Journal of Biotechnology 126 (2006) 475 487
Start-up of the Anammox process in a membrane bioreactor
"
C. Trigo, J.L. Campos, J.M. Garrido , R. Méndez
Department of Chemical Engineering, Faculty of Engineering, University of Santiago de Compostela,
Avda Lope Gómez de Marzoa, E-15782 Santiago de Compostela, Galicia, Spain
Received 30 December 2005; received in revised form 20 April 2006; accepted 4 May 2006
Abstract
The start-up of an Anammox process was studied in a membrane sequencing batch reactor (MSBR) in which a submerged
hollow fibre membrane module was used to retain the biomass. The reactor was seed with Anammox biomass and fed using the
Van de Graaf medium. During a first operating stage, salt precipitation was observed and interfered with microbial activity and
caused a decrease of the nitrogen removal rate of the reactor from 100 to only 10 mg l-1 per day. Salt precipitation was avoided
by diminishing adequately the Ca and P concentrations of the Van de Graaf medium during the last operating stage. This action
increased quickly the activity of the system, and nitrogen removal rate reached up to 710 mg l-1 per day with almost full nitrite
removal. Sporadic flotation of the sludge was observed in the MSBR. The use of the membrane avoided biomass wash-out from
the system. Moreover, a surprising fact was that Anammox biomass did not grow in flocs in the MSBR, but in granules. This
fact showed that this kind of microorganisms have a trend to grow in aggregates. Results indicated that the use of the MSBR
could be a suitable system for nitrogen removal by using the Anammox reaction.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Anammox; Denitrification; Granule; Membrane; MSBR; Wastewater
1. Introduction bon, but it results expensive in those wastewaters with
low biodegradable matter and high nitrogen concen-
The removal of the nitrogen present both in munic- trations, such as, e.g. old landfill leachates or effluents
ipal and industrial wastewaters, mainly as ammonium, from the anaerobic digestion of sludge in wastewa-
is carried out conventionally by means of the combina- ter treatment plants (WWTP). In this case the addi-
tion of two biological processes, nitrification and deni- tion of an external organic matter source (methanol
trification. This procedure is suitable for the treatment or acetic acid) for the denitrification stage is neces-
of nitrogenous wastewaters rich in biodegradable car- sary in order to obtain the nitrogen removal. This
increases the operating costs in the wastewater treat-
ment plant due to the cost of the chemicals added
"
Corresponding author. Tel.: [phone]x16778;
and the treatment of the additional sludge that is
fax: [phone].
E-mail address: [e-mail] (J.M. Garrido). generated.
0168-1656/$  see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.05.008
476 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
An alternative, to these conventional processes, is The development of reactors using the Anammox
the combination of two biological processes: the par- process is still recent. The first Anammox reactors were
tial nitrification of ammonium to nitrite by means of biofilm reactors, e.g. fixed bed reactor, fluidised bed
nitrifying bacteria and the denitrification of nitrite to reactors and gas lift reactor (Van de Graaf et al., 1996;
dinitrogen gas by using ammonium as electron donor, Strous et al., 1997; Sliekers et al., 2003; Dapena-Mora
the Anammox process (Mulder et al., 1995; Van de et al., 2004a). However, some of these systems did not
Graaf et al., 1995). The Anammox process is a biolog- show as the most adequate to avoid the Anammox
ical mediated reaction in which ammonia is oxidized biomass wash-out. In order to improve the biomass
to nitrogen gas using nitrite as the electron acceptor retention and the stability process, the sequencing
under anaerobic conditions. In Eq. (1) the stoichiom- batch reactor (SBR) was successfully used to grow
etry of this reaction proposed by Strous et al. (1998) Anammox biomass (Strous et al., 1997, 1999; Dapena-
is shown, in which ammonium and nitrite, in almost Mora et al., 2004a). These reactors were operated with
equimolar ratio, react to produce dinitrogen gas and a an additional mechanical stirring in order to improve
small quantity of nitrate. the biomass retention and prevent the entrapment of
nitrogen bubbles, therefore increasing the stability of
NH4+ + 1.32NO2- + 0.066HCO3- + 0.13H+ the process. Other systems that were used with suc-
cess were a reactor containing non-woven media for
1.02N2 + 0.26NO3-
biomass immobilisation (Furukawa et al., 2003) and
+ 0.066CH2O0.5N0.15 + 2.03H2O (1) an upflow system seed with anaerobic granular sludge
(Imajo et al., 2004). However, a fraction of the gener-
The Anammox process should be combined with a pre- ated biomass is inevitably washed out with the effluent
vious partial nitrification stage, in which around 50% of in all these systems, especially during unstable peri-
ammonia should be converted to nitrite. This could be ods due in many cases to overloads, which provoke the
obtained either by manipulating the temperature and biomass flotation.
