This document summarizes research on using aerobic granular sludge to treat wastewater from azo dyes. Aerobic granular sludge was cultivated and its ability to simultaneously remove color and degrade aromatic amines through anaerobic and aerobic processes was investigated. The effects of granule size, dissolved oxygen, biomass concentration, and organic loading on treatment performance were evaluated. Under optimized conditions, over 60% equivalent dye removal and 80% removal of color and aromatic amines were achieved within 48 hours. Future work may focus on controlling granule size and applying the process to real textile wastewater.

1. Introduction to Azo Dye Treatment
Aerobic Granular Sludge - A Possible Alternative
Project Objectives and Goals
Methods
Results & Discussion
Summary and Future Work
1
Outline
HKUST BIOENGINEERING GRADUATE PROGRAM

2. 2

3. INTRODUCTION TO AZO DYE TREATMENT – Azo Dyes’ Structures
3
Mordant Orange 1
Coloring Azo Bond
Azo Dyes
Simple synthesis
Good technical
performance
Wide color spectrum
70 % of synthetic
colorants used in the
textile industry

4. INTRODUCTION TO AZO DYE TREATMENT – Azo Dye Pollution
4
Dyeing
Process
Water
UncoloredProducts
Colored
Products
Azo Dyes
Wastewater containing 2-
50% of original azo dyes
Deeply
Colored
Potentially
Carcinogenic
and Mutagenic

5. INTRODUCTION TO AZO DYE TREATMENT – Azo Dyes Wastewater Treatment
AzoDyeWastewater
Treatments
Physicochemical
Methods
Ozonation
Photochemical
Treatments
Sonolysis
Fenton’s Reagent
Oxidation
Adsorption
Chemical Coagulation/
Flocculation with
Sedimentation
Biological Methods
Biosorption
Biodegradation
Advanced Oxidation
Process
Economical and
Environmentally
Friendly Option
Require Expensive
Equipment and
Chemicals
Generate
Secondary Waste
5

6. CO2, H2O, N2 …
Colorless,
Harmless
Aerobic Amine Mineralization
INTRODUCTION TO AZO DYE TREATMENT – Conventional Activated Sludge System
6
Conventional Activated Sludge System
Anaerobic
Tank
Sedimentation Tank
Sludge SedimentationColor Removal Aromatic Amine Removal
Aerobic
Tank
N
N
R1
R2
NH2 R2
R1 NH2
Azo Dye Aromatic Amines
Colored Colorless, Toxic
Chromophore
Anaerobic Decolorization

7. 7

8.  Aerobic Sludge Granulation:
 A process of microbial self-
aggregation, by means of
biological, physical and
chemical phenomena, to
help the community
achieving better survivability
 Microbial cell organized into
dense and fast settling pellets
with diameter 0.2 mm to
around 5 mm
8
AEROBIC GRANULAR SLUDGE – Introduction to Sludge Granulation

9. 9
AEROBIC GRANULAR SLUDGE – Formation Mechanisms
Aerobic sludge granulation
usually occurs in sequencing
batch reactors with air aerated
at a high superficial velocity

10. 10
AEROBIC GRANULAR SLUDGE – Granulation Process
Hydraulic
Shearing
Forces
Flagella
Movements
Gravitational
Forces
Extracellular Polymeric
Substances (EPS)
Chemical Forces
Formation of ionic pairs or triplets
with divalent/ trivalent ions
Physical Forces
van der Waals Forces, opposite
charge attraction
Biochemical Forces
Surface Dehydration,
Membrane Fusion
Step 1
Initiation of bacterium-
bacterium contact by physical
movements
Step 2
Maintaining stable bacterium-
bacterium contact with the
establishment of attractive
forces
Step 3
Maturation of aerobic granule
structures by microbial forces
Step 4
Formation of stable 3-
dimensional structure with
granules shaped by
hydrodynamic shearing forces

11. 11
AEROBIC GRANULAR SLUDGE – Replication Mechanisms
Sludge
growth and
aggregation
Shearing forces
acting on the
granules cause
fragmentation
Internal
decay of
biomass
resulting in
voids and
cavities
Reformation of
stable granule
structure

12. 12
AEROBIC GRANULAR SLUDGE – Anaerobic Zones in Aerobic Granules
Dead Cells
Polysaccharides,
Lipids,
Proteins
Aerobic
Zone
Anaerobic
Zone
Living
Anaerobic
Bacteria
Living
Aerobic
Bacteria
Modified from: Y. Li, Y. Liu, L. Shen and F. Chen, "DO diffusion profile in aerobic granule and its microbiological implications," Enzyme
and Microbial Technology, pp. 349-354, 2008.

