Wat. Res. Vol. 33, No. 2, pp.

555±561, 1999
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(98)00206-1 0043-1354/98 $19.00 + 0.00

SIMULTANEOUS CARBON AND NITROGEN REMOVAL IN

A MIXED CULTURE AEROBIC RBC BIOFILM
A. B. GUPTA and S. K. GUPTA*
Centre for Environmental Science and Engineering, Indian Institute of Technology, Bombay 400 076,
India

(Received February 1995; accepted April 1998)

AbstractÐA mixed culture bio®lm was developed with a sulfur oxidising, heterotrophic bacterium Thio-
sphaera pantotropha, autotrophic nitri®ers and other heterotrophs in a three stage rotating biological
contactor (RBC). Speci®c bene®ts due to peculiar properties of T. pantotropha of simultaneous hetero-
trophic nitri®cation and aerobic denitri®cation were derived in the reactor investigated for a combined
carbon and nitrogen removal from a synthetic domestic sewage. The ®rst stage bio®lm which contained
T. pantotropha showed high COD and NH+ 3
4 -N removal rates of 5.8±14.1 g COD/m
d and 0.47±1.1 g
N/m2
d for the corresponding loading rates of 6.9±20.7 g COD/m3
d and 0.69±2.09 g N/m2
d, respect-
ively. Contrary to the conventional units designed for a concurrent carbon removal and nitri®cation,
the nitri®cation rates increased linearly with an increase in organic loading rate before stabilising at
about 1 g N/m2
d corresponding to a COD loading rate of about 15 g/m2
d and a nitrogen loading rate
of 1.5 g N/m2
d showing a change in the order of reaction from ®rst to zero. A simultaneous nitrogen
removal of 20±68% was also obtained. The system's performance indicated that a single stage aerobic
bio®lm can be developed to meet the increasingly stringent regulations on e‚uent nitrogen discharges
a€ording several advantages over the conventional systems, e.g. low bu€er requirements, no need for
external carbon source for denitri®cation, etc., which may result in substantial reduction in the treat-
ment cost. # 1998 Elsevier Science Ltd. All rights reserved

Key wordsÐThiosphaera pantotropha, mixed culture bio®lm, carbon removal, nitrogen removal, auto-
trophic, nitri®cation, heterotrophic nitri®cation, aerobic denitri®cation

INTRODUCTION exceed the ability of traditional wastewater treat-

Ammonia nitrogen is not easy to remove completely ment plants to produce high quality e‚uents at
by conventional high-rate wastewater treatment reasonable costs. Consequently, economical and
techniques and frequently remains in domestic innovative wastewater treatment techniques are
wastewater treatment plant e‚uents. The adverse needed to be evolved in order to meet the demand
environmental impacts associated with ammonia for nitrogen control.
nitrogen include promotion of eutrophication, tox- Biological nitri®cation±denitri®cation is one of
the most economical processes for nitrogen removal
icity to aquatic organisms and depletion of dis-
from municipal wastewaters. The design and econ-
solved oxygen in receiving streams due to bacterial
omy of such a system are closely related to process
oxidation of ammonia to nitrate (Klees and
kinetics, i.e. to the rates at which nitri®cation and
Silverstein, 1992). Improper disposal of human
denitri®cation proceed. These rates, in turn, depend
wastes may result in enhanced nitrate levels in
on the microbial population, the composition and
groundwater raising possibilities of infant methae-
concentration of wastewater and a wide variety of
moglobinemia and gastric cancers (Shuval and
physical and chemical parameters (Eckenfelder and
Gruner, 1977).
Argaman, 1978).
Due to these adverse impacts, the need to restrict
Rotating biological contactors have been tried
the ammoniacal nitrogen discharges has been
extensively for single stage carbon removal and
increasingly felt. In the European directive on
nitri®cation as well as for separate stages in series
urban wastewater treatment (Cooper et al., 1994),
for BOD removal and nitri®cation from municipal
the maximum allowable nitrogen concentration
wastewaters (Weng and Molof, 1974; Stover and
including ammonia and organic nitrogen, nitrite
Kincannon, 1975; Murphy et al., 1977; Huang,
and nitrate is 20 mg N/l (daily average). A further
1982). E€ects of various operating parameters like
reduction to 10 mg/l is possible (Lefevre et al.,
turbulence (Kugaprasatham et al., 1992), disc ro-
1993). Stringent limitations on ammoniacal nitrogen
tation speed (Friedman et al., 1979), hydraulic con-
discharge, while necessary for pollution control, can
ditions (Kugaprasatham et al., 1991), organic
particulate matter (Figueroa and Silverstein, 1992)
*Author to whom all correspondence should be sent. and recirculation (Klees and Silverstein, 1992) on
555
556 A. B. Gupta and S. K. Gupta

