Biochemical Engineering Journal 77 (2013) 214–219

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Performance of a hybrid activated sludge/biofilm process for

wastewater treatment in a cold climate region: Influence
of operating conditions
Daniele Di Trapani a,∗ , Magnus Christensson b , Michele Torregrossa a ,
Gaspare Viviani a , Hallvard Ødegaard c
a
Dipartimento di Ingegneria Civile, Ambientale, Aerospaziale, dei Materiali, Università degli Studi di Palermo, Viale delle Scienze, 90128 Palermo, Italy
b
Anoxkaldnes AB, Klosterängsvägen 11A, Lund, Sweden
c
NTNU – Norwegian University of Science and Technology, Department of Hydraulic and Environmental Engineering, 7491 Trondheim, Norway

a r t i c l e i n f o a b s t r a c t

Article history: The main aim of the study was to investigate a hybrid MBBR process, mostly in terms of organic matter
Received 21 March 2013 removal and nitrification, when operating with different values of the mixed liquor sludge retention time
Received in revised form 23 May 2013 (SRT), and highlighting the influence of temperature on the process. Based on experience in practice it
Accepted 15 June 2013
was hypothesized that nitrification could be maintained at far lower SRT’s than in conventional activated
Available online 24 June 2013
sludge systems and with high organic loading rates applied. A field gathering campaign has been carried
out on a hybrid activated sludge/biofilm. The obtained results highlighted that the pilot plant was capable
Keywords:
to remove the organic matter at loading rates up to 3.00 kg TCOD m−3 day−1 , also showing very high
Biofilm
Hybrid reactors
nitrification activity. Ammonia uptake rate (AUR) batch test showed that biofilm nitrification activity
MBBR increased when the mixed liquor SRT decreased. The final suggestion is that it is possible to run a hybrid
Nitrification reactor with low mixed liquor SRT values, as well as low temperatures, still having a high ammonium
Integrated Fixed-film Activated Sludge removal efficiency, since a large fraction of nitrification activity will take place in the biofilm.
Biological wastewater treatment © 2013 Elsevier B.V. All rights reserved.

1. Introduction
Secondary treatment of municipal wastewater is usually accom-
plished by biological processes that can be classified as being
either suspended or attached growth. The conventional activated
sludge (CAS) process can indeed present some shortcoming when
exposed to high hydraulic and organic loading rates. To increase
the performances of CAS systems it would be necessary building
new aerobic volumes or increasing the amount of biomass inside
the aerobic reactor. However, one of the major plant limitations
could be related to the clarifier capacity, since additional clarifier
volumes are usually difficult and expensive to obtain. As a con-
sequence, in the last years it was proposed to combine the two
different biomasses by introducing suspended carriers into the aer-
ation/anoxic tank for biofilm attachment and growth. This is often
referred to IFAS (Integrated Fixed-film Activated Sludge) process
[1–3].
One of the main advantages of IFAS processes is represented
by the increase of the total amount of biomass inside the sys-
tem, but without a significant increase of the solid load to the
∗ Corresponding author. Tel.: +39 091 23896552; fax: +39 091 6657749.
E-mail address: daniele.ditrapani@unipa.it (D. Di Trapani).
final settling tank. Indeed, the biofilm is naturally retained inside
the reactor, attached on the carrier elements, except the detached
portion, which usually is negligible compared to the mixed liquor
suspended solid (MLSS) concentration. Further, the high residence
time characterizing the biofilm enhances the development of a
nitrifying community [4,5] and therefore nitrification may be main-
tained throughout the winter, without the need of additional
volumes. Indeed, the high sludge retention time (SRT) values of
biofilm leads to a favorable environment for the growth of nitrifying
bacteria [1]. Consequently, an IFAS process can be a suitable alter-
native for biological nitrogen removal and as a cost-effect option
for retrofitting wastewater treatment plants (WWTPs) to sustain
nitrification under low temperatures [2].
In the last years many studies have been carried out on hybrid
systems in order to investigate the process efficiencies and to evalu-
ate different carrier media performances, in terms of organic carbon
and nitrogen removal [6–8]. Concerning the carrier media, it can be
fixed [1,9] or freely moving inside the reactor volume [10–13]. One
of the mostly used alternative is to couple the KaldnesTM moving
bed biofilm reactor (MBBR) process with a CAS system. In the MBBR
process the biofilm grows attached on small carrier elements that
are kept in constant motion throughout the entire volume of the
reactor [14,15]. The carriers are kept inside the reactor through a
sieve arrangement at the reactor outlet. Compared to fixed biofilm

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 D. Di Trapani et al. / Biochemical Engineering Journal 77 (2013) 214–219 215

Table 1 Table 2
Average operational conditions during the experimental periods. Average influent wastewater characteristics in the experimental periods.

