Wastewater Purification: Aerobic Granulation in Sequencing Batch Reactors - Chapter 13 potx

236 13.1 INTRODUCTION A high calcium content has been reported in acetate-fed aerobic granules even though the calcium concentration in the synthetic wastewater was very low Qin, Liu, an

in Acetate-Fed Aerobic Granules

Zhi-Wu Wang, Yong Li, and Yu Liu

CONTENTS

13.1 Introduction 223

13.2 Effect of Calcium on Aerobic Granulation 224

13.3 Calcium Accumulation in Acetate-Fed Aerobic Granules 225

13.4 Chemical form of calcium in acetate-fed aerobic granules 226

13.5 Calcium Distribution in Acetate-Fed Aerobic Granules 227

13.6 Granule Size-Dependent CaCO3Formation in Acetate-Fed Aerobic Granules 227

13.7 Mechanism of Calcium Accumulation in Acetate-Fed Aerobic Granules 229

13.7.1 Ionic Equilibrium of Carbonate Ion 230

13.7.2 Diffusion Kinetics in Aerobic Granules 231

13.7.3 Distribution of pH and CO32-in Acetate-Fed Aerobic Granules 233

13.7.4 Size-Associated Formation of CaCO3in Acetate-Fed Aerobic Granules 234

13.8 Conclusions 235

Symbols 236

References 236

13.1 INTRODUCTION

A high calcium content has been reported in acetate-fed aerobic granules even

though the calcium concentration in the synthetic wastewater was very low (Qin,

Liu, and Tay 2004; Wang, Liu, and Tay 2005) Extensive accumulation of calcium

was also found in biofilms and anaerobic granules (Batstone et al 2002; Kemner

et al 2004) Two hypotheses have been put forward to explain the calcium

accumu-lation: (1) calcium links with extracellular polymeric substances (EPS) and forms an

EPS-Ca2+-EPS cross-linkage; and (2) calcium is present in the form of CaCO3(Yu,

Tay, and Fang 2001; Wloka et al 2004) This chapter thus explores the mechanism

behind the accumulation, chemical form, and spatial distribution of calcium ion in

acetate-fed aerobic granules

13.2 EFFECT OF CALCIUM ON AEROBIC GRANULATION

Ca2+has been reported to enhance the formation of anaerobic granules and

acido-genic biofilms (Huang and Pinder 1995; Yu, Tay, and Fang 2001) Jiang et al (2003)

studied the effect of calcium on aerobic granulation in sequencing batch reactors

(SBRs) For this purpose, two SBRs were operated at the respective Ca2+

concentra-tions of zero and 100 mg L–1 It was found that aerobic granules were formed in both

SBRs, and granule sizes were stabilized at around 2 mm and 2.8 mm in the

calcium-free and calcium-added SBRs, respectively, after 2 months of operation (figure 13.1)

These results indicate that aerobic granulation may not depend on calcium ion, that

is, calcium ion is not essential for aerobic granulation in SBRs Mahoney et al (1987)

investigated anaerobic granulation in two upflow anaerobic sludge blanket (UASB)

reactors fed with aero and 100 mg Ca2+L–1, respectively Similar to the results shown

in figure 13.1, successful anaerobic granulation was achieved in both reactors,

indi-cating that calcium is not an essential element for anaerobic granulation either

Compared to aerobic granules grown on calcium-free medium, aerobic granules

cultivated with addition of calcium showed better settleability and higher strength

(figure 13.1) It is thought that the Ca2+ion should bind to negatively charged groups

A

B

FIGURE 13.1 Aerobic granules cultivated at different calcium concentrations, 0 mg Ca2+ L –1

(a) and 100 mg Ca 2+ L –1(b) (From Jiang, H L et al 2003 Biotechnol Lett 25: 95–99.

