Journal of Membrane Science 286 (2006) 202–212

Effect of oxygen concentration on biological nitrification and

microbial kinetics in a cross-flow membrane bioreactor (MBR) and
moving-bed biofilm reactor (MBBR) treating old landfill leachate
Roberto Canziani a,∗ , Valeria Emondi b , Massimiliano Garavaglia a ,
Francesca Malpei a , Eleonora Pasinetti b , Gianluigi Buttiglieri a
a Politecnico di Milano, Environmental Section, Piazza Leonardo da Vinci 32, I-20133 Milan, Italy
b SIAD S.p.A., Laboratory of Environmental Biology and Chemistry, Via Pasubio 5, 24040 Dalmine–Bergamo, Italy
Received 14 November 2005; received in revised form 22 September 2006; accepted 26 September 2006
Available online 7 October 2006

Abstract
Ammonium nitrogen concentration in leachate from old Italian landfills ranges from 0.5 to as high as 3 g L−1 . In this paper biological nitrogen
removal from leachate has been achieved by partial nitrification to nitrite in a pure-oxygen membrane bioreactor (PO-MBR) and by subsequent
denitrification in a moving-bed biofilm reactor (MBBR). When ammonium is biologically oxidized to nitrite, only 75% of the oxygen required
for full nitrification is needed. Moreover, denitrification can be performed by saving 30–40% of the carbon required. The process was carried out
by an MBR oxidation tank of 500 L equipped with an UF ceramic membrane followed by a 540-L post-denitrification tank filled with moving
plastic support media. The best operational conditions to achieve partial nitrification were analyzed. TKN loading rate was variable from 50 to
120 g TKN (kg TSS day)−1 with an influent ammonia concentration between 1000 and 1500 mg L−1 . When DO concentration in the MBR was
kept in the range 0.2–0.5 mg L−1 , 90% oxidation of ammonia to nitrite was achieved, with stable inhibition of nitrite-oxidizing bacteria even at
sludge retention time higher than 45 days.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Pure oxygen membrane bioreactor (PO-MBR); Landfill leachate treatment; Carbon and nitrogen removal; Partial nitrification; Stable inhibition of
nitrite-oxidizing bacteria

1. Introduction brane separation and evaporation processes. As a matter of fact,

Treatment of landfill leachate can be performed by chemical–
physical processes such as ultrafiltration and reverse osmosis
[1–3], evaporation (thermal compression, single or multiple-
stage, thin film or vacuum reactors) [4,5] or chemical and photo-
chemical oxidation [6–11]. Combined treatment processes have
also been proposed in which chemical oxidation is followed
by physical treatments, such as membrane separation or evap-
oration or biological ones [12,13]. Finally, technical literature
provides examples of electrochemical leachate oxidation [14]
which may be followed by a biological process [15]. How-
ever, high ammonium concentration, which is typical in landfill
leachate, can reduce the purification efficiency in both mem-
∗ Corresponding author. Tel.: +39 02 23996410; fax: +39 02 23996499.
E-mail address: roberto.canziani@polimi.it (R. Canziani).
even multi-stage reverse osmosis can not remove ammonium
under permit limits for discharge into natural waters or even
into sewers when influent concentrations are higher than 1 g L−1
[16]. Moreover, ammonium is concentrated in the brine and this
makes it difficult to perform inertization processes successfully,
such as immobilization into concrete. More often, ammonia
stripping is usually performed as a pre-treatment stage, at an
additional cost; however, the high operational pH (>11, [17])
requires further neutralization and this increases salinity.
Biological aerobic or anoxic treatment processes are much
simpler and cheaper than a sequence of combined chemical–
physical treatments. However, they could not achieve high and
reliable ammonium and COD removal efficiencies [18]. Mem-
brane bioreactors (MBR) can be operated at very long sludge
ages and can extend greatly the field of application of biolog-
ical processes for concentrated streams, such as leachate [19],
particularly for the biological removal of nitrogen and of recal-

0376-7388/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2006.09.044
 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212 203

Table 1
Kinetic coefficients at T = 20 ◦ C
Coefficient Definition NH4 -oxididation NO2 -oxidation