Parameter Units 1st period 2nd period 3rd period 1st period 2nd period 3rd period

Duration days 55 50 40 TCOD [mg L−1 ] 214.50 ± 24.25 178.92 ± 68.80 205.88 ± 77.7
Influent flow L h−1 24.98 23.63 26.58 FCOD [mg L−1 ] 111.75 ± 34.62 88.31 ± 43.98 114.20 ± 55.28
RAS L h−1 29.23 29.25 28.30 CODsol [mg L−1 ] 91.41 ± 32.11 87.03 ± 47.07 76.65 ± 48.22
HRT h 3.47 3.83 3.26 BOD7 [mg L−1 ] 88.5 ± 81.60 85.00 ± 63.32 105.70 ± 61.25
SRT days 5.73 8.51 3.42 TN [mg L−1 ] 36.20 ± 9.06 33.73 ± 9.35 36.19 ± 7.90
MLSS mg TSS L−1 3.30 3.76 2.40 NH4 -N [mg L−1 ] 28.04 ± 8.55 21.41 ± 9.06 25.56 ± 7.24
F/M kg COD kgTSS−1 day−1 0.45 0.31 0.66 NO3 -N [mg L−1 ] 0.19 ± 0.16 0.59 ± 0.43 0.83 ± 0.30
DO mg O2 L−1 2.16–3.25 3.37–4.66 2.96–5.02 NO2 -N 0.19 ± 0.20 0.11 ± 0.07 0.17 ± 0.10
◦
T C 11.54 9.72 13.83 TP [mg L−1 ] 4.30 ± 1.33 4.66 ± 0.92 6.15 ± 0.97
pH – 7.25 7.15 7.05 TSS [mg L−1 ] 94.7 ± 16.11 70.03 ± 31.71 72.24 ± 34.19