nitrifying bio®lms have been studied in details. Table 1. Summary of the dimensions of RBC experimental unit
Some literature is also available on simultaneous Parameter Units Value
nitri®cation and denitri®cation in microaerobic
No. of stages number 3
®lms (Masuda et al., 1991; Watanabe et al., 1992) No. of disks/stage number 14
but there have been no reports on simultaneous Disk diameter cm 25.0
organics and nitrogen removal in a fully aerobic Disk thickness cm 0.2
Disk spacing cm 1.5
bio®lm. Shaft diameter cm 5.0
Robertson and Kuenen (1983) isolated a bacter- E€ective surface area/stage m2 1.2
Total e€ective surface area of the m2 3.6
ium Thiosphaera pantotropha from a sulfur oxidis- unit
ing wastewater which was capable of simultaneous E€ective trough capacity/stage l 8
heterotrophic nitri®cation and aerobic denitri®ca- Total trough capacity/stage l 24
Submergence % 32
tion. Ample literature has been published on the Rotational speed rpm 5
peculiar enzyme system to explain its properties of
heterotrophic nitri®cation (Robertson and Kuenen,
1988; Robertson et al., 1988, 1989) and aerobic
Reports of the use of this bacterium in suspended
denitri®cation (Robertson and Kuenen, 1984a,b;
and immobilised forms in axenic cultures for carbon
Robertson et al., 1988; Bell et al., 1990; Bell and
removal, nitri®cation and denitri®cation are avail-
Ferguson, 1991; Berks et al., 1993; Moir et al.,
able (Robertson et al., 1988; Geraats et al., 1990;
1993). It has been reported (Robertson and
Hooijmans et al., 1990) but no work on mixed cul-
Kuenen, 1988; Robertson et al., 1988) that the
tures is reported which could be of application to
speci®c nitrifying activity of the heterotrophs is
environmental systems. This paper describes a single
103±104 times lower than that of autotrophs.
stage aerobic RBC system for simultaneous carbon
However, the ammonia oxidising rates of T. panto-
removal, nitri®cation and denitri®cation by using T.
tropha are only 10±103 times lower than the auto-
pantotropha, in mixed bacterial bio®lm.
trophs (Geraats et al., 1990). While growing as a
heterotroph the growth rates of this bacterium tend
to be much higher than those for the autotrophs
(the mmax for Nitrosomonas europea is about 0.03± MATERIALS AND METHODS
0.05 hÿ1 and that of T. pantotropha can be as high The experimental set up
as 0.4 hÿ1 under the same growth conditions) giving A three stage RBC each comprising of 14 circular plates
it a competitive advantage (Robertson et al., 1988). of acrylic sheet, 25 cm in outer diameter, was used. The
The aerobic denitri®cation rates were much higher shaft was rotated at 5 rpm with a 0.25 hp motor and re-
than heterotrophic nitri®cation rates in chemostat duction gear system. Sampling ports were provided in
studies with axenic cultures of T. pantotropha at all each stage for e‚uent collection. Table 1 lists the salient
dimensions of the RBC and Fig. 1 gives a sketch of the
dilution rates indicating extra capacity of this bac- set up.
terium to take nitrate or nitrite coming from other
routes apart from its nitri®cation path (Robertson Composition of the medium
et al., 1988). A synthetic sewage was prepared with acetate as carbon
The above considerations may be utilised in a source and ammonium chloride as nitrogen source in con-
centrations which were close to those found in domestic
mixed culture having autotrophic nitri®ers and T. e‚uents (Stover and Kincannon, 1975). The wastewater
pantotropha along with other heterotrophs to evolve composition is shown in Table 2 while Table 3 shows
an optimum carbon and nitrogen removal system. average characteristics of the synthetic feed.

