Recent Advances in Characteristics and Technologies of Denitrifying
Phosphorus-accumulating Organisms
Xiaohong Hong
, Bohan Chen
, Haixia Feng
3,* c
and Xianqiong Hu
Sewage Treatment Operation Supervision Center of Shenzhen Longgang District, Shenzhen, Guangdong, 518172, China
Civil and Environmental Engineering, Harbin Institute of Technology, Shenzhen, Shenzhen, Guangdong, 518055, China
Shenzhen Municipal Engineering Consulting Center CO., LTD, Shenzhen, Guangdong 518028, China
Keywords: Denitrifying Phosphorus-accumulating Organisms, Characteristics, Technology, Carbon Source, Nitrate.
Abstract: Denitrifying phosphorus-accumulating organisms (DPAOs) have attracted continuous attention from
researchers because of that they could achieve simultaneously nitrogen and phosphorus removal. So far, many
new technologies about DPAOs have been developed in recent years. At present, there are some contradictions
about the utilization of nitrate by DPAOs and the interaction between DPAOs and other organisms. In addition,
new technologies based on DPAOs have some difficulties in practical operation. This paper reviews recent
advances in characteristics and technologies of DPAOs. The influences of carbon source and electron acceptor
on DPAOs are also discussed.
In wastewater treatment plants (WWTPs), Enhanced
biological phosphate removal (EBPR) technique is
the main phosphate removal method. The key
mechanism of EBPR is the excessive phosphorus
absorption by phosphorus-accumulating organisms
(PAOs) in active sludge (Mino et al., 1998). However,
the difficulties of adjusting parameters and the sludge
age contradiction between PAOs and nitrifying
bacteria lead to the instability of EBPR in practical
operation (Shukla et al., 2020). Longer sludge age is
conducive to the growth of nitrifying bacteria, and the
increase of the abundance of nitrifying bacteria can
effectively improve the removal efficiency of
ammonia nitrogen, but too long sludge age will
reduce the abundance of PAOs, resulting in high
phosphorus concentration in effluent.
In the 1990s, researchers found some PAOs could
utilize nitrate as electron acceptor to achieve
orthophosphate uptake in anoxic phase (Kuba et al.,
1993). This kind of PAOs was named denitrifying
phosphorus-accumulating organisms (DPAOs).
According to the features of simultaneous
denitrification and phosphorus uptake of DPAOs,
researchers have developed denitrification phosphate
removal (DPR) process and applied it to WWTPs .
Compared to EBPR, DPR reduces the need for
aeration and carbon source and less sludge production
due to the low growth rate of DPAOs. Hence,
compared to EBPR, DPR is more energy efficient.
Recently years, many technologies based on DPR
have been proposed and applied to different scenarios
(Zhang et al., 2020). However, there are drawbacks to
the DPR process. In DPR process, heterotrophic
denitrification bacteria are always dominant in the
process of carbon source competition due to different
mechanisms of carbon source utilization, that is,
denitrification and nitrogen removal is preferred, thus
affecting the efficiency of denitrification and
phosphorus removal. Therefore, in recent years,
many researches have optimized DPR process and
developed new technology.
This paper reviewed current studies on
characteristics of DPAOs and its coupling process
and provided guidance for further research.
Hong, X., Chen, B., Feng, H. and Hu, X.
Recent Advances in Characteristics and Technologies of Denitrifying Phosphorus-accumulating Organisms.
DOI: 10.5220/0011311600003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 867-871
ISBN: 978-989-758-595-1
2022 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
2.1 The Metabolic Mechanism of
Since the discovery of DPAOs, researchers have done
many studies on its metabolic mechanism and
denitrifying phosphorus removal characteristics.
The metabolic mechanism of DPAOs can be
divided into two phases as shown in Figure 1 (Mino
et al., 1998). In anaerobic phase, DPAOs produce
acetyl-CoA through the degradation of stored
glycogen to provide reducing power (NADH
) and
break polyphosphates (poly-P) to generate energy
(ATP) and store as polyhydroxyalkanoates (PHAs)
(Ahn et al., 2001). At the macro level, this metabolic
process is represented by the reduction of organic
matters and the increase of orthophosphates in
wastewater. In addition, DPAOs uptake carbon
sources in both active and passive transportation.