13. 13
AEROBIC GRANULAR SLUDGE – Comparing to Conventional Activated Sludge
Conventional
Activated
Sludge
Granular Sludge

14. 14
AEROBIC GRANULAR SLUDGE – Proposed Azo Dye Degradation Mechansim
Living Cells
Living
Anaerobic
Bacteria
Living
Aerobic
Bacteria
Dead Cells
Polysaccharides,
Lipids,
Proteins
Mass
Transfer
Dye
Anaerobic
Decolorization
Aromatic
Amine Aerobic
Aromatic
Amines
Mineralization
Harmless
End Products
Fast Settling
Velocity

15. 15

16. PROJECT OBJECTIVES & GOALS
• To devise a method towards successful cultivation of
aerobic granules for azo dye wastewater treatment;
• To demonstrate the developed granules capabilities
in performing simultaneous anaerobic decolorization
and aerobic aromatic amines mineralization;
• To investigate the effects of different operating
parameters to the process performances, including:
• 1. Sludge granular size
• 2. Saturated bulk dissolved oxygen concentration
(DO)
• 3. Biomass concentration
• 4. Organics loading concentration
16

17. 17

18. METHODS – Schematic Diagram of the Experiment
18
Granules
Characterization
Morphology and
Size
Particle Density
Volatile
Solid/Solid
Dry Mass/Wet
Mass
Sludge Granular
Size
Biomass
Loading
Bulk Dissolved
Oxygen
Organic Loading
Color
Aromatic Amine
COD
Cultivation and
Acclimation of
aerobic granules
Performance
Tests

19. METHODS – Conventional Granulation of Aerobic Sludge
19

20. METHODS – Revised Method of Acclimation and Sludge Granulation
20

21. METHODS – Photo of Aerobic Granules Cultivated in the Project
21

22. METHODS – Granular Size Control
22

23. METHODS – Dissolved Oxygen Control
23

24. METHODS – Verification of the Dissolved Oxygen Control
24
To verify the method of
dissolved oxygen control
 Mixed gases with different
compressed air to nitrogen ratios
were used to maintain a certain
oxygen content (%) in it.
 The dissolved oxygen
concentration of the reactor
content was measured by a ODO
probe (YSI ProODO™)
 For the verifying the method,
dissolved oxygen concentrations
of different oxygen contents in the
mixed gases were also predicted
using the Henry’s Law.

25. METHODS – Color Removal Monitoring by UV-Vis Spectrometry
25
To measure the color
intensity
The absorbance at 371.5nm, which
is the characteristic peak of the
model pollutant Mordant Orange 1,
was measured using a UV-Vis
spectrophotometer

26. METHODS – Aromatic Amines Monitoring by Diazonium Coupling Reaction
26
To measure the aromatic amines
concentration
• Diazonium coupling reaction was
employed to measure the colorless
aromatic amines concentration generated
during the anaerobic decolorization. The
reaction couples the colorless primary
aromatic amines with coupling agent and
form purplish-pink products, which was then
measured using a UV-Vis Spectrometer.
• With different aromatic amines, the
reaction is known to produced products
with slightly different intensities at different
wavelengths of maximum absorbance.

27. METHODS – Diazonium Coupling Reaction Modification
27
To make accurate
measurement of aromatic
amines concentration:
The theoretical reduction products of
mordant orange 1, 4- Nitroaniline (4-NA)
and 5- Aminosalicylic acid (5 –ASA), were
used to produce a calibration curve for
amines monitoring.

28. 28

29. RESULTS & DISCUSSSIONS – Size and Morphology
29
Diameter and shape descriptors of different sizes granules.
Size Category
(a)
0.3 – 1.0 mm
(b)
1.0 – 1.7 mm
(c)
1.7 – 2.4 mm
(d)
> 2.4 mm
Area-averaged Diameter (mm) 0.73±0.22 1.51 ± 0.33 2.42±0.46 4.12±0.93
Aspect Ratio 1.69±0.48 1.49±0.38 1.38±0.27 1.30±0.20
Roundness 0.63±0.15 0.70±0.14 0.75±0.12 0.79±0.10
Size and morphology of
granules
• Granules developed in this study had
area-averaged diameters of 0.3 – 5 mm
• The decreasing aspect ratios and
increasing roundness with increases in
granular size indicate larger granules
were more spherical in shape.

