Development of Nitrifying Granules

Yang et al. (2005) cultivated nitrifying granules in at various substrate N/COD ratios in the range of 5/100-30/100 by weight. It was found that nitrifying granules could form at all the tested substrate N/COD ratios and the characteristics of nitrifying granules were found to be substrate N/COD ratio-dependent.

The Formation of Nitrifying Granules

Nitrifying granulation was found to be a gradual process from the dispersed seed sludge through the tiny aggregates to the mature spherical granules as shown in Fig. 8.1 (Yang et al., 2005). After about four weeks of operation, nitrifying granules with a dense structure formed at the substrate N/COD ratios studied. The biomass concentrations in the reactors were increased up to 10 g/l of total solids (TS) upon the formation of nitrifying granules. The granules developed at all tested N/COD ratios exhibit compact structure compared to the seed sludge (Fig. 8.1).

Characteristics of Nitrifying Granules

Granule Size

Compared to the seed activated sludge with a floc size of 0.09 mm, mature nitrifying granules had a mean size of 1.9, 1.5, 0.5, and 0.4 mm in the reactors operated at substrate N/COD of 5/100-30/100, respectively (Fig. 8.1). These seem to indicate that small nitrifying granules would be cultivated at high substrate N/COD ratios and vice versa.

p n/cod: 10/100 |

i M

• t» •

Fig. 8.1. Morphology of mature nitrifying granules developed at different substrate N/COD ratios. Bar: 1 mm (Yang et al., 2005).

Fig. 8.1. Morphology of mature nitrifying granules developed at different substrate N/COD ratios. Bar: 1 mm (Yang et al., 2005).

Settleability

In the environmental engineering field, sludge settleability has been commonly described by sludge volume index (SVI). Figure 8.2 shows changes of SVI in the course of granulation observed at the different substrate N/COD ratios (Yang et al., 2005). It appears that the SVI of microbial granules exhibited a decreasing trend with the increase of substrate N/COD ratio, e.g. the lowest SVI was found at the highest substrate N/COD ratio of 30/100. These results imply that the substrate N/COD ratio would have a significant effect on the structure of microbial granules, i.e. a more compact microbial structure could be expected at higher substrate N/COD ratio. Such information is important because one may expect to manipulate the structure of aerobic granules by adjusting substrate N/COD ratios.

Fig. 8.2. Change of SVI during aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

Fig. 8.2. Change of SVI during aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

In addition, compared to the seed activated sludge with a SVI of 265 ml/g, the settleability of microbial granules was improved noticeably.

It should be realized that the settling velocity of aerobic granules cultivated at different substrate N/COD ratios was greater than 60 m/h (Yang et al., 2005), while the settling velocity of conventional activated sludge was around 5 m/h (Giokas et al., 2003). Compared to the conventional activated sludge flocs, the excellent settleability of aerobic granules can ensure quick and effective separation of biosolids from the effluent and high biomass retention can be achieved in the reactors. These are very attractive from the point of view of industrial application as they may help to solve the problems encountered in conventional nitrogen removal processes, such as sludge bulking, washout of nitrifying biomass, and so on.

Specific Gravity

The specific gravity can be used to describe the compactness of a microbial community. Figure 8.3 shows the specific gravities of the mature aerobic granules developed at different substrate N/COD ratios (Yang et al., 2003). It can be seen that the specific gravity of granules tends to increase with

1.07

1.01

0.10 0.20 Substrate N/COD

0.30

Fig. 8.3. Specific gravity of aerobic granules developed at different substrate N/COD ratios (Yang et al., 2003).

0.05

0.10 0.20 Substrate N/COD

0.30

Fig. 8.3. Specific gravity of aerobic granules developed at different substrate N/COD ratios (Yang et al., 2003).

the increase of substrate N/COD ratio, i.e. a high substrate N/COD ratio would result in a more compact structure of aerobic granules. Such a trend is indeed consistent with the changes in SVI as illustrated in Fig. 8.2. Compared to the seed sludge with a specific gravity of 1.002, it is obvious that the aerobic granules had a much denser and more compact microbial structure.