Fig. 1. Sketch of the experimental set-up.

 Simultaneous carbon and nitrogen removal 557

Table 2. Composition of synthetic wastewater Development of the bio®lm and the reactor operation
Compound Concentration in mg/l T. pantotropha pure culture was obtained from Dr L. A.
Robertson, University of Delft, The Netherlands. The bio-
NH4Cl 112.5
CH3COONa
3H2O 525
®lm development on acrylic plates was achieved using a
Na2HPO4 75 high strength medium four times as concentrated as the
KH2PO4 37.5 above composition and running the reactor, ®rst in a
MgSO4 25 batch mode for 15 days and then shifting over to the con-
tinuous mode at 2 days HRT as described elsewhere
(Gupta et al., communicated). A fresh liquid culture of T.
pantotropha was added twice a week in the trough during
Table 3. Wastewater characteristics the bio®lm development. Thereafter no addition of pure
cultures was made to the system. After operating the sys-
Parameter Value* tem on two di€erent strength media of four and two times
COD 250 the above concentration, it was switched to the compo-
NH+4 -N 27.5 sition described in Table 2. The reactor was initially run at
C/N 3.32 a HRT of 18 h corresponding to a nitrogen loading rate
Total PO3ÿ
4 -P 25.5
pH 7.3
of 0.23 g N/m2
d. The loading rate was increased slowly in
Alkalinity (as CaCO3) 200 steps. At every set of experimental conditions the reactor
was run for 2 weeks. It was found that the e‚uent par-
*All values are in mg/l except pH.
ameters yielded consistent values within 4±5 days.

Table 4. Average monitored pseudo steady-state values of various parameters

HRT (h) NH+4 -N NOÿ
2 -N NOÿ
3 -N COD (mg/l) pH Alkalinity S.S. (mg/l) Temp. (8C) D.O. (mg/l)
Sample (mg/l) (mg/l) (mg/l) (mg/l)

I 18 26.18 259.8 7.25 199.92 19

E1 8.35 4.83 8.10 41.23 7.67 152.32 5.0
E2 6.03 5.49 9.29 41.23 7.74 104.72 6.0
E3 4.82 5.24 10.72 41.23 7.35 104.72 10 6.3

I 15 26.18 259.8 7.25 196.00 23

E1 9.66 3.82 3.57 41.23 7.73 184.00 4.0
E2 4.52 5.49 7.86 41.23 7.71 138.00 5.6
E3 1.90 3.49 12.15 41.23 7.72 108.00 12 5.9

I 12 26.18 259.8 7.41 239.00 24

E1 9.46 7.46 2.98 41.23 7.81 154.00 3.8
E2 6.64 8.26 5.24 41.23 7.69 131.00 5.5
E3 2.81 7.73 8.46 41.23 7.72 122.00 16 6.1

I 10 26.18 259.8 7.26 216.00 22

E1 10.06 7.33 1.19 n 7.61 172.00 4.5
E2 6.24 10.32 2.50 n 7.51 128.00 6.9
E3 2.93 10.66 6.31 n 7.50 112.00 22 7.0

I 9 26.18 259.8 7.26 209.00 24

E1 10.06 5.92 1.90 41.23 7.75 161.00 3.8
E2 3.42 5.79 9.54 41.23 7.60 127.00 5.9
E3 1.40 4.99 11.63 41.23 7.60 61.00 20 6.0

I 8 26.18 259.8 7.33 216.00 25.5

E1 8.45 2.46 2.38 20.61 7.72 136.00 3.6
E2 1.40 2.26 9.89 20.61 7.70 120.00 4.9
E3 0.90 1.73 10.96 20.61 7.76 120.00 24 5.8

I 7 26.18 259.8 7.35 204.00 24.5

E1 10.06 3.62 0.95 41.23 7.71 188.00 3.9
E2 3.92 4.57 3.04 20.61 7.66 128.00 5.8
E3 1.81 2.36 4.05 n 7.73 120.00 20 5.9

