MECHANISM OF PHOSPHORUS REMOVAL BY SBR

SUBMERGED BIOFILM SYSTEM

BAOZHEN WANG*
*M
, JUN LI, LIN WANG
*M
, MEISHENG NIE and JI LI
Water Pollution Control Research Center, Harbin University of Architecture and Engineering,
66 West Dazhi Street, Harbin 150006, PR China
(First received January 1997; accepted in revised form September 1997)
AbstractThe mechanism of phosphorus removal in SBR submerged biolm system was studied in the
research. The DNP and nuclear magnetic resonance methods were employed to verify the mechanism
of phosphorus removal by bacteria with the phosphorus release in anaerobic phase and uptake in oxic
and anoxic phases. The mathematical models of phosphorus releases in anaerobic phase and phos-
phorus uptake in oxic phase were deduced and evaluated by comparison between the theoretically cal-
culated values and the test data. # 1998 Elsevier Science Ltd. All rights reserved
Key wordssequencing batch reactor, submerged biolm system, biological phosphorus removal,
phosphorus release, phosphorus uptake
INTRODUCTION
It has been well proved by many researches that the
submerged biolm systems including those with
xed and suspended carriers like Lindpor processes
(Morper and Wildmoser, 1990; Morper, 1994) are
very eective and ecient in organic and nitrogen
removal by means of the attached growth biolm,
which exhibits lots of advantages as compared with
activated sludge process such as stability and long
retention time of microorganisms, much higher bio-
mass content in terms of MLSS and MLVSS, much
less surplus biomass or sludge because of longer
food chains exist in biolm consisting of abundant
amount and various species of metozoa, protozoa,
bacteria and fungi (Wang et al., 1991). However,
the continuous ow submerged biolm systems have
been proved by many studies to be only ecient in
nitrogen removal, the ammonia removal by nitrica-
tion in particular (Wang et al., 1991, 1992; Lee and
Welander, 1994; Liu and Capdeville, 1994; Mik et
al., 1995), but not ecient to remove phosphorus,
which is mainly due to the uneven distribution of bio-
mass either in quantity or in microbial species along
the ow path in the continuous ow submerged bio-
lm reactor (CFSBR), which are usually divided into
two sections, i.e. anoxic and oxic (A/O) zones, in
which the microbial community is dominated by het-
erotrophic species of bacteria responsible for organic
degradation and denitrication in anoxic zone and
the autotrophic nitrifying bacteria are dominant
species for nitrication in oxic zone. In the A/O sub-
merged biolm system there is no room for P
removal or accumulating bacteria growth because
there exists no alternative anaerobic/oxic environ-
mental conditions for their growth, which is hardly
developed in the CFSBF. However, the submerged
biolm sequencing batch reactor (SB-SBR) that oper-
ates with an alternate anaerobic/oxic procedure devel-
oped by the authors have been proved very ecient in
phosphorus removal (Li, 1996). Two methods were
employed in the study to verify the biological removal
of phosphorus, of which the 2,4 dinitrophenol (DNP)
was added as a xenobiotic to observe its inhibitory
eect on biological P removal and the
31
P nuclear
magnetic resonance spectroscopic technique (Hill et
al., 1989) was used to verify the phosphorus release
and uptake biologically in anaerobic, oxic and anoxic
phases, respectively, by observation of the variation of
peaks of ortho-phosphate and polyphosphate in the
31
P NMR spectrograms under anaerobic, anoxic and
oxic conditions. Some mathematical models for simu-
lating phosphorus release in the anaerobic phase and
phosphorus uptake in the oxic phase have been
developed as well.
EXPERIMENTAL METHOD
Experimental equipment
As shown in Fig. 1, the experimental equipment was
made of a plexiglass column with an inner diameter of
15 cm and a volume of 22 l, of which the eective volume
was 16 l, in which the biolm carriers occupied 6 l and the
settling zone 2 l.
Test process, parameters and synthetic wastewater
The test process was employed as follows: Inuent 4
anaerobic phase, with HRT of 3 h 4oxic phase, 6 h 4
sedimentation, 1 h. The raw wastewater was prepared by
the mixing of a certain amount of peptone, ammonia
chloride magnesium sulfate, calcium chloride and sodium
chloride and potassium dihydrogen phosphate. The main
Wat. Res. Vol. 32, No. 9, pp. 26332638, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0043-1354/98 $19.00 +0.00 PII: S0043-1354(97)00413-2
*Author to whom all correspondence should be addressed.
2633
Fig. 1. Schematic diagram of experimental equipment.
Fig. 2. Eect of DNT on P release in anaerobic phase and
P uptake in oxic phase.
Table 1. The synthetic wastewater quality
Parameter Concentration (mg/l)
COD 250400
BOD
5
180300
TN 3060
NH
4
+
N 1020
NO
3

