Environ Sci Pollut Res (2018) 25:1243–1256
https://doi.org/10.1007/s11356-017-0448-6
RESEARCH ARTICLE
Strict anaerobic side-stream reactor: effect of the sludge
interchange ratio on sludge reduction in a biological nutrient
removal process
Roberta Ferrentino 1 & Michela Langone 1 & Roberta Villa 1 & Gianni Andreottola 1
Received: 10 April 2017 / Accepted: 9 October 2017 / Published online: 30 October 2017
# Springer-Verlag GmbH Germany 2017, corrected publication November/2017
Abstract In order to investigate the impact of the sludge inter-
change ratio (IR) on both the sludge reduction process and the
carbon and nutrient removal efficiencies, an anaerobic side-
stream reactor (ASSR) at 20 °C and − 400 mV was operated
for 300 days coupled to a sequencing batch reactor (SBR) for
urban wastewater treatment. It was found that a 100% inter-
change rate, corresponding to an anaerobic solid retention time
(SRTASSR) of 2.5 days, was the most suitable case in terms of
sludge reduction and wastewater treatment process, achieving a
66% sludge reduction compared to a control system simulated
as an SBR. Chemical oxygen demand (COD), ammonium ni-
trogen, total nitrogen, and phosphate removal efficiencies of
86.1 ± 7.2, 82.5 ± 11.2, 81.7 ± 12.0, and 62.6 ± 15.0%, respec-
tively, were achieved. When the interchange rate was increased,
more ammonium nitrogen and soluble extracellular polymeric
substance concentrations were released in the ASSR. This im-
plies that cell lysis and hydrolysis of particulate organic matter
The correct Fig. 1 is shown in this paper.
The original version of this article was revised.
Responsible editor: Gerald Thouand
* Roberta Ferrentino
roberta.ferrentino@unitn.it; ferrentino.roberta@gmail.com
Michela Langone
michela.langone@unitn.it
Roberta Villa
roberta.villa@unitn.it
Gianni Andreottola
gianni.andreottola@unitn.it
1
Department of Civil, Environmental and Mechanical Engineering,
University of Trento, Via Mesiano 77, 38123 Trento, Italy
in the ASSR were processes of fundamental importance with the
increasing mass of sludge cycled to the ASSR. Compared to the
release of ammonia, soluble COD release was detected to a
lesser extent, due to its consumption by microorganisms in the
ASSR. There was also a simultaneous increase in slow-growing
microorganisms which use organic carbon for metabolic activ-
ities, above all sulfate-reducing bacteria and denitrifying
phosphate-accumulating organisms. This increase contributed
significantly to sludge reduction in the SBR–ASSR system.
Keywords ASSR . Cell lysis . EPS destruction .
Phosphate-accumulating organisms . Sludge interchange
ratio . Sludge reduction
Introduction
Conventional activated sludge (CAS) treatment is the most
commonly used biological process for urban wastewaters.
However, this technology produces excess sludge accounting
for about 25–65% of total operational costs in a wastewater
treatment plant (WWTP) (Sid et al. 2017). Consequently, new
technologies are being investigated to reduce the amount of
sludge in WWTPs.
There are many techniques for sludge reduction. However,
several are costly (Pérez-Elvira et al. 2006), and others nega-
tively affect the effluent quality of the process due to the forma-
tion of by-products, such as the oxidation with strong oxidants
(Foladori et al. 2010). Biological treatments are a challenging
strategy for sludge reduction in WWTPs (Wei et al. 2003).
An effective way of reducing sludge is to deploy an anaero-
bic side-stream reactor (ASSR) in the sludge return line of a
CAS system, where a portion of, or in some cases, all the acti-
vated sludge can be recycled (Chon et al. 2011b; Semblante
et al. 2014). The process consists of an activated sludge cycling
1244
between aerobic, anoxic, and anaerobic conditions. The rate of
solids that pass through the ASSR from the mainstream reactor
is usually expressed as a percentage per day of the total mass
present in the water line, and is known as the interchange rate
(IR). Many configurations and schemes have been proposed,
different with each other for configurations, operative
conditions, and design parameters, as reviewed by Ferrentino
et al. (2016b), Semblante et al. (2014) and Ragazzi et al. (2015).
Chudoba et al. (1992) were the first to create an oxic settling
anaerobic (OSA) process that treated organic pollutants from
synthetic wastewater. The OSA is composed of a modified ac-
tivated sludge system with the anaerobic treatment of all the
returned sludge (CAS–OSA). Chudoba et al. (1992) showed
that the periodic passage of facultative aerobic activated sludge
microorganisms through the anaerobic zone (T = 18–20 °C;
IR = 100%; anaerobic sludge retention time (SRTASSR) = 3 h)
created uncoupled growth, due to the anaerobic starvation, indi-
cated by the adenosine triphosphate (ATP) stock depletion, and
resulted in a 40% reduction of the activated sludge production.
Chen et al. (2003) argued that the sludge decay mechanism
in the ASSR (T = 20 °C; IR = 100%; SRTASSR = 11, 15,
17 days) could be the major cause of the reduction of the cell
mass, when oxidation-reduction potential (ORP) levels lower
than − 100 mV are achieved. Saby et al. (2003) investigated
the importance of ORP levels in the ASSR (T = 20 °C;
IR = 100%; SRTASSR = 11, 15, 17 days). The authors reported
that operating at an ORP level of − 250 mV could reduce
the excess sludge by 51% as compared to a control CAS
process. However, there is no clear ORP threshold. In fact,
there have been studies with ASSRs conducted both in anoxic
and anaerobic conditions (Ye et al. 2008; Saby et al. 2003;
Torregrossa et al. 2012).
Compared with a CAS system with aerobic or anaerobic
digesters performed at a 10-day SRT, Chon et al. (2011a)
observed a solid reduction of about 40–50% less in a CAS
system with an ASSR (T = 19 ± 1.5 °C; IR = 10%;
SRTASSR = 10 days). Furthermore, Khursheed et al. (2015)
observed that increasing the ratio of sludge exposed to
anaerobic and aerobic conditions may lead to higher levels
of sludge reduction.
Novak et al. (2007) deployed an ASSR (T n.a.; IR = 4–7%;
SRTASSR = 10 days) in a CAS system. They found 60% fewer
solids than the control CAS system were generated, without
any negative effect on the effluent quality or the settling char-
acteristics of the activated sludge. They also observed the
release of protein into the ASSR and the release of calcium,
magnesium, and polysaccharides into the aerobic sequencing
batch reactor (SBR) in the water line.
Although promising results have been achieved, there
have been very few full-scale experiments, mainly due
to knowledge gaps about the mechanisms governing the
sludge reduction process (Ferrentino et al. 2014, 2016b;
Khursheed et al. 2015).
Environ Sci Pollut Res (2018) 25:1243–1256
In most cases, the systems have been fed with synthetic
wastewaters (Ferrentino et al. 2016b), and clear design param-
eters and operational conditions are lacking.
This study proposes the IR as a design parameter. We stud-
ied effect of the IR, and the related SRTASSR, on the sludge
reduction in a sequencing batch reactor (SBR) for nutrient
removal coupled with an ASSR under strict anaerobic condi-
tions (− 400 mV). To ensure strict anaerobic conditions in the
ASSR, a denitrifying side-stream reactor (DSSR) was intro-
duced. The SBR–ASSR was fed with a real municipal waste-
water in order to provide results that can be implemented at a
full scale. We also focused on the mechanisms underlying the
reduction of sludge production.
Materials and methods
Experimental setup and system operation
Figure 1 shows the experimental setup of the wastewater treat-
ment systems used in this study. During the experiment, the
influent to the laboratory scale system was a fresh primary
wastewater effluent collected from a municipal wastewater
treatment plant (Trento, Italy). The characteristics of the influ-
ent are reported in Table 1.
The laboratory-scale system consisted of an oxic and an-
oxic SBR for nutrient removal from real municipal wastewater
which included a continuously mixed ASSR at ambient tem-
perature for a side-stream treatment of the recycled sludge.
Prior to introducing the recycled sludge to the ASSR, a
denitrifying side-stream reactor (DSSR) was introduced in
order to complete the nitrate removal. This ensured a tightly
anaerobic environment in the ASSR (ORP = − 400 mV). The
DSSR was also designed to increase the solid concentration of
the sludge, thus reaching a high solid concentration in the
ASSR (in the range of 8–9 g TSS L−1). The sludge from the
ASSR was then cycled back to the SBR during the anoxic
phase. A schematic representation of the system operations
can be found in our previous study (Ferrentino et al. 2016a).
The SBR was a circular type reactor with a working vol-
ume of 10 L (diameter and height were 19.0 and 43.0 cm,
respectively). The hydraulic retention time (HRT) of the
SBR was maintained at 0.6 day. Disc diffusers were placed
for the aeration at the bottom of the SBR. A mechanical stirrer
was used to mix the activated sludge. The SBR had six cycles
a day, and each cycle (4 h) consisted of anoxic feeding
(20 min), anoxic phase (30 min), aeration (50 min), anoxic
feeding (20 min), anoxic phase (30 min), aeration (50 min),
settling (30 min), supernatant extraction (5 min), and settled
sludge extraction (5 min). Concentrations of dissolved oxygen
(DO) during the aeration phases were all maintained at 2.0–
2.5 mgL-1. The settled sludge was fed from the SBR to the
DSSR with a 2-L working volume, designed as a sludge
Environ Sci Pollut Res (2018) 25:1243–1256
Fig. 1 The experimental setup.
QIN wastewater influent flow rate
to the SBR, QEFF wastewater
effluent flow rate to the SBR, QW
excess sludge flow rate, QIN, DSSR
sludge influent flow rate to the
DSSR, QIN, ASSR sludge influent
flow rate to the ASSR, QOUT, ASSR
sludge effluent flow rate from the
ASSR, QSUP supernatant flow
rate from the DSSR
thickening unit (HRTDSSR = 3 h) (Metcalf and Eddy 2013).
Finally, a certain quantity of the thickened sludge was sent fed
six times per day from the DSSR to the ASSR, and an equal
volume of mixed sludge was cycled back from the ASSR to
the SBR. The ASSR had a 10-L working volume (diameter
and height were 19.0 and 43.0 cm, respectively). The ASSR
was continuously mixed, whereby the SRTASSR was equal to
the HRT, as there was no sludge wastage from the ASSR.
The plant was extensively monitored with chemical analy-
ses and using pH, ORP, temperature, and DO on line signals.
The SBR–ASSR operation was mainly based on timers. The
pH and the ORP were left free to vary. The DO in the SBR was
controlled and maintained above 1.5–2.0 mg O2L-1 during the
aerobic reaction phases. The whole experimental setup was
operated under a temperature of 20 ± 2 °C by means of a
thermostatic water bath.
The activated sludge (TSS = 3.5 ± 0.5 g L−1) obtained from
a local aeration treatment unit (Trento, Italy) and the anaerobic
sludge (TSS = 5.5 ± 0.5 g L−1) obtained from a local anaerobic
sludge treatment unit (Levico Terme, Italy) were used as in-
oculum of the SBR and ASSR, respectively.
The entire experimental study consisted of three dif-
ferent periods (Table 1).
During the Period I, the IR was set at 27%. It was increased
to 50 and 100% in the Periods II and III, respectively, by
increasing the QIN,ASSR. The IR was calculated following
the Eq. (1):
QIN;ASSR  X IN;ASSR
IRð%Þ ¼ ; ð1Þ
V SBR  X SBR
1245
where QIN,ASSR is the ASSR influent flow rate (L day−1),
XIN,ASSR is the TSS concentration in the influent flow of the
ASSR (g TSS L−1), VSBR is the volume of SBR (L), and XSBR
is the TSS concentration in the SBR (g TSS L−1).
The ASSR influent flow rates, and thus the sludge cycled
back to the SBR, were maintained at 1, 2, and 4 L day−1,
respectively, in the Periods I, II, and III. The related
SRTASSR values were 10, 5, and 2.5 days, respectively.
The SRT of the whole system was evaluated as the ratio of
the total mass of sludge inside the system and the mass rate of
sludge leaving the system (Eq. 2).
X SBR V SBR þ X ASSR V ASSR
SRT ¼ ; ð2Þ
X SBR QSBR;waste þ X ASSR QASSR;waste þ X eff Qeff
where XSBR and XASSR are the TSS concentration in the SBR
and in the ASSR (g TSS L−1); VSBR and VASSR are the volumes
of SBR and ASSR (L); and QSBR,waste, QASSR,waste, and Qeff are
the flow rates of wastage from SBR, from ASSR, and from
effluent (L day−1), respectively. In this study, the SBR–ASSR
system was operated with no solid wastage except for sampling.
Chemical analyses
Total suspended solid (TSS), chemical oxygen demand
(COD), ammonium nitrogen (NH4+-N), nitrate nitrogen
(NO 3 − -N), nitrite nitrogen (NO 2 − -N), and phosphate
(PO 4 -P) concentrations were quantified according to
Standard Methods (APHA 2005). Soluble COD (sCOD),
NH4+-N, NO3−-N, NO2−-N, and PO4-P were measured
1246 Environ Sci Pollut Res (2018) 25:1243–1256

