Chemosphere 61 (2005) 10321041

www.elsevier.com/locate/chemosphere

Microbial activity in a combined UASBactivated

sludge reactor system
J.S. Huang
a

a,*

, C.S. Wu b, C.M. Chen

Department of Environmental Engineering, Kun Shan University of Technology, Tainan County 710, Taiwan, ROC
b
Department of Resource and Environment, Leader University, Tainan City 709, Taiwan, ROC
c
Department of Environmental Engineering, National Cheng Kung University, Tainan City 701, Taiwan, ROC
Received 29 August 2004; received in revised form 3 March 2005; accepted 7 March 2005
Available online 26 April 2005

Abstract
A combined upow anaerobic sludge bedactivated sludge (UASBAS) reactor system with consistently wasting of
excess biomass was used to treat suspended-solids pre-settled piggery wastewater (COD = 2000 mg l
1, total Kjeldahl
nitrogen TKN = 400 mg l
1, suspended solids = 250400 mg l
1). Thus, the activity of nitrogen-related microbial
groups in each individual bioreactor was investigated. When the granules retention time (GRT) of 2050 d in the UASB
reactor, the solids retention time (SRT) of 1025 d in the AS reactor and the recycle-to-inuent ratio (Re) of 1 were
maintained, the combined system removed 9597% of chemical oxygen demand (COD), 100% of TKN and 5455%
of total nitrogen (TN). Denitrication and methanogenesis occurred in the UASB reactor so that both biochemical processes contributed to most of the COD removal and, complete nitrication (most of the TKN removal) occurred in the
AS reactor. Compact granules with good settling abilities developed in the UASB reactor, and rapid rates of granulation of break-up granules in the UASB reactor were conrmed by experiments. The activity of nitriers and denitriers
(an = 0.680.87; adn = 0.550.70) and the calculated specic nitrication and denitrication rates (qn = 0.26

1
1

1
1
0.47 mg NH
d ; qdn = 0.0460.076 mg NO

d ) signicantly increased with decreasing
4 N mg VSS
x N mg VSS
SRT and GRT, respectively. Accordingly, the combined UASBAS reactor system should be regarded a promising
alternative for the removal of organic carbon and nitrogen from piggery wastewater.

2005 Elsevier Ltd. All rights reserved.
Keywords: Combined system; UASB; Activated sludge; Microbial activity; Piggery wastewater

1. Introduction
A separate-sludge system consisting of an anaerobic
denitrifying/methanogenic reactor and an additional
aerobic nitrifying reactor has been proven ecient in

*
Corresponding author. Tel.: +886 6 2051331; fax: +886 6
2050540.
E-mail address: huangjsd@mail.ncku.edu.tw (J.S. Huang).

removing organic carbon and nitrogen from wastewater

(Borzacconi et al., 1999; Bernet et al., 2000). Borzacconi
et al. (1999) demonstrated that a denitrifying/methanogenic UASB reactor, combined with a nitrifying rotating
biological contactor with euent recycle, could successfully remove organic carbon and nitrogen from sanitary
landll leachate. Bernet et al. (2000) found that a combined anaerobicaerobic system (two sequencing batch
reactors with euent recycle) could eectively remove
organic carbon and nitrogen from piggery wastewater.

0045-6535/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2005.03.027

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

In the separate-sludge system, appropriate conditions

can be purposely established in each individual bioreactor to favor denitrication/methanogenesis and
nitrication (Chen and Lin, 1993; Lin and Chen, 1995;
Hendriksen and Ahring, 1996; Borzacconi et al., 1999;
Bernet et al., 2000). Accordingly, denitrication/methanogenesis proceeds in the anaerobic reactor to utilize