μmax Maximum specific growth rate 0.77 day−1 1.08 day−1

kSH Half-velocity constant for the unionized substrate 2.8 × 10−2 mg NH3 -N L−1 3.2 × 10−5 mg HNO2 -N L−1
kIH Inhibition coefficient for the unionized substrate 540 mg NH3 -N L−1 0.26 mg HNO2 -N L−1
kO2 Oxygen saturation coefficient 0.3 mg O2 L−1 1.1 mg O2 L−1

citrant COD. Biomass can be kept in the system regardless of where:

its ability to settle. Under these conditions, slow-growing bac-
terial species can develop and much time is also left to degrade
refractory compounds.
The nitrification process is well known (e.g.: [20,21]), ammo-
nia is converted to nitrite and then nitrite is converted to nitrate:
ammonia oxidizing bacteria
NH4 + + 23 O2 → NO2 − + H2 O + 2H+
nitrite oxidizing bacteria
NO2 − + 21 O2 → NO3 −
From the stoichiometry of the reactions it can be seen that
2 mol of oxygen are required for the complete oxidation of 1 mol
of ammonia to nitrate. However, when nitrification is stopped to
nitrite, only 1.5 mol of oxygen per mol of ammonia are required
by the oxidation process with a 25% oxygen saving.
Heterotrophic reduction of nitrite, moreover, requires less
organic substrate. As an example, the stoichiometry of catabolic
heterotrophic nitrates or nitrites denitrification, with methanol
as a carbon source is:
nitrates removal process
6NO3 − + 5CH3 OH + CO2 → 3N2 + 6HCO3 − + 7H2 O
nitrites removal process
6NO2 − + 3CH3 OH + CO2 → 3N2 + 6HCO3 − + 3H2 O
Therefore, denitrification starting from nitrite rather than
nitrate may yield 40% savings of methanol. Partial ammonia
oxidation is achieved by favouring the growth of ammonia-
oxidizing bacteria over nitrite-oxidizing bacteria. The main
parameters involved are temperature, DO and pH(a) [20–22]:
ammonia oxidizing bacteria
[NH4 -N] × 10pH
[NH3 -N] = (3)
e6344/(273+T )
[NO2 − -N] × 10−pH
[HNO2 -N] = (4)
e−2300/(273+T )
Indexes: AOB refers to ammonia oxidizing bacteria; NOB refers
to nitrite oxidizing bacteria.
Table 1 summarizes kinetic coefficients for ammonia and
nitrite oxidation. All coefficients are temperature dependent.
These relationships are not considered here but may be found
elsewhere [21].
Eqs. (1)–(4) indicate that the dependence of the kinetics from
temperature and pH is different for the two bacterial groups.
Ammonia-oxidizing bacteria show higher growth rates than
nitrite oxidizing bacteria at high temperatures (35–40 ◦ C, [23]).
Moreover, ammonia oxidizers are less affected by low oxygen
concentration than nitrite oxidizers. The optimal pH values are
between 7.9 and 8.2 for ammonia oxidizers and between 7.2 and
7.6 for nitrite oxidizers [24].
These ranges are due to:
• nutritional limitation related to alkalinity;
• free ammonia and nitrous acid inhibition.
Free ammonia is reported to be a much stronger inhibitor for
nitrite oxidizers (0.1–1.0 mg NH3 L−1 , [25]) than for ammonia
oxidizers (10–150 mg NH3 L−1 , [26]). In this study a term KI
for ammonia inhibition has been added to the Eq. (2a) and cal-
ibrated using experimental data. Therefore, expression (2a) can
be written as follows:
μNOB = μmax NOB

μAOB = μmax [HNO2 -N]

AOB ×
[NH3 -N] KSH NOB + [HNO2 -N] + ([HNO2 -N] /KIH
2
NOB )
× [O2 ] KI
KSH AOB + [NH 3 -N] + ([NH3 -N]2 /KIH AOB ) × (2b)
[O2 ] KO2 NOB + [O2 ] KI + [NH3 -N]
× (1)
KO2 AOB + [O2 ]
Complete biological conversion of highly concentrated
nitrite oxidizing bacteria ammonium to nitrite has been achieved in the SHARON pro-
cess at particular T, pH and DO. This process operates as a
μNOB = μmax NOB completely stirred tank reactor (CSTR) with a short SRT (about
[HNO2 -N] 1–1.5 days) and achieves partial ammonia oxidation by means of
× the wash-out of nitrite oxidizing bacteria, which grow at slower
KSH NOB + [HNO 2 -N] + ([HNO2 -N] /KIH
2
NOB )
rates under the selected environmental conditions [25–28]. A
[O2 ] membrane bio-reactor with high sludge retention time (SRT)
× (2a)
KO2 NOB + [O2 ] has been also used to achieve partial conversion of ammo-
204 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212