the HRT using the Sharon process (Hellinga et al., For these reasons, further investigation is needed
1998), by inhibition of nitrite oxidizing bacteria by free to increase biomass retention inside the reactor espe-
ammonia (Balmelle et al., 1992) or by manipulating the cially in those cases where the Anammox activity of the
dissolved oxygen concentration (Garrido et al., 1997). inoculum is very low. This is an important challenge
The Anammox process presents advantages for the in order to scale up Anammox systems from labora-
treatment of effluents with deficiency in organic mat- tory to industrial scale, in which the start-up could be
ter, compared to the nitrification denitrification pro- done using secondary sludge from WWTP that is even
cess. This process allows the reduction of the oxygen bioaugmented with the Anammox biomass generated
requirements and carbon dioxide emission to the atmo- in small scale laboratory units. In fact, there is only
sphere and less production of sludge in the WWTP. one reference of an industrial scale unit in Rotterdam
However, the practical application of the Anammox WWTP (Van Loosdrecht and Salem, 2005).
process is still limited by its long start-up periods due An alternative for obtaining full biomass retention
to the very low growth rates (0.072 d-1 measured at in Anammox systems might be the use of membrane
ć%
32 C) and biomass yield generated per ammonia nitro- biological reactors (MBR) for the treatment of the
gen consumed (0.088 g g-1) of these microorganisms wastewaters. In the last 20 years, membrane technol-
(Jetten et al., 1997). Moreover, an additional problem ogy has been utilized to promote biomass retention
is caused by loss of a fraction of the sludge washed out instead of secondary clarifiers in WWTPs. MBR sys-
with the effluent. For this reason, an efficient system or tems are compact reactors, which may operate with
operation strategy in order to avoid biomass wash-out high biomass concentrations and an absolute control of
with the effluent is required. To achieve high biomass solids and hydraulic retention times. Limitations inher-
retention it is very important during the start-up, which ent to MBR processes are the cost of membranes and
can take months or even a year in laboratory scale reac- operative costs due to fouling and higher energy con-
tors. Even a slight loss of biomass supposes a delay in sumption compared to traditional WWTPs. Since the
the time required to obtain the desired loading rate. MBR retains all organisms, it could ideally be suited
C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487 477
ć%
to slow growing cultures such as Anammox bacteria, at a fixed temperature of 35 C by means of ther-
enhancing the start-up of the process. The use of an mostated jacket. The mixture inside the reactor was
Anammox MBR was referred for first time by other achieved with a mechanical stirrer. Norprene tubing
authors (Wyffels et al., 2004). However, there is still a and connections were used in order to avoid the dif-
limited knowledge concerning the behaviour of Anam- fusion of oxygen. The hollow fibre membrane was
mox MBRs during the start-up, the factors that would provided by Zenon, Environmental Inc. The ultrafiltra-
inferred in the process, the kind of aggregates formed tion membrane module with a pore size of 0.04 mwas
by Anammox biomass, or the recovery of the capacity used to ensure the complete retention of the suspended
of these systems after episodes of nitrite build-up. The solids into the reactor. The small dimensions of the
aim of the present work is to present the results obtained reactor and the presence of a mechanical stirrer forced
in a membrane sequencing batch reactor (MSBR) used to design a module with the membranes arranged in
to promote the growth of Anammox biomass, which a circular configuration with the extremes joined to a
could be an alternative to other suspended or biofilm tube where permeate was collected.
systems. The MSBR was seeded at the operating day 0
with enriched anaerobic ammonium-oxidising granu-
lar sludge from a laboratory scale SBR (Dapena-Mora
et al., 2004b). The initial biomass concentration in
2. Materials and methods
the membrane bioreactor was 0.125 g l-1. The specific
Anammox activity of this biomass was 0.3 g g-1 per
2.1. Reactor
day.
In Fig. 1 is depicted the MSBR used during the
experiments, containing a submerged ultrafiltration 2.2. Feeding media and strategy of operation
hollow fibre membrane module. The system had a max-
imum working volume of 5 l. The reactor was operated The reactor was fed with three different synthetic
media (synthetic media 1, 2 and 3) during the three
main operating stages (stages 1, 2 and 3) of the research
(Table 1). The feeding synthetic medium 1 was the one
described by Van de Graaf et al. (1996) which is, at the
moment, the most used synthetic medium to operate
the Anammox process (Strous et al., 1997; Kuai and
Table 1
Composition of the three feeding synthetic media fed to the MSBR,
in mg l-1
Compound Synthetic Synthetic Synthetic
medium 1a medium 2 medium 3
(NH4)2SO4 75.3 283.4 66.4 79.7 66.4 1727
NaNO2 83.7 315.4 73.9 88.7 73.9 1823
NaNO3 8.5
KHCO3 1000
KH2PO4 50 10 10
CaCl2·2H2O 226 22.6 5.65
MgSO4·7H2O 58.6
FeSO4 6.25
EDTA 6.25
Trace elementsa 1.25 ml/l
Fig. 1. Scheme of the experimental system. (1) MSBR, (2) stirrer, Composition of all compounds in media 2 and 3 were the same as
(3) membrane module, (4) influent tank, (5) peristaltic pump of the synthetic medium 1, except for the concentrations of (NH4)2SO4,
feeding media, (6) permeate pump, (7) permeate tank and (8) pro- NaNO2, KH2PO4 and CaCl2·2H2O that are indicated.
a
grammable logic controller. Described by Van de Graaf et al. (1996).