Hence, when the concentration of organic matter in
the water is low, such as in a continuous flow reactor,
DPAOs have the advantage in carbon sources uptake
(Tu & Schuler, 2013). In anoxic phase, DPAOs
uptake orthophosphates excessively to regenerate
poly-P and synthesis glycogen by utilizing the
internal PHAs. In the meanwhile, DPAOs reduce
nitrate to nitrogen gas. Because DPAOs uses nitrate
or nitrite as electron acceptor in the process of
phosphorus absorption, the nitrogen removal effect of
DPAOs is affected by phosphorus concentration. The
more phosphorus released by DPAOs, the better the
nitrogen removal.
Figure 1: The metabolic mechanism of DPAOs (The solid
line presents the metabolism in anaerobic condition and
dotted line presents the one in anoxic condition).
The general metabolic pathways of DPAOs are
described above. However, different carbon sources
have a significant impact on PHAs storage of DPAOs
and the distinction of electron acceptors utilization is
also visible between different strains under anoxic
2.2 Carbon Source
Since DPAOs can only use volatile fatty acids (VFAs)
as energy sources, researchers generally use
propionate and acetate to grow DPAOs. DPAOs
cultured with acetate and propionate as a single
substrate or mixed substrate show different
denitrification and phosphorus removal performance
and substrate absorption rate (Lu et al., 2006). It was
found that acetate-based DPAOs collapsed after the
aerobic phase was cancelled, while propionate-based
DPAOs remained stable. By analyzing the types of
PHA and their contents, acetate-based DPAOs
synthesized more polyhydroxybutyrate (PHB) and
propionate-based DPAOs mainly synthesized
polyhydroxyvalerate (PHV) and 3-hydroxy-2-
methylvalerate (3H2MV) but not PHB. The glycogen
content also showed differences between acetate-
based DPAOs and propionate-based DPAOs. The
former was usually higher than the latter (Carvalho et
al., 2007). The glycogen required to store acetate as
PHA was higher than that required for propionate,
which means acetate-based DPAOs need to produce
more glycogen for VFA uptake and the elimination of
aerobic phase limited the amount of glycogen stored
in acetate-based DPAOs. Hence, propionate maybe is
a better substrate for DPAOs than acetate.
Using acetate and propionate simultaneously is
more favourable to DPAOs enrichment and does not
need to adjust pH, while a pH above 7.5 is required
for DPAOs cultivation when using acetate . It may be
related to the increasing concentration of the acetic
acid when pH below 7.5 because the acetic acid form
of the acetic acid/acetate acid−base pair may
negatively affect the growth and metabolism of some
organisms (Tu & Schuler, 2013). Another strategy of
DPAOs enrichment is to alternate the sole carbon
sources between acetate and propionate, which
achieved up to 90% biological abundance of DPAOs
in a lab-scale reactor (Lu et al., 2006).
Although there are many studies on the effects of
carbon source types on DPAOs metabolism, most
carbon sources used in these researches are simple
short-chain organic compounds. However, the carbon
source in actual sewage is mostly long-chain organic
matter, and the utilization process of carbon source by
DPAOs is more complicated. Therefore, future
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
research should pay more attention to the utilization
of organic matter in actual sewage by DPAOs.
2.3 Electron Acceptor
In early study, researchers argued DPAOs could only
utilize nitrate in anoxic phase and nitrite was
considered as an inhibitor for denitrifying phosphorus
removal of DPAOs. However, later researchers
successfully cultured nitrite-based DPAOs in the lab,
showing a great phosphorus uptake performance at
the nitrite concentration of 45mg/L (Zhang et al.,
2010). This contradiction may be due to the free
nitrous acid (FNA) which restrains DPAOs uptake
orthophosphates in anoxic phase (Pijuan et al., 2010).
Moreover, the presence of FNA can inhibit PHA
oxidation, glycogen regeneration and cell growth.
The concentration of FNA is related to the
concentration of nitrite and pH value of water, and a
small amount of FNA can seriously inhibit the
metabolic growth of DPAOs. Therefore, when nitrite
acts as the electron acceptor of DPAOs, the pH must
be strictly controlled to ensure that the concentration
of FNA is kept at a very low level. With the
development of metagenomics, DPAOs have been
classified more carefully depending on the
denitrification capability.