Table 2
BOD5 , ammonium nitrogen (N-NH4 + ) and COD concentrations in raw leachate
COD (mg L−1 ) BOD5 (mg L−1 ) N-NH4 + (mg L−1 ) BOD5 /COD (◦ ) N-NH4 + /COD (◦ )

Average 6,316 2,950 1,497 0.43 0.32

Standard deviation 5,877 3,537 1,190 0.12 0.20
Minimum 247 99 215 0.17 0.12
Maximum 25,600 13,900 4,130 0.76 0.98

Number of data = 70 (from January 2001 to October 2003); (◦ ) average of the single ratios.

Fig. 1. Schematic flow-sheet of the pilot plant.

nium to nitrite as a pre-treatment of an Anammox process The pilot plant included a 500-L stainless steel tank for bio-
[29]. logical oxidation.
The aim of this research was to achieve complete conversion The membrane module was a tubular zirconium ceramic
of ammonium to nitrite in an MBR with high sludge retention MEMBRALOX® model P19-40 (Table 3).
time (SRT more than 45 days) and by controlling T, DO and pH. The membrane was operated at 1.5 × 105 Pa (1.5 bar). The
chemical cleaning procedure, which was performed only twice
2. Material and methods during the experimental period (days 306 and 406), was per-
formed according to the instructions provided by the producer.
2.1. Leachate Commercial membrane cleaners (Ultrasil, both acid and alka-
line solutions) were used. In the first washing the sequence was
The leachate used in the experimentation came from an old
as follows: (i) alkaline solution (NaOH, 1.5%), (ii) acid solution
municipal solid waste (MSW) sanitary landfill located in North-
(Ultrasil 0.5%) and (iii) NaClO solution (1.5%). Between each
ern Italy. Table 2 shows BOD5 , ammonium nitrogen (N-NH4 + )
washing step, the membrane was rinsed with tap water. In the
and COD concentrations in the raw leachate (in mg L−1 ). Con-
second washing, the sequence was different: at first NaClO (5%)
centrations are highly variable even eight years after closure.
was used, followed by an acid wash at pH 2 and a final alkaline
This variability, as well as the high variations in BOD5 /COD and
wash at pH 12.3. Again, tap water was used for rinsing after
N-NH4 + /COD ratios, is not due to biochemical processes inside
each washing step.
the mass of wastes, but to the collection systems of leachate:
Mixed liquor was pumped at a pressure of 4 × 105 Pa (4 bar)
in fact subsurface drainage is active only during wet weather
into the external ceramic membrane module at a flow rate of
periods, and deep wells are active during dry periods.
4.25 m3 h−1 , which corresponded to a cross-flow velocity of
The biodegradable fraction in the leachate fed to the pilot
4.94 m s−1 . Mixing was ensured by recirculating the retentate
plant was even lower, as it came from an anaerobic fixed-bed
biofilter, used as a pre-treatment prior to discharge into a munic-
ipal wastewater treatment plant (MWWTP). Table 3
Membrane technical data
2.2. Pilot plant
Parameter Value Unit
The flow-sheet of the pilot plant is reported in Fig. 1. Leachate Number of tubes 19 –
was stocked in a 1000 L tank and kept mixed by a recirculat- Tube diameter 4 mm
ing pump (Q = 1.8–4.8 m3 h−1 , Pmax = 4 × 105 Pa (4 bar)) and a Length 1020 mm
hydraulic ejector. The recirculating pump was also used to feed Pore size 50 nm
Total cross-sectional area 2.4 × 10−4 m2
the pilot plant, by opening an actuated solenoid valve for a fixed Filtering area 0.24 m2
time.
 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212 205

Table 4
Average characteristics of the influent during the experimental runs A, B and C (data in mg L−1 )
Run (days) DO TSS Total solids (TS) Total volatile solids (TVS) CODin N NH4, in + TKNin