Table 1 Characteristics of the

influent wastewater and Influent characteristics
operational parameters during Parameter Period I Period II Period III
each experimental period Total COD (mg TCOD L−1) 200 ± 63 247 ± 51 285 ± 86
Soluble COD (mg sCOD L−1) 82 ± 34 107 ± 2 9 116 ± 43
Ammonium nitrogen (mg NH4+-N L−1) 38 ± 14 53 ± 5 47 ± 13
Nitrate (mg NO3--N L−1) 1.6 ± 1.2 1.5 ± 1.2 0.5 ± 0.5
Nitrite (mg NO2--N L−1) 0.26 ± 0.7 0.39 ± 0.1 0.30 ± 0.5
TN (mg TN L−1) 43 ± 19 54 ± 5 47 ± 15
Soluble phosphorous (mg PO4-P L−1) 2.9 ± 1.1 4.0 ± 0.6 3.5 ± 1.1
Sulfate (mg SO42−-S L−1) 40.0 ± 6 46 ± 3 47 ± 15
Total suspended solids (TSS) (g TSS L−1) 0.16 ± 0.1 0.15 ± 0.1 0.20 ± 0.05
pH 7.4 ± 0.5
sCOD/NH4+ 2.1 ± 0.5 2.0 ± 0.5 2.5 ± 1.1
COD/NH4+ 5.7 ± 2.2 4.7 ± 0.8 6.3 ± 2.1
Operational parameters
Parameters Period I Period II Period III
Experimental days 110 90 100
Temperature (°C) 20 20 20
SBR
Influent flow rate (L day−1) 18 18 18
TCOD loading rate (g TCOD L−1 day−1) 0.36 ± 0.11 0.44 ± 0.09 0.51 ± 0.15
sCOD loading rate (g sCOD L−1 day−1) 0.15 ± 0.06 0.19 ± 0.05 0.21 ± 0.08
TN loading rate (g N L−1 day−1) 0.077 ± 0.035 0.979 ± 0.093 0.084 ± 0.027
HRTSBR (day) 0.6 0.6 0.6
SRTSBR (day) 22 31 42
TSS (g L−1) 2.8 3.2 3.0
DSSR
Influent flow rate (L day−1) 2 4 8
HRTDSSR (h) 3 3 3
ASSR
Influent sludge flow rate (L day−1) 1 2 4
IR (%) 27 50 100
SRTASSR = HRTASSR (day) 10 5 2.5
TSS (g L−1) 8.5 8.5 8.8