478 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
Verstraete, 1998; Imajo et al., 2004; Dapena-Mora et formed using a scanning electron microscope (SEM;
al., 2004a). The other two synthetic media were based Leica 440). Elemental analysis of C, H, N and S of a
in the Van de Graaf et al. (1996) medium. The con- biomass sample was done by using Fisons EA-1108
centration of calcium salt was diminished from 226 to elemental analyser and O content by using Carlo Erba
22.6 and 5.65 mg l-1 in the synthetic medium 2 and 1108 elemental analyser.
synthetic medium 3, respectively, and the concentra- The content of Anammox bacteria in the biomass
tion of phosphorus was set at 10.0 mg l-1 for these two samples from the reactors was followed by fluores-
synthetic media. cence in situ hybridisation (FISH) (Amann, 1995).
The control of the SBR was carried out with a PLC This analysis was performed with a set of fluorescent-
system (CPU224, Siemens). The reactor was operated labelled 16S rRNA-targeted probes according to the
in cycles of 6 h (Dapena-Mora et al., 2004b). Each cycle procedure described by Amann (1995). Probes used
comprised four stages. During the first one, the reac- for FISH and the formamide concentrations used dur-
tor was continuously fed and mixed for 330 min. In the ing hybridisation were the mixture EUB 338I, EUBII
second stage, the stirrer was put off during 9 min allow- and EUBIII for all the eubacteria (Daims et al., 1999),
ing the biomass to settle. The third stage consisted of the probe PLA 46 for the Planctomycetales and the
the permeation of the supernatant liquid, which was probe Amx 820 for the Candidatus  Brocardia anam-
removed from the reactor by creating a membrane moxidans and Candidatus  Kuenenia stuttgartiensis
under-pressure with a peristaltic pump, during 18 min. (Strous et al., 1998) labelled with fluos and Cy3 flu-
Finally, in the fourth stage, part of the permeated liquid orochromes. For analysis of the slides, an epifluores-
was backwashed during 3 min to minimise the mem- cence microscope (Axioskop 2 plus, Zeiss) in combina-
brane fouling. The operational cycle of the reactor is tion with a digital camera (Coolsnap, Roper Scientific
detailed in Fig. 2. Photometrics) were used.
The operational strategy of the reactor consisted of Batch experiments to determine the specific anam-
increasing the nitrogen loading rate (NLR) applied to mox activity (SAA) were performed according to the
the reactor by means of increasing the ammonium and methodology described elsewhere (Dapena-Mora et
nitrite concentrations in the influent media, once the al., 2004a), based on the measurement along time of
nitrite concentration was close to 0 in the effluent. The the overpressure generated in closed vials by the nitro-
pH of the feeding medium was adjusted around 8.0 by gen gas produced.
means of H2SO4 (1 M) addition. The HRT was fixed at
1 day.
3. Results
2.3. Analytical methods
The MSBR was operated during 375 days. From the
Nitrate, nitrite and ammonium concentrations were obtained results (Fig. 3), the experimental period can
determined spectrophotometrically and biomass con- be divided in three different experimental stages, which
centrations were determined as volatile suspended were coincident with the periods in which the three
solids (VSS), according to Standard Methods (APHA, different synthetic media were fed: stage 1, from oper-
1985). The sludge volumetric index (SVI) was deter- ating day 0 till day 80, in which the original medium
mined according to Ramalho (1991). The elemental developed by Van de Graaf was fed; stage 2, from
analysis of the surface of biomass aggregates was per- operating day 80 till day 183, in which was used a
Fig. 2. Operational cycle strategy of the MSBR. The time length of each operating phase is indicated in the grey box (min).