carrier systems, one of the interesting advantages of hybrid reac-
tors with MBBR is the low head loss, no filter channeling and no
need of periodic backwashing [16]. When used in a hybrid process,
the carriers are kept in the whole of or part of the reactor volume,
depending on the desired level of the treatment. The main aim of
the present study was to investigate the performances of a hybrid
MBBR pilot plant, when operated at different SRT values and under
different temperatures, focusing in particular on the nitrification
process. Based on experience in practice it was hypothesized that,
with low SRT values, it would result in less nitrification taking place
in the mixed liquor and more on the biofilm and that nitrification
could be maintained at far lower SRT’s than in CAS systems, at low
temperature and with high organic loading rates applied. Ammo-
nia uptake rate (AUR) batch tests have been performed to evaluate
nitrification rate in both suspended and attached biomass in order
to evaluate their respective role in the nitrification process. In this
paper in particular, the results of an experimental campaign on a
hybrid MBBR pilot plant are presented and discussed.
2. Materials and methods
2.1. Pilot plant description
The pilot plant was built at the Department of Hydraulic and
Environmental Engineering of the Norwegian University of Science
and Technology (NTNU) in Trondheim, Norway, and was fed with
municipal wastewater subject to primary clarification. The pilot
plant consisted of three tanks, each of 30 L, and a final 65 L set-
tler; the layout scheme is represented in Fig. 1. The first and third
aerobic reactors (in the following respectively referred to AS1 and
AS2) were conventional pure suspended biomass systems, while
the second aerobic reactor (in the following referred to HYB) was
filled with the KaldnesTM K1 carriers, with a 60% filling fraction, cor-
responding to a net surface area in the HYB reactor of 300 m2 m−3 ;
for further details on the characteristics of the adopted carriers, the
reader is referred to the literature [12]. Mixing in the reactors was
provided by coarse-bubble aeration systems, while special sieve
arrangements were adopted to retain the carriers inside the HYB
reactor.
The municipal wastewater was pumped from the city sewer sys-
tem and stored into a load equalization basin, in order to secure a
quite constant pollutants concentration during the day. Further, the
wastewater was pumped into a primary settling tank from which
it was fed to the pilot plant. The overall field campaign, which
duration was nearly 150 days, was divided into three experimental
periods, each characterized by a different SRT value; in Table 1, the
average operational conditions in the three experimental periods
are reported.
In order to enhance biofilm development on carrier media,
the pilot plant was continuously fed for about one month before
starting the field campaign. No particular problems were experi-
enced during the experimental periods, except for some foaming
at the beginning of the start-up phase, completely disappeared after
almost one week of operations. In Table 2, the average influent
wastewater characteristics are reported.
2.2. Analytical methods
Grab samples were taken three times a week and analyzed for
total nitrogen (TN), ammonium nitrogen (NH4 -N), nitrite nitrogen
(NO2 -N), nitrate nitrogen (NO3 -N), total COD (TCOD), filtered COD
(FCOD), truly dissolved COD (CODsol ) and every working day for
total suspended solids (TSS) and volatile suspended solids (VSS).
All the analyses were carried out according to Standards Methods
[17] except CODsol that was analyzed according to the protocol pro-
posed by Mamaïs et al. [18]. Periodically, BOD7 was determined as
a control of the pilot plant behavior.
Periodic test on carrier samples were carried out, in order to
establish the biofilm growth on the carriers; briefly, a 20 carriers
sample was taken from HYB reactor, dried in a oven for one night
at 105 ◦ C and then weighted (W1 ). After biofilm was removed, the
carriers were dried another night at 105 ◦ C and then weighted again
(W2 ); the amount of the attached biomass was then calculated as
W1 − W2 .
Ammonium uptake rate (AUR) batch tests were performed to
evaluate the nitrification rates of both suspended biomass and
biofilm from the HYB reactor, by adopting a modified procedure
derived by Kristensen et al. [19]; for further details referring to
biofilm measurements as well as AUR test the reader is addressed
to the literature [12].
Sludge settleability was determined by the sludge volume index
(SVI) test, carried out on biomass samples taken from HYB reactor,
according to the protocol reported in the technical literature [20].
Microscopic observations were carried out for the evaluation
morphological floc characteristics as well as to observe the poten-
tial effects caused by carriers on floc structure. Observations were
made at 100× and 1000× magnifications.
3. Results and discussion
3.1. Organic carbon removal
The pilot plant showed high COD removal efficiencies through-
out the experimental campaign, even in the 2nd period,
characterized by the lowest temperature values, lower than 9 ◦ C
for many days and by lower pollutant concentrations, likely due
to the snow melting occurring in that period. Nevertheless, it has
to be stressed that the SRT variation did not affect in a consider-
able way the pilot plant behavior in terms of COD removal (total
and filtered), as well as of BOD7 . In Fig. 2 the COD loading rates
applied and removed are reported, referring to each experimen-
tal period. The bisector line indicates the 100% removal efficiency.
It can be noticed that the pilot plant was capable to remove the
substrate also at higher loading rates in terms of TCOD, up to
216 D. Di Trapani et al. / Biochemical Engineering Journal 77 (2013) 214–219

Primary Settling tank AS1 HYB AS2 Final Settling tank

Inlet Outlet

Waste sludge

RAS flow

Sludge
C
Air blower

Fig. 1. Configuration of the experimental pilot plant layout.