after filtration of the sample on a 0.45-mm membrane. Sludge reduction was evaluated by comparing the observed
DO, pH, temperature, and ORP were measured with sludge yield of the SBR–ASSR system (Yobs, SBR − ASSR) with
probes connected to a multimeter (Multimeter 44, the observed sludge yield of the control system (Yobs, SBR),
Crison). Sulfates (SO42--S) were analyzed by an ion chro- following the Eq. (4):
matograph (DIONEX ICS-100) equipped with an AS9-
Y obs;CAS −Y obs;SBR−ASSR
HC column. Sludge reduction ð%Þ ¼  100: ð4Þ
Y obs;CAS

Evaluation of the observed sludge yield and sludge
reduction
The sludge production of the SBR–ASSR system was evalu-
ated following Chon et al. (2011b) (Eq. 3).
Y obs ¼ CumulativeGeneratedSludge=CumulativeConsumedSubstrate ;
where Yobs is the overall observed sludge yield (g TSS/g sCOD).
The control system consisted of an SBR with the same
characteristics, in terms of geometry and operational setup,
as the SBR used in the laboratory application. The system
simulated with WEST® using the ASM1 model. In order to
calculate the sludge production in each period, a fractionation
of influent COD performed in each period was used as input
ð3Þ
data for the simulation. The reported value is an average of the
three experimental periods. The COD fractionation was
Environ Sci Pollut Res (2018) 25:1243–1256
developed according to the ASM definition, based on biode-
gradability and using respirometric tests (data not shown).
EPS extraction
Extracellular polymeric substances (EPS) are complex high-
molecular-weight extracellular polymers which are produced
by microbial aggregates, including polysaccharides, proteins,
and nucleic acids (Sheng et al. 2010). Two kinds of EPS were
analyzed in this study, namely the soluble and attached frac-
tions. The soluble fraction, also referred to as soluble micro-
bial products (SMPs), was obtained following Guglielmi et al.
(2007). For the extraction of the attached EPS, the cation
exchange resin (CER) and the base extraction methods were
adopted following Chon et al. (2011a). In order to determine
the polysaccharide and protein content, both the soluble and
the attached fractions of EPS were analyzed in a spectropho-
tometer. The polysaccharide concentration was measured ac-
cording to DuBois et al. (1956), using glucose as a standard.
Proteins were measured according to Frolund et al. (1995),
using bovine serum albumin as the reference protein.
Batch test procedure
As a high phosphorus removal efficiency was observed in the
SBR–ASSR system, phosphorus uptake batch tests were used
to facilitate the monitoring of the two different families of
phosphorus-accumulating organisms (PAOs): total (TPAOs)
and denitrifying (DPAOs) phosphorus-accumulating organ-
isms, which are able to release phosphorus in anaerobic con-
ditions and uptake phosphorus in oxic and anoxic conditions.
The batch tests were performed according to Cyganecka et al.
(2011). Reactors were used with a total volume of 2.0 L and a
volume of sludge of 1.2 L, mixed with a magnetic stirrer. The
reactors were inoculated with the anaerobic sludge from the
ASSR. The concentration of the total suspended solid was
approximately 8.0 ± 1.0 g TSSL-1. The temperature was main-
tained at 20 °C by means of a thermostatic jacket. The batch
tests were sampled every 30 min. The samples were immedi-
ately vacuum filtered on 0.45-μm membrane filters and the
concentration of PO4-P was evaluated.
The specific phosphorous uptake rate of TPAOs (SPURtot)
was determined in a 2-stage batch assay, initially under strictly
anaerobic conditions (first stage) and then under aerobic con-
ditions (second stage). The sludge was flushed with nitrogen
gas for 10 min before using, in order to maintain anaerobic
conditions in the reactors. The DO was continuously verified
to be zero. Analyses were performed to ensure that there was
no nitrate in the ASSR sludge. No carbon sources were added
at the beginning of the anaerobic stage, because they were
already present in the ASSR due to cell lysis. Aerobic condi-
tions were ensured by sparging air through the bulk liquid
using an aquarium air stone. The resulting DO concentration
1247
was about 5.5 mg O2L-1. The anaerobic phase was maintained
in the reactor for about 100 min. The aerobic phase lasted at
least 240 min and was carried out until no further changes in
concentration of PO4-P were observed.
The specific phosphorous uptake rate of DPAOs
(SPURDPAO) was determined in a 2-stage batch assay, initially
under strictly anaerobic conditions (first stage) and then under
anoxic conditions (second stage). Anaerobic conditions were
ensured as reported for the TPAO activity assays. Again, no
carbon sources were added at the beginning of the anaerobic
stage. Anoxic conditions were achieved by dosing a nitrate-
rich solution. The resulting nitrate concentration was 30.0 mg
NO3−-NL-1. The anaerobic phase was maintained in the reac-
tor for about 120 min. The anoxic phase lasted at least 220 min
and was carried out until no further changes in concentration
of PO4-P were observed.
Both the specific TPAO and DPAO activities were calcu-
lated from the slope of the initial section of PO4-P curves in
the first and second stages, divided by the concentration of
TSS in the test. The results of the batch tests were used to
determine the fraction of DPAO over TPAO, according to
Hu et al. (2003). The nitrate consumption rate in the anoxic
phase of the batch test was determined from the slope of the
regression line in order to calculate the activity of DPAO in
terms of nitrate consumption.
Statistical analysis
Each sample was measured in duplicate. One-way ANOVA
and Tukey’s HSD multiple comparison test were performed to
verify statistical significance in the datasets at a 95% confi-
dence interval. Statistical analysis was performed with
Minitab version 18. The results are presented as means ±
standard deviation.
Results and discussion
Wastewater treatment performance
The SBR–ASSR system performance was monitored for
300 days, during which the operational parameters were var-
ied in the Periods I, II, and III as shown in Table 1.
When treating real wastewater, the organic carbon and nu-
trient components in the stream fluctuated throughout the
study period. The statistical evaluation of the characteristics
of the influent stream showed that there was a statistically
significant difference between the influent characteristics of
the Period I as compared to the Periods II and III. However,
the one-way ANOVA results showed that the differences be-
tween the three periods in terms of the carbon to nitrogen ratio
were not statistically significant. During the whole experimen-
tal period, the organic carbon and total nitrogen loading rates
1248
were 0.43 ± 0.13, 0.18 ± 0.07, and 0.09 ± 0.03 approximately
kg TCOD m−3 day−1, kg sCOD m−3 day−1, and kg TN
m −3 day −1 , respectively. A reasonable sludge age was
established in the SBR (SRTSBR) to allow both carbon and
nitrogen removal (Li and Wu 2014). An SRTSBR equal to
22, 31, and 42 days was calculated in the Periods I, II, and
III, respectively, which increased overall due to a slight rise in
the total solid concentration in the SBR.
Figure 2 shows the characteristics of influent and effluent
of the SBR–ASSR system during each experimental period.
As shown in Fig. 2(a)), the three periods were almost effective
in COD removal, with a consistent average total COD remov-
al efficiency of 81.9 ± 10.5, 80.4 ± 7.1, and 86.1 ± 7.2% in the
Periods I, II, and III, respectively. The organic carbon removal
rates were 0.30 ± 0.11, 0.36 ± 0.9, and 0.45 ± 0.16 kg COD
day−1 m−3. The effect of increasing IR was statistically signif-
icant on both the COD removal efficiency and organic carbon
removal rate in the Period III (IR = 100%), when the whole
sludge in the SBR was subjected to the anaerobic conditions
in the ASSR and then, to the aerobic/anoxic alternate condi-
tions in the SBR. The anaerobic conditions in the ASSR im-
proved the organic matter hydrolysis processes and the sludge
decay, thus releasing soluble organic matter that could be
more easily degraded in the SBR, by means of both aerobic
heterotrophs and denitrifies.
With the NH 4 + -N concentration in the influent of
38 ± 14 mg L−1, the Period I with an IR of 27% was charac-
terized by a poor ammonia removal efficiency (60.8 ± 19.5%).
Later, in the Periods II (IR = 50%) and III (IR = 100%), with
the NH4+-N concentration in the influent of 53 ± 5 and
47 ± 13 mg L-1, the ammonia removal increased up to
71.4 ± 15.3 and 82.5 ± 11.2%, respectively (Fig. 2(b)). The
ammonia removal rates were 0.041 ± 0.019, 0.069 ± 0.019,
and 0.070 ± 0.020 kg NH4+-N day−1 m−3, in the Periods I, II,
and III, respectively. The one-way ANOVA results showed
that there was a significant difference in the ammonia removal
rate between the Periods I and II. On the other hand, there was
no significant difference between the Periods II and III, thus
indicating that an IR higher than 50% has a positive impact on
the nitrification of the SBR. This could be linked to the lower
decay rate of nitrifying biomass under anaerobic conditions as
compared to the values under aerobic and anoxic conditions
(Zhou et al. 2015a).
Figure 2(c) depicts the variations in TN concentrations in
the influent and effluent of the three periods. The nitrogen
removal efficiency of the three periods with IR of 27, 50,
and 100% increased from 67.7 ± 15.9 to 72.5 ± 15.3% and
to 81.7 ± 12.0%. The total nitrogen removal rates were
0.051 ± 0.025, 0.071 ± 0.019, and 0.069 ± 0.022 kg TN
day−1 m−3 in the Periods I, II, and III, respectively.
Again, ANOVA showed that there was significant differ-
ence in total nitrogen removal rate between the Periods I and
II, while there was no significant difference between the
Environ Sci Pollut Res (2018) 25:1243–1256
Periods II and III, indicating that an IR higher than 50% pro-
moted a higher denitrification contribution in the SBR–ASSR.
Other studies have shown that by inserting an ASSR, denitri-
fication is enhanced (Cheng et al. 2017; Zhou et al. 2015b).
Finally, Fig. 2(d)) shows variations in the orthosphate con-
centrations in the influent and effluent of the three periods.
Orthosphate removal increased from 46.6 ± 14.8% in the
Period I to 56.0 ± 16.0% in the Period II, and finally to
62.6 ± 15.0% in the Period III, while the orthosphate removal
rates were 0.0023 ± 0.0014, 0.0040 ± 0.0013, and
0.0039 ± 0.0014 kg PO4-P day−1 m−3, respectively. As obtain-
ed for ammonia and total nitrogen removal rates, the one-way
ANOVA results showed that there was a significant difference
in the orthophosphate removal rates between the Periods I and
II, while there was no significant difference between the
Periods II and III, indicating that phosphorous removal can
be improved in an SBR–ASSR system by applying an IR
higher than 50%.
The best results in terms of carbon and nutrient removal
were obtained in the Period III, when the IR was the maximum
(100%), and the related SRTASSR was the minimum (2.5 days),
i.e., the whole sludge in the SBR was anaerobically treated for
a short period.
The TSS effluent concentration (average values 42 ± 0.1,
31 ± 0.1, and 16 ± 0.1 mg TSS/L) and the sludge settling
properties (average values 200 ± 1.7, 145 ± 1.0, and
130 ± 1.0 mL/g) improved switching from the Period I to
Period III (data not show).
During each experimental period, the influent and ef-
fluent of the DSSR were also observed. The nitrate influ-
ent concentrations of the DSSR were 1.4 ± 0.8 in the
Period I, 1.5 ± 0.5 in the Period II, and 0.8 ± 0.5 mg
NO3−-N L−1 in the Period III. The nitrate effluent concen-
tration of the DSSR fluctuated in a narrow range and was
always less than 0.1 mg NO3--N L−1, except for a few
samples during the Period I. The DSSR certainly appears
to have contributed to the completion of the nitrate deg-
radation and to ensuring a strict anaerobic environment in
the ASSR. In fact, throughout experimental period, the
ORP in the ASSR ranged between – 400 ± 25 mV, with-
out showing significant variations. The pH in the ASSR
was in the range of 7.0 ± 0.5.
Reduction of excess sludge production
The observed growth rate (Yobs) in the SBR–ASSR system in
the Periods I, II, and III were 0.2092 g TSS g −1 COD
(R2 = 0.9935), 0.1432 g TSS g−1 COD (R2 = 0.9926), and
0.117 g TSS g−1 COD (R2 = 0.9955), respectively, as com-
pared to the 0.36 g TSS g−1 COD of the control system. Thus,
the SBR–ASSR system used in this study with an IR of 27, 50,
and 100% reduced sludge production by 42, 61, and 66%
compared to the control system. The SRTs of the whole
Environ Sci Pollut Res (2018) 25:1243–1256 1249