the inow organics and the recycled NO

3 and NO2
from the aerobic reactor and, nitrication proceeds in
the aerobic reactor at a low concentration level of
organic materials.
Although the mechanism of granulation in upow
anaerobic sludge bed (UASB) reactors is not well understood, some inuencing factors, including wastewater
constituents (Grotenhuis et al., 1991), growth environments (Schimidt and Ahring, 1996), and supercial velocity (us) (Tay and Yang, 1996; Huang et al., 2003; Jih et al.,
2003) have been claimed. In recent studies, granulation in
UASB denitrifying reactors has been observed (Borzacconi et al., 1999; Rouse et al., 1999; Bhatti et al., 2001;
Eiroa et al., 2004). Bhatti et al. (2001) found that the
UASB reactor (with periodically wasting of excess biomass granules) could successfully remove nitrate from
soft ground water. Nonetheless, attempts to carry out
denitrication and/or methanogenesis in UASB reactors
with consistently wasting of excess biomass granules were
not found in the literature. Such an operation mode for
UASB reactors may be regarded one of the measures that
can be taken to improve the activity of microbial groups
in UASB reactors; other measures include the elevation
of operating temperature (Lin and Yang, 1991; Chou
et al., 2004) and the maintenance of favorable growth
environments (Grady et al., 1999). If a UASB reactor
with wasting of biomass granules is to be adopted, a
sludge granulation experiment should be conducted to
ensure granulation in the UASB reactor is rapid enough.
In this work, a laboratory-scale combined UASB
activated sludge (AS) reactor system with consistently
wasting of excess biomass granules was used to treat suspended-solids pre-settled piggery wastewater. Thus, the
activity of nitrogen-related microbial groups in each
individual bioreactor was investigated. The biogas contents and biogas production rate over the UASB reactor
and the alkalinity change over the AS reactor were monitored. The batch reactors, which were provided with
growth environments similar to the AS and UASB reactors, were used to study nitrication and denitrication
kinetics, respectively. A reliable method was also used
to estimate the variations in the activity of nitriers
and denitriers with dierent solids retention times
(SRTs) and granules retention times (GRTs) of the AS
and UASB reactors, respectively. Moreover in this
article, the specic nitrication and denitrication rates
together with relative percentage enhancement of microbial activity in each individual bioreactor are presented
as well.

1033

2. Materials and methods

2.1. Piggery wastewater
Liquid wastewater generated in pig farms in Taiwan
generally contained fairly high concentrations of suspended solids (20003000 mg SS l
1). Thus, it has been
dicult to evaluate the performance and associated
nitrication and denitrication kinetics of biological
processes on suspended solids removal. In engineering
practice, piggery wastewater is suggested to remove
most of suspended solids by using the dissolved air otation process prior to biological treatment. Accordingly, piggery wastewater after being shipped to the
laboratory was rst sieved through 1 mm diameter mesh
and then placed in a cooling room (4 1 C) for 6 h to
settle most of the suspended solids. Thereafter, the
supernatant was stored in the cooling room before use.
After settling, the chemical oxygen demand (COD)
and total Kjeldahl nitrogen (TKN) of the supernatant
were 16002700 and 320416 mg l
1, respectively. To
maintain stable inuent water quality (COD =
2000 mg l
1, TKN = 400 mg l
1), piggery wastewater
used in this work was either diluted with tap water or
supplemented with methanol and ammonium chloride.
Other wastewater characteristics included pH (7.57.6),
alkalinity (12501550 mg CaCO3 l
1), organic nitrogen
(4080 mg l
1), total phosphorus (90130 mg l
1), and
suspended solids (250400 mg l
1). The substrate-feed
tank was placed in a water bath at 8 2 C.
2.2. Experimental system
A schematic diagram of the combined UASBAS
reactor system is presented in Fig. 1.
2.2.1. UASB reactor
The UASB reactor, with dimensions of 6.0 (length)
6.0 (width) 105 cm (height) and six-equal distance
sampling ports along the reactor height, had an active
liquid volume of 3.78 l. The dimensions of the gas
liquidsolids separator of the UASB reactor were
6.0 (length) 6.0 (width) 18 cm (height). The UASB
reactor was seeded with 2.0 l of anaerobic sludge, which
was obtained from the anaerobic reactors of a local piggery wastewater treatment plant and a laboratory single
sludge (nitricationdenitrication) system. The temperature was maintained at 30 1 C by circulating hot
water in the outer-water jacket.
2.2.2. AS reactor
The AS reactor consisted of an aeration tank and a settler with active liquid volumes of 6 and 6 l, respectively.
The AS reactor was seeded with aerobic sludge, which
was obtained from the activated sludge reactors of a local
piggery wastewater treatment plant and a laboratory

1034

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

1. Thermistor
2. UASB reactor

Biogas

3. Outer-water jacket
4. Gas-liquid-solids separator
5. Internal recycle

6. Sampling port
7. Excess biomass
8. Activated sludge reactor
9. Automatic heating rod
10. Settler
11. Sludge return

3 2 3

12. Effluent holding tank

13. Effluent recycle

7
9
5

5
10

Effluent

8
6

12

7
11
Thermometer

1
13
Influent

Fig. 1. Schematic diagram of the combined UASBAS reactor system.