I 6 26.16 259.8 7.40 224.00 26.5

E1 13.14 1.47 0.39 82.46 7.77 200.00 2.9
E2 4.76 3.94 6.04 41.23 7.73 129.00 4.4
E3 2.01 1.62 8.74 41.23 7.80 122.00 12 5.1
I: In¯uent; E1, E2 and E3: e‚uent from stage 1, 2 and 3 respectively; n: not detectable.
558 A. B. Gupta and S. K. Gupta

4 -N removed after correction for alkalinity generated during denitri®cation assuming that the rate of alkalinity generation during denitri®cation is half the rate
Sampling and analysis
overall

8.88
10.66
13.33
16.00
17.77
20.00
22.85
26.66
Hydraulic loading

E‚uent samples were collected from the sampling ports

rate (l/m2
d)

provided at the bottom of the trough of the three stages

and were analysed for various parameters according to
APHA (1989). All parameters were measured on alternate
®rst stage

26.66
32.00
40.00
48.00
53.33
60.00
68.56
80.00
days except for pH and D.O. which were monitored daily.
Ammonia-nitrogen was measured by Nesslerization,
nitrate-nitrogen by brucine±sulphate, nitrite-nitrogen by
(g COD removed/
4 -N removed)

the diazotization method and COD by the closed re¯ux

in ®rst stage

method. D.O. was measured by an YSI D.O. probe, pH

12.24
13.22
13.06
16.11
13.55
13.47
13.53
13.69
by a pH meter and alkalinity by titrating against a stan-
dard HCl solution. Suspended solids in the e‚uent were
also measured once in 4 days.
NH+
removal
First stage organics

12.47
11.65
14.34
14.96
14.18
5.82
6.99
8.74

RESULTS AND DISCUSSION

(g COD/m2
d)

A pseudo-steady-state was assumed to have been

achieved when the e‚uent NH+ 4 -N from all the
loading

10.39
12.47
13.85
15.58
17.79
20.78
6.92
9.31

compartments did not vary signi®cantly for the

three consecutive observations. Table 4 gives aver-
age pseudo-steady-state values of various monitored
NAD2

6.23
5.17
7.14
5.05
5.45
5.90
5.1
4.4

parameters at di€erent loading rates corresponding

to HRTs of 18 to 6 h. Table 5 lists all derived par-
ameters from Table 4.
NAD1

4.45
3.62

4.47
5.97
3.79
3.44
4.22
5.0

The e‚uent NH+ ÿ

4 -N, NO2 -N and NO3-N con-
centrations varied between 2.0±4.8, 1.6±10.6 and
Table 5. Derived parameters from Table 4

nitrogen (mg/l)
Total e‚uent

4.0±12.1 mg/l, respectively. The D.O. reduced from

5.0 to 2.9 mg/l in the ®rst stage as the HRT
20.78
17.64
19.00
19.90
18.02
13.59

12.37
8.22

decreased from 18 to 6 h but in the second and

third stages it remained high (4.4±7.0 mg/l)
throughout. The pH in the reactor varied between
Overall % Overall %

removal

7.3±7.8 while the wastewater temperature during

total N

20.60
33.00
27.40
23.90
31.10
48.00
68.6
52.7

the study ranged from 19±26.58C. E‚uent S.S. was

between 10 and 22 mg/l at various HRTs. The e‚u-
ent phosphate was also measured occasionally but
removal
4 -N

81.53
92.70
89.20
88.80
94.60
96.50
93.00
92.30

no signi®cant di€erence was observed in the in¯uent

NH+

and the three stage values.

Most of the carbon was consumed in the ®rst
4 -N removed from the system.
0.1989
0.2589
0.3116

0.4405
0.5056
0.5569
0.6445
0.372

stage. The organic removal rates in the ®rst stage

overall

varied between 5.8±14.1 g COD/m2
d for the corre-

4 -N removal rate g N/m
d

sponding loading rates of 6.9±20.7 g COD/m2 d.

3

0.0838
0.1532
0.1588
0.1077

0.1446

The nitri®cation rates in this stage varied between

0.032

0.030

0.220
stage 3

0.47±1.10 g N/m2
d for nitrogen loading rates 0.69±

2.09 g N/m2 d. The ratio of COD removed to
0.06186
0.1645
0.1128
0.1833
0.3541
0.4230
0.4209
0.6704
stage 2

NAD2: Net overall alkalinity destroyed per unit of NH+

NAD1: Net overall alkalinity destroyed per unit of NH+
NH+

0.4754
0.5286
0.6688
0.7737
0.8597
1.0638
1.1053
1.0432
stage 1

its destruction during nitri®cation.