Fig. 2 Profile of sCOD, NH4+-N, a)

TN, and PO4 -P removal Period I Period II Period III
700 100
efficiency
600
80
500

Removal efficiency [%]

TCOD [mg/L]
60
400

300
40

200
20
100

0 0
0 50 100 150 200 250 300
Time [d]
Influent Effluent Removal efficiency

b)
Period I Period II Period III
200 100
180
160 80
140

Removal efficiency [%]

NH+4 -N [mg/L]

120 60
100
80 40
60
40 20
20
0 0
0 50 100 150 200 250 300
Time [d]
Influent Effluent Removal efficiency
c)
200
Period I Period II Period III 100

Removal efficiency [%]

80
150
TN [mg/L]

60
100
40

50
20

0 0
0 50 100 150 200 250 300
Time [d]
Influent Effluent Removal efficiency

d)
IPeriod I Period II Period III 100
20
18
16 80
Removal efficiency [%]

14
12 60
PO4-P [mg/L]

10
8 40
6
4 20
2
0 0
0 50 100 150 200 250 300
Time [d]
Influent Effluent Removal efficiency
1250 Environ Sci Pollut Res (2018) 25:1243–1256

Table 2 Literature review of sludge reduction in biological nitrogen removal systems (adapted from Ferrentino et al. 2016b)

References Type of HRT in the ASSR ORP (mV) T (°C) IR (%) Yobs TSS/COD Sludge
wastewater CAS + ASSR reduction (%)