A: 328–396 <0.5 5,920 14,441 10,120 3,818 1,254 1,360

B1 : 397–406 >1.5 5,860 18,785 12,560 4,224 1,547 1,628
B2 : 407–439 >1.5 5,177 17,213 11,683 3,765 1,603 1,703
C: 440–455 <0.5 7,891 21,010 15,500 3,900 1,470 1,561

flow from the ceramic membrane into the bioreactor. Pure liq-
uid oxygen was kept in a pressurized tank and was fed to the
oxidation tank through a solenoid valve which was automatically
actuated and controlled by an oxygen probe that was immersed
in the oxidation tank; pH control in the oxidation-nitrification
tank was performed by dosing a sodium hydroxide solution.
Before process start-up, it was observed that temperature
increased in the MBR tank, due to the energy dissipated by
the recirculating pump; therefore a cooling hydraulic circuit
was installed, to maintain a constant temperature in the process
reactor.
The permeate came to an overflow tank and then was pumped
by a peristaltic pump to the post-denitrification tank (540 L
volume). Filtrate in excess was recirculated into the oxidation
tank. As much as 37.5% of the denitrification tank volume was
filled with floating plastic media that were used as supports for
biofilm growth (ANOX KALDNES MBBR® carrier K1). Mix-
ing was ensured by a mechanical mixer. The external carbon
source for post-denitrification was provided by dosing a hydro-
alcoholic mixture named BITOLEA® Liquifeed 25, a mixture
of alcohols with 1–4 carbon atoms (methanol to butanol), with
methanol less than 20% and COD content of 415,000 mg O2 L−1
(7.2–12 Lmixture added /kg Ntreated ).
2.3. Analytical methods
Determinations of TKN, nitrate, nitrite, COD, total sus-
pended solids (TSS), total solids (TS) were performed according
to Standard Methods, 20th Ed. [30]. Phosphate was determined
by spectrophotometry using Dr. Lange procedure and kit. Bac-
teria quantification and identification were performed by plate
counts according to the Italian Standard Methods for Sludge [31]
and by optical microscopic observation [32,33].
concentration in the leachate was near 200 mg L−1 and was mea-
sured only occasionally. Total and volatile solids were measured
regularly; inorganic solids were over 1 g L−1 , mostly dissolved.
Average phosphorus concentration in the leachate was always
lower than required as nutrient for biomass growth. There-
fore, phosphoric acid was continuously added to the influent, in
order to maintain a concentration of phosphorus in the permeate
>5 mg P L−1 . The ratio BOD5 /COD in the influent was around
1:3. pH adjustment in the MBR by addition of a 0.2 M sodium
hydroxide solution was required since alkalinity of the leachate
was not sufficient to buffer the acidity produced by nitrification.
Temperature of the mixed liquor was always kept between
30 and 37 ◦ C and pH between 7.8 and 8.2 in order to enhance
the growth of ammonium oxidizing bacteria.
During the first experimental run (run A, days 328–396), dis-
solved oxygen was kept low (0.2–0.5 mg L−1 ) and free ammonia
was kept high (about 40 mg NH3 L−1 ) in order to study the
inhibitory effect on nitrite-oxidizing biomass.
After day 396, run B, the oxygen concentration was increased
to 1.5 mg L−1 and above in order to test the inhibitory effect on
the biomass caused only by high free ammonia concentration.
At day 406 an accidental biomass chlorination during mem-
brane cleaning obliged to decrease for a few days the TKN sludge
loading rate to recover bacterial growth (run B1 before and run
B2 after chlorination).
After day 440, during the last experimental run (run C), DO
concentration was decreased again between 0.2 and 0.5 mg L−1
and free ammonia concentration was maintained at about
2–5 mg NH3 L−1 .
3. Results and discussion
3.1. Start-up

2.4. Influent data and operating experimental conditions The process of partial nitrification was started up after 328
days of conventional full nitrification (ammonium to nitrate)
Influent data and operating parameters during the experimen- [34]. At the start-up of the new process, the MBR sludge con-
tal programme are listed in Tables 4 and 5. Suspended solids centration was as high as 10 g TSS L−1 .

Table 5
Average operating parameters
Run (days) Flow rate O2 Sludge loading rate MLTSS T (◦ C) pH Free NH3
(L h−1 ) (mg L−1 ) (mg L−1 ) (mg L−1 )
kg COD (kg TSS−1 day−1 ) g TKN (kg TSS−1 day−1 )

A: 328–396 6.9 <0.5 0.258 90.4 5,920 33 7.8 42.6

B1: 397–406 5.1 >1.5 0.195 76.4 5,860 32.1 8 0.1
B2: 407–439 3.5 >1.5 0.147 66.5 5,177 32.6 8 23.6
C: 440–455 6 <0.5 0.156 63.6 7,891 33.6 7.9 3.3
206 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212

Fig. 2. COD specific loading rate (F/M, left axis) and removal efficiency (right
axis).