3.00 kg TCOD m−3 day−1 , and FCOD, up to 1.80 kg FCOD m−3 day−1 Table 3
Average ammonium loading rates applied, removal efficiencies and temperature
(Fig. 2a and b, respectively).
values.
During the whole experimental campaign a relationship
between FCOD loading rate and removal efficiency could be estab- Ammonium loading Ammonium T [◦ C]
rate [g NH4 -N day−1 ] removal [%]
lished, as shown in Fig. 3a, without any influence of the SRT, based
on the suspended biomass only. 1st period 16.80 81.19 11.54
From these results, the suggestion is to load this process at high 2nd period 12.15 83.89 9.72
3rd period 16.30 96.93 13.83
rates since there is still an efficient utilization of the inlet organic
substrate. Previous studies on hybrid reactors have showed anal-
ogous results [21]. This behavior confirms that the high process during the second period characterized by the lowest temperature
treatment capacity in terms of organic substrate could be useful in values. Indeed, compared to a CAS process, a hybrid system is
the up-grading of overloaded CAS plants, no longer able to guaran- less influenced by the temperature decrease, due to the fact that
tee adequate removal efficiencies [22]. The average FCOD removal the nitrification process in biofilm systems is strongly influenced
efficiencies were equal to 62%, 60% and 69% during the 1st, 2nd and by oxygen diffusion, which is related to oxygen concentra-
3rd period, respectively, where the major part was removed in AS1 tion in the bulk liquid, and oxygen solubility increases when
reactor, whereas the removal percentages in HYB and AS2 reactors the temperature decreases [7,14,23]; such aspect could likely
were lower, even if a dilution effect from the return activated sludge mask the natural reduction of nitrifiers biological activity due
(RAS) flow exists in AS1. In Fig. 3b the FCOD profile (referring to to decreasing temperatures. In Fig. 4, the influence that organic
average values) is shown. Indeed, in AS1 reactor, the carbonaceous (Fig. 4a) and ammonium (Fig. 4b) loading rates had on nitrification
substrate was non-limiting and consequently the organic removal efficiencies is reported. From Fig. 4, it can be pointed out that
rates were really high. both the organic and the ammonium loading rates exerted a
significant influence on the nitrification process, similarly to what
3.2. Ammonium removal observed in previous experiences with pure MBBR systems [24];
in particular, threshold values could be highlighted, over which
The average ammonium loading rates, removal efficiencies and the nitrification efficiency decreased abruptly. However, such
temperatures for the three experimental periods are reported in threshold values were different in the experimental periods; it
Table 3. The pilot plant was able to guarantee high ammonium is worth noting that in the 3rd period, characterized by a 3 days
removal throughout the whole experimental campaign, even SRT, it could be possible to sustain complete nitrification even at

(a) 3,50 (b) 2,00

Loading rate removed [kgTCOD m d ]

1st Period
Loading rate removed [kgFCOD m d ]
-1

-1

3,00 1st Period

2nd Period
-3

-3

3rd Period 1,50 2nd Period

2,50
3rd Period
2,00
1,00
1,50

1,00
0,50
0,50

0,00 0,00
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 0,00 0,50 1,00 1,50 2,00
-3 -1
Loading rate applied [kgTCOD m d ] Loading rate applied [kgFCOD m d ]
-3 -1

Fig. 2. TCOD (a) and FCOD (b) volumetric loading rates applied and removed (based on total volume).
 D. Di Trapani et al. / Biochemical Engineering Journal 77 (2013) 214–219 217

100 120
a b 1st Period
Period
90
100 2nd Period
Period
80 3rd Period
80

FCOD [mg L-1]

FCOD removal [%]

70

60 60

50
40
40 SRT 6
SRT 8 20
30
SRT 3
20 0
0,00 0,50 1,00 1,50 2,00 Influent AS1 HYB AS2

Organic loading rate [kgFCOD/m d]3 Stages

Fig. 3. FCOD removal efficiency vs FCOD loading rate at different SRT calculated on suspended biomass only (a) and FCOD profiles in the pilot plant compartments (b).

higher organic (2.50 kg TCOD m−3 day−1 ) as well as ammonium
(0.25 kg NH4 -N m−3 day−1 ) loading rates, compared to that of the
other periods. Such apparent unexpected result can be explained
by the temperature increase as well as by the biofilm growth
inside the system. Indeed, during the third period, temperature
values continuously increased, reaching values equal to 18 ◦ C in
the last experimental days, confirming the significant influence of
temperature on nitrifiers activity; furthermore, in this period the
biofilm biomass concentration (biofilm biomass divided by avail-
able surface) reached its maximum values (close to 20 gTS m−2 ),
thus exerting a decisive influence on nitrification, as better out-
lined in the following. Another interesting aspect which relates
nitrification efficiency with the organic loading rate is represented
by the increasing nitrite concentration with organic loading rate
applied to the system, as reported in Fig. 5. Indeed, as the organic
loading rate increased, it was observed a consequent increase of
nitrite effluent concentration, even over the threshold value of
0.6 mg NO2 -N L−1 , which represents the Italian discharge limit.
In particular, such threshold value was reached at an organic
loading rate almost equal to 1.50 kg TCOD m−3 day−1 ; previous
experiences on pure MBBR systems showed that at an applied
load of 0.6 kg TCOD m−3 day−1 nitrification efficiency decreased
remarkably and nitrite concentration rapidly increased over
0.6 mg NO2 -N L−1 [25]. Thus, the suggestion is that hybrid systems
are capable to treat higher organic loading rates with a reduced
nitrite accumulation inside the biological reactor.
3.3. AUR batch test