The Candidatus Accumulibacter phosphatis
(Accumulibacter) are the most common PAOs in
study. Accumulibacter Type I and Accumulibacter
Type II differ in the use of electron acceptors. The
former can utilize both nitrate and nitrite whereas the
later can only reduce nitrite due to the lack of gene
responsible for the encodification of the respiratory
nitrate reductase (Martín et al., 2006). However,
recent research showed that in a highly enriched
Accumulibacter Type I bioreactor a small amount of
phosphorus uptake occurred in anoxic phase when
using nitrate as electron acceptor (Rubio-Rincon et
al., 2017). Accumulibacter Type I were cultured with
nitrite as electron acceptor in a reactor, and mixed
carbon source was used to eliminate glycogen-
accumulating organisms (GAOs) which competed
with PAOs for carbon source in anaerobic phase.
After successful enrichment of Accumulibacter Type
I, the electron acceptor was replaced with nitrate and
almost no phosphorus uptake occurred. On the
contrary, when denitrifying glycogen-accumulating
organisms (DGAOs), which could reduce nitrate to
nitrite, coexisted with Accumulibacter Type I in
another reactor, orthophosphates and nitrate were
obviously removed in anoxic phase. This difference
implied that when DPAOs and DGAOs coexisted,
DGAOs first converted nitrite into nitrite and then
DPAOs used nitrite to achieve phosphorus uptake.
Hence, the relationship between DPAOs and DGAOs
cannot be simply concluded as a competitive
relationship. The relationship between them may
change from competition to co-existence with the
change of environmental conditions. Figure 2
illustrates the possible nitrogen metabolic patterns in
the coexistence of DPAOs and DGAOs.
Figure 2: The potential nitrogen metabolic pathways of
DPAOs and DGAOs system.
Many new technologies of DPAOs have been
developed for the past few years with the deep
understanding of DPAOs characteristics. In order to
achieve deep removal of the nutrient, Fu et al.
enriched DPAOs and heterotrophic nitrifying bacteria
in a single-stage biofilter to treat secondary effluent
(Fu et al., 2019). The abundance of heterotrophic
nitrifying bacteria decreased with the increase of
reactor height, while DPAOs increased gradually.
Therefore, heterotrophic denitrification mainly
occured in the middle and lower part of the reactor,
while denitrification phosphorus removal occured in
the upper part. Nevertheless, this treatment effect was
based on the effluent COD up to 120mg/L, which
means additional carbon sources need to be added.
And whether an excessively long anoxic period (40
hours) affects DPAOs metabolism remains to be
studied in a longer term.
Wang et al. enriched DPAOs and DGAOs in an
anaerobic/anoxic/short micro-aerobic sequencing
batch reactor (SBR) and achieved 75.3% nitrate-to-
nitrite transformation rate and 92.3% phosphorus
removal efficiency (Wang et al., 2019a). In this
system, DPAOs and DGAOs stored PHA anaerobic
Recent Advances in Characteristics and Technologies of Denitrifying Phosphorus-accumulating Organisms
and in the anoxic phase DPAOs completed
denitrification and phosphorus uptake while DGAOs
achieved partial denitrification. In the meanwhile, the
excess organic matter was used for denitrification by
heterotrophic denitrifying bacteria. In the micro-
aeration stage, PAOs utilised oxygen to achieved
excessive phosphorus uptake, reducing the remaining
phosphorus concentration. On this basis, they
combined anammox with this technology to reduce
the total nitrogen and total phosphorus to 6mg/L and
0.2mg/L, respectively (Wang et al., 2019b). On the
one hand, this process can save the aeration, on the
other hand, it can realize the deep removal of nitrogen
and phosphorus under the condition of low organic
matter. However, there are still many difficulties in
realizing the mainstream anammox technology in
Some researchers enriched DPAOs/AOB and
anammox in an anaerobic/anoxic/aerobic continuous
flow reactor and a moving bed biofilm reactor,
respectively (Zhang et al., 2020). The effluent total
nitrogen and phosphorus were 10.26mg/L and
0.21mg/L, respectively. In addition, this process
reduced carbon source requirement by 16% and
oxygen requirement by 15%. This indicates that
phosphorus removal via nitrite can maximize carbon
sources than nitrate, which is very important for many
urban sewage plants with low organic matter
concentration influent.
Recently, a new technology was proposed, called
SNADPR, for simultaneous nitrification,
denitrification and phosphorus removal, which
enriched anammox, DPAOs and