3.2. COD removal

COD load and removal efficiency are shown in Figs. 2 and 3.

It can be seen that low oxygen concentration did not deteriorate
the removal efficiency, which levelled out around 45%. This
trend seemed to be more related to the low BOD5 /COD value
of the influent, which was as low as 0.30 in the last 2 months of
the experimentation.

3.3. Inhibition of nitrite oxidizers

From day 358, after an acclimation period of about 30 days,

a sharp and stable nitrite build-up was observed. Starting from
day 354 an increase of nitrite concentration was observed and, at
the same time, nitrate concentration decreased from more than
1500 to less than 50 mg L−1 (Fig. 4a).
This was initially ascribed to inhibition of nitrite oxidizers
(see later) due to dissolved oxygen limitation (at values as low
as 0.2–0.5 mg L−1 ) and to high free ammonia in the reactor (up
to 40 mg NH3 L−1 , Fig. 4b), which was due to the high TKN
loading (up to 120 g TKN kg TSS−1 day−1 ) and to the initial
low efficiency of TKN oxidation (Fig. 4c). However, the TKN
oxidation efficiency rapidly increased and kept steadily over
80% after 40 days from start-up, even at peak loads. At the
same time, nitrite were always the dominant oxidized nitrogen
form.
Fig. 3. COD removal efficiency (ηCOD) at different dissolved oxygen concen-
tration in the MBR. Oxygen concentration was <0.5 mg L−1 during period A
and C and >1.5 mg L−1 during period B.
Fig. 4. (a) NO2 -N and NO3 -N concentrations during experimental run A (days
328–396); (b) free NH3 and NH4 + -N during experimental run A; (c) TKN spe-
cific loading rate and removal efficiency during experimental run A.
Experimental run B1 started on day 397, when dissolved
oxygen concentration was increased (the lower limit was set
to 1.5 mg L−1 ). In the following 10 days, a constant increase
of the NO3 -N to NO2 -N ratio was observed (Fig. 5a). Due to
membrane cleaning, an accidental chlorination caused a partial
inhibition of both nitrifying populations.
However, in run B2 ammonium oxidizers recovered much
quicker than nitrite oxidizers. This event confirmed selective
inhibition of nitrite oxidizers due to chlorine compounds as
observed by Peng et al., 2004 [35].
To quickly recover the biological activity, the loading rate
was lowered from around 100 g TKN kg TSS−1 day−1 to around
30 g TKN kg TSS−1 day−1 for a few days and then gradually
increased to previous values. This lead to a quick increase of
the nitrate to nitrite ratio after day 420. Starting from day 440
(run C), oxygen was reduced again (the average DO concentra-
tion was between 0 and 0.5 mg L−1 ) and the loading rate was
increased to 65 g TKN kg TSS−1 day−1 . In these new operat-
ing conditions ammonium increased (as well as free ammonia,
Fig. 5b) and a rapid decrease of the nitrate to nitrite ratio
was observed; at the same time the concentration of NO3 -N
decreased from 300 to around 100 mg L−1 (Fig. 5c).
 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212 207

Fig. 5. (a) NO3 -N to NO2 -N ratio in the permeate. Dotted lines: changes in
dissolved oxygen control; grey area: accidental chlorination; (b) free ammo-
nia (right axis) and ammonium nitrogen (left axis) in the MBR after lowering
the dissolved oxygen to below 0.5 mg L−1 (day 440); (c) nitrate nitrogen vs.
dissolved oxygen in the MBR.

3.4. Process kinetics

3.4.1. Heterotrophs
The decay constant of the heterotrophic bacteria (kd ) was Fig. 6. Regression of (TSS/TSScumulated ) vs. time during: (a) periods A, (b)
period B1 , (c1 ) period B2 , (c2 ) final period B2 ; during B2 , the effect of the
estimated by measuring the decrease of suspended solids in the chlorination is evident.
MBR after stopping the feed for 8 days. It resulted as low as
0.03 day−1 at a reactor temperature of 25 ◦ C and pH 7.9. In spite
suspended solids grown in the reactor and the corresponding
of applying the lowest correction coefficient for temperature
COD removed (Fig. 6). The actual yield coefficient (Y) has been
reported by [36], i.e. θ T = 1.03, the value of the decay rate at
calculated by taking into account the decay rate (kd ) as follows:
20 ◦ C (0.026 day−1 ) resulted well below the lower limit of the
range reported in the literature (0.06–0.2 day−1 , [36]).
The observed yield coefficient of heterotrophic bacteria (Yobs ) kd
Yobs · ν = Y · ν − kd or Y = Yobs +
has been calculated as the slope of the regression between the ν
208 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212

