Temperature Effects on the Activity of Denitrifying Phosphate
Accumulating Microorganisms and Sulphate Reducing Bacteria in
Anaerobic Side-Stream Reactor
Roberta Ferrentino, Michela Langone* and Gianni Andreottola
Department of Civil, Environmental and Mechanical Engineering, University of Trento, Trento, Italy
Received date: 26 Jul 2017; Accepted date: 11 Sep 2017; Published date: 15
*Corresponding author: Michela Langone, Department of Civil, Environmental and
Mechanical Engineering, University of Trento, Trento, Italy, Tel: +39 328 6828225;E-mail: email@example.com; firstname.lastname@example.org
Four different temperatures, 5, 10, 15 and 20°C were tested in batch assays to investigate temperature effects on denitrifying phosphate
accumulating organisms (DPAOs) and sulphate reducing bacteria (SRBs), showed to play a main role in the biological sludge reduction
process. The effect of temperature on aerobic PAOs was also investigated for reason of completeness. Tests were performed using a selected
biomass from a laboratory scale anaerobic side-stream reactor (ASSR) process coupled in a sequencing batch reactor (SBR) for nutrient removal.
Results showed that the phosphate release and uptake kinetics of PAOs and DPAOs were influenced by the variation of the temperature,
while temperature did not influence significantly the anaerobic and the anoxic stoichiometry of the process.
In general, decreasing the temperature, a decreasing in the P-uptake and P-release rates was observed. The temperature had a moderate
effect on SRBs activity.
Arrhenius temperature coefficients, θ, for anaerobic, aerobic and anoxic PAOs metabolism were found to be 1.114, 1.121 and 1.165,
respectively, while θ for SRBs metabolism was 1.087. Those results indicated that DPAO activity was more affected by lower temperatures
than aerobic PAOs and SRBs activities, representing the limiting step of the biological sludge reduction process at low temperature.
Keywords: Temperature; PAO; DPAO; SRB; ASSR
Abbreviations: PAO: Phosphate Accumulating Organisms; DPAO: Denitrifying Phosphate Accumulating Organisms; SRB: Sulphate
Reducing Bacteria; ASSR: Anaerobic Side-Stream Reactor
Temperature changes could strongly affect microbial activity.
This dependence is linked to the kind of bacteria used. Any species’
response to temperature is characterized by upper and lower limits of
temperature for growth . When the temperature increases the rate of
denaturation of particular cell components increases as well, with the
consequent disruption of cellular function. On the contrary, when the
temperature is too low a loss of efficiency of transport proteins embedded
in the membrane, and thus a loss of affinity for substrates, occurs .
Temperatures below the optimum typically have a more significant effect
on growth rate than temperatures above the optimum .
Fermenting bacteria, able to release extracellular polymeric substances
and hydrolyze organic matter, and slow growing bacteria, involved in
nutrient removal, could have an important role in the biological sludge
reduction process, where an anaerobic side-stream reactor (ASSR) is
implemented in an activated sludge system [4,5]. The selection of sulphate reducing bacteria (SRBs) and denitrifying phosphate accumulating
organisms (DPAOs) has been detected in the ASSR coupled in a
sequencing batch reactor (SBR) for nutrient removal . Most of the
literature studies regarding the implementation of an ASSR in a activated
sludge system were performed at controlled temperature ranging between
18-20°C. However, the effects of temperature on the ASSR performance
have not yet investigated in literature . Thus, one of the main questions
that may arise is how the temperature could affect the activity of two
important bacteria selected in the ASSR: SRBs and DPAOs.
SRBs are anaerobic microorganisms that are widespread in anaerobic
habitats. They use sulphate as a terminal electron acceptor for the
degradation of organic compounds, producing sulphide. Although
limited studies have been performed so far, the presence and the activity
of SRBs in urban wastewater treatment plants has been clearly observed
[7, 8]. Further, they are of increasing importance in wastewater treatment
because of their ability to remove COD in the presence of sulphate with
low excess sludge production (0.3 gVSS/gCOD) . The effect of the
temperature on SRBs has been little studied, with contrasting results.