single sludge (nitricationdenitrication) system. To

maintain the dissolved oxygen (DO) concentrations at
34 mg l
1 and the temperature at 30 1 C, the AS reactor was thoroughly aerated with a diuser and installed
with a completely submersible automatic heating rod
(Visi-Therm, Aquarium System, Italy), respectively.
2.3. Combined system operations
To ensure acclimatization of anaerobic and aerobic
seed sludge in each individual bioreactor, the combined
UASBAS system was continuously fed with piggery
wastewater with an inow rate of 6 l d
1, a recycle-toinuent ratio (Re) of 1, and the COD and TKN concentrations of 500 and 100 mg l
1 for one month. Then the
combined system was gradually fed with piggery wastewater with increasing COD and TKN concentrations
shown in run 1 (Table 1) for two more months. To begin
granulation of the sludge, the us of the UASB reactor
was gradually increased from 0.069 to 2.0 m h
1 by controlling the inow rate of 6 l d
1 and the step-wise
increasing internal-recycling ow rate. Granulation in

the UASB reactor was allowed to proceed for two

months. The GRT of the UASB reactor was controlled
by wasting a designated amount of biomass granules
(= sludge-bed volume/GRT) daily from the lower-, middle-, and upper-part of the sludge-bed zone. Each of a
total of four test runs of the combined system was conducted (Table 1) and continuously operated for at least


three months. The COD, TKN, NO

2 N, and NO3 N
concentrations in the euent of each individual bioreactor were monitored. Steady state was assumed to be
reached after test results of the four chemical parameters
were within 10% deviation for four consecutive samples
(sampling twice weekly).
2.4. Determination of granule size
When the combined system reached steady state,
approximately 510 ml of biomass granules were randomly removed from the lower-, middle-, and upperpart of the sludge-bed zone of the UASB reactor. Then
the granule diameter was determined using the method
of image analysis (Chou et al., 2004).

Inuent COD = 2000 mg l
1; inuent TKN = 400 mg l
1; inuent alkalinity in test runs 1, 2, 3, and 4 were 1250, 1250, 1450, and 1550 mg CaCO3 l
1, respectively. The inow rate
(Q), recycle-to-inuent ratio (Re), and hydraulic retention times of the UASB and AS reactors were maintained at 6 l d
1, 1, 15.1 h, and 24.0 h, respectively.
a
Expressed in mg CaCO3 l
1.

200
190
205
330
183
182
180
184
183
182
180
183
ND
ND
0.2
0.6
0.1
0.1
0.2
0.2
58
65
85
102
1420
1350
1550
1680
187
190
184
188
ND
ND
ND
ND
ND
ND
ND
ND
187
190
184
188
180
182
205
254
12.5
10.7
8.2
7.1
43.3
37.6
31.6
26.5
2080
1790
1370
1180
15 440
12 900
11 100
9200
25
20
15
10
50
40
25
20
1
2
3
4

TKN
COD
NO

2 N
TKN
COD
AS

AS
UASB
UASB

UASB

AS

UASB-euent
(mg l
1)
Biomass
(g)
Xi (weighted mean)
(mg VSS l
1)
GRT (SRT)
(d)
Test run

Table 1
Operating conditions and treatment performance of the combined UASBAS reactor system

NO

3 N

TN

Alka

AS-euent
(mg l
1)