Table 6
Observed (Yobs ) and net (Y) yield coefficients during the three experimental phases
Period (days) Yobs (g TSS T (◦ C) kd,T COD loading rate (kg COD COD removal ␯ (kg COD kg Y (g TSS g
g COD−1 ) (day−1 ) kg TSS−1 day−1 ) efficiency TSS−1 day−1 ) COD−1 )

A: 328–396 0.168 33.0 0.03808 0.253 0.577 0.146 0.429

B: 397–439a 0.189 32.1 0.03714 0.159 0.449 0.071 0.712
C: 440–452 0.210 33.3 0.03841 0.150 0.388 0.058 0.870
Average 0.189 32.8 0.03788 0.187 0.488 0.092 0.670
St. dev. 0.021 0.62 0.00066 0.057 0.095 0.048 0.223
a Data corresponding to the low loading period after biomass chlorination were not considered (days 407–430).

Table 7
Theoretical and experimental growth rates of heterotrophic bacteria during the
three experimental periods
Period (days) μobs (day−1 ) μ (day−1 ) μcalc (day−1 )

A: 328–396 0.0239 0.062 0.063

B: 397–439a 0.0073 0.044 0.051
C: 440–452 0.0125 0.051 0.050
Average 0.014 0.051 0.054
St. dev. 0.006 0.006 0.007
a Data corresponding to the low loading period after biomass chlorination

were not considered (days 407–430).

Fig. 7. Regression of (TSS/TSScumulated ) vs. time during period C (low oxygen
as in period A).
where “ν” is the daily COD removal rate per unit mass of sus-
pended solids (kg CODrem kg TSS−1 day−1 ). The observed and
actual yield coefficients calculated for the three experimental
periods are reported in Table 6. The VSS/TSS ratio of the mixed
liquor was fairly constant during the experimental runs (run A:
0.72 ± 0.05; run B: 0.68 ± 0.02; run C: 0.73 ± 0.05).
The observed growth coefficient (μobs ) has been calculated
during the three periods as the slope of the regression of
(TSS/TSScumulated ) versus time (Figs. 6 and 7).
The observed growth rate includes the decay rate:
μobs = μ − kd and, therefore: μ = μobs + kd .
The growth rate can also be calculated (μcalc ) from the actual
removal rate (ν) and growth yield (Y) as μcalc = Y · ν. In Table 7
values of μobs , μ and μcalc are reported.
Theoretical and calculated estimations of growth rates agree
very well either in period A or in period C, thus confirming the
consistency of the experimental trials. As the experimental peri-
ods were short if compared with SRT, it is not possible to state
that steady-state conditions were achieved. However, removal
efficiency was stable and this allows to state that stable process
conditions were achieved (Fig. 4, period A). The same does not
apply to period B, which was affected by an accidental chlori-
nation. It can be observed that low-oxygen conditions did not
affect negatively the growth of heterotrophs.
3.4.2. Autotrophs
Theoretical growth rates (μ) for autotrophic organisms have
been estimated from Eqs. (1) to (4) by adopting a μMAX value of
0.77 day−1 for ammonium oxidizers and 1.08 day−1 for nitrite
oxidizers [21]. Results of these calculations are summarized in
Table 8 and show that the growth rate of ammonium oxidizers
is always higher than that of nitrite oxidizers, confirming the
experimental observations (e.g.: NO3 -N/NO2 -N ratio, Fig. 5a).
In particular, low oxygen concentration increases the difference
between the two growth rates, as it has also been observed by
others [29].
As observed by Wyffels et al. [29], nitrate can be kept at low
concentration in the reactor by lowering the dissolved oxygen
concentration. Also, dissolved oxygen was shown to be more
important than temperature and free ammonia as nitrate con-
troller. This assumption is confirmed by the results obtained in
the present work: in fact, Wyffels et al. performed partial nitri-
tation as the preceding step of an ANAMMOX process and free
ammonia concentration in their reactor was much higher than in
the experiments presented here.