With permission.)

of extracellular polysaccharides present on bacterial surfaces, and act as a bridge to

interconnect these components, so as to promote bacterial aggregation and further

enhance the structural stability of aerobic granules, anaerobic granules, and

bio-films (Costerton et al 1987; van Loosdrecht et al 1987; Bruus, Nielsen, and Keiding

1992) It should be pointed out that such a view is still debatable

13.3 CALCIUM ACCUMULATION IN ACETATE-FED

AEROBIC GRANULES

Wang, Li, and Liu (2007) systematically investigated the calcium accumulation in

acetate-fed aerobic granules harvested from a column SBR after 2 months of

opera-tion, while calcium concentration in influent was as low as 4.65 mg L–1 It was found

that acetate-fed aerobic granules had a high calcium content of 225 mg Ca2+mg g–1,

contributing to 37% of granule ash content Compared to acetate-fed aerobic granules,

aerobic granules grown on ethanol showed very low calcium and ash contents

(figure 13.2) This seems to suggest that calcium accumulation is a phenomenon

closely associated with the substrate applied

0 50 100 150 200 250

Acetate Ethanol

0.0 0.1 0.2 0.3

FIGURE 13.2 Calcium and ash contents in ethanol- and acetate-fed aerobic granules (Data

on ethanol from Liu, Yang, and Tay 2003 and on acetate from Wang, Li, and Liu 2007.)

13.4 CHEMICAL FORM OF CALCIUM IN ACETATE-FED

AEROBIC GRANULES

To investigate the chemical form of calcium ion accumulated in acetate-fed aerobic

granules, Wang, Li, and Liu (2007) quantified the elemental composition (Ca, Mg, P,

Fe, Al) of fed aerobic granules The amount of carbonate ion in the

acetate-fed aerobic granules was also analyzed For this purpose, 3 ml of 1 M hydrochloric

acid solution was added to 50 ml of 2 g soluble solids (SS) L–1acetate-fed aerobic

granules, and the carbon dioxide gas produced was online measured by the carbon

dioxide sensor equipped with the respirometer (figure 13.3) Changes in inorganic

carbon in the liquid phase were determined by total organic carbon analyzer before

and after the experiment (Wang, Li, and Liu 2007) Thus, the content of carbonate in

acetate-fed aerobic granules was calculated from the sum of produced carbon dioxide

gas and increased inorganic carbon in the liquid phase Figure 13.4 shows the major

inorganic components of acetate-fed aerobic granules As can be seen, both Ca2+and

CO32- are dominant over the other inorganic components, such as Mg, P, Fe, and

4°C

6 4

5 2

1

3

FIGURE 13.3 Respirometer system for analysis of carbonate in the acetate-fed aerobic

granule: 1 computer for data collection; 2 respirometer; 3 fridge; 4 shaker; 5 acid containing

vial; 6 reaction bottle (From Wang, Z.-W., Li, Y., and Liu, Y 2007 Appl Microbiol Biotechnol

74: 467–473 With permission.)

CO32–

0.0 0.3 0.6 0.9 1.2 1.5 1.8

FIGURE 13.4 Ionic composition of acetate-fed aerobic granules (Data from Wang, Z.-W.,

Li, Y., and Liu, Y 2007 Appl Microbiol Biotechnol 74: 467–473.)

calcium to carbonate was estimated as 1:1.16, indicating that most calcium ions in

aerobic granules exist in the form of calcium carbonate In terms of chemistry, this

also implies that the concentration product of Ca2+and CO32-in acetate-fed aerobic

granules should be larger than the solubility product constant of calcium carbonate

13.5 CALCIUM DISTRIBUTION IN ACETATE-FED

AEROBIC GRANULES

The calcium distribution in acetate-fed aerobic granules was investigated using a

scan-ning electron microscope (SEM); meanwhile, energy dispersive x-ray spectroscopy

(EDX) was also employed for mapping of calcium distribution (Wang, Li, and Liu

2007) The carbonate localization was determined by chemical titration method, that

is, 1 M hydrochloric acid solution was dropped on a sliced granule cross section, and

the origin of bubbles was visualized by image analysis technique (Wang, Li, and Liu

2007) Fresh acetate-fed aerobic granules with a specific oxygen uptake rate (SOUR)

of 64 mg O2g–1volatile solids (VS) h–1; sludge volume index (SVI) of 52 mL g–1, and

of the aerobic granule, while the granule shell was nearly calcium free The image

analysis further showed white deposits localized at 300 µm beneath the granule

sur-(figure 13.5c), gas bubbles were immediately generated sur-(figure 13.5d) The gas phase

analysis confirmed that the bubbles generated were carbon dioxide (figure 13.5d)