Sahinkaya et al.  reported an unsuccessful reactor operating at 8°C
with ethanol as electron donor for SRBs. On the contrary, Ingvorsen et al.
 reported that at 5°C SRBs in activated sludge still show an exponential
growth profile, although the rate was lower than that at 20°C.
Phosphate accumulating organisms (PAOs) have been identified
in the biological phosphorus removal (BPR) process. Aerobic PAOs
are capable to release phosphate under anaerobic conditions, while
consuming it only under aerobic conditions . Further, Jørgensen
and Pauli  demonstrated that, a part from aerobic conditions,
also under anoxic conditions, with nitrate as the electron acceptor,
denitrifying phosphate accumulating organisms (DPAOs) are capable
of polyphosphate accumulation. Thus, the following PAOs metabolic
pathways have been identified : (i) under anaerobic conditions, acetate
or other low molecular weight organic compounds are converted to
polyhydroxyalkanoates (PHAs), while intracellular stored polyphosphate
(poly-P) and glycogen are degraded, and phosphate, generated from
Poly-P degradation, is released into the bulk liquid; (ii) under aerobic or
anoxic conditions, the stored PHAs are converted to glycogen, phosphate
is assimilated, and poly-P is intracellular produced using oxygen and
nitrate/nitrite as electron acceptor, respectively for aerobic or anoxic
environment. Thus, bacterial growth and phosphate uptake are regulated
by the energy released from the breakdown of PHAs.
Up to now, the temperature effect on PAOs activity had been
investigated under anaerobic conditions (phosphate release process) and
aerobic conditions (phosphate uptake process). On the contrary, studies
on the effect of temperature on the anoxic DPAOs activity are missing in
Concerning PAOs, conflicting results have been achieved. Brdjanovic
et al. [15, 16] suggested that the contrasting results obtained so far on
the temperature effects on BPR could be explained by the use of different
substances, activated sludge and measurement methods. The authors,
probably for the first time, studied the temperature effects (over a range 5 -
30 °C) on stoichiometry and kinetics of aerobic PAOs. Results showed that
the stoichiometry of the anaerobic process was insensitive to temperature
changes while several effects on aerobic stoichiometry were observed. On
the contrary, the temperature had a strong impact on the kinetics of the
process under anaerobic as well as under aerobic conditions. Based on
these results, the authors calculated the anaerobic and aerobic temperature
coefficients θ from short-term steady state experiments that were found to
be 1.055 and 1.065, respectively . Temperature coefficients obtained
by Brdjanovic et al.  were used in Meijer  to create a new extended
model for BPR. Henze et al  reported that this model was successfully
applied for simulation of enhanced BPR (EBPR) systems [19,20] and for
developing an anaerobic and aerobic metabolic models that incorporated
carbon source, temperature and pH dependences of PAOs and (glycogenaccumulating
organisms) GAOs [21, 22]. The results of this study showed
that, for pH of 7.5 and temperature lower than 20°C, PAOs tended to be
the dominant microorganisms and, therefore, beneficial for the BPR.
In this light, the main goal of this study was to establish the short term
temperature effects on anaerobic, aerobic and anoxic metabolisms of
aerobic- and denitrifying- PAOs and anaerobic metabolism of SRBs. The
effects of temperature on the stoichiometry and kinetics of the biological
processes have been evaluated. Four different temperatures, 5, 10, 15 and
20°C were tested in batch assays using a selected biomass from a laboratory
scale ASSR process performed at room temperature.