Table 8
Theoretical growth rates (μ) for autotrophic organisms
Period (days) No. of data Ammonium oxidizers μNH (day−1 ) No. of data Nitrite oxidizers μNO (day−1 )

A: 328–396 19 0.445 ± 0.030 21 0.312 ± 0.023

B1 : 397–406a 2 0.624 ± 0.012 5 0.559 ± 0.022
B2 : 430–439 1 0.639 3 0.575 ± 0.017
C: 440–452 5 0.456 ± 0.106 8 0.306 ± 0.970
a Data corresponding to the low loading period after biomass chlorination were not considered (days 407–430).
 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212 209

Fig. 8. Sludge flocs in the MBR (200×, 400×, 1000×).

Fig. 9. (a) Left: Opercularia sp.; (b) Right: H. Hydrossys bridging between sludge flocs (1000×).

3.5. Microscopy observation and bacterial counts
Optical microscopic observation showed that sludge aggre-
gates were generally smaller than 20 ␮m with few zoogleal
flocs around 50–100 ␮m (Fig. 8). Sessile ciliate Protozoa (Oper-
cularia sp.) were always present at a concentration around
4 × 104 L−1 (Fig. 9a). These organisms are very resistant even
in industrial wastewater treatment plants because of their strong
resistance to sub-optimal environmental conditions.
After the operation was switched from full to partial nitri-
fication, the abundance of filamentous organisms, mainly H.
hydrossis, increased. Since sludge separation was operated by a
membrane, their presence did not affect the performance of the
process.
The evolution of the main bacterial populations in the MBR
has been monitored with classical methods (CFU: colony form-
ing units; MPN: most probable number) providing a result in
terms of single bacterial units even if they could represent bac-
terial aggregates, especially in the case of nitrifying organisms.
As a matter of fact several clusters have been observed, as shown
in Fig. 8 above.
The results of the counts are shown in Table 9. What mat-
ters is that the total bacterial count did not show any substantial
difference among the different experimental periods. On the con-
trary, the ratio of ammonium oxidizers to nitrite oxidizers passed
from around 1:6 (full nitrification) to around 2000–4000:1 after
switching to the partial nitrification mode of operation.
The ratio of the two populations was estimated by MPN
(Table 9).
Also, the ratio of the two populations has been calculated as
the ratio between their net growth, by assuming that the system
was at steady state:
XNH YAOB (NH4 -N/(μAOB − kD ))
=
XNO YNOB (NO3 -N/(μNOB − kD ))
where μNOB were calculated according to formula (2b).
Results are shown in Table 10 and Fig. 10:
The great discrepancy between the two series of results can be
explained by the error of the MPN method. This method, in fact,
can be affected by an error of one order of magnitude or more
when applied to river water samples and even higher values can
be expected when this method is applied to samples of mixed

Table 9
Evolution of the bacterial counts over the experimental periods
Period Sample taken on day no. Total bacterial count (CFU mL−1 ) AOB (MPN mL−1 ) NOB (MPN mL−1 ) AOB/NOB

Full nitrification 313 6.4E+08 1,500,000 9,500,000 0.16

Period A 369 1.0E+06 1,500,000 4,500 333
384 2.8E+07 950,000 200 4,750
398 8.4E+07 950,000 450 2,111
Period B 432 2.0E+08 450,000 250 1,800
Period C 453 8.0E+07 250,000 950 263

Legend: CFU: colony forming units; AOB: ammonium oxidizers; NOB: nitrite oxidizers.
210 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212

Table 10
Calculated and measured ratios between AOB and NOB populations (KI = 20 mg L−1 )
Experimental period μAOB (day−1 ) μNOB (day−1 ) Ratio XAOB /XNOB Notes

Calculated Measured (MPN)

233–328 0.625 0.555 2.96 1.6E−01 Full nitrification (ammonia to nitrate)

358–369 0.450 0.129 16.54 3.3E+02 Lower oxygen (0–0.5 mg L−1 )
370–383 0.468 0.192 25.02 2.5E+03
384–396 0.474 0.256 42.43 3.4E+03
397–405 0.632 0.395 31.66 2.1E+03 Higher oxygen (0.5–1.5 mg L−1 )
420–433 0.582 0.275 18.97 1.8E+03
432–456 0.477 0.277 15.72 2.6E+02 Low oxygen (0–0.5 mg L−1 )

Fig. 12. Specific flux related to total suspended solids (TSS) in the aerated tank.