N 0.2
NO
2

_N 0.1
TP 10
SP 8
pH 7.3 (log[H
+
]
1
)
Alkalinity 380440
quality parameters of the synthetic wastewater are shown in
Table 1.
Tests and results
Addition of DNP. 20 mg/l DNP (2,4 dinitrophenol) was
added to the raw wastewater and the submerged biolm
SBR was operated according to the alternate A/O pro-
cedure as mentioned in Section 1. The result is shown in
Fig. 2. The phosphorus release was still observed in the
anaerobic phase, but the phosphorus uptake was not
observed in the oxic phase, which indicated that DNP is a
decoupling agent that inhibits phosphorylation respiratory
chains and ATP formation, thus preventing phosphorus
accumulation in the oxic phase, but does not aect the
phosphorylation beside the oxidation respiratory chain. In
the metabolism process of polyphosphate decomposition
in the anaerobic phase, the ATP is produced by phos-
phorylation at the substrate level which was not inhibited
by DNP, thus causing PO
4
3
release and PHB formation,
However, in the oxic phase in the presence of oxygen the
ATP was not formed due to the inhibition to electron
transfer in the respiratory process, which led to no phos-
phorus uptake and polyphosphate formation and therefore
no phosphorus removal in the oxic phase because of the
DNP inhibitory eect.
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPIC
TECHNIQUE
Test method
Material. Bacteria strains used in the test were
contained in the biolm taken from the carrier in
the submerged biolm SBR for phosphorus removal,
which were cultured by many times of alternate A/O
operation, thus resulting in the maturation of the P-
removing bacteria strains. The biolm containing the
bacteria strains was diluted with NO
3

free distilled
water with the addition of beef paste as culture med-
ium to a nal solution with BOD
5
of 600 mg/l.
Test equipment. An AC 80 type nuclear magnetic
resonance spectrometer produced by Brucrer Co. in
Switzerland was employed for analysis at 36.43 Mhz.
A few drops of D
2
O and then HMPA (hexamethyl
phosphoramide) were added onto the culture med-
ium, which exhibited a peak at +25 ppm and served
as an external standard of phosphorous compounds
in biomass.
The scanning range was 6000 Hz, the capture time
was 0.17 s, the pulse was 808, decay was the multi-
plicity of the exponential function of 6 Hz, the ac-
cumulative rotation number was from 10,000 r
to 20,000 r, the temperature was 278C, chemical
Baozhen Wang et al. 2634
position moving unit ppm, 85% ortho-phosphate
was used as reference sample. The removed biolm
of 3000 mg after 5 min of centrifugal separation was
put into a 10 ml sample tube.
Results and discussion. The variation of spectral
peaks in the anaerobic phase are shown in Fig. 3.
At the beginning of the anaerobic phase there
existed three peaks, of which the peak at +25 ppm
was caused by HMPA, the peak at +2 ppm by
PO
4
3
and that at 22 ppm by polyphosphate. The
peaks varied with operating time in the anaerobic
phase with such a typical tendency that the peak at
22 ppm decreased gradually with the decrease of
polyphosphate content until the peak disappeared
after a 28 h operation, which indicated that the
polyphosphate contained in the bacterial cells was
decomposed into phosphate. The peak at +2 ppm
increased gradually with the increase of ortho-phos-
phate content from the degradation of polypho-
sphate, which took place completely after a 28 h
operation in the anaerobic phase as clearly shown
in Fig. 3.
The variation of the
31
P-NMP spectrogram in the
anaerobic/oxic phases in turn is shown in Fig. 4.
The three spectrograms express the original state of
biolm at the beginning of the anaerobic phase,
after a 5 h operation in the anaerobic phase and the
state after 5 h aeration in the oxic phase, respect-
ively, from which it was found that the peak of
ortho-phosphate at +2 ppm increased signicantly
in a 5 h operation in the anaerobic phase than that
at beginning, but decreased sharply in the oxic
phase and dropped to a very small peak after 5 h
aeration, while the peak of polyphosphate at
22 ppm increased sharply, which clearly indicated
that the biolm took up phosphate and carried out
the polyphosphorylation, which resulted in the
decrease of ortho-phosphate content and the
increase of polyphosphate content in biolm under
oxic conditions.
As shown in Fig. 5, under anoxic conditions, the
inorganic phosphorus was also converted to poly-
phosphate with the NO
3