0.2327
0.2792
0.3490
0.4188
0.4654
0.5236
0.5983
0.6981
overall
4 -N loading
(gN/m3-d)

0.6981
0.8376

1.2564
1.3962
1.5708

2.0943
1.047

1.794
stage 1
NH+

HRT (h)

Fig. 2. Variation of nitrogen in the ®nal e‚uent at di€er-

ent HRTs.
18
16
12
10
9
8
7
6
 Simultaneous carbon and nitrogen removal
Fig. 3. Variation of nitrogen in e‚uent from stage 1 at
di€erent HRTs.
NH+ 4 -N removed was close to 13 in this stage.
Figure 2 depicts the e‚uent NH+ ÿ
4 -N, NO2 -N and
ÿ
NO3 -N values vs HRT for the ®nal e‚uent. It can
be seen that while the NH+ 4 -N values were almost
consistent at all HRTs, the NOÿ 2 -N and NO3 -N
ÿ Any increase in the organic loading rate would
varied greatly at di€erent HRTs. A possible expla-
nation of this is the di€erence in behaviour of the
®rst stage from that of the other two stages. It can
be seen that most of the in¯uent carbon gets con-
sumed in the ®rst stage itself at almost all the
HRTs and as such T. pantotropha survives only in
the ®rst stage being a heterotroph (it is a facultative
autotroph if given reduced sulfur compounds as
energy source). The second and third stages are
comprised of only autotrophic nitri®ers, as a very
low amount of organic carbon reaches them. The
®rst stage dominated by T. pantotropha shows a
consistent e‚uent NH+ 4 -N up to 8 h HRT below
which it starts increasing as seen in Fig. 3. The
D.O. in this stage decreases as the HRT decreases
resulting in increased heterotrophic nitri®cation
rates which probably compensate for the decrease
Fig. 4. Relation between overall NH+
4 -N loading and
removal rates.
559
Fig. 5. Relation between NH+ 4 -N loading and removal
rates for stage 1.
in autotrophic contribution at higher loading rates.
favour the growth of T. pantotropha as against that
of autotrophic nitri®ers as it essentially would mean
a higher organic loading and shorter detention
time. The NOÿ ÿ
2 -N and NO3 -N decrease as HRT (as
a consequence the D.O.) decreases in this stage.
The increase in heterotrophic nitri®cation and
aerobic denitri®cation rates with a depletion of
D.O. to about 25% air saturation level is well docu-
mented in the literature (Robertson et al., 1988).
Figure 4 plots the overall NH+ 4 -N removal rate
vs nitrogen loading rate which gives a straight line
relation. Figure 5 shows the same relation graphi-
cally for stage 1 which indicates a probable change
in the order of reaction from ®rst to zero above a
nitrogen loading rate of about 1.5 g N/m2
d.
Figure 6 indicates percent NH+ 4 -N and total N
removal vs HRT. Total N removals increase at low
HRTs due to a higher denitri®cation rate in the
®rst stage.
The overall analysis indicates a vast superiority
of the present system over the conventional ones,
Fig. 6. Percent removal of nitrogen at di€erent HRTs.
560 A. B. Gupta and S. K. Gupta
Table 6. Nitrogen balance across the reactor
Total nitrogen in mg/l
HRT (days) in¯uent stage 1 stage 2
18 26.18 21.28 20.81
15 17.05 17.87
12 19.90 20.14
10 18.58 19.06
9 17.58 18.75
8 13.29 13.55
7 14.63 11.53
6 15.00 14.74
attained due the introduction of T. pantotropha.
Boongorsrang et al. (1982) observed that the COD
loading rate must be reduced to below 2.54 g/m2
d
for nitri®cation to occur in a rotating disc contac-
tor. In the present system, stage 1 showed a high
nitri®cation rate of 0.47±1.10 g NH+ 2
4 -N/m
d
despite a very high COD loading rate of 6.9±20.7 g
COD/m2
d. Similarly, Bovendeur et al. (1990) in a
bio®lm system indicated a nitri®cation rate of
0.575 g NH+ 2
4 -N/m
d at zero COD which decreased
by 0.015 g NH4 -N/m2
d at every g/m2
d of COD
+
removed. In the present study the nitri®cation rates
in the ®rst stage continuously increased despite a
simultaneous increase in the COD loading rate
which levelled o€ at a high value of about 1 g
NH+ 2
4 -N/m