These results clearly indicate that both calcium and carbon ions coexist in the same

zone of acetate-fed aerobic granules, that is, calcium exists mainly in the form of

CaCO3in the acetate-fed aerobic granules, which is in good agreement with the

stoichiometric analysis (figure 13.4)

The accumulation of calcium was observed in biofilms and anaerobic granules,

and Ca2+has been often considered to bridge negatively charged sites on extracellular

biopolymers, thus enhancing the matrix stability of attached microbial

communi-ties (Bruus, Nielsen, and Keiding 1992; Korstgens et al 2001; Batstone et al 2002;

Kemner et al 2004; Wloka et al 2004) According to such a hypothesis, excessive

calcium has often been introduced into the medium for enhanced formation of

bio-film and anaerobic granules (Huang and Pinder 1995; Yu, Tay, and Fang 2001)

However, it appears from figures 13.4 and 13.5 that calcium detected in acetate-fed

aerobic granules was mainly in the form of calcium carbonate rather than in

associa-tion with extracellular polymeric substances

13.6 GRANULE SIZE-DEPENDENT CACO 3 FORMATION IN

ACETATE-FED AEROBIC GRANULES

It should be pointed out that the accumulation of calcium in the form of CaCO3in

acetate-fed aerobic granules was found to be granule size-dependent (Wang, Li, and

Liu 2007) As can be seen infigure 13.6, the calcium content of acetate-fed aerobic

granules was proportionally related to the granule size, for example, the calcium

Al, which are indeed marginal According tofigure 13.4, the molar ratio of granule

a mean diameter of 1.4 mm were used for the above-mentioned analyses (figure 13.5)

face (figure 13.5c) After hydrochloric acid was added to the zone of white deposits

Figure 13.5a and b clearly show that calcium was mainly accumulated in the core part

Sludge Radius Range (mm) 0.1-0.2 0.3-0.4 0.5-0.6 0.7-0.8 0.8-1.0 1.4-2.0

0 50 100 150 200 250

FIGURE 13.6 Calcium contents in aerobic granules with different radius (Data from Wang,

Z.-W., Li, Y., and Liu, Y 2007 Appl Microbiol Biotechnol 74: 467–473.)

FIGURE 13.5 (a) Cross-section view of the acetate-fed aerobic granule by SEM; (b) the

EDX mapping for calcium indicated by white color; bar: 100 µm; (c) image analysis

cross-section view of the acetate-fed aerobic granule; (d) generation of gas bubbles during the

acid-granule reaction; scale bar: 200 µm (From Wang, Z.-W., Li, Y., and Liu, Y 2007 Appl

Microbiol Biotechnol 74: 467–473 With permission.)

content in big aerobic granules with radius of 1.4 to 2.0 mm was nearly ten times

higher than that in small aerobic granules with radius of 0.1 to 0.2 mm (figure 13.6)

In the course of aerobic granulation, it was found that the ash content was very low at

the initial stage of aerobic granulation, but it sharply increased on the eighth day in

response to a significant increase in granule size, and gradually stabilized at the level

of about 0.4 g g–1SS after 40 days of operation (figure 13.7) This implies that the

con-tent of CaCO3or so-called ash content was indeed very low in small aerobic granules,

but it tended to increase with the growth in size of acetate-fed aerobic granules

13.7 MECHANISM OF CALCIUM ACCUMULATION IN

ACETATE-FED AEROBIC GRANULES

As discussed earlier, calcium ion is not an essential element necessary for

success-ful aerobic granulation (figure 13.1), and the extensive accumulation of calcium was

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Time (days)

0.0 0.3 0.6 0.9 1.2 1.5

FIGURE 13.7 Ash content and corresponding mean radius of acetate-fed aerobic granules

in the course of aerobic granulation (Data from Wang, Z.-W., Li, Y., and Liu, Y 2007 Appl

Microbiol Biotechnol 74: 467–473.)