Material and methods
PAOs and SRBs enriched sludge
A sludge enriched with PAOs and SRBs was developed in a laboratory
scale ASSR integrated in a nutrient removal activated sludge system as
previously described [4, 23]. The experimental set-up consisted of a
sequencing batch reactor (SBR) and an ASSR where the produced sludge
was treated. A denitrifying side-stream reactor (DSSR) was introduced
in the treatment scheme both to increase the solid concentration in the
sludge to cycled back to the ASSR and to complete the nitrate removal in
order to ensure a tightly anaerobic environment in the ASSR (oxidation
- reduction potential (ORP = -400 mV). The SBR had a working volume
of 10 liters and operated with six cycles a day in alternate denitrification/
nitrification mode to remove carbon and nutrient from real urban
The ASSR, having a working liquid volume of 10 liters, was completely
mixed and equipped with a mechanical stirrer. The anaerobic solid
retention time (SRTASSR) of the ASSR was 2.5 d and the sludge interchange
ratio (IR) was equal to 100%, meaning that the whole sludge in the SBR
was subjected to the anaerobic conditions in the ASSR. Figure 1 shows the
SBR-ASSR experimental set-up and the system mode of operation.
The lab-scale system operated at room temperature ranged between 18
and 21°C. The ORP and the pH in the ASSR were monitored and left to
vary. The characteristics of the ASSR biomass are reported in Table 1.
Batch phosphate accumulating bacteria assays
The assays were performed in a double jacked laboratory reactor with
a maximal operating volume of 2.0 L and a working volume of sludge
of 1.5 L. The reactor was mixed with a magnetic stirrer (150 rpm). The
total suspended solid (TSS) concentration in the batch test reactor was
approximately 8.5 gTSS / L. Each test lasted at least 12 hours, and it was
composed of three different phases: anaerobic (240 min), aerobic (at least
240 min), and anoxic (at least for 240 min). The initial pH value was equal
to 7.4. The final pH value of each phase was always measured in order
to check that it was maintained in the optimal range for the biological
activity (7.0 - 7.5). The batch tests were sampled every 30 minutes. The
samples were immediately vacuum filtered on 0.45 μm membrane filters
and analyzed. A summary of operating conditions is reported in Table 2.
Anaerobic phase: The anaerobic phase was performed to evaluate
the total release of phosphate by total (PAOs), both aerobic PAOs and
DPAOs. Nitrogen gas was injected into the reactor at the beginning of the
anaerobic experiments. DO and ORP were continually monitored. Nitrate
was also monitored during the experiment to ensure that anaerobic
conditions were present. Working temperatures (5, 10, 15 or 20 °C) in
the batch reactor were set 1 h before the beginning of the test, in order to
acclimatize the biomass. After this period, acetate was added to the batch
reactor as substrate for TPAOs. In accordance to Brdjanovic et al. , the
amount of acetate added to the batch reactor was less, but not limiting,
at lower temperature (from 350 mg COD/L at 20°C to 250 mg COD/L
at 5°C) in order to obtain a similar acetate uptake at each temperature
without changing the duration of the anaerobic phase. At the beginning
of the anaerobic phase, sulphate was further added in order to enhance
the activity of SRBs. A concentration of 50 mg SO4
2-S/L was ensured at the beginning of the tests. Anaerobic conditions were maintained for 240
min. For each temperature, the anaerobic phase was double performed in
two reactors (test A, B).
Aerobic phase: In the Test A, the aerobic phase was performed to
evaluate the aerobic phosphate uptake rate of TPAOs. Aerobic conditions
were ensured by sparging air through the bulk liquid using an aquarium
air stone and an air compressor (Schego M2K3350). The resulting DO
concentration was about 5.5 mg O2 / L. The aerobic phases were carried
out until no further changes in concentration of PO4-P could be observed
and lasted at least for 240 min.
Anoxic phase: In the Test B, the anoxic phase was performed in
order to evaluate the denitrifying phosphate uptake rate of DPAOs.
Anoxic conditions were obtained by adding nitrate to the bulk solution
at a concentration of 30 mg NO3--N / L. DO and ORP values were
continuously monitored during the entire experimental test. The anoxic
phase was carried out until no further changes in concentration of PO4-P
could be observed and lasted at least for 240 min (Table 2)
Figure 1: Schematic representation of the experimental setup system and time sequence in each SBR cycle
Sulphate reducing bacteria assays
The SRB assay was performed simultaneously with the evaluation of
the total release of phosphate by TPAOs in the anaerobic phase. Tests
were conducted at 5, 10, 15 and 20°C. At the beginning of the phase a
concentration of 50 mg/L of sulphate was added in order to enhance the
activity of SRBs. Anaerobic conditions were maintained for 240 min.
