core of aerobic granule would be easily biodegradable.
production of soluble EPS in the granule core (Figure 2). It is a reasonable consideration that soluble EPS would be less important in constructing and maintaining the structural stability of the aerobic granule, i.e. EPS property and distribution in aerobic granules instead of their quantity would play a crucial role in improving the stability of aerobic granules. In addition, it appears from Figures 2 and 3 that the high hydrophobicity of the outer shell of aerobic granule result from the insoluble (-linked polysaccharides accumulated in the shell. In fact, insoluble (-linked EPS has been found to serve as the backbone of the biofilm structure.