Ferrentino et al. 2016a Urban 10 days − 400 20±2 27 0.209 42

5 days 50 0.143 61
2.5 days 100 0.117 66
Coma et al. 2013 Urban 34.5 h − 150 20 10 0.55 a, b 0.2
11.8 h 50 0.51 a, b 8.8
a, b
5.9 h 100 0.45 18.3
Zhou et al. 2015c Urban 6h − 150 n.a. 100 0.212 32
− 100
Torregrossa et al. 2012 Urban 9h − 180 n.a. 30 0.36 a,b 35
Chon and Park 2012 Urban 10 days n.a. 21 10 0.35 43
2.5 days 21 0.25 59
2.5 days 37 0.23 61
2.5 days 55 0.26 57
Chon et al. 2011c Urban + synthetic 10 days n.a. 19±1.5 10 0.15 49 - 39
Chudoba et al. 1992 Synthetic 3h − 250 18±5 100 0.21 50
Saby et al. 2003 Synthetic 11 days + 100 20 100 0.29 28
15 days − 100 0.21 48
17 days − 250 0.17 58
Ye et al. 2008 Synthetic 5.5 h n.a.c 25±1 100 n.a. 33
7.6 h 23
11.5 h 14
Novak et al. 2007 Synthetic 10 days n.a. n.a. 7 0.15 a, b 60
4 0.21 a, b 46
Sun et al. 2010 Synthetic 10 days n.a. n.a. 10 (IT = 4) 0.12 77.4
10 (IT = 1) 0.25 52.8
Goel and Noguera 2006 Synthetic 10 days n.a. n.a. 10 0.19 62 - 63
Datta et al. 2009 Synthetic 10 days n.a.d n.a. 10 0.17 63

n.a. not applicable

a
Calculated
b
1 g VSS = 1.42 g COD; VSS/TSS = 0.72 (Novak et al. 2003); 1 g TSS = 1.02 g COD
c
Nitrate reduction did not take place in the study; thus, ORP values were presumed to be positive
d
Low ORP values were ensured as the nitrate concentration in the effluent of the SBR–ASSR was lower than 5 mg/L

SBR–ASSR system were found to be 47, 60, and 83 days in
the Periods I, II, and III, respectively.
The results obtained with this configuration were compared
to those reported in the literature (Table 2), where sludge re-
duction had been achieved in biological nitrogen removal
processes.
In line with Coma et al. (2013), our findings showed that
when treating urban wastewater, the higher the IR was, the
more sludge was reduced. Our results with an IR of only
27% are comparable with those obtained in other studies with
an IR in the range of 10–30% and when urban wastewater was
treated (Chon and Park 2012; Chon et al. 2011b; Torregrossa
et al. 2012). With an IR of 100%, a significant reduction in
sludge production was achieved both by Coma et al. (2013)
and Zhou et al. (2015c) who worked at ORP values of − 100/
− 150 mV and SRTASSR in the range of 6–24 h using real
wastewater. Their results highlight that a high IR and strict
anaerobic conditions (ORP < − 150 mV) in the ASSR are
fundamental in order to improve the sludge reduction in a
full-scale plant. However, the minimum SRTASSR needs fur-
ther optimization, thus reducing the volume of the ASSR.
On the other hand, in studies that are carried out treating
synthetic wastewater, where there were no inert solids, it
seems that the IR is not a key factor in reducing sludge.
Applications with an IR of 100% with negative ORP values
only produced a 50–58% sludge reduction (Chudoba et al.
1992; Saby et al. 2003). A higher sludge reduction efficiency,
in the range of 58–77%, was achieved also working with an IR
of only 10% and SRTASSR of 10 days (Datta et al. 2009; Goel
and Noguera 2006; Novak et al. 2007; Sun et al. 2010).
Environ Sci Pollut Res (2018) 25:1243–1256 1251

Fig. 3 Profile of NH4+-N (a) and

soluble COD (b) during the
a) Period I Period II Period III
100
experimental study. The open and 90
solid symbols represent the
80
influent and effluent values of the
ASSR, respectively 70

NH4 -N[mg/L]
60
50
40
30
20
10
0
0 50 100 150 200 250 300
Time [d]

b) Period I Period III

180 Period II
160

140

120
sCOD [mg/L]

100

80

60

40

20

0
0 50 100 150 200 250 300
Time [d]