C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487 479
Fig. 3. Evolution of the ammonium concentration in the influent ( )
and the effluent (©) and the nitrite concentration in the influent ( )
and the effluent ( ) along the operational period (mg N/l).
modified Van de Graaf medium with a lower concen-
tration of calcium and phosphorus; and stage 3, from
operating day 183 to the end of the experiments in
which another modified Van de Graaf medium with
a lower concentration of calcium than the two others
was fed. Results showed that the nitrogen removal rate
(NRR) of the MSBR diminished during the period 1
Fig. 4. Total nitrogen (©), ammonium ( ) and nitrite ( ) removal
and did not vary in period 2. NRR is expressed here
rates in the reactor during experimental stage 1 (A) and experimental
as the loss of nitrogen from ammonia and nitrite due
stage 3 (B).
to the Anammox reaction and is indicated in terms
of mass per unit of reactor volume and unit of time.
the reason of the loss of activity of the system, and the
During stage 3 NRR increased with time till a max-
stirring rate was reduced from 75 to 45 rpm on the oper-
imum value of 710 mg l-1 per day. Throughout the
ating day 29. However, the activity still diminished, in
operation, both the ammonium and nitrite concentra-
spite of diminishing the stirring speed in order to reduce
tions were gradually increased from 10 to 390 mg l-1,
shear stress on the biomass. Another cause that was
as the capacity of nitrogen removal of the system
considered as possibly responsible for the low activity
increased.
of the biomass was the presence of inhibitory concen-
trations of oxygen in the reactor. In order to prevent
3.1. Stage 1 (from day 0 till day 80) the presence of oxygen in the reactor, the reactor was
hermetically closed and argon gas was fluxed to avoid
During the first operating days, after the inoculation any accidental air entrance in the system. No improve-
the system showed almost full nitrite removal an even ment of the Anammox activity was observed, but even
near increase in the nitrogen removal rate (NRR) up to the nitrogen removal rate decreased until it reached a
100 mg l-1 per day. However, after operating day 10, minimum value of 5 mg l-1 per day, at around day 70.
NRR started to decrease (Fig. 4). The NRR obtained The seed was composed by small red colour gran-
on the day 18 was around 17 mg N l-1 per day. These ules, but biomass colour gradually changed to light
facts coincided with an important increase in the non- chestnut colour with time (Fig. 6) that occurred simul-
volatile suspended solids (NVSS) concentration from taneously with the observed NVSS accumulation. Cal-
50 to 150 mg l-1 and an increase of the percentage of cium phosphate precipitation was considered as a fea-
NVSS in the biomass from 26 to 49% (Fig. 5), as well sible reason of the observed increase in the NVSS
as with the breakage of the granules (Fig. 6). Due to concentration, and a cause of loss of biomass activity
this last fact, an excess of agitation was considered to be in the reactor during this period.
480 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
Fig. 5. Evolution of the total suspended solids ( ) and NVSS ( ) in the MSBR (primary y-axis) and NVSS percentage ( ) referred to TSS
during the experiment (secondary y-axis). Arrow points out the increase in the NVSS that took place along stage 1.
3.2. Stage 2 (from day 80 till day 183) system was a consequence of the corresponding bio-
logical mediated reaction.
In order to avoid the NVSS accumulation observed During this stage, an increase in biomass concentra-
in the previous stage, phosphorus and calcium concen- tion, in terms of volatile suspended solids concentration
trations in the feeding medium were diminished on (VSS), was observed from 0.2 to 0.4 g l-1. Despite
the operating day 80. The concentration of calcium the diminution of a 90 and 80% for Ca and P con-
salt was lowered 10 times (from 226 to 22.6 mg l-1) centrations, respectively, the NVSS concentration in
and the phosphorus salt one, 5 times (from 50 to the reactor increased achieving around 0.4 g l-1. The
10 mg l-1). The activity remained very low, 5 mg l-1 fraction of NVSS in the biomass did not vary and
per day, through this stage and did not vary during remained around 50% during the whole experimental
almost 100 experimental days. The diminution of the stage. Taking these data into account it was verified that
concentration of calcium and phosphorus in the influent the precipitation was still present, which indicated that
did not increase the capacity of the reactor. In order to the selected phosphorus and calcium concentrations in
determine whether there was still active biomass inside medium 2 were not the adequate ones in order to avoid
the reactor, a FISH assay was carried out. It could be salt precipitation in the system. For this reason, an ele-
appreciated that although in slow proportion, there was mental analysis of the surface and the microscopically
still active Anammox biomass. This indicated that the observation of the surface of the biomass was carried
residual anaerobic ammonia oxidation capacity of the out by means of SEM (Fig. 7A). Mass percentage of
Fig. 6. Evolution of the appearance of the Anammox biomass during the first operating days of stage 1.
C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487 481
centration of calcium in the feeding medium both the
activity and the nitrogen uptake of the system increased
quickly. During the first 45 operating days of this stage,
the nitrogen capacity of the system increased 5 times,
from 4.8 to 25.6 mg l-1 per day (Fig. 4). This fact was
observed again during the next 45 days, and NRR of
the system increased till 142.7 mg l-1 per day. A max-
imum NRR of 710 mg l-1 per day was obtained in this
system, 185 days after the second reduction in the cal-
cium concentration. The doubling time of Anammox
biomass in this system was estimated in 18 days. These
values were calculated as the experimental time needed
to double the NRR in the reactor during this stage.