NO

2 N

NO

3 N

TN

Alka

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

1035

2.5. Determination of biokinetic constants

Prior to determining the biokinetic constant of nitriers (kn), the enrichment of nitriers in a 5-liter batch
reactor proceeded. First, biomass removed from the
AS reactor was loaded into the batch reactor. Then
the batch reactor was added into 1.19 g NH4Cl,
1 g KH2PO4, 8 g NaHCO3, and adequate amounts of
trace metals (together with tap water to make an active
liquid volume of 5 l) twice a day, and maintained at the
DO concentrations of 34 mg l
1. To keep an SRT of
the batch reactor at 25 d (i.e., close to the SRT of the
AS reactor in run 1), 0.2 l of mixed liquor was wasted
daily prior to decanting of the supernatant. The above
procedures were repeated for three months.
To determine kn, an adequate amount of enrichment
culture (nitriers) was taken from the previous batch
reactor and loaded into a 1-liter batch reactor. The
batch reactor was then added into 0.38 g NH4Cl,
0.2 g KH2PO4, 1.6 g NaHCO3, and adequate amounts
of trace metals, and maintained at the DO concentration
of 34 mg l
1. In a mixing state, the initial biomass concentration in the batch reactor was measured, while the
NH
4 N concentration remaining in the solution was
measured for every 20 min. Thereafter, linear regression
was applied to search for kn which would t in experimental data.
Prior to determining the biokinetic constant of denitriers (kdn), the enrichment of denitriers in a 5-liter
batch reactor proceeded. First, biomass granules (removed from the UASB reactor) together with a few glass
beads were loaded into a 500-ml serum vial, followed by
placing the serum vial on a shaker (30 1 C; 80 rpm)
for two hours to break up biomass granules. The size
distribution of the obtained dispersed sludge was determined using a laser particle size distribution analyzer
(Horiba, model LA-920, Japan). More than 86% of
the particles were found within the size range of 0.26
200 lm. Thereafter, the dispersed sludge was loaded into
the 5-liter batch reactor installed with a mixing device
(approximately 80 rpm) and equipped with a cover but
provided with a gas vent to strip o oxygen using nitrogen gas. The batch reactor was added into 7 g KNO3
and piggery wastewater (approximately 700 mg COD)
twice a day. To maintain an SRT of the batch reactor
at 50 d (i.e., close to the GRT of the UASB reactor in
run 1), 0.1 l of mixed liquor was wasted daily prior to
decanting of the supernatant. The above procedures
were repeated for ve months and biogas was monitored
to ensure that no methane production occurred in the
batch reactor.
To determine kdn, an adequate amount of enrichment
culture (denitriers) was taken from the previous batch
reactor and loaded into a 500-ml serum vial. The serum
vial was then placed on a shaker (30 1 C; 80 rpm) and
added into 0.3 g KNO3 and piggery wastewater to make

1036

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

an active liquid volume of 500 ml. In an agitation state,

the initial biomass concentration in the serum vial was


measured, while the NO

3 N and NO2 N concentrations remaining in the solution were measured for every
30 min. Thereafter, linear regression was applied to
search for kdn.
2.6. Determination of activity of nitrogen-related
microbial groups
When the combined system reached steady state, biomass was removed from the AS reactor and loaded into
a 1-liter batch reactor. The batch reactor was thoroughly
aerated with a diuser and added into the euent of the
UASB reactor. At a temperature of 30 C and in an agitation state, the initial biomass concentration (XB,i) in
the batch reactor was measured, while the NH
4 N
concentration remaining in the solution was measured
for every 30 min. Thereafter, the initial rate method
(Jih et al., 2003; Chou et al., 2004) was used to determine
the mass fraction of nitriers (fn) in the AS reactor
in run 1 (SRT = 25 d) and the activity of nitriers
(an) in the AS reactor in runs 14, as shown in Eqs.
(1)(3):
k 0i

S B;
1
S B;1
2X B;i Dt

fi

k 0i
ki

for run 1; SRT 25 d and GRT 50 d

2

ai

k 0i
ki

for runs 14

where i denotes n and dn; ki and k 0i represent the maxi

mum specic NH
4 N oxidation (NOx N reduction)
rate of the enrichment- and mixed-culture, respectively;
SB,
1, SB,0 and SB,+1 represent the bulk concentration


of NH
4 NNOx N in the initial three consecutive samples; XB,i represents biomass concentration in the batch
reactor; and Dt represents the time interval.
Similarly, biomass granules were removed from the
UASB reactor and loaded into a 250-ml serum vial, followed by placing the serum vial on a shaker (30 1 C;
80 rpm) for 2 h to break up granules. The serum vial was
added into a mixture of piggery wastewater and the
euent of the AS reactor with a proportion just the
same as the recycle-to-inuent ratio (Re) of 1. In an agitation state, the initial biomass concentration (XB,i) in
the serum vial was measured, while the NO

3 N and
NO

2 N concentrations remaining in the solution were
measured for every 20 min. Thereafter, the initial rate
method was used to determine the mass fraction of denitriers (fdn) in the UASB reactor in run 1 (GRT = 50 d)
and the activity of denitriers (adn) in the UASB reactor
in runs 14, as shown in Eqs. (1)(3).