Specific uptake rate: The uptake and release rates were determined
as the maximum slope of the substrates profile through linear regression. The specific uptake and production rates were calculated
as the ratios of uptake and release rates and the biomass concentration
Temperature coefficients: The temperature coefficient θ was calculated
for each reaction rate using the simplified Arrhenius equation for the
temperature dependency (Equation 1):
where T is the temperature in °C,rTis the kinetic parameter at
temperature T, T20 is the reference temperature (20°C), r20 is the kinetic
parameter at a temperature equal to 20°C, and θ is the Arrhenius
temperature coefficient. This expression was used to describe the
effect of the temperature both on phosphorus release and on sulphate
consumption in anaerobic tests, and on phosphorus uptake in aerobic and anoxic tests
TSS was measured according to the Standard Methods . The
samples were filtered through 0.45 μm membrane filters. The filtrate
was analyzed for soluble COD (sCOD), ammonium nitrogen (NH4
+-N),nitrate as nitrogen (NO3- -N), soluble orthophosphate (PO4-P) and
sulphate as sulphur (SO42--S). NH4+-N, NO3--N and PO4-P concentrations
were determinate according to the Standard Methods . Sulphates were
analyzed by an ion chromatograph (DIONEX ICS-100) equipped with
an AS9-HC column. COD in the form of sodium acetate (CH3COONa),
nitrate in the form of sodium nitrate (NaNO3) and sulphate in the form
of sulphuric acid (H2SO4) were added at the required final concentration.
Effect of temperature on P release by total PAOs
Figure 2 shows the distribution of PO4-P, NH4
+-N, SO42--S and sCOD during the anaerobic period for all culture series. Nitrate concentration
was monitored during the entire experimental period and its value was
always equal to zero. The orthophosphates profiles in the anaerobic phase
have been related to the anaerobic metabolism of total PAOs, as they store
acetic acids as PHB through the cleavage of poly-P with the associated
release of phosphate in the bulk solution .
The graphs clearly indicated that, both the P- release and the sCOD
uptake changed largely with a different temperature. In each assay, the
added acetate was partially consumed, corresponding to sCOD removal
efficiencies of 2%, 3%, 9% and 10% at 5, 10, 15 and 20°C, respectively
At 5°C (Figure 2a) and 10°C (Figure 2b) the specific P- release rates were very low and equal to 0.06 mg PO4-P / (g TSS h) and 0.08 mg PO4-P
/ (g TSS h), respectively. At 15°C (Figure 2c) the specific P release rate
increased up to 0.20 mg PO4-P / (g TSS h), reaching the maximum value
of 0.30 mg PO4-P / (g TSS h) at 20°C. Thus, data showed that the P release
rate at 15, 10 and 5°C was 33, 73 and 81% lower than that measured at
Similarly, the rate of sCOD uptake was very low both at 5 and 10°C,
accounting for 0.23 mg sCOD / (gTSS h) and 0.27 mg sCOD / (gTSS h),
respectively. The rate of sCOD uptake increased at 15 and 20°C, reaching
values of 0.89 and 1.15 mg sCOD / (gTSS h). The sCOD uptake rate at 15,
10 and 5°C was 23, 77 and 80% lower than that measured at 20°C, respectively.
The stoichiometric ratios of the PO4-P released and the sCOD
consumed at each experimental temperature were listed in Table 3.
Smolders et al.  obtained similar results from a MBR sludge, where
for 1 C-mol of acetate 0.5 P-mol phosphate was released in anaerobic
conditions, obtaining a PO4-P /acetate ratios of about 0.25. Brdjanovic
et al.  showed that under anaerobic conditions, P release activity
increased from 5°C to 20°C, reaching a maximum at 20°C.
In this study, results from short-term tests confirmed that the
stoichiometry of the anaerobic phase was relatively insensitive to
temperature changes. However, the stoichiometric ratios measured in
this study were lower than the values reported by Brdjanovic et al. 