the regression was not statistically significant if no inhibition was

Fig. 10. AOB/NOB ratios assuming NOB growth rate is inhibited by free ammo-
nia (KI = 20 mg NH3 -N L−1 ).
liquor. As a result, the ratio of two MPN measurements can be
affected by an error of a factor of 100 or more. Fig. 10 refers to
the fit between the two ratios at a KI value of 20 mg NH3 -N L−1 .
However, what matters here is to check whether a correla-
tion exists between the two series and to which extent (if any)
ammonia inhibited the growth of NOB. The inhibition constant
of ammonia (KI ) for estimating μNOB has been taken as the sole
fitting parameter, ranging from zero to infinity.
It has been tested whether a statistically significant regression
could be found between the two series of results, and which
was the value of the inhibition constant which gave the best
regression. The regression of the two series yielded the best R2 by
assuming an ammonia inhibiting constant KI = 18 mg L−1 , while
assumed (Fig. 11).
3.6. Specific flux
Specific flux varied over a large range (from 15 to
120 × 10−5 L h−1 m−2 Pa−1 (15 to 120 L h−1 m−2 bar−1 )) dur-
ing the whole experimental period, including the previous con-
ventional nitrification phase and, as depicted in Fig. 12, it gen-
erally decreased as TSS concentration increased.
It is interesting to observe the outcome of cleaning at
day 406, and of the related accidental chlorination (Fig. 13).
After a first short recovery of the flux, there was a rapid and
marked decrease from 80 to 40 × 10−5 L h−1 m−2 Pa−1 (80 to
40 L h−1 m−2 bar−1 ). This was not investigated in detail and
more experimental trials should be needed. However, it might

Fig. 11. The regression with the highest R2 was found at an inhibition constant Fig. 13. Specific flux related to total suspended solids (TSS) in the aerated tank.
for free ammonia KI = 18 mg L−1 . Effect of chlorination after cleaning at day 406.
 R. Canziani et al. / Journal of Membrane Science 286 (2006) 202–212 211

be hypothesized that Extracellular Polymeric Substances (EPS)

concentration has increased due to the bacterial stress [37]. In
MBR membrane bioreactor
fact the content of EPS is suggested as a probable index for mem-
MPN most probable number
brane fouling in activated sludge MBR systems as the higher the
MSW municipal solid waste
content of EPS, the greater the membrane fouling [38].
MWWTP municipal wastewater treatment plant
NOB nitrite oxidizing bacteria
4. Conclusions
PO-MBR pure-oxygen membrane bioreactor
SHARON single reactor for high activity removal over
Compared to conventional nitrification, the process tested
nitrite
during this experimentation (ammonia oxidation stopped to
SRT sludge retention time (days)
nitrite) allows up to 25% savings in oxygen demand and up
T temperature (◦ C)
to 40% in COD demand for denitrification.
TKN total Kjehldahl nitrogen (mg L−1 )
The process tested during this experimental programme was
TS total solids (mg L−1 )
very different from the SHARON process because sludge reten-
TSS total suspended solids (mg L−1 )
tion time (SRT) was not a control parameter. Typical SRT values
UF ultrafiltration
for SHARON process are 1–11 days [28,29], while here SRT was
VSS volatile suspended solids (mg L−1 )
always kept higher than 45 days.
XNH /XNO ratio of ammonia and nitrite oxidizing bacteria
This work evidenced that dissolved oxygen concentration
Y actual yield coefficient (g TSS g COD−1 )
should be kept lower than 0.5 mg L−1 in order to achieve a
YOBS observed yield coefficient (g TSS g COD−1 )
stable inhibition of nitrite-oxidizing organisms. This leads to
high nitrite accumulation even at high sludge retention time. Greek letters
Temperature higher than 30 ◦ C and free ammonia concentration ηCOD COD removal efficiency (%)
higher than 2.5 mg L−1 enhance this effect, but dissolved oxygen μ theoretical growth rates (day−1 )
remains the main controlling factor. Under these conditions it μCALC calculated growth coefficient (day−1 )
was possible to oxidize more than 95% of influent TKN to nitrite μmax maximum specific growth rate (day−1 )
at effluent ammonium nitrogen concentration always lower than μOBS observed growth coefficient (day−1 )
50 mg L−1 . ν COD removal rate per unit mass of suspended
solids (kg CODrem kg TSS−1 day−1 )
Acknowledgement θ␶ correction coefficient for temperature

The authors wish to thank SOGEIVA S.p.A. (Varese, Italy)

for hosting and partially funding the experimental programme.
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