Nitrite accumulation took place in the effluent
between the operating days 290 and 350. Concen-
trations around 10 and 20 mg l-1 in terms of nitrite
nitrogen were detected. This fact caused a partial inhi-
bition of the biomass. As a result, during this period
the nitrogen-loading rate was not increased in order to
avoid accumulations of higher nitrite concentrations.
Nitrite accumulation also was the cause of the spo-
radic sludge flotation events that were observed in the
reactor. In this respect, the use of a membrane avoided
the wash-out of the biomass with the effluent from the
system and the consequent loss of capacity of the sys-
tem that was referred for SBR and gas lift systems
during nitrite accumulation periods (Dapena-Mora et
al., 2004a,b). Nitrite accumulation was sorted out at
Fig. 7. Elemental analysis carried out with the SEM system, indi-
the operating day 355, when the feeding was stopped
cating the percentage composition by mass of the most abundant
during an operating cycle. This strategy make feasible
elements, of the surface of biomass samples during stage 2 at the
to diminish the concentration of both ammonium and
operating day 170 (A) and during stage 3 at the operating day 300
(B), respectively.
nitrite in the reactor. The diminution of the concentra-
tion of the inhibitory nitrite made feasible to recover
17.3% calcium and 7.8% phosphorus were detected in
the activity and the NRR of the reactor. In fact nitrite
the biomass surface using the SEM. The molar rela-
concentration after operating day 350 was almost fully
tionship between Ca and P was 1.71, which is close to
removed and its concentration was below 1 mg l-1.
that of 1.5 of calcium phosphate, this indicated that the
It was possible to avoid the precipitation of cal-
precipitates were formed by calcium salts, especially
cium phosphate, due to the second reduction of the
by the calcium phosphate salt.
concentration of calcium. This fact was demonstrated
not only by determining the concentration of NVSS
3.3. Stage 3 (from day 183 on) (Fig. 5) but also analysing chemically the surface of
the biomass. Fig. 7B shows clearly the reduction of P
At the beginning of this period, an additional reduc- and Ca percentage in the biomass surface with regard to
tion in the concentration of calcium salts in the medium those observed during period 2. Moreover, the concen-
was assayed. The concentration of calcium salt was tration of NVSS kept approximately constant around
reduced 75%, from 22.6 to 5.65 mg l-1 in order to avoid 0.2gl-1 indicating that no additional salt precipitation
or at least to reduce the precipitation observed in the took place. Additionally, biomass colour varied from
two previous stages. Few days after reducing the con- light chestnut to red colour and a mass percentage of
482 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
12.7% nitrogen was detected in the surface (Fig. 7B). It
was observed an increase in the concentration of VSS
from 0.27 to 0.96 g l-1. These facts cause a gradual
decrease of the percentage of NVSS in the biomass
from 53 till 18%.
Elemental analysis of whole biomass samples was
realised indicating that the molecular formula and
biomass yield (in terms of g of biomass per g of
ammonia) were CH2.04O0.49N0.16 and 0.070 g g-1,
respectively. These results were very similar to those
obtained by Strous et al. (1998) of CH2O0.5N0.15 and
0.088 g g-1. The stoichiometry of the reaction was also
determined, indicating that 1.22 mol of nitrite were
consumed and 0.22 mol of nitrate produced by mol of
ammonia consumed, which are lower than the values
of 1.32 and 0.26, respectively, indicated in Eq. (1). The
obtained production of nitrate per mol of ammonia was
similar to that referred to by Wyffels et al. (2004) of
0.20 in an Anammox MBR system. However, these
authors indicated a stoichiometry of only 1.05 mol of
nitrite consumed per mol of ammonia consumed.
FISH assays indicated that most of the active
biomass that grew in the MSBR was composed by
Anammox microorganisms. With regard to the activ-
ity of the sludge, the activity assays indicated a gradual
increase of SAA till values between 0.35 and 0.45 g g-1
per day, that are similar to those obtained by other
authors in an SBR system (Dapena-Mora et al., 2004a).
During the period in which nitrite accumulation took
Fig. 8. Photograph of granules of biomass (A) and microphotograph
place, between operating days 290 and 350, the activ-
of a granule obtained in the SEM (B), during the operating stage 3.