Figure 2: Temperature effects in anaerobic phase of batch phosphate accumulating bacteria assays: a) 5°C; b) 10°C; c) 15°C; d) 20°C
who measured PO4-P / acetate ratios of about 0.4. The lower ratios
measured in this study have been related to the simultaneous SRBs and
total PAOs metabolic activity in the tests, that cause a higher consumption
of the sCOD.SRBs consumed sulphate for the degradation of organic
compounds (Figure 2). The additional sCOD consumption by SRBs
during the anaerobic conditions caused a lower PO4-P / acetate ratio.
At the end the tests the TSS concentrations at 5, 10, 15 or 20°C were
8.5, 8.4, 8.5 and 8.4 g / L, respectively. These data showed that bacteria
did not grow during the batch tests. Further, ammonia concentration
was monitored and was always constant during the anaerobic phase in
all batch assays. These results further indicated that the sludge decay and
extracellular polymeric substances (EPS) destructuration processes were
negligible during the short term experiments.
Effect of temperature on P uptake in aerobic phase by total PAOs
Figure 3: Temperature effects on P -uptake in aerobic phase of batch
phosphate accumulating bacteria assays at 5, 10, 15 and 20°C
Figure 3 shows the aerobic phosphate uptake at 5, 10, 15 and 20°C,
which has been related to the aerobic metabolism of TPAOs (Figure 3).
The phosphate uptake at 20, 15 and 10°C had an exponential trend. On
the contrary, at 5°C the phosphate uptake had a fairly linear trend. The
maximum specific P uptake rate was 4.53 mg PO4-P / (g TSS h) at 20°C.
At 15°C, the P uptake rate decreased down to 2.41 mg PO4-P / (g TSS h).
At 10°C and 5°C, the P uptake was 1.47 mg PO4-P / (g TSS h) and 0.95 mg
PO4-P / (g TSS h), respectively. In aerobic conditions, TPAOs activity was
influenced by temperature as well as in anaerobic conditions, but in a less
significant way at 10 and 5°C. Indeed, a reduction of 47%, 67% and 79%
was observed at 15, 10 and 5°C, respectively. After 3.5 h the same PO4-P
concentration of 3.0 mg PO4-P /L was reached both at 20 and 15°C. On the
contrary, at 10°C and 5°C, the final PO4-P concentrations were higher, and
equal to 5.0 and 10.0 mg PO4-P /L, respectively. These findings confirmed
the results of Brdjanovic et al.  who reported that at 5 and 10°C an
incomplete P-uptake was observed in the aerobic phase, while a complete
P-uptake at 20 and 30°C was measured.
Effect of temperature on P uptake in anoxic phases
Figure 4 shows the anoxic phosphate uptake at 5, 10, 15 and 20°C,
linked to the anoxic metabolism of DPAOs. The added nitrate was totally
consumed only at 20°C, while it was partially consumed up to 44%, 68%
and 84% at 5, 10 and 15°C, respectively (Figure 4).
Results showed that the anoxic P- uptake kinetic was influenced by the
temperature. It had an exponential trend at 20 and 15°C, which became
linear at 10 and 5°C. At 5 and 10°C the specific P- uptake rate was equal
to 0.24 mg PO4 -P / (g TSS h) (Figure 4a) and 0.53 mg PO4 -P / (g TSS
h) (Figure 4b), respectively. At 15°C specific P -uptake rate was 1.55 mg
PO4 -P / (g TSS h) (Figure 4c), increasing up to 3.01 mg PO4 -P / (g TSS
h) at 20°C. Thus anoxic P -uptake rate was 48, 82 and 92% lower than
the value measured at 20°C. The trends of the phosphorus uptake at
different temperatures showed that the anoxic DPAOs metabolism could
be negatively affected by the low temperature, more than the aerobic PAOs
Figure 4: Temperature effects on P and nitrate uptake in anoxic phase of batch phosphate accumulating bacteria assays: a) 5°C; b) 10°C; c) 15°C; d) 20°C
The stoichiometric ratios of the PO4 -P uptake and the NO3
--N consumed at each experimental temperature were listed in Table 3.