only found in aerobic granules grown on acetate (figure 13.2) Furthermore, most

accumulated calcium actually existed in the form of CaCO3, and it was mainly

centralized in the core part of acetate-fed aerobic granules (figures 13.4and13.5)

One necessary condition for CaCO3formation at the low calcium ion concentration

of 4.65 mg Ca2+L–1is the presence of high CO32-concentration at the core of the

acetate-fed aerobic granule so that the ionic product of Ca2+and CO32-can be higher

than the solubility product constant of calcium carbonate

13.7.1 I ONIC E QUILIBRIUM OF C ARBONATE I ON

In terms of chemistry, the CaCO3formation is determined by its ionic

concentra-tion product:

[Ca2 ][CO ] K sp CaCO,

3 2

3

where K sp CaCO, 3is the CaCO3solubility product constant Calcium carbonate will

form only when the concentration product of calcium and carbonate is greater than

K sp CaCO, 3 Acetate can be oxidized in a way such that:

Dissolution of carbon dioxide can be expressed as follows:

CO2H O2 jHHCO3

(13.3) and

CO a1

3 2

and

HCO

2 3

The overall reaction for carbonate can be expressed as:

It should be pointed out that CO2produced in equation 13.2 can be dissolved into

liquid phase according to Henry’s law:

P CO2 K h CO, 2[CO2] (13.8)

where P CO2is the partial pressure of CO2in gas phase, [CO2] is molar concentration

of CO2in the liquid phase, and K h CO, 2is the Henry’s constant for CO2

13.7.2 D IFFUSION K INETICS IN A EROBIC G RANULES

It was assumed in chapter 8 that (1) an aerobic granule is isotopic in physical,

chemical, and biological properties; (2) an aerobic granule is ideally spherical; (3)

no anaerobic reaction occurs in the process; (4) aerobic granule responses to the

change of bulk substrate concentration occur so quickly that the response time can

be ignored As presented in chapter 8, the mass balance equations for a substance

between the two layers in granule whose radiuses are, respectively, r and r + dr can

be written as:

ds

2 2

2

¥

§

´

¶

where D s and R sare, respectively, the diffusion coefficient and mass conversion rate of the

substance According to equation 13.9, Wang, Li, and Liu (2007) proposed the following

mass diffusion balance equations for O2, H+, HCO3-, and CO32-in aerobic granules:

dC

O

O

2

2

2 2

2

¥

§

¦

´

¶

dC

H

H

¥

§

¦

´

¶

µ  2

2

2

(13.11)

D

d C

dC

HCO

HCO

3

3

2 2

2

¥

§

¦

¦

´

¶

µ

D

d C

dC

CO

CO

3

3

2 2

2

¥

§

¦

¦

´

¶

µ

Li and Liu (2005) showed that dissolved oxygen would be a rate-limiting factor

in the growth of aerobic granules, and the oxygen utilization rate can be described

by the Monod equation:

R Y

C

O x

x O

O

2 2

2

R M /

in which Rx is biomass density, Y x O/ 2 is the dissolved oxygen-based growth yield,

K O2is the dissolved oxygen-associated half-rate constant, and µmaxis the maximum

specific growth rate

According to equation 13.2, the oxygen utilization rate and the H+consumption

rate are interrelated by equation 13.15, that is:

R O2 2R H (consumption) (13.15)

Similarly, the following relationship can be obtained from equations 13.3 and

13.7 for H+, HCO3-, and CO32-:

R H(production) R HCO R CO

Thus, the net consumption rate of H+, namely R Hin equation 13.11, is given by

equation 13.17:

R H R H (consumption) R H(production) (13.17)

The dissolved oxygen (DO) concentration at the granule surface can be

reason-ably assumed to be equal to its bulk concentration and its rate of change in the

gran-ule center would be close to zero in consideration of the DO symmetrical distribution

in the granule center (Li and Liu 2005), that is:

dC dr O r

2

0

0

Likewise, C H at the granule surface is assumed to be equal to the bulk H+

con-centration, and the derivative of C H at the center of the granule is zero (Wang, Li,

and Liu 2007):

dC dr H r

 0

Equations 13.10 to 13.13 were solved numerically by Matlab™ 7.0 based on the

finite differentiation principle as described inchapter 8