as oxygen source, which

indicated that the uptake and polyphosphorylation
of inorganic phosphorus could take place in bac-
terial cells under anoxic conditions as it did under
oxic conditions but with NO
3

as oxygen source,
which is similar to another study carried out in an
activated sludge system with biolm nitrication
(Sorm et al., 1996)
It has been concluded from the study of
31
P-
NMR spectrograms of biolm under anaerobic,
oxic and anoxic conditions that the P-removing
bacteria contained in submerged biolm released
phosphorus due to the degradation of polypho-
sphate into ortho-phosphate under anaerobic
Fig. 3.
31
P-NMP spectrogram of biolm in anaerobic
phase.
Fig. 4.
31
P-NMR spectrogram of biolm in anaerobic/oxic
phase.
Fig. 5.
31
P-NMP spectrogram of biolm in anoxic/oxic
phases.
Mechanism of phosphorus removal by SBR 2635
conditions and took up phosphorus and carried
out polyphosphorylation under oxic and anoxic
conditions.
MATHEMATICAL MODELS ON PHOSPHORUS REMOVAL
IN AN SBR SUBMERGED BIOFILM SYSTEM
Dynamic model development
Phosphorus release in anaerobic phase. Two deci-
sive factors aecting the concentration distribution
in biolm are mass transfer rate and reaction rate,
which were also aected by the concentration distri-
bution, Therefore, the rst order reaction equation
was employed to describe the phosphorus release
and organic uptake in anaerobic phase as follows:
dPadt = K
1
(P
m
P) (1)
dLadt = K
2
(L L
m
) (2)
in which P
m
is the maximum value of phosphorus
release, mg/l; P is the instant value of phosphorus
concentration in biolm SBR at time t, mg/l; K
1
is
the phosphorus release rate constant, h
1
; dP/dt is
the phosphorus release rate, mg/lh; dL/dt is the
substrate uptake rate, mg/lh; L
m
is the nal value
of substrate, mg/l; L is the instant value of sub-
strate concentration at time t in biolm SBR and
K
2
is the substrate uptake rate constant, h
1
.
The values of P
m
and L
m
were determined by
test. Equations (1) and (2) are integrated to achieve
the time functions of P and L as follows:
P = P
m
(Pm P
0
)exp(K
1
t) (3)
L = L
m
(L
m
L
0
)exp(K
2
t) (4)
in which P
m
P
0
=DP
m
: maximum probable amount
of phosphorus release, mg/l; L
m
L
0
=DL
m
: maxi-
mum probable amount of COD removal, mg/l; P
0
:
initial TP concentration, mg/l; L
0
: initial COD con-
centration, mg/l.
In order to achieve the amounts of phosphorus
release and organic uptake at any time, two vari-
ables were dened as follows:
P
/
= P P
0
(5)
L
/
= L L
0
(6)
in which P' is the TP release at time t, mg/l; L' is
the COD removal at time t, mg/l.
Substituting equations (5) and (6) into
equations (3) and (4), the following equations are
obtained:
P
/
= DP
m
[1 exp(K
1
t)] (7)
L
/
= DL
m
[1 exp(K
2
t)] (8)
which after simplication, achieve
exp(K
1
t) = (DP
m
P
/
)aDP
m
(9)
exp(K
2
t) = (DL
m
L
/
)aDL
m
(10)
which after processing logarithmically become
K
1
t = ln[(DP
m
P
/
)aDP
m
] (11)
K
2
t = ln[(DL
m
L
/
)aL
m
] (12)
equation 11 is divided by equation 12 and then sim-
plied, thus obtaining the following equations:
P
/
= DP
m
[1 (1 L
/
aDL
m
)
K1aK2
] (13)
PaDP
m
= 1 (1 L
/
aDL
m
)
K1aK2
(14)
Estimation of parameters of the models
The peptone was used as a substrate, inuent
COD was 340380 mg/l, TP was 9.510.0 mg/l, the
total HRT in the reactor was 9 h, of which the an-
aerobic phase took 3 h and the oxic phase 6 h and
the temperature was maintained at 208C, at which
the average values of DL
m
and DP
m
were obtained
as 175.0 and 4.05 mg/l, respectively, by test.
The estimation of parameters was carried out by
computer scanning calculation-gradient search
method (Li and Wang, 1989), by which the phos-
phorus release rate constant was obtained from
equations (3) and (4) as K
1
=0.750 h
1
and the or-
ganic uptake rate constant as K
2
=0.910 h
1
.
In order to verify the correctness of the devel-
oped models, the track test values are used to make
comparisons between the theoretically calculated
and test values, which are shown in Figs 68, from
which it is evident that the calculated values from
the developed equations coincide with correspond-
ing test values very well, which indicates that
equations (3)(5) can describe phosphorus release
and COD removal satisfactorily under anaerobic
conditions in submerged biolm SBR.
Phosphorus uptake in oxic phase
Similar to organic uptake in the anaerobic phase,