Cell Hydrophobicity

Changes in cell hydrophobicity of microbial granules cultivated at different substrate N/COD ratios are presented in Fig. 8.4 (Yang et al., 2005). The cell hydrophobicity gradually increased until a stable value was achieved after a 40-day operation, while the cell hydrophobicity at steady state exhibits a positive relation to the substrate N/COD ratio. Increasing evidence shows that cell hydrophobicity plays a crucial role in the formation of biofilm, anaerobic granules as well as aerobic granules (Mahoney et al., 1987; Rouxhet and Mozes, 1990; Tay et al., 2000; Liu et al., 2004a). In a thermodynamic sense, the increase of cell hydrophobicity would simultaneously cause a decrease in the excess Gibbs energy of the surface, which in turn favors the self-aggregation of bacteria from liquid

Fig. 8.4. Changes in cell hydrophobicity during aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

Fig. 8.4. Changes in cell hydrophobicity during aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

phase to form a new solid phase, namely microbial aggregates (Liu and Tay, 2002). In fact, the cell hydrophobicity of bacteria has been recognized as a decisive affinity force in cell immobilization process (Rouxhet and Mozes, 1990; Bossier and Verstraete, 1996; Zita and Hermansson, 1997; Liu et al., 2004a). It seems certain that hydrophobic binding force has a prime importance for the cell-to-cell approach and interaction, and the hydrophobicity of bacterial surface can act as a driving force for the initiation of cell-to-cell aggregation, which is the first step towards aerobic granulation, and further keep bacteria aggregated tightly together.

It should be pointed out that increased substrate N/COD ratio would lead to an enriched nitrifying population in aerobic granules. It had been shown that nitrifying bacteria had a higher hydrophobic interaction than that of activated sludge microorganisms (Sousa et al., 1997; Kim et al., 2000). Thus, high cell hydrophobicity of aerobic granules developed at high substrate N/COD ratio, in part, could be attributed to the enriched nitrifying population in granules.

Production of Extracellular Polysaccharides

Extracellular polysaccharides can mediate both cohesion and adhesion of cells, and play a crucial role in building and maintaining structural a

Fig. 8.5. Changes in the PS/PN ratio in the course of aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

Fig. 8.5. Changes in the PS/PN ratio in the course of aerobic granulation at different substrate N/COD ratios (Yang et al., 2005).

integrity in a community of immobilized cells (Fletcher and Floodgate, 1973; Schmidt and Ahring, 1996; Lopes et al., 2000; Liu et al., 2004b; Wang et al., 2005). Figure 8.5 shows changes in the ratio of extracellular polysaccharides (PS) to extracellular proteins (PN) in the course of aerobic granulation at different substrate N/COD ratios (Yang et al., 2005). The salient points from Fig. 8.5 can be summarized as follows: (i) the PS/PN ratio increased in a very significant way with the formation of aerobic granules, e.g. the PS/PN ratio increased from an initial value of 0.57 for the seed sludge to 4.0-5.0 for the aerobic granules. These may indicate that microbial aggregation would be partially related to the production of extracellular polysaccharides; (ii) with increasing the substrate N/COD ratio, the PS/PN ratio shows a decreasing trend. In fact, this is in line with previous finding showing that a reduced substrate N/COD ratio would stimulate the production of extracellular polysaccharides, resulting in improved bacterial attachment to solid surfaces (Schmidt and Ahring, 1996; Durmaz and Sanin, 2001). In addition, Tsuneda et al. (2003) found that extracellular polysaccharides exhibited good correlation with cell adhesion, while no protein was related to cell adhesion.

Extracellular polysaccharides are produced by most bacteria out of cell wall with the purpose of providing cells with the ability to compete in a variety of environments, providing a mode for adhesion to surfaces

(Sutherlan, 2001). The PS/PN ratios of aerobic granules tend to decrease with the increase of the substrate N/COD ratio (Fig. 8.5). As the nitrifying populations in aerobic granules were greatly enriched at high substrate N/COD ratios, the lower production of extracellular polysaccharides at higher substrate N/COD ratio can be explained in a way such that nitrifying bacteria cannot utilize organic carbon for microbial growth, and only 11-27% of energy generated goes to biosynthesis (Laudelout et al., 1968). Thus, less extracellular polysachharides would be synthesized in aerobic granules developed at high substrate N/COD ratio.

It was also found that the production of cell polysaccharides is quasi-linearly dependent on the respiration activity of heterotrophic bacteria present in the aerobic granules, i.e. a high catabolic activity favors the production of cell polysaccharides. These are consistent with other research showing that the production of extracellular polysaccharides was energy-dependent (Robinson et al., 1984; Wuertz et al., 1998). In fact, there is evidence that cell carbohydrate content increased and protein content decreased significantly, the way as the substrate N/COD ratio decreased (Durmaz and Sanin, 2001). It seems reasonable to consider that nitrifying bacteria would produce much less extracellular polysaccharides than heterotrophs. In addition, Tsuneda et al. (2001) used extracellular polysac-charides produced by heterotrophic bacteria to enhance the formation of nitrifying biofilm. As shown in Fig. 8.5, the content of aerobic granule-polysaccharides at steady state was at about 3-fold higher than that of proteins. Vandevivere and Kirchman (1993) also found that the content of exopolysaccharides was 5-fold greater for attached cells than for free-living cells. These might imply that cell proteins would less contribute to the structure and stability of immobilized microorganisms. A more recent research further showed that the structural stability of aerobic granules was closely related to the content and distribution of insoluble extracellular polysaccharides (Wang et al., 2005).