Hydrolysis and sludge decay
The hydrolysis and sludge decay were studied by mon-
itoring the concentrations of NH4+-N and soluble COD
(Fig. 3) of the influent and the effluent of the ASSR, as
these are the result of the non-biotic organic matter
hydrolysis and the disruption of bacterial cells.
Starting from the NH4+-N concentration in the influent
of about 9.4 mg NH4+-NL-1, a value of 42.7 mg NH4+-
NL-1 had been reached at the end of the Period I. During
the Period II, the effluent concentration of NH4+-N in-
creased again up to 70.0 mg NH4+-NL-1. The same in-
creasing trend was repeated in the Period III, reaching
the value of 90.0 mg NH4+-NL-1. Figure 3(a) shows an
increasing solubilization of organic nitrogen to NH4+-N
by increasing the IR from 27 to 100%.
An increase of the sCOD in the ASSR was also found. The
average influent concentrations of sCOD during the Periods I,
II, and III were 24.3 ± 11, 31.6 ± 9, and 27.8 ± 10 mg sCODL-1,
respectively, which reached the average effluent value of
63.3 ± 30, 80.5 ± 27, and 71.2 ± 15 mg sCODL-1 (Fig. 3(b)).
The COD solubilization measured in the ASSR was at its max-
imum at the beginning of each experimental period, while it
decreased thereafter. Figure 2(b) highlights that there was no net
increase in COD solubilization because this substrate is likely
used by microorganisms for their maintenance function.
The sCOD and NH4+-N profiles confirmed that under an-
aerobic conditions in the ASSR, organic matter hydrolysis and
cell lysis occurred, thus enhancing the solubilization of the
organic matter and nutrients that are substrates available for
degradation, both in the ASSR and in the water line. The
mechanisms observed were also related to the higher COD
and TN removal efficiency obtained in the Period III in the
SBR–ASSR system. Organic matter hydrolysis and cell lysis
contributed to the sludge reduction process and have been
reported by Wang et al. (2008), Chon et al. (2011b), and
Novak et al. (2003).
EPS destructuration
Figure 4 shows the concentrations of proteins and polysaccha-
rides in both soluble (SMP) and attached EPS.
Concerning the concentration of SMP, our results showed a
strong increase in the concentrations of both proteins
(Fig. 4(a)) and polysaccharides (Fig. 4(b)), passing from the
Period I to Period III, possibly because of the higher amount
1252
Fig. 4 Proteins (a) and
polysaccharides (b) extracted by
a)
80
CER and base methods for each
experimental period
Protein [mg/ g VS]
60
40
20
0 0
Period 1
of sludge sent to the ASSR. This evidence is consistent with
the increase in NH4+-N and sCOD concentrations detected in
the ASSR effluent. Throughout the experiments, the SMP of
ASSR sludge contained more proteins than polysaccharides,
similarly to previous research on ASSR (Chon et al. 2011a).
As expected, at the same time, the bound EPS in the ASSR
(both CER and base extracted) decreased from the Period I to
III. The increasing percentage of sludge cycled to the ASSR
led to the destruction of the attached EPS. CER and base
extraction were employed to target and extract two different
extracellular biopolymer fractions (Novak et al. 2003).
Base extraction hydrolyzes a large amount of aluminum
and iron, releasing aluminum- and iron-bound floc materials,
while CER mainly removes divalent cation (Ca2+ and Mg2+)
from sludge and extracts divalent cation-associated EPS
(Chon et al. 2011a). During the Period I, proteins and poly-
saccharides were closely linked within the sludge floc, as can
be seen from the high values of both base- and CER-
extractable EPS, named EPS–base and EPS–CER, respective-
ly, in Fig. 4.
In the Period II, the concentration of protein EPS–base
extracted was slightly lower than that in the Period I, while
the EPS–CER decreased significantly. Likewise, the decrease
in the polysaccharide EPS–CER concentration was still higher
than that in the EPS–base, confirming that the release of diva-
lent cations was the main EPS destruction mechanism in the
Period II (SRTASSR = 5 days), as compared to the release of
Fe- and Al-associated EPS.
In the Period III, when the maximum IR was applied, the
EPS–CER was almost similar to that in the Period II; however,
the EPS–base decreased, achieving the lowest protein and
polysaccharide concentration in the flocs. Thus, in the
Period III, the iron- and aluminum-associated materials main-
ly accounted for the increase in SMP, and the degradation of
iron- and/or aluminum-associated materials was greater than
the release of divalent cations. Similar results were achieved
by Chon et al. (2011a), who showed that the degradation of
base-extractable EPS accounts for the lower sludge yield in
AS + ASSR.
The different composition of the EPS destructuration de-
tected in the Periods II and III may be explained considering
Environ Sci Pollut Res (2018) 25:1243–1256
b)
12 SMP
Polysaccharide [mg/ gVS]
SMP EPS - BASE
EPS - BASE
EPS - CER
EPS - CER 9
6
3
Period 2 Period 3 Period 1 Period 2 Period 3
the variation in the microbial community structure of the sys-
tem, as previously reported in (Ferrentino et al. 2016a).
However, further studies are needed in order to better under-
stand these mechanisms.
Selection of slow-growing bacteria
During the SBR–ASSR operation, two biological processes
were detected in the ASSR, i.e., phosphorous release and sul-
fate removal, both of which were facilitated by bacteria char-
acterized by a low growth yield, 0.10 and 0.13 g VSSg-1
COD, respectively for SRB (van den Bosch et al. 2007) and
DPAO (Hu et al. 2002).
Figure 5 shows the PO4-P concentrations in the influent
and effluent of the ASSR. The average influent concentrations
during the Periods I, II, and III were 2.7 ± 0.9, 3.3 ± 1.9, and
2.0 ± 1.8 mg PO4-P L−1, respectively. The effluent concentra-
tions reached values of 4.2 ± 1.9, 15.4 ± 8.7, and 32.8 ± 7.1 mg
PO4-P L−1.
A gradual accumulation of the phosphorus in the ASSR
effluent was thus detected. This suggests that the increasing
mass of sludge cycled through the ASSR influenced the mi-
crobial activity, thus causing the selection of those bacteria
that are able to accumulate polyphosphates (poly-P) under
aerobic and anoxic conditions and subsequently release them
under anaerobic condition, such as phosphate-accumulating
organisms (PAO). The selection of phosphate-accumulating
organisms (PAOs) was confirmed by molecular microbial
analyses (Ferrentino et al. 2016a) and activity tests
(Ferrentino et al. 2017) conducted in our previous studies.
The presence of poly-P bacteria in configurations that in-
volved an ASSR has been noted in previous studies
(Chudoba et al. 1992; Goel and Noguera 2006). Due to the
cycling between aerobic, anoxic, and anaerobic conditions in
the SBR–ASSR carried out in our study, both the presence of
aerobic PAO and denitrifying phosphate-accumulating organ-
ism (DPAO) was hypothesized, and batch experiments were
performed to evaluate their contribution to the phosphorous
removal process.
The results of batch tests on the total PAO (TPAO) showed
that the specific phosphorous uptake rate (SPURtot) in aerobic
Environ Sci Pollut Res (2018) 25:1243–1256 1253

Fig. 5 Profile of PO4-P

concentration. The open and solid 50 Period I Period II Period III
symbols represent the influent 45
and effluent values of the ASSR, 40
respectively
35

PO4 -P [mg/L]
30
25
20
15
10
5
0
0 50 100 150 200 250 300
Time [d]

conditions increased from an initial value of 0.77 mg PO4-P (g phosphate concentration at the beginning and the end of each
TSS h)−1 in the Period I to 1.41 mg PO4-P (g TSS h)−1 in the experimental period was determined and was used for the
Period II and up to 1.75 mg PO4-P (g TSS h)−1 in the Period III estimation of the phosphate release.
(Fig. 6(a–c)). These results confirmed the growing activity However, the batch test conducted on DPAO showed their
and the selection of TPAO in the ASSR process. Correlating selection over the aerobic PAO. The specific phosphorous
the release of orthophosphate in the ASSR (y) and the SPURtot uptake rate of DPAO (SPURDPAO) in anoxic conditions in-
(x) measured in the three experimental periods reveals a linear creased as well from 0.11 mg PO4-P (g TSS h)−1 in the
correlation (y = 28.174x − 22.109, R2 of 0.89), indicating the Period I to 0.89 mg PO4-P (g TSS h)−1 in the Period II and
correctness of the reported data. The difference of the to 1.64 mg PO4-P (g TSS h)−1 in the Period III (Fig. 6(d–f)).

a) Anaerobic Aerobic b)60 Anaerobic Aerobic c)60 Anaerobic Aerobic

60
y = -0.1083x + 21.367 50 y = -0.2x + 50.962 50
50 y = -0.2579x + 74.258
PO 4 - P [mg/l]

R² = 0.9694 R² = 0.9762 R² = 0.9353

PO 4 - P [mg/l]

40
PO4 - P [mg/l]

40
40
30 30
30
20 20
20

10 10 10

0 0 0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
Time [min] Time [min] Time [min]

d) 60 Anaerobic Anoxic e) 60 Anaerobic Anoxic f) 60 Anaerobic Anoxic

PO4 - P; NO3 - N [mg/l]

PO 4 - P ; NO3 - N [mg/l]

y = -0.0153x + 9.4948 y = -0.1232x + 45.499 50 y = -0,2207x + 71,279

50 50
PO 4 - P, NO3 - N [mg/l]

R² = 0.9838 R² = 0.9331 R² = 0,9801

40 40 40

30 30 30

20 20 20

10 10 10

0 0
0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
0 50 100 150 200 250 300 350
Time [min] Time [min] Time [min]