30. RESULTS & DISCUSSSIONS – Physical Properties
30
Physical properties of the
cultivated aerobic
granules
• By Stokes Law, V ∝ 𝜌 and d2
• Where V= settling velocity of
a spherical particle;
• 𝜌 and d = the particle
density and particle
diameter;
• The particle density of aerobic
granules (1.01 to 1.04 g/cm3) is
similar to the density of activated
sludge ((1.01-1.06 g/cm3)
• The improved settling
velocity is due to the
increase in particle diameter
instead of the improvement
of particle density

31. 31

32. RESULTS & DISCUSSSIONS – COD Removal
32
% of COD removal of
different size granules under
different DO
• DO ↑ , % of COD removal ↑
• Activation of oxygen dependent
metabolic pathway that is more
energetically efficient.
• Granular size ↑ , No significant impact
to the % of COD removal in 48 hours,
but with ↓ COD removal rate.
• Smaller mass transfer resistance
in smaller granules
COD removal kinetics of different sizes granules under a dissolved
oxygen of 1 ppm

33. RESULTS & DISCUSSSIONS – General Trends of Dye Removal Kinetics
33
General Trends of Dye
Removal Kinetics
• Dye Equiv. ppm =Dye conc. +
Aromatic amines
D
Where D = dye content of the
commercial dye
• Dye equiv. is a collective
measurement of the total amount of
dye related compound in the
wastewater
• Sudden drop of dye and dye equiv.
conc. (120 to 100 ppm) immediately
after aerobic granules added
• Initial rapid biosorption
• Continuous decolorization with
aromatic amines generation
• Reductive decolorization
• Aromatic amines concentration built
up faster at the beginning, but
dropped later in the experiment
• Simultaneous production and
consumption of aromatic amines
Dye remediation kinetics using different sizes granules under different
dissolved oxygen levels (▲ represents the Dye Equiv. concentration;
 represents the color;  represents the aromatic amines
concentration)
Dye removal kinetics of granules in the size of 1.7 – 2.4 mm
under a DO conc of 1 ppm
Sudden drop

34. RESULTS & DISCUSSSIONS – Color Removal
34
% of Color removal of
different size granules under
different DO
• DO↑, % of color removal ↓
• Reductive decolorization is
favored by the –ve redox
potential provided by anaerobic
condition
• Oxygen is better e- acceptor
than azo bond
• Granular size ↑, % of color removal ↑
• Better synergy in more mature,
larger granules
• Granular size ↑, % of color removal is
less sensitive to the change in DO
concentration
• Smaller change in the anaerobic
region to aerobic region ratio

35. RESULTS & DISCUSSSIONS – Color Removal (cont.)
35
Anaerobic zone in aerobic
granules
• The thickness of aerobic layer on
aerobic granules is independent to
the sludge granular size (Li et. al.
(2008))
• Larger granules have larger portions of
anaerobic zone in a high DO conc.;
• With the change of the bulk DO conc.,
smaller granules may change from
completely anaerobic to completely
aerobic, while larger granules might
only change to partially aerobic
• Larger granules have a more
stable ratio of anaerobic zone to
aerobic zone

36. RESULTS & DISCUSSSIONS – Aromatic Amine Removal
36
% of Aromatic amines removal
of different size granules under
different DO
• Granular size ↑, % of aromatic amines
removal is less sensitive to the change in
DO concentration
• Smaller change in the anaerobic
region to aerobic region ratio
• DO ↑, % of aromatic amines removal ↑
• Oxygen is required for the
destruction of aromatic structures
by the enzymes hydroxylase and
oxygenase;
• In low DO, larger granules showed better
aromatic amines removal;
• Better synergy in the bacterial
community;
• In high DO, smaller granules showed
better aromatic amines removal;
• Smaller granules have larger portion
of aerobic region in high DO

37. RESULTS & DISCUSSSIONS – Equivalent Dye Removal
37
% of Equivalent dye removal
of different size granules
under different DO
• DO ↑, Decolorization rate ↓ and
aromatic amines mineralization rate ↑;
• In low DO, the dye removal is limited
by the aromatic amines mineralization;
In high DO, the dye removal is limited
by the decolorization;
• To achieve an optimized dye removal,
the decolorization rate and aromatic
amines mineralization rate have to be
balanced.