the organic degradation in the oxic phase can be
Fig. 6. Comparison between the test and calculated values
of phosphorus release at three COD loading rates.
Baozhen Wang et al. 2636
also described as follows:
dLadt = K
3
(L L
e
) (15)
in which K
3
is the organic degradation rate con-
stant, h
1
and L
e
is the euent organic concen-
tration measured by COD in the oxic phase, which
was determined by test and can be regarded as non-
degradable COD. After integration equation 15 is
converted into the following equation:
L = L
e
(L
0
L
e
)e
K3T
(16)
in which L
0
is the inuent organic (COD) concen-
tration, mg/l.
A track test was carried out by using peptone as
a substrate, from which the euent COD value in
the oxic phase was determined as 38.0 mg/l.
The parameter was also estimated by the compu-
ter scanning calculation-gradient search method and
the organic degradation rate constant was obtained
as K
3
=1.300 h
1
, which was much higher than that
of organic uptake. K
2
=0.90 h
1
in the anaerobic
phase. The theoretically calculated values expressed
in a curve and the test values expressed in circular
points are shown in Fig. 9.
The eect of three dierent substrates such as
peptone, glucose and acetic acid on the value of the
organic degradation rate constant was studied as
well and the results are shown in Table 2, which
shows that the K
3
values of glucose and acetic acid
are remarkably higher than that of peptone, which
means that the microorganisms rst utilize the more
readily degradable organic compounds with smaller
molecular weight.
The correlation between phosphorus release in
the anaerobic phase and phosphorus uptake in the
oxic phase was also researched with the results
shown in Fig. 10, which clearly indicated that the
maximum P uptake, DP
m
, in the oxic phase was
proportional to the maximum P release in the an-
aerobic phase, from which after linear regression
processing, the following equation was derived:
DP
m
(oxic) = 0X93P (anaerobic) 9X24 (mgal) (17)
with a correlation coecient of 0.94.
The maximum anaerobic P release and aerobic P
uptake values with three dierent substrates are
shown in Table 3. As a result, the theoretically cal-
culated values of P release and uptake rate con-
stants and organic removal rate constant coincided
with respective test ones very well.
CONCLUSION
The 2.4 dinitrophenol (DNP) which has the
characteristics does not inhibit phosphorus release
in the anaerobic phase but does inhibit phosphorus
uptake in the oxic phase, was employed to verify
that the phosphorus was removed biologically in
the submerged biolm SBR.
31
P-NMR spectrograms were used to explain the
mechanism of phosphorus removal, from which the
polyphosphate was decomposed and converted into
Fig. 7. Comparison between the test and calculated value
of COD removal at three COD loading rates.
Fig. 8. Correlation between phosphorus release and COD
uptake.
Fig. 9. Comparison between theoretical and test values of
organic removal in anaerobic and oxic phases.
Table 2. COD removal rate constant values with three dierent
substrates
Substrate L
0
(mg/l) L
e
( mg/l) K
3
(h
1
)
Peptone 192.9 38.0 1.300
Glucose 180.0 25.2 1.560
Acetic acid 195.2 22.6 1.520
Mechanism of phosphorus removal by SBR 2637
inorganic phosphorus in the form of ortho-phos-
phate in the anaerobic phase and the inorganic
phosphorus was converted to polyphosphate and
storage in bacterial cells in the oxic and anoxic
phases.
Mathematical models have been developed on P
release in the anaerobic phase and P uptake in the
oxic or anoxic phase, in which the optimum values
of parameters were calculated by the computer
scanning calculation and gradient search method,
by which the calculated values of P release and
uptake and organic removal coincide with the
respective test ones very well.
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31
-
NMR Spectroscopy characterization of polyphosphates
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Fig. 10. Correlation between the anaerobic P release and
aerobic P uptake with peptone as organic substrate.
Table 3. Maximum anaerobic P release and aerobic P uptake
values, DP
m
(mg/l)
Substrate In. TP In. COD
Anaerobic
DP
m
Oxic DP
m
Peptone 9.3 102.0 0.8 9.1
9.6 272.7 1.6 10.3
10.2 372.0 4.0 13.5
9.1 649.8 4.7 13.6
11.1 942.1 5.2 16.3
Glucose 8.0 427.0 7.5 15.3
Ethanoic acid 8.2 395.7 10.4 18.4
Baozhen Wang et al. 2638