PO4-P concentrations during anaerobic ( ), and aerobic/anoxic ( ) phases, NO3-N concentration during anoxic phase ( )
Fig. 6 Batch test under anaerobic-aerobic condition: Period I (a), Period anaerobic (black circle) and aerobic/anoxic (white circle) phases; NO3--N
II (b), and Period III (c). Batch test under anaerobic-anoxic condition: concentration during anoxic phase (black triangle)
Period I (d), Period II (e), and Period III (f). PO4-P concentrations during
1254
Fig. 7 Profile of SO42--S
concentration. The open and solid 90 Period I
symbols represent the influent
and effluent values of the ASSR, 80
respectively 70
SO4-S [mg/L]
60
50
40
30
20
10
0
0 50
In terms of nitrate, the specific denitrification rate (SDNR) in
anoxic conditions increased from 0.34 mg NO3−-N (g TSS h)−1
in the Period I to 0.62 mg NO3−-N (g TSS h)−1 in the Period III
with the increasing selection of DPAO. Moreover, when the
phosphate had almost completely been removed, the nitrate
concentration did not decrease further. Thus, in anoxic con-
ditions, the decrease in phosphate concentration would
seem to be closely related to the presence of nitrate in
the solution. During each test on DPAO activity, sCOD
was evaluated in anoxic conditions, and no differences
between the beginning and the end of each test were
found (data not shown), confirming that nitrate reduction
could be due to DPAO activity.
Data collected by aerobic and anoxic batch tests enabled us
to calculate the percentage of DPAO over the TPAO. At the
beginning of the study, the DPAO accounted for only 15% of
the TPAO; thus, the 27% IR did not affect their selection
significantly. In the Period II, the percentage of DPAO in-
creased to 63%. In the Period III, when all the sludge of the
SBR had been anaerobically treated in the ASSR (IR = 100%)
and the related SRTASSR was 2.5 days, the fraction of DPAO
present in the sludge was approximately 94% of the TPAO
population.
The increasing phosphorous and total nitrogen removal
efficiencies in the SBR–ASSR system with the increasing IR
were in line with the increasing selection of DPAOs, which are
able to simultaneously remove phosphate and nitrate in the
anoxic conditions in the water line, using the organic matter
internally stored during anaerobic conditions in the ASSR.
DPAOs tend to take up acetate, store it internally, and then
release phosphate in anaerobic conditions. The internally
stored acetate can then form long-chain carbon molecules of
PHA that could be used for DPAO energy generation and for
the growth of new cells in anoxic conditions, together with
nitrate reduction in the water line (Seviour et al. 2003).
In this study, in order to assess the sulfate removal efficien-
cy in the ASSR, we investigated the importance of sulfate-
Environ Sci Pollut Res (2018) 25:1243–1256
Period II Period III
100 150 200 250 300
Time [d]
reducing bacteria (SRB), which are able to incompletely de-
grade organic matter and volatile fatty acids (VFAs), such as
propionic acid, to acetate and to improve the sludge disinte-
gration thanks to the sulfide production (Lens et al. 1998). The
influent and effluent SO2−4 −S concentrations of the ASSR are
reported in Fig. 7. The average sulfate influents during the
Periods I, II, and III were 40.9 ± 7.3, 45.8 ± 2.7, and
4 −S L , respectively. The effluent concen-
-1
48.2 ± 4.0 mg SO2−
trations progressively decreased, achieving values of
4 −S L . Thus,
-1
28.3 ± 10, 8.6 ± 2.5, and 9.8 ± 2.3 mg SO2−
the reduction of the sulfate concentration in the ASSR in-
creased during the experiment from 65 ± 0.25% in the
Period I to 81 ± 0.07% in the Period II and then remained
constant during the Period III.
These results revealed that by increasing the IR, the
selection of DPAO and SRB, which are both slow-
growing microorganisms, was enhanced in the SBR–
ASSR system. This evidence is consistent with the micro-
bial results reported in our previous study (Ferrentino et al.
2016a). The higher selection of DPAO and SRB is related
to maximum sludge reduction together with the high car-
bon and nutrient removal efficiencies achieved in the
Period III.
Conclusions
The interchange ratio in an SBR–ASSR is an important
operative parameter. In this study, three different IRs were
tested: 27, 50, and 100% corresponding to SRTASSR of 10,
5, and 2.5 days, respectively. Results revealed that an in-
crease in the IR improves the performances of the SBR–
ASSR system in terms of carbon and nutrient removal ef-
ficiency. Treating the whole mass of the sludge of the SBR
with an SRTASSR of 2.5 days allowed us to achieve a sig-
nificant percentage (66%) of sludge reduction. The main
Environ Sci Pollut Res (2018) 25:1243–1256
effects of the IR variations in an SBR–ASSR, where bio-
logical nitrogen process is carried out, are as follows:
– an enhancement of sludge decay and organic matter hy-
drolysis in the strict anaerobic side-stream reactor
(ASSR),
– the destructuration of the EPS, and
– the selection of slow-growing bacteria involved in organ-
ic matter degradation and phosphorous and nitrate remov-
al processes, such as SRBs and DPAOs.
Slow-growing microorganisms play a key role in sludge
reduction mechanisms. Their selection is higher when the
whole mass of the sludge of the SBR is sent to the ASSR. In
this study, an SRT ASSR of 2.5 days was applied with an IR of
100%. However, further research is needed to decrease the
SRT ASSR and thus the required volume of the ASSR.
Acknowledgments The first author is grateful for the financial support
of the Fondazione Caritro, Trento (Research Project and Economic
Development, Grant 2016). The second author was funded by a grant
from the Fondazione Caritro, Trento (Young Researcher, Grant 2015).
References
APHA, AWWA, WEF, 2005. Standard methods for the examination of
water and wastewater. Am Public Health Assoc
van den Bosch PLF, van Beusekom OC, Buisman CJN, Janssen AJH
(2007) Sulfide oxidation at halo-alkaline conditions in a fed-batch
bioreactor. Biotechnol Bioeng 97:1053–1063
Chen G-H, An K-J, Saby S, Brois E, Djafer M (2003) Possible cause of
excess sludge reduction in an oxic-settling-anaerobic activated
sludge process (OSA process). Water Res 37:3855–3866. https://
doi.org/10.1016/S0043-1354(03)00331-2
Cheng C, Zhou Z, Niu T, An Y, Shen X, Pan W, Chen Z, Liu J (2017)
Effects of side-stream ratio on sludge reduction and microbial struc-
tures of anaerobic side-stream reactor coupled membrane bioreac-
tors. Bioresour Technol 234:380–388. https://doi.org/10.1016/j.
biortech.2017.03.077
Chon D-H, Park C (2012) Method to reduce sludge generation in waste-
water treatment systems. US20120152812 A1
Chon DH, Rome M, Kim H-S, Park C (2011a) Investigating the mecha-
nism of sludge reduction in activated sludge with an anaerobic side-
stream reactor. Water Sci Technol 63:93–99
Chon DH, Rome M, Kim YM, Park KY, Park C (2011b) Investigation of
the sludge reduction mechanism in the anaerobic side-stream reactor
process using several control biological wastewater treatment pro-
cesses. Water Res 45:6021–6029. https://doi.org/10.1016/j.watres.
2011.08.051
Chudoba P, Morel A, Capdeville B (1992) The case of both en-
ergetic uncoupling and metabolic selection of microorganisms
in the OSA activated sludge system. Environ Technol 13:
761–770
Coma M, Rovira S, Canals J, Colprim J (2013) Minimization of sludge
production by a side-stream reactor under anoxic conditions in a