2.7. Chemical analyses

Nitrate and nitrite were determined using the chromotropic acid method and the colorimetric method, respectively (APHA, 1995). The close reux, titrimetric method
(APHA, 1995) was used to determine COD. N2 and CH4
were analyzed using a Shimadzu gas chromatographic
column system with a 150 cm 3 mm stainless-steel column (packed with 45/60 Carboxen 1000), a thermal conductivity detector, and helium carrier gas. Injector,
column, and detector temperatures were 130, 130, and
150 C, respectively. Biomass (expressed in VSS), alkalinity, TKN, and NH
4 N were measured according to
Standard Methods (APHA, 1995).

3. Results and discussion

3.1. Performance of combined UASBAS
reactor system
The operating conditions and performance data of
the combined UASBAS reactor system treating piggery
wastewater are presented in Tables 1 and 2. As shown in
Table 1, when the inuent TKN and COD concentrations of 400 and 2000 mg l
1, respectively, the hydraulic
retention time (HRT) of 15.1 h and the GRT of 2050 d
in the UASB reactor, the HRT of 24.0 h and the SRT of
1025 d in the AS reactor, and the Re of 1 were maintained, biomass measured in the UASB and AS reactors
decreased (43.326.5 g; 12.57.1 g) with decreasing GRT
and SRT, respectively.
As shown in Table 2, the COD, TKN, and TN removal eciencies of the combined system varied slightly
(4597%; 100%; 5455%, respectively) with dierent
GRTs and SRTs. According to material-balance calculations using the performance data over the combined system, the removal of COD primarily occurred in the
UASB reactor (8085%); the removal of TKN mainly
took place the AS reactor (9295%); and the removal
of TN primarily occurred in the UASB reactor (5153%).
Although the inuent COD/TKN ratio of as high as
5.0 was maintained, complete nitrication occurred in
the AS reactor at low COD bulk concentrations of 58
102 mg l
1 (Table 1); nitrate was generally the main
product of nitrication (Table 1). This was primarily because synchronous complete denitrication (utilizing the


recycled NO

3 and NO2 as electron acceptor; Table 1)
and methanogenesis occurred in the UASB reactor,
resulting in the utilization of most of inow organics.
A relative lower TN removal eciency (compared with
TKN removal eciency) found in this work was because
in the combined system the AS reactor was placed after


the UASB reactor. Thus, a part of NO

3 and NO2 generated in the AS reactor was discharged from the combined system. Accordingly, the combined UASBAS

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

1037

Table 2
COD, TKN and TN removal eciencies of each individual bioreactor and the combined UASBAS reactor system
Test run

Removal (%)
COD

1
2
3
4

TKN

TN

UASB

AS

Combined system

UASB

AS

Combined system

UASB

AS

Combined system

85
85
84
80

12
12
12
15

97
97
96
95

7
5
8
6

93
95
92
94

100
100
100
100

52
51
53
52

2
4
2
2

54
55
55
54

reactor system was proven to be an eective alternative

for the removal of organic carbon and nitrogen from
piggery wastewater.
3.2. Granule characteristics in UASB reactor
As shown in Table 3, a shorter GRT resulted in smaller
granule size (dp) and lower biomass concentration (Xi)
(weighted mean) in the UASB reactor. Even at the
shortest GRT of 20 d, granulation of sludge (i.e., good settling properties) was also observed in the UASB reactor,
and the measured dp and Xi were 1.05 mm and
9200 mg VSS l
1 (weighted mean), respectively. At a
GRT range of 20 to 50 d, the average dp and Xi in the
lower-part of the sludge-bed zone was the largest (1.06
1.90 mm; 985019 040 mg VSS l
1), compared with the
average dp and Xi in the middle-part of the sludge-bed
zone (1.031.63 mm; 948014860 mg VSS l
1) and the
average dp and Xi in the upper-part of the sludge-bed zone
(1.061.13 mm; 825011 720 mg VSS l
1). This is a typical case of the distribution of granule size and biomass
along the sludge-bed height (H) of UASB reactors (Grotenhuis et al., 1991; Jih et al., 2003; Chou and Huang,
2005). The specic gravity (sp gr) and microbial density
(Xf) of the granule in the lower-part of the sludge-bed zone
(1.061.07; 101 500133 200 mg VSS l
1) tended to be
greater than those in the middle-part and upper-part of
the sludge-bed zone (1.021.06; 83 900109 500 mg
VSS l
1). The sp gr of granules primarily depends on the
occurrence of inorganic precipitation (Schimidt and Ahring, 1996). Typical sp gr of granules in UASB reactors ranged from 1.03 to 1.08 (Schimidt and Ahring, 1996; Jih
et al., 2003). According to calculations, sludge-bed porosity [e = 1
(Xi/Xf)] ranged from 0.85 to 0.92 (weighted
mean). Accordingly, compact granules could develop in
the UASB reactor with fairly large sludge-bed porosity.
Moreover, a granulation experiment was conducted
after all the analytical work required for run 1 had been
completed. First, all the biomass granules (i.e., granule
sizes ranged from 0.25 to 2.0 mm) were removed from
the UASB reactor and loaded into four 1-liter serum
vials. The serum vials were then placed on a shaker
(30 1 C; 80 rpm) for over 6 h to break up biomass
granules to the sizes ranging from 0.25 to 0.75 mm.