ity suffered a decrease till a value of 0.2 g g-1 per day,
that was a consequence of the partial inhibition of the
biomass by nitrite. Once the nitrite accumulation dis-
appeared, SAA value was recovered. 3.4. Membrane fouling
One surprising fact in the MBR was the aspect and
settling properties of the sludge. The value of the SVI An additional objective of this research was to study
gradually decreased during 73 operating days of stage the effect of the biological process on the behaviour
3 from around 125 till 60 ml g-1, which indicates an of the membrane. In this sense to study the behaviour
increase of the biomass density. Moreover, biomass of the membrane permeability during the operation
growth did not occur as suspended biomass but in gran- and its fouling was very important. The monitoring
ules with an irregular cauliflower appearance (Fig. 8A of the membrane fouling was made by following the
and B). The former fact was similar to those results evolution of the transmembrane pressure. The foul-
observed by Dapena-Mora et al. (2004b) in a pre- ing of the membrane module was very low during the
vious research done with a SBR Anammox system. whole experimental period. Membrane fouling control
They observed that SVI changed from 108 to 63 ml g-1 involved only one method that was backwashing with
after around 80 operating days. However, these permeate after the end of the permeation cycle. Due to
authors indicated that the biomass grew as flocculent the characteristics of the reactor, neither coarse bubble
sludge. gasification at the bottom of the membranes nor the
C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487 483
utilization of chemicals were applied. Moreover, the The influent concentrations and the NRR treated in
external chemical washing of the membrane was nec- this study are comparable to those previously obtained
essary only after approximately 3 months of operation. by Dapena-Mora et al. (2004b) and by Van Dongen
This probably was a consequence of the characteristics et al. (2001) of 600 and 700 mg l-1 per day, respec-
of the solids retained in the reactor, a mixture of NVSS tively, in SBR systems. Moreover, NRR was in between
and biomass that tended to grow in granules with low the values of 650 and 1100 mg l-1 per day referred by
fouling capacity. Transmembrane pressure increased Wyffels et al. (2004) in an Anammox MBR system
progressively during every permeation period, proba- fed with pre-filtered reject water from the dewater-
bly as result of the deposition of an outer layer of solids ing of digested sludge in a wastewater treatment plant.
on the membrane. Backwashing had a positive effect Values of NRR higher than those of the MSBR were
to remove solids deposition, since the value of trans- referred by Dapena-Mora et al. (2004a) and by Strous
membrane pressure at the beginning of each cycle was et al. (1997) of 2000 and 1800 mg l-1 per day, respec-
recovered, this being around 32 kPa. Once the maxi- tively, in fluidised bed systems. Futhermore, Fux et al.
mum transmembrane pressure attained a value of 60 (2002) obtained NRR of 1800 mg l-1 per day in an
or 70 kPa at the end of the permeation period, the SBR. However, it is important to indicate that these sys-
membrane was replaced by another and was externally tems underwent many problems of stability in periods
cleaned with commercial sodium hypochlorite, diluted of overload, provoked both clogging of dinitrogen gas
to a concentration of 250 mg l-1. and sludge flotation. These problems of stability could
be prevented by means of the use of membrane biore-
actors, since the wash-out would be avoided owing to
4. Discussion
the presence of the membranes. Consequently, lower
times to recover the activity of the system would be
4.1. Application of membrane bioreactors to the
necessary. Furthermore, it is important to point out
operation of the anammox process
that the operation of these systems was carried out
The operation of the MSBR was unsuccessful dur- with high concentrations of biomass, in many cases
ing the first two stages, but successful during the inoculated from other systems, which made feasible
third. The maximum NRR reached during stage 3 was to achieve high NRR since the reactor s start-up. In the
approximately of 700 mg l-1 per day. The average total fluidised bed reactor, concentrations of biomass of 10 g
nitrogen removal efficiency was of 73.6% in stage 3. VSS/l would be necessary to reach NRR of 1500 mg l-1
Nevertheless, this was a consequence of the use of an per day. Nevertheless, in the MSBR it was possible
operation strategy, in which ammonia nitrite ratio was to remove 700 mg l-1 per day with less than 2 g l-1
higher than the ratio suggested by stoichiometry. The biomass.
kinetic and stoichiometric parameters of the Anammox Granule formation has been referred for both aero-
process, found in the membrane system are similar bic (Beun et al., 1999) and anaerobic systems (Hulshoff
to those found in other reactors. The stoichiometric Pol et al., 2004). Granule formation in aerobic sys-
parameters found, nitrate produced to ammonia con- tems depends in different parameters as e.g. loading
sumed and nitrite consumed to ammonia consumed rate, shear stress, oxygen concentration. However the
were 0.22 and 1.22 mol mol-1, respectively, similar most important selection mechanism to promote gran-
to those found by Strous et al., 1999. FISH analysis ulation is hydraulics, especially when settling is used to
showed the presence of Anammox microorganisms in promote the separation of the biomass and the treated
the sludge. These facts demonstrate that the process water. Microorganisms in granules have higher settling
was carried out by Anammox microorganisms. On the rates than those growing in dispersed form or in flocs
other hand, the use of a membrane did not shorten the than are more sensitive to be washed out with the efflu-
period of time that is necessary to operate at high NLR. ent. For the case of the anaerobic systems as UASB
Moreover, the doubling time observed in the MSBR reactors, granulation has been also based on the selec-
was 18 days which was similar to 19 days obtained by tive wash-out of dispersed sludge or the retention of
Dapena-Mora et al. (2004b) in a SBR, but higher than biomass in, resulting in an increased growth of retained
the 11 days referred by Strous et al. (1999). heavier sludge agglomerates.