d corresponding to in¯uent COD and
+
NH4 -N loading rates of about 15 and 1.5 g/m2
d,
respectively. These nitri®cation rates were compar-
able to the values obtained by Krup et al. (1980)
which ranged from 0.27±1.1 g N/m2
d for loading
rates of 0.29±2.14 g N/m2
d for the treatment of the
primary e‚uent of West±Southwest sewage treat-
ment plant; Greater Chicago. These were also
higher than the nitri®cation rates 0.2±0.6 g N/m2
d
reported by Surampalli and Baumann (1989) for a
loading range of 0.88±2.4 g N/m2
d for primary
treated sewage.
In a conventional RBC described by Gonenc and
Harremoes (1985) designed for carbon removal and
nitri®cation the nitrogen balances indicated that an
average of 8% of the total in¯uent nitrogen did not
appear in the e‚uent. Table 6 gives the nitrogen
balance across the RBC which indicates that 18±
49% nitrogen was unaccounted in the ®rst stage
e‚uent which could primarily be attributed to
aerobic denitri®cation by T. pantotropha. There was
little loss (<5%) in the subsequent stages except
for the last two observations. The increase in unac-
counted nitrogen in the second and third stages for
these observations may be due to the leakage of
carbon from the ®rst stage at high loading rates
thereby helping T. pantotropha to survive and deni-
trify. The amount of ammonium-nitrogen removed
by assimilation was not accounted for as the maxi-
mum nitrogen removal by assimilation for co-cul-
ture of T. pantotropha and Nitrosomonas in a
chemostat experiment was reported to be 15% as a
% Nitrogen lost
stage 3 stage 1 overall
20.76 18.71 20.70
17.54 34.87 33.00
19.00 25.42 27.42
19.90 29.02 23.98
18.02 31.70 31.16
13.59 49.23 48.09
8.22 44.11 68.60
12.37 42.70 52.75
C:N ratio of 3.8 (Robertson and Kuenen, 1992). At
a lower C:N ratio of 3.2 used in our system and
considering the low suspended solids in the e‚uent,
the nitrogen lost in assimilation can be neglected.
The overall nitrogen removals of 44±63% due to a
simultaneous aerobic denitri®cation in the present
study further establish its superiority over conven-
tional ones.
The net alkalinity consumed per unit of ammo-
niacal nitrogen removed varied between 3.62 to
5.97. If the generation of alkalinity during denitri®-
cation is considered as equal to half of that
destroyed during nitri®cation then the above ratio
assumes values between 4.4 and 7.14 as shown in
Table 3. This re¯ects a saving in terms of chemicals
required for maintaining the desired alkalinity levels
in a separate nitri®cation unit.
The presence of T. pantotropha was con®rmed
qualitatively following the biochemical tests pre-
scribed for its identi®cation (Kuenen and
Robertson, 1989).
CONCLUSION
Based on the ®ndings of this study the following
conclusions can broadly be drawn:
(1) The ®xed ®lm system developed in the present
work could derive the speci®c advantages a€orded
by T. pantotropha in mixed biomass for nitrogen
removal.
(2) The high COD removal (5.8±14.1 g COD/
m2
d) and NH+ 4 -N removal (0.47±1.1 g N/m
d)
2
rates and a simultaneous aerobic denitri®cation in
the ®rst stage indicated the importance of the intro-
duction of T. pantotropha in the bio®lm.
(3) The second and third stages worked as auto-
trophic nitrifying reactors in the absence of organics
and hence the T. pantotropha.
(4) With a higher ®rst stage surface area, certain
changes in the ¯ow regulation, e.g. recirculation of
a part of e‚uent, a simultaneous two stage feeding,
the system may help in meeting e‚uent nitrogen
regulations without adding anything to the cost of
the presently employed combined carbon removal
and nitri®cation systems or with a little extra cost
to the systems designed for only carbon removal.
 Simultaneous carbon and nitrogen removal
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