Thereafter, the break-up granules were re-loaded into

the UASB reactor to proceed with the granulation
experiment. As shown in Fig. 2, the average dp (surface-area basis) of the break-up granules in the upperpart, middle-part, and lower-part of the sludge-bed zone
restored to close to the average dp of the original granules approximately at the sixth-, ninth-, and tenth-day,
respectively. From an engineering perspective, the
UASB reactor having an advantage of rapid granulation
rates (i.e., time-saving for the start-up of the bioreactor)
should be regarded a promising alternative in treating
piggery wastewater.
3.3. Biogas production in UASB reactor
Biogas production and percent contents of N2 and
CH4 in the UASB reactor were measured. At a GRT
range of 2050 d, the biogas produced was mainly composed of N2 (55.860.1%; mean = 57.4%) and CH4
(37.541.6%; mean = 39.7%). The monitored biogas
production rate ranged from 1.41 to 1.55 l d
1 (mean =
1.51 l d
1). The abundance of nitrogen gas generated in
the UASB reactor suggested that denitrication was the
main biological process occurred in this reactor. The
presence of methane in the biogas also indicated that
methanogenesis occurred with denitrication so that
both processes contributed to the removal of organic
carbon (COD) from piggery wastewater.
From the stoichiometry of nitrate reduction 15 NO

3

6


1
3
H

e
!
N

H
O,
the
consumption
of
one
2
2
5
10
5
mole of NO

3 N generates 0.5 mole of nitrogen gas. By
using material-balance calculations together with
NO

3 N data over the UASB reactor (Table 1), the calculated N2 production rates ranged from 0.94 to 0.96 l d
1
(mean = 0.95), which showed fairly good agreement with
the experimental data (0.850.88 l d
1). This suggested
that the biogas (N2) data could be used for monitoring
and control of the combined UASBAS reactor system.
3.4. Alkalinity change in AS reactor
Nitrication in the AS reactor is accompanied by the
consumption of alkalinity. According to the stoichiometry of ammonia nitrication, 1 g of NH
4 N consumes

1.06
1.05
1.04
1.04
1.04
1.04
1.03
1.02
78
81
79
80
1
2
3
4

1.13
1.08
1.07
1.06

1.57
1.35
1.34
1.05

1.07
1.07
1.06
1.06

1.06
1.05
1.04
1.04
1.63
1.49
1.48
1.03
1.90
1.51
1.47
1.06

Weighted
mean
Upper

Granule diameter (mm)

106 400
96 800
95 000
111 300
83 900
90 600
86 200
108 700
96 300
96 100
96 700
109 500
14 860
11 340
11 600
9480
19 040
16 220
13 200
9850

11 720
10 640
8300
8250

15 440
12 900
11 100
9200

133 200
103 300
101 500
115 000

Weighted
mean
Upper
Middle
Lower
Lower
Middle
Lower

Middle

Granule sp gr ()

H
(cm)
Test
run

Table 3
Granule characteristics of UASB reactor

2.0

1.5

1.0
During granulation
Upper
Middle
Lower

Original granules
Upper
Middle
Lower

0.5

0.0
0

10

12

Time (d)

Fig. 2. Time course of granulation of break-up granules along

the sludge-bed height of UASB reactor.