484 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
Table 2
On the other hand, Cicek et al. (1999) has shown
Ca to P ratio (g g-1), pH value measured in the reactor and pH that
that in systems with submerged membranes, biomass
may cause phosphate precipitation throughout the three different
tended to grow in suspension instead of in granules.
operating stages
These researchers justify this because of the better
Stage Ca/P Reactor pH Precipitation pH Fact
access to the substrate by the biomass and the lesser
1 5.45 7.8 8.7 <8.0 Salt precipitation
problems of diffusion than those of the granules. In this
favoured
respect, formation of granular biomass in the MSBR
2 2.71 8.0 8.7 From 8.5 to 9.0 Salt precipitation
is an unexpected result of this research. Anammox
favoured
granule formation was previously referred by other
3 0.68 7.8 8.4 9.0 No precipitation
authors in an upflow sludge blanket system (Imajo et
al., 2004) and gas lift system (Dapena-Mora et al.,
inert material, either because the feeding medium con-
2004a), in which the selective wash-out of dispersed
tains these inorganic compounds or these precipitate
biomass favoured the growth of granules. However,
in the reactor. The effect of accumulation of inorganic
from our results, it seems that other mechanisms, not
material in a biological reactor with membranes may be
the hydraulic, would be responsible for granule forma-
very different. Moreover, it is not too clear the conse-
tion for Anammox biomass. Fernández et al. (2006),
quences that to the microorganisms will have the total
operating a SBR with flocculent Anammox biomass,
retention of this inert material in the system. This can
observed a change in the aspect of the sludge from
be especially outstanding in systems with low grow-
flocculent to granular when they added 5 g l-1 NaCl.
ing microorganisms, such as Anammox biomass, since
The floc formation indicated by other authors could be
the inorganic material may be, in some way, an obsta-
influenced by an interference of the granulation for-
cle to the correct development of the microorganisms
mation by other processes as solids precipitation, or
(Wagner and Rosenwinkel, 2000).
by the same definition of what a granule is and what
The reasons of calcium and phosphorus salts precip-
a floc is. In this respect, granule formation by Anam-
itation during the first two operating stages can be stated
mox biomass could be a consequence of an intrinsic
from the work of Song et al. (2002). These authors indi-
tendency of these microorganisms to grow into aggre-
cated that calcium phosphate precipitation relies on the
gates (biofilms or granules).
pH value and the Ca to P ratio of the water. In fact the
The growth of biomass as granules might be very
optimum pH at which precipitation occurs depended
positive for promoting the use of membrane Anam-
on the Ca to P ratio of the water as suggested below:
mox reactors. This grow could be a reason of the low
membrane fouling observed in our system. Granular
" optimum pH value for precipitation higher than 9 if
biomass could have better conditions of filterability
Ca/P ratio is 1.67;
than flocculent biomass. The low SVI of 60 ml g-1
" optimum pH value for precipitation higher than 8.5
VSS implies that it could be able to retain biomass
if Ca/P ratio is 3.33;
concentrations of up to 16 g l-1, which implies that the
" optimum pH value for precipitation higher than 8 if
maximum NRR that could be achieved in this system
Ca/P is 5.00;
would be limited to 5000 or 6000 mg l-1 per day.
" optimum pH value for precipitation higher than 7.5
if Ca/P is 6.67.
4.2. Precipitation of calcium phosphate salts
The evolution of the pH value throughout the oper-
As was above-mentioned, the system lost its activity ation of the reactor is shown in Table 2. It can be
owing to the precipitation of calcium phosphate salts in concluded that during the first two stages precipitation
the mixed liquor, when the synthetic medium described occurred due to the pH and Ca to P ratio used, as the
by Van de Graaf et al. (1996) was used. Rosenberger et conditions were optimum for precipitation. However,
al. (2000) indicated that the utilization of membranes during the operating stage 3 the operating conditions,
for the treatment of wastewater with high cellular reten- in which Ca to P ratio was 0.68 and pH was between
tion times can lead to the accumulation of non-volatile 7.8 and 8.4, did not favour precipitation. These facts are
material into the reactor, partly by the accumulation of according to the evolution of NVSS and the results of
C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487 485
the elementary analysis of the surface of the biomass, activity and caused a decrease of the nitrogen removal
during the three experimental stages. rate (NRR) of the reactor from 100 to only 10 mg l-1
The Van de Graaf et al. (1996) medium that caused per day during operating stage 1. Experimental results
the formation of precipitates in the MSBR, was used have shown that the precipitation was a result of the use
successfully during the operation of other Anammox of the Van de Graaf et al. medium (1996) in the MSBR.