On the contrary of the anaerobic phase, stoichiometry of the anoxic
metabolism was strongly influenced by temperature changes. In
particular, the PO4 -P uptake/ NO3
--N consumed ratio became greater as
the temperature increases.
Effect of temperature on S uptake by SRBs
Sulphate concentration was monitored during each batch test as a
marker of the SRBs activity. Results from the short term experiments
showed that low temperatures affected SRBs activity. The specific sulphate
uptake rate was 0.05, 0.17, 0.33 and 0.38 mg SO42--S/ (gTSS h) at 5, 10,
15 and 20°C. For temperature equal to 20 and 15°C, the sulphate uptake
differed only for 13%. However, decreasing the temperature to 10 and
5°C, the percentage increased significantly, accounting for 55% and 87%,
Comparing these results with those directly measured in the laboratory
scale ASSR , a higher value (0.74 mg SO42--S / (gTSS h)) was found in
the ASSR conducted at room temperature than the value obtained in the
present study at 20°C. This result could be explained considering that in
the laboratory scale ASSR more than one type of electron donor for SRBs
could be present, obtaining higher rates than batch tests feed with a single
electron donor . Indeed, SRBs can be divided into two main groups:
those that degrade organic compounds incompletely to acetate and those
that degrade organic compounds completely to carbon dioxide, which
commonly also use acetate as a growth substrate .
In this study, acetate was selected as a carbonaceous substrate because
readily biodegradable by most heterotrophic populations, among them
PAOs. Nevertheless, Ferrentino et al.  found in a laboratory scale
ASSR the presence of bacteria able to hydrolyze complex organic matter
producing propionic acid in anaerobic environments. The expected
variety of volatile fatty acids (VFAs) in the laboratory ASSR, of acetate
and propionate for instance, therefore, is beneficial for sulphate reduction
From short term experiments, the temperature coefficients for the
anaerobic, aerobic and anoxic metabolisms of PAOs were equal to
1.114, 1.121 and 1.165, respectively. Brdjanovic et al. [15, 16] for shortterm
steady state experiments, showed that the temperature had a
moderate impact on the anaerobic P-release process rate (θ=1.071) and
on the aerobic P-uptake process rate (θ=1.032). In this study, a stronger
temperature effect has been evaluated on both the anaerobic P-release
process rate (θ=1.114) and on the aerobic P-uptake process rate (θ=1.121).
However, similarly to Brdjanovic et al. [15, 16], the aerobic metabolisms of
PAOs was higher influenced by temperature as compared to the anaerobic
metabolism of total PAOs.
Concerning the anoxic metabolism of DPAOs, data showed that the
DPAOs were more sensitive to the temperature than aerobic PAOs.
Further, the temperature had a moderate effect on SRBs activity. The
temperature coefficient for the sulphate reduction process in the anaerobic
phase was 1.087.
Temperature coefficients of DPAOs (θ=1.165) were higher than
those of others bacteria found to be essential in the biological sludge
reduction process [4 ] such as fermenting bacteria (θ=1.070, ) and
SRBs (θ=1.087, this study). Thus, it can be hypothesized that the biological
phosphorus removal process is the limiting step of the biological sludge
reduction process at low temperature.
Results achieved showed that the stoichiometry of the anoxic
metabolism of DPAOs was strongly sensitive to the temperature; on
the contrary temperature changes seem to have no negative effects
on the anaerobic metabolism of total PAOs. Concerning the kinetic
aspects, anaerobic, aerobic and anoxic metabolisms of PAOs seem to
be significantly affected by low temperatures. Above all, the anoxic
metabolism of DPAOs highlighted the highest sensitivity to the lowering
temperature. Further, results showed that the temperature had a moderate
effect on SRBs activity.
Given these results, the decrease in temperatures mainly compromised
the activities of DPAOs, and supposedly the efficiencies of the reduction
of sludge in an SBR-ASSR process. However, future studies directly
performed running an SBR -ASSR at low temperatures are necessary to
evaluate the long term effect of temperature on microbial activity, EPS
destructuration and cell lysis, and thus on the whole sludge reduction
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).
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