Elemental Compositions of Nitrifying Granules

The characteristics of aerobic granules seem to be substrate N/COD ratio-dependent. It has been known that changes in characteristics are usually related to the changes in chemical compositions of microorganisms (Pitryuk et al., 2002). In mixed microbial culture, chemical compositions of microorganisms may reflect the changes of microbial community and growth conditions (Duboc et al., 1995; Heldal et al., 1996). Table 8.1 shows the elemental compositions of aerobic granules developed at different substrate N/COD ratios (Liu et al., 2003a). These data indicate that aerobic granules mainly comprised six major elements, i.e. C, H, O, N, S, and P. The substrate N/COD ratio displays a profound effect on the respective ratio of cell oxygen, nitrogen, and calcium normalized to cell carbon. Cell N/C ratio increased with the increase in the substrate N/COD ratio, whereas cell O/C ratio decreased. Heldal et al. (1996) observed a marked reduction in cell O/C level when the conditions changed from nitrogen-limitation to carbon-limitation. Vrede et al. (2002) also reported that elemental composition of bacterioplankton was closely related to the substrate carbon and nitrogen and the lowest cell carbon content was found in carbon-limited cells. Microorganisms are often found to differ in their relative contents of C, H, N, O, and other elements when they experience the shift of microbial community and the change of growth conditions (Duboc et al., 1995; Pitryuk et al., 2002). Obviously, information on chemical composition of microorganisms is essential for a sound understanding of the behaviors of microbial community.

The accumulation of calcium in anaerobic granules had been reported (Yu et al., 2001). However, it appears from Table 8.1 that no accumulation of cell calcium occurred in aerobic granules cultivated at different substrate N/COD ratios. In fact, the cell Ca/C ratio of aerobic granules is even lower than that of the seed sludge (7.5 mmolmol-1). Increasing evidence shows that the accumulation of calcium in aerobic granules would be related to organic substrate used, e.g. aerobic granules reported Table 8.1 were grown on ethanol, however, aerobic granule grown on acetate had high calcium content (Qin et al., 2004a).

Microbial Diversity of Nitrifying Granules

To remove organics and nitrogen from wastewater, nitrifying, denitrifying, and heterotrophic populations should co-exist in microbial granules. It had been reported that substrate with different N/COD ratios would lead to significant shift among various populations in both suspended and attached cultures (Moreau et al., 1994; Ohashi et al., 1995; Princic et al., 1998; Ballinger et al., 2002). A variation of the relative substrate composition

Table 8.1. Elemental compositions of aerobic granules developed at different substrate N/COD ratios, in terms of percentage by dry weight of granules (Liu et al., 2003a)

Element

Substrate N/COD ratio

Table 8.1. Elemental compositions of aerobic granules developed at different substrate N/COD ratios, in terms of percentage by dry weight of granules (Liu et al., 2003a)

Element

Substrate N/COD ratio

5/100

10/100

20/100

30/100

C

41.960

42.820

41.890

43.023

H

7.380

7.020

7.160

7.300

O

38.770

38.740

36.540

36.200

N

8.490

9.080

9.220

9.480

P

0.750

0.810

0.850

0.830

S

1.010

0.890

0.900

0.990

Ca

0.420

0.230

0.290

0.540

Fe

0.180

0.043

0.049

0.160

Mg

0.130

0.070

0.110

0.140

Al

0.022

0.003

0.042

0.250

Mn

0.005

0.002

0.003

0.002

Co

0.001

0.000

0.000

0.000

Cu

0.150

0.008

0.019

0.098

Ni

0.011

0.011

0.075

0.055

Zn

0.093

0.010

0.016

0.034

Na

0.330

0.110

0.480

0.430

K

0.300

0.160

0.360

0.480

Formula

C5.8H12.2O4.0NP0.04

C5.4H10.8O3.7NP0.04

C5.3H10.9O3.4NP0.04

C5.3H10.8O3.3NP0.04

S"

TO Ci

TO S

in the bulk fluid can result in rapid and drastic changes of the relative abundance and spatial distribution of organisms in biofilms (Fruhen et al., 1991). Zhang et al. (1995) found that heterotrophs, supported by soluble microbial products or metabolic products, could exist in nitrifying biofilms. Nitrifiers, however, have difficulty to survive in heterotrophic biofilms because they are likely to be out-competed by heterotrophs for dissolved oxygen and space. Inhibition or elimination of nitrifying populations by interspecies competition usually leads to a decline in nitrification efficiency, or even a failure of the process. Thus, an understanding of the effects of substrate N/COD ratio on the dynamic changes of microbial species in microbial granules is an important need.