pilot plant. Bioresour Technol 129:229–235. https://doi.org/10.
1016/j.biortech.2012.11.055
1255
Cyganecka A, Podedworna J, Żubrowska-sudoł M (2011) Analysis of
methods for determining DPAO fraction in phosphorus accumulat-
ing organisms. Polish J Environ Stud 20:303–309
Datta T, Liu Y, Goel R (2009) Evaluation of simultaneous nutrient re-
moval and sludge reduction using laboratory scale sequencing batch
reactors. Chemosphere 76:697–705. https://doi.org/10.1016/j.
chemosphere.2009.02.040
DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)
Colorimetric method for determination of sugars and related sub-
stances. Anal Chem 28:350–356. https://doi.org/10.1021/
ac60111a017
Ferrentino R, Langone M, Andreottola G, Rada EC, (2014) An anaerobic
side-stream reactor in wastewater treatment: a review. 1435–1446.
https://doi.org/10.2495/SC141212
Ferrentino R, Langone M, Gandolfi I, Bertolini V, Franzetti A,
Andreottola G (2016a) Shift in microbial community structure of
anaerobic side-stream reactor in response to changes to anaerobic
solid retention time and sludge interchange ratio. Bioresour Technol.
https://doi.org/10.1016/j.biortech.2016.09.07
Ferrentino R, Langone M, Merzari F, Tramonte L, Andreottola G (2016b)
A review of anaerobic side-stream reactor for excess sludge reduc-
tion: configurations, mechanisms and efficiency. Crit Rev Environ
Sci Technol 46:382–405. https://doi.org/10.1080/10643389.2015.
1096879
Ferrentino R, Langone M, Ancdreottola G (2017) Temperature effects on
the activity of denitrifying phosphate accumulating microorganisms
and sulphate reducing bacteria in anaerobic side-stream reactor. J
Environ Bio Res 1 (1)
Foladori P, Andreottola G, Ziglio G (2010) Sludge reduction technologies
in wastewater treatment plants. IWA Publishing
Frolund B, Griebe T, Nielsen PH (1995) Enzymatic activity in the
activated-sludge floc matrix. Appl Microbiol Biotechnol 43:755–
761. https://doi.org/10.1007/s002530050481
Goel RK, Noguera DR (2006) Evaluation of sludge yield and
phosphorus removal in a cannibal solids reduction process.
1331–1337
Guglielmi G, Saroj DP, Chiarani D, Andreottola G (2007) Sub-critical
fouling in a membrane bioreactor for municipal wastewater treat-
ment: experimental investigation and mathematical modelling.
Water Res 41:3903–3914. https://doi.org/10.1016/j.watres.2007.
05.047
Hu ZR, Wentzel MC, Ekama G a (2002) Anoxic growth of phosphate-
accumulating organisms (PAOs) in biological nutrient removal acti-
vated sludge systems. Water Res 36:4927–4937. https://doi.org/10.
1016/S0043-1354(02)00186-0
Hu JY, Ong SL, Ng WJ, Lu F, Fan XJ (2003) A new method for charac-
terizing denitrifying phosphorus removal bacteria by using three
different types of electron acceptors. Water Res 37:3463–3471.
https://doi.org/10.1016/S0043-1354(03)00205-7
Khursheed A, Sharma MK, Tyagi VK, Khan AA, Kazmi AA (2015)
Specific oxygen uptake rate gradient—another possible cause of
excess sludge reduction in oxic-settling-anaerobic (OSA) process.
Chem Eng J 281:613–622. https://doi.org/10.1016/j.cej.2015.06.
105
Lens PNL, Vissera A, Janssena AJH, Hulshoff Pola LW, Lettingaa G
(1998) Biotechnological treatment of sulfate-rich wastewaters. Crit
Rev Environ Sci Technol 28:41–88
Li B, Wu G (2014) Effects of sludge retention times on nutrient removal
and nitrous oxide emission in biological nutrient removal processes.
Int J Environ Res Public Health 11:3553–3569. https://doi.org/10.
3390/ijerph110403553
Metcalf & Eddy (2013) Wastewater engineering: treatment and resource
recovery, 5th edn. AECOM, Los Angeles
Novak JT, Sadler ME, Murthy SN (2003) Mechanisms of floc destruction
during anaerobic and aerobic digestion and the effect on condition-
ing and dewatering of biosolids. Water Res 37:3136–3144
1256
Novak JT, Chon DH, Curtis B-A, Doyle M (2007) Biological solids
reduction using the cannibal process. Water Environ Res 79:2380–
2386. https://doi.org/10.2175/106143007X183862
Pérez-Elvira SI, Nieto Diez P, Fdz-Polanco F (2006) Sludge minimisation
technologies. Rev Environ Sci Biotechnol 5:375–398. https://doi.
org/10.1007/s11157-005-5728-9
Ragazzi M, Rada EC, Ferrentino R (2015) Analysis of real-scale experi-
ences of novel sewage sludge treatments in an Italian pilot region.
Desalination Water Treat 55(3):783–790. https://doi.org/10.1080/
19443994.2014.932717
Saby S, Djafer M, Chen GH (2003) Effect of low ORP in anoxic sludge
zone on excess sludge production in oxic-settling-anoxic activated
sludge process. Water Res 37:11–20
Semblante GU, Hai FI, Ngo HH, Guo W, You SJ, Price WE,
Nghiem LD (2014) Sludge cycling between aerobic, anoxic
and anaerobic regimes to reduce sludge production during
wastewater treatment: performance, mechanisms, and implica-
tions. Bioresour Technol 155:395–409. https://doi.org/10.1016/
j.biortech.2014.01.029
Seviour RJ, Mino T, Onuki M (2003) The microbiology of bio-
logical phosphorus removal in activated sludge systems.
FEMS Microbiol Rev 27:99–127. https://doi.org/10.1016/
S0168-6445(03)00021-4
Sheng G-P, Yu H-Q, Li X-Y (2010) Extracellular polymeric substances
(EPS) of microbial aggregates in biological wastewater treatment
systems: a review. Biotechnol Adv 28:882–894. https://doi.org/10.
1016/j.biotechadv.2010.08.001
Sid S, Volant A, Lesage G, Heran M (2017) Cost minimization in a full-
scale conventional wastewater treatment plant: associated costs of
biological energy consumption versus sludge production. Water Sci
Technol wst2017423. https://doi.org/10.2166/wst.2017.423
Environ Sci Pollut Res (2018) 25:1243–1256
Sun L, Randall CW, Novak JT (2010) The influence of sludge in-
terchange times on the oxic-settling-anoxic process. Water
Environ Res 82:519–523. https://doi.org/10.2175/
106143009X12487095236711
Torregrossa M, Di Bella G, Di Trapani D (2012) Comparison between
ozonation and the OSA process: analysis of excess sludge reduction
and biomass activity in two different pilot plants. Water Sci Technol
66:185–192
Wang J, Zhao Q, Jin W, Lin J (2008) Mechanism on minimization of
excess sludge in oxic-settling- anaerobic (OSA) process. Frontiers of
Environmental Science & Engineering. 2(1):36–43. https://doi.org/
10.1007/s11783-008-0001-4
Wei Y, Van Houten RT, Borger AR, Eikelboom DH, Fan Y (2003)
Minimization of excess sludge production for biological wastewater
treatment. Water Res 37:4453–4467. https://doi.org/10.1016/S0043-
1354(03)00441-X
Ye F, Zhu R, Li Y (2008) Effect of sludge retention time in sludge holding
tank on excess sludge production in the oxic-settling-anoxic ( OSA )
activated sludge process. J Chem Technol Biotechnol 114:109–114.
https://doi.org/10.1002/jctb
Zhou Z, Hu D, Jiang L, Xing C, Zhu Y, Jiang M (2015a) Nitrification
kinetics of a full-scale anaerobic/anoxic/aerobic wastewater treat-
ment plant. Desalination Water Treat 56:2054. https://doi.org/10.
1080/19443994.2014.958105
Zhou Z, Qiao W, Xing C, An Y, Shen X, Ren W, Jiang L, Wang L (2015b)
Microbial community structure of anoxic–oxic-settling-anaerobic
sludge reduction process revealed by 454-pyrosequencing. Chem
Eng J 266:249–257. https://doi.org/10.1016/j.cej.2014.12.095
Zhou Z, Qiao W, Xing C, Wang C, Jiang L-M, Gu Y, Wang L (2015c)
Characterization of dissolved organic matter in the anoxic–oxic-set-
tling-anaerobic sludge reduction process. Chem Eng J 259:357–363.
https://doi.org/10.1016/j.cej.2014.07.129
Reproduced with permission of copyright owner. Further reproduction
prohibited without permission.