38. 38

39. RESULTS & DISCUSSSIONS – Effect of Biomass Concentration
39
Effect of biomass
concentration
• Biomass conc. ↑, process
performances ↑;
• From 1.25 g/L to 5 g/L, significant
improvements was observed in all
parameters
• More degradation enzymes can
be produced with higher
biomass concentration.
• Similar % of pollutant removal after
5g/L
• The process may be limited by
other factors, including the trace
element concentration in the
wastewater

40. RESULTS & DISCUSSSIONS – Equivalent Dye Removal
40
Effect of organic loading
concentration
• Organic loading played two roles on
the bacterial dye removal, which are:
• (a) Carbon, nitrogen and energy
source;
• (b) Electron donors for the
reductive decolorization.
• Organic loading conc. ↑, % of color
removal ↑, % of aromatic amines
removal ↓;
• More e- generated for reductive
decolorization;
• Preferential utilization of organic
loadings with simpler structures,
instead of aromatic amines
• The optimized organic loading
concentration was determined to be
4000 ppm, as which balance the
organic loading effect on color and
aromatic amines removal.

41. 41

42. Conclusions
42
1. An integrated acclimation and granulation scheme was devised to cultivate aerobic
granules for simultaneous anaerobic decolorization/ aerobic aromatic amines mineralization
with its performances optimized by varying sludge granular size, bulk dissolved oxygen
concentration, biomass concentration and organics loading concentration.
2. A good equivalent dye mineralization (61 ± 2%), decolorization (88 ± 1%), aromatic amines
removal (70 ± 3%) and COD removal (88 ± 2%)within 48 hours reaction was obtained by
using 5 g/L, 1.0 mm – 1.7 mm granules under a bulk dissolved oxygen concentration of 1
ppm, supplemented with 4000 ppm organic loadings from nutrients.

43. Future Work
43
1. Although many factors may affect the size of granules cultivated in the reactor, no
technique is currently available to control granular size. The design of sludge granular size
controlling method may be important towards a better performances of aerobic sludge
granulation system using in azo dye wastewater treatment.
2. Only a single model pollutant, Mordant Orange 1, was used in this study. In real life textile
wastewater, other constituents, including surfactants, may exist and damage the
developed granulation system. A further investigation of using the developed system for
treating real textile wastewater may also be necessary.
3. The techniques of sludge granulation may not only applicable in wastewater treatment, but
also in mass production of other bacterial metabolite. The faster settling velocity, higher
biomass concentration and easier separation of the biomass from the liquid content may
lead to the development of production process with more efficient conversion and easier
downstream processes.

44. 44

45. Acknowledgement
45
Supervisor
Prof. Ka Ming NG
Thesis examination committee
Prof. Guohua CHEN and Prof. Henry LAM
Thesis supervision committee
Prof. Xi Jun HU and Prof. David HUI
Wastewater treatment team
Dr. Kelvin FUNG, Dr. Judy Zhang, Ms. Pinky Sin
Technical Staff in CBME
Mr. Hoi Yau CHENG, Mr. Wing Li LEUNG, Mr. Kam Tim TANG
Technical and administrative Staff in BIEN
Ms. Inez TSUI, Ms Zoei CHU, Ms Winnie LEUNG
Labmates and Friends
My family members

46. 46

47. METHODS – Selective Pressure Theory
47
By Stokes Law:
The free settling velocity of a
spherical sediment is given by:
V =
𝐷 𝑝
2
𝑔 ρ 𝑝 − ρ
18𝜇
=
𝐿
𝑇𝑠
Where V = the settling velocity of the
sediment
𝑇𝑠= the allowed Settling Time
L = the liquid level above the
disposal port
i.e. Volumetric Exchange Ratio =
1
4
𝜋𝐷2 𝐿
1
4
𝜋𝐷2 𝐻
=
𝐿
𝐻
Dp = the diameter of the sediment
particle
𝜌 𝑝 = the density of the sediment
𝜌 = the density of the settling medium
𝜇 = the viscosity of the settling
Medium
• Settling time and volumetric exchange ratio were
known to be influential to the granule formation and
thus be regarded as the selective pressures of the
aerobic sludge granulation,
• As short settling time and high volume exchange
ratio will result in a larger wash out of reactor
content, such conditions were also known as with
high selective pressures