7.14 g of alkalinity (expressed in CaCO3). Thus, materialbalance calculations together with TKN data over the AS
reactor (Table 1) were used to calculate the change in
alkalinity and compared with the measured values. The
ratios of DAlkcal/DAlkexp over the AS reactor ranged
from 0.94 to 1.14 (mean = 1.04), showing good agreement
between the predicted and measured values. This suggests
that the alkalinity data could be used for monitoring and
control of the combined UASBAS reactor system.
3.5. Evaluation of kinetic parameters

dp (mm)

Upper

Weighted
mean

Xi (mg VSS l
1)

Upper

Weighted
mean

Lower

Middle

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

Xf (mg VSS l
1)

1038

Ammonia nitrication in the batch reactor (with

enrichment culture originally taken from the AS reactor
and initial biomass and NH
4 N concentrations of
1430 mg VSS l
1 and 97 mg l
1, respectively) followed
zero-order kinetics. By performing linear regression of
the experimental data, the estimated kn value was

1
1
0.71 mg NH
d . Typical values of kn
4 N mg VSS
reported in the literature were 0.701.15 mg NH
4 N
mg VSS
1 d
1 (Shieh and LaMotta, 1979; Neufeld
et al., 1980; Dincer and Kargi, 2000).
Denitrication of NO

x (i.e., piggery wastewater was
added to supply carbon source) in the batch reactor
(with enrichment culture originally taken from the
UASB reactor and initial biomass and NO

3 N concentrations of 1200 mg VSS l
1 and 79 mg l
1, respectively)
followed zero-order kinetics. By performing linear
regression of the experimental data, the estimated kdn

1
1
value was 0.33 mg NO

d . The estix N mg VSS
mated kdn value in the present study was close to that
of Dincer and Kargi (2000) who utilized glucose as

1
1
carbon source (kdn = 0.23 mg NO

d ).
x N mg VSS
3.6. Activity of nitrogen-related microbial groups
When the combined system reached steady state, biomass was removed from the AS reactor and loaded into
batch reactors to determine the mass fraction of nitriers

0.26
0.31
0.40
0.47
0
+16
+25
+28
0.68
0.79
0.85
0.87
60
55
117
108

RPE (relative percentage enhancement) was calculated by selecting the activity of nitriers (an) and specic nitrication rate (qn) determined at the SRT of 25 d as reference value.
Calculated by using the mass fraction of nitriers (fn) of 0.68 together with the performance data of the AS reactor.
c
RPE (relative percentage enhancement) was calculated by selecting the activity on denitriers (adn) and specic denitrication rate (qdn) determined at the GRT of 50 d as
reference value.
d
Calculated by using the mass fraction of denitriers (fdn) of 0.55 together with the performance data of the UASB reactor.

0
+16
+22
+27
0.55
0.64
0.67
0.70
60
57
81
57
0
+19
+54
+81

1790
2530
3250
3290

Initial NO

3 N
(mg l
1)
Biomass conc.
(mg VSS l
1)
Initial NH
4 N
(mg l
1)
Biomass conc.
(mg VSS l
1)

910
920
1000
940
1
2
3
4

0
+15
+35
+65

1039

0.046
0.053
0.062
0.076

qdnd (mg NH
4 N
mg VSS
1 d
1)
RPE for
adnc (%)
adn ()
UASB

Batch test
RPE for
qna (%)
qnb (mg NH
4 N
mg VSS
1 d
1)
RPE for
ana (%)
an ()

AS

Batch test

If the fn and fdn values were multiplied by the biomass measured in the AS and UASB reactors, together
with the use of performance data shown in Tables 1
and 2, the specic nitrication and denitrication rates
(qn; qdn) could be calculated, respectively (Table 4).
The calculated qn and qdn values (0.260.47 mg NH
4

1
N mg VSS
1 d
1;
0.0460.076 mg NO

N
mg
VSS
x

1
d ) and the RPE values for qn and qdn [0(+81)%;
0(+65)%] increased with decreasing SRT (of the AS
reactor) and GRT (of the UASB reactor). Thus, a
shorter SRT resulted in higher activity of nitriers in
the AS reactor, while a shorter GRT gave higher activity
of denitriers in the UASB reactor.
The qn values of the present study were relatively
lower than those of Campos et al. (1999) (qn = 0.5

1
1
0.7 mg NH
d ), because they conducted
4 N mg VSS
nitrication in an AS reactor at high ammonia loading

3
1
d and without the
rates of up to 7.5 kg NH
4 N m

Test run

3.7. Specic nitrication and denitrication rates

Table 4
Activity of nitrogen-related microbial groups and specic nitrication and denitrication rates in the combined UASBAS reactor system