Evolution of Heterotrophic Activities

To investigate the microbial activities and distributions of respective population, Yang et al. (2004a) determined the activities of heterotrophic, ammonia-oxidizing, and nitrite-oxidizing bacteria by the respective specific oxygen utilization rate (SOUR)H, (SOUR)nh4, and (SOUR)NO2. Figure 8.6 shows the activities of heterotrophic populations in aerobic granules developed at different substrate N/COD ratios in the course of the reactor operation. The activity of heterotrophs in granules slightly decreased over operation time at the substrate N/COD ratios of 10/100, 20/100, and 30/100, only with the exception at the substrate N/COD ratio of 5/100 at which it remained unchanged with the operation time.

Evolution of Nitrifying Activities

The respective respirometric activities of ammonia oxidizers and nitrite oxidizers were described by the specific ammonium oxygen utilization rate (SOUR)nh4 and the specific nitritation oxygen utilization rate (SOUR)NO2. The activity of both ammonia oxidizers and nitrite oxidizers tended to increase with the operation time (Yang et al., 2004a). It is a reasonable consideration that the sum of (SOUR)NH4 and (SOUR)NO2, namely (SOUR)N may represent the overall activity of nitrifying populations in microbial granules. Figure 8.7 shows the values of (SOUR)N at different operation time. As can be seen, after 86 days of operation, the overall activity of

Substrate N/COD ratio

Fig. 8.6. Respirometric activities of heterotrophs in aerobic granules (Yang et al., 2004a).

Substrate N/COD ratio

Fig. 8.6. Respirometric activities of heterotrophs in aerobic granules (Yang et al., 2004a).

Substrate N/COD ratio

Fig. 8.7. Respirometric activities of nitrifiers in aerobic granules (Yang et al., 2004a).

Substrate N/COD ratio

Fig. 8.7. Respirometric activities of nitrifiers in aerobic granules (Yang et al., 2004a).

nitrifying populations was approaching respective stable levels at different N/COD ratios.

These results imply that the substrate N/COD ratio would have remarkable effects on the activity distribution of ammonium-oxidizing and nitrite-oxidizing bacteria in the microbial granules, i.e. both the ammonium-oxidizing and nitrite-oxidizing activities were significantly increased with the increase of the substrate N/COD ratio, while the het-erotrophic activity in the aerobic granules decreased. At high substrate N/COD ratios, heterotrophs became much less dominant, whereas nitrifying populations would be able to compete with heterotrophs, and became an important component of the aerobic granules (Figs 8.6 and 8.7). Similar phenomenon was also observed in biofilm culture (Moreau et al., 1994; Ohashi et al., 1995; Ochoa et al., 2002).

Interactions between Heterotrophic and Nitrifying Populations

The fraction of active biomass in a culture would be proportionally related to the respirometric activity (Ochoa et al., 2002). Thus, the relative abundance of nitrifying populations over heterotrophic populations can be proportionally represented by (SOUR)N/(SOUR)H. The relative abundance of nitrifying populations over heterotrophic populations in the aerobic granules can be accordingly calculated with activities values obtained (Yang et al., 2004a). The value of (SOUR)n/(SOUR)h in the SBR run at the substrate N/COD ratio of 30/100 was 0.6 on day 60 and further increased to about 1.1 on day 86 onwards. Interactions between heterotrophic and nitrifying populations in the SBRs operated at the respective substrate N/COD ratio of 10/100 and 20/100 followed the similar pattern, i.e. (SOUR)N/(SOUR)H gradually stabilized at a certain level. However, (SOUR)N/(SOUR)H in the SBR operated at the substrate N/COD ratio of 5/100 almost remained constant. These seem to imply that a balance between two populations could be finally achieved in aerobic granules.

Nitrifying populations are commonly found in activated sludge and biofilms, while their quantity is generally insufficient because they would be out-competed by heterotrophs (Moreau et al., 1994). It appears from Figs 8.6 and 8.7 that nitrifying populations continued to build up over heterotrophic population in the aerobic granules until a balance between heterotrophic and nitrifying populations was reached on day 86 onwards. Aerobic granules appear to provide a protective matrix for nitrifying bacteria to grow peacefully without the risk of being washed out from the system. It may be expected that aerobic granule-based compact and efficient bioreactor for simultaneous organic removal and nitrification would be developed in near future.

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