Several cycles of AUR test were performed in order to determine

the nitrification rates in both biofilm and suspended biomass sam-
ples from HYB reactor during the different experimental periods, in
order to evaluate how the mixed liquor SRT values could influence
the nitrification rates. The basic hypothesis was that decreasing the
mixed liquor SRT would result in more nitrification taking place
on the biofilm and that nitrification could be maintained at far
lower SRTs than in CAS systems. At the same time, nitrification
activity of suspended biomass, related to the biofilm presence, was
investigated. Since the AUR batch tests were run at the real water
temperature of the pilot plant, the results presented in Table 4 are
temperature corrected to a reference value of 10 ◦ C, using a tem-
perature coefficient of 1.06. By analysing the experimental data,
it can be noticed that an increase of the biofilm nitrification rate
occurred with the decrease of the mixed liquor SRT. The obtained

4,0

3,5

3,0
NO2-N [mgNO2-N L ]
-1

2,5

2,0

1,5

1,0

0,5

0,0
0,00 0,50 1,00 1,50 2,00 2,50 3,00
Organic loading rate [kgTCOD m-3 d-1]
Fig. 4. Nitrification efficiency vs organic (a) and ammonium (b) volumetric loading
rates, during the three experimental periods. Fig. 5. Effluent nitrite concentration vs applied organic loading rate.
218 D. Di Trapani et al. / Biochemical Engineering Journal 77 (2013) 214–219
Table 4
Nitrification rates for biofilm and suspended biomass corrected to 10 ◦ C.
1st period 2nd period
SRT [days] 5.73 8.51
Temperature [◦ C] 10 10
Dissolved oxygen [mg O2 L−1 ] 5.00 5.00
Biofilm nitrification rate 0.92 0.71
[g NH4 -N m−2 day−1 ]
Biofilm nitrification rate 0.93 0.72
[g NOx -N m−2 day−1 ]
AS nitrification rate 2.05 1.27
[mg NH4 -N g−1 VSS h−1 ]
values are well in the range of that ones derived in previous stud-
ies on hybrid reactors [7,26]. The observed nitrification rates were
coherent with what expected, confirming that nitrification can be
maintained at far lower SRTs than in CAS systems, since the biofilm
nitrification activity is quite high; furthermore, the biofilm nitrifi-
cation rates were in good agreement with what reported in other
investigations with IFAS systems [27,28].
On the other hand, the nitrification rates of the suspended
biomass showed a much higher nitrification activity than what
would be normal in a pure activated sludge reactor working under
the same operative conditions, particularly during the 3rd period.
This apparently unexpected result can be explained by the “seed-
ing” effect of nitrifiers from the biofilm to the mixed liquor, due
to biofilm detachment from the carriers that likely contributed to
enrich the mixed liquor by nitrifying population; such aspect was
confirmed in previous experimental investigations reported in the
technical literature [28]. Indeed, this aspect could be favored by
the adopted layout, which was chosen in order to avoid competition
between heterotrophic and autotrophic bacteria for the availability
of dissolved oxygen in the AS1 reactor, thus allowing a high organic
matter removal in such compartment, enhancing at the same time
the growth of autotrophs on biofilm in HYB reactor, and the con-
sequent “seeding” effect of nitrifiers from the biofilm to the mixed
liquor.
Furthermore, the high nitrification activity of suspended
biomass in the last experimental period could be also enhanced
by the increased temperatures which likely enhanced the nitrifiers
activity.
Based on AUR test results, the percentages of nitrification which
took place on biofilm and suspended biomass respectively were
evaluated, basing on nitrification rates at the actual water temper-
ature of the pilot plant and the ammonium loading rates removed
in the corresponding period. In detail, during the first period (SRT
6 days), the nitrification percentages were 60% for the biofilm and
40% for the activated sludge; in the second period (SRT 8 days),
the nitrification percentages were 55% for the biofilm and 45%
for the activated sludge, respectively. Finally, in the third period
(SRT 3 days), the nitrification percentages were quite similar for