(fn) and the activity of nitriers (an) in the AS reactor in

runs 14. As shown in Table 4, the biomass and initial
NH
4 N concentrations measured in the batch tests

1
were 9101000 mg VSS l
1 and 55117 mg NH
4 N l ,
respectively; and the estimated fn value was 0.68 [i.e., calculated by dividing k 0n by kn (SRT = 25 d)]. This implied
that nitriers appeared to be the predominant microbial
groups in the AS reactor. Although the yield coecient
of aerobic heterotrophic bacteria was generally higher
than that of aerobic autotrophic bacteria (nitriers)
(Metcalf & Eddy, 2003), most of the inow organics
(COD) in the present study were consumed in the UASB
reactor, causing nitriers to be the predominant microbial groups in the AS reactor (at low COD concentration
levels). The obtained an and relative percentage enhancement (RPE; an determined at the SRT of 25 d was
selected as reference value) values were 0.680.87 and
0(+28)%, respectively; and a shorter SRT resulted in
higher an and RPE values, implying that the activity of
nitriers was increased with decreasing SRT.
Moreover, the mass fraction of denitriers (fdn) and
the activity of denitriers (adn) in the UASB reactor in
runs 14 were determined using batch reactors. As shown
in Table 4, the biomass and initial NO

3 N concentrations
measured in the batch tests were 17903290 mg VSS l
1

1
and 5781 mg NO

3 N l , respectively; and the estimated fdn value was 0.55 [i.e., calculated by dividing k 0dn
by kdn (GRT = 50 d)]. This revealed that denitriers appeared to be the predominant microbial groups in the
UASB reactor. The obtained adn and RPE (i.e., adn determined at the GRT of 50 d was selected as reference value)
values were 0.550.70 and 0(+27)%, respectively; and a
shorter GRT gave higher adn and RPE values, showing
that the activity of denitriers in the UASB reactor was
increased with decreasing GRT.

RPE for
qdnc (%)

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

1040

J.S. Huang et al. / Chemosphere 61 (2005) 10321041

addition of organic carbon. In addition, transportation

of ammonia from the bulk liquid to the nitrier cells
could be hindered by the crowded heterotroph cells (Hanake et al., 1990), which were actually present in our
activated sludge reactor. The qdn values of the present
study were generally higher than those of Nyberg et al.
(1992), and Lemmer et al. (1997) (qn = 0.014

1
1
0.052 mg NO

d ), who conducted denix N mg VSS
trication in xed-lm reactors. In other words, the
activity of denitriers in the biomass granule was relatively higher than that of denitriers in the biolm.
4. Conclusions
The results of the present study showed that the combined UASBAS reactor system (with consistently wasting of excess biomass and at the recycle-to-inuent ratios
(Re) of 1) achieved ecient removal of COD (9597%),
TKN (100%) and TN (5455%) from suspended-solids
pre-settled piggery wastewater. Denitrication and methanogenesis occurred in the UASB reactor, contributing to
most of the COD removal for a GRT range of 2050 d
and, complete nitrication (most of the TKN removed)
occurred in the AS reactor. Compact granules with good
settling abilities developed in the UASB reactor, yielding
average granule sizes (dp) of 1.051.57 mm, granule specic gravity of 1.041.06, biomass concentration (Xi) of
920015 440 mg VSS l
1, microbial densities (Xf) of
95 000111 300 mg VSS l
1, and sludge-bed porosity (e)
of 0.850.92. The estimated mass fractions of nitriers
(fn) and denitriers (fdn) were 0.68 and 0.55, respectively,
reecting the fact that nitriers and denitriers were the
predominant microbial groups in the AS and UASB reactors, respectively. Specic nitrication and denitrication
rates signicantly increased (0.260.47 mg NH
4 mg

1
1
VSS
1 d
1; 0.0460.076 mg NO

mg
VSS
d
,
respecx
tively) with decreasing SRT (2510 d) and GRT
(5020 d), respectively. Consequently, the combined
UASBAS reactor system should be regarded a promising alternative for the removal of organic carbon and
nitrogen from piggery wastewater.
Acknowledgment
Financial support of this research from Grant Number COA 90-AST-1.5.2-AID-U1 (1) from the Council of
Agriculture of the Republic of China is greatly
appreciated.
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