reactors fed with synthetic media (Strous et al., 1997; The membrane acted as a barrier that may retain the
Kuai and Verstraete, 1998; Imajo et al., 2004; Dapena- inorganic precipitation nuclei more efficiently than in
Mora et al., 2004a,b). Dapena-Mora et al. (2004b) conventional SBR and other biological systems, caus-
referred NRR up to 600 mg l-1 per day in an Anam- ing the accumulation of around 50% NVSS in the
mox SBR system operating with an inorganic solids biomass. Modification of the Ca and P concentration
content between 15 and 25% TSS. Moreover, we have of this medium were necessary to avoid precipitation.
also detected the presence of a high fraction of cal- The nitrogen loading rate could be increased during
cium phosphate salts in the Anammox SBRs fed with stage 3, by avoiding salt precipitation in the system,
the Van der Graaf medium in our laboratory (data not and nitrogen removal rate was up to 710 mg l-1 per
published). In the present study inorganic solids con- day. NVSS percentage in the biomass diminished dur-
tent was higher, up to 50% TSS. A possible reason of ing period 3 till 18%. On the other hand, the use of a
the higher accumulation of NVSS in the MBR system membrane did not shorten the period of time that is nec-
could be the presence of the membrane. This bar- essary to obtain a system operating at high NLR. In this
rier may retain the inorganic precipitation nuclei more respect, this could be explained because the doubling
efficiently than in conventional SBR and other bio- time observed in the MSBR of 18 days was similar to
logical systems, causing the accumulation of a higher that observed by other authors as SBRs and biofilm
inorganic solids percentage in the system that cov- systems subjected to biomass losses with the effluent.
ered the biomass surface. Salt precipitation interfered Moreover, the stoichiometric parameters of the reac-
with microbial activity and caused a decrease of the tion found in the membrane system were similar to
NRR of the reactor from 100 to only 10 mg l-1 per those found in the bibliography.
day observed during the operating stages 1, and the The MSBR could be a suitable system for nitro-
low NRR of around 5 mg l-1 per day obtained during gen removal using Anammox biomass. Either biomass
stage 2. wash-out or contact with air were avoided by the
Salt precipitation might limit the use of MSBR sys- use of the membrane. The system maintained a good
tems for some wastewaters. Nevertheless, it could be activity even during periods in which a little amount
feasible to perform a pre-treatment stage of the water of nitrite accumulated and sporadic sludge buoyancy
to avoid the possible precipitation in the MSBR. Some was detected. Nitrite accumulation was removed by
authors also recommended for membrane systems stopping the feeding during an operating cycle. In
operational strategies based on low purges of biomass fact nitrite concentration after that action was below
in order to avoid accumulation of inorganic material 1mgl-1.
(Rosenberger et al., 2000; Wagner and Rosenwinkel, The behaviour of the aspect and settling properties
2000). In this sense, a feasible solution for Anammox of the sludge were good. The value of the SVI decreased
MSBR treatment could be to promote simultaneous during stage 3 from around 125 to 60 ml g-1 VSS, indi-
precipitation of salts in the previous partial nitrification cating an increase of the biomass density. Moreover,
stage to nitrite that is carried out before the Anammox biomass growth did not occur as suspended biomass but
reactor. in granules with an irregular cauliflower appearance.
This could be a result of the tendency of Anammox
microorganisms to grow into biofilms or granules.
5. Conclusions The growth of biomass as granules can be very posi-
tive membrane reactors. This growth could be a reason
The operation of the MSBR was unstable during the of the low membrane fouling observed in our system.
first two stages due to salts precipitation. Salts precipi- Granular biomass could have better conditions of fil-
tation on the biomass surface interferes with microbial terability than flocculent biomass.
486 C. Trigo et al. / Journal of Biotechnology 126 (2006) 475 487
Acknowledgements Garrido, J.M., van Benthum, W.A.J., van Loosdrecht, M.C.M., Hei-
jnen, J.J., 1997. Influence of dissolved oxygen concentration
on nitrite accumulation in a biofilm airlift suspension reactor.
To the Spanish Ministry of Science (CTQ2005-
Biotechnol. Bioeng. 53, 168 178.
04935) and the Xunta de Galicia through the GRAFAN
Hellinga, C., Schellen, A.A.J.C., Mulder, J.W., Van Loosdrecht,
project (PGIDIT04TAM26500PR). Authors also want
M.C.M., Heijnen, J.J., 1998. The SHARON process: an inno-
to thank the Zenon, Environmental Inc. for the kind vative method for nitrogen removal from ammonium-rich waste
water. Water Sci. Technol. 37, 135 142.
supply of the membrane fibres.
Hulshoff Pol, L.W., de Castro Lopes, S.I., Lettinga, G., Lens, P.N.L.,
2004. Anaerobic sludge granulation. Water Res. 38, 1376
1389.
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