the two biomasses, 52% for the biofilm and 48% for the suspended
biomass. Both in the first and second periods, the obtained results
highlighted the significant influence of biofilm on nitrification;
on the other hand, referring to the last experimental period, the
nitrification percentages confirmed the above mentioned explana-
tion about the biofilm “seeding” effect to the mixed liquor which
contributed to enhance the apparent suspended biomass nitrifica-
tion activity. Furthermore, with higher temperatures and thicker
biofilm, there should be a reduction in ammonium penetration
depth, thus reducing the contribution of biofilm to nitrification [28].
To conclude, the final suggestion is that a hybrid biofilm/activated
sludge process is characterized by a huge nitrification activity,
mainly related to the high residence time of the biofilm attached
on the suspended carriers.
350
300
3rd period
3.42 250
SVI [mL g-1TSS]
10
5.00 200
0.92
150
1.02
100
2.57
50
0
0 25 50 75 100 125 150
Time [day]
Fig. 6. Sludge volume index values during the overall experimental campaign.
3.4. Microbiological analysis and sludge settleability
Qualitative microscopic observations were carried out on mixed
liquor as well as on detached biofilm samples; they revealed at the
beginning of the experimental campaign (Period 1) good floc struc-
ture and a relative high number of higher life forms, such as sessile
ciliated colonial protozoa, amoebas, which presence could suggest
enough aeration and significant nitrification activity. However, in
the following experimental days, and in particular in Period 3, it was
noticed a reduction of superior forms in the mixed liquor samples,
coupled to a reduction (qualitative estimate) in floc size likely due
to the organic loading rate fluctuations and/or SRT reduction; on the
other hand, microscopic analysis carried out on detached biofilm
samples revealed a significant presence of microfauna, such as car-
nivorous swimming ciliates and ciliated protozoa, even in Period
3.
The characteristic of sludge samples could explain the worsen-
ing of sludge settleability during the experimental campaign, which
is clearly visible from the SVI values reported in Fig. 6. Indeed, the
reason for the high SVI could be explained by the high organic
loading rates compared to dissolved oxygen concentrations (in par-
ticular in the Period 3), which could enhance the worsening of
sludge characteristics. Such result is confirmed by the presence of
Vorticella microstoma, a sessile ciliate organism, which growth is
favored by high organic load and oxygen deficit conditions [29].
4. Conclusions
The main aim of the study was to investigate the performance of
a hybrid biofilm/activated sludge pilot plant operated at different
mixed liquor SRTs and temperatures. In terms of organic removal,
the pilot plant showed a good behavior during the overall exper-
imental campaign; the obtained results highlighted that the pilot
plant was capable to remove the substrate also at loading rates up to
3.00 kg TCOD m−3 day−1 and 1.80 kg FCOD m−3 day−1 , respectively.
It seemed that the SRT variation did not affect significantly the pilot
plant behavior in terms of COD removal (total and filtered).
Concerning the nitrification activity, the pilot plant showed
good removal efficiencies, except some days during the 2nd, char-
acterized by temperature values lower than 9 ◦ C. The suggestion
is that temperature plays a key role in the nitrification activity,
even if the increased oxygen solubility with low temperatures could
likely hinder the reduction of nitrifiers biological activity due to
decreasing temperatures. The highest nitrification performances
were recorded during the last period, characterized by 3 days SRT,
likely due to higher temperature values as well as to highest biofilm
concentrations. Furthermore, it was found out that the biofilm nitri-
fication activity increased when the mixed liquor SRT decreased,
thus confirming that nitrification could be maintained at far lower
 D. Di Trapani et al. / Biochemical Engineering Journal 77 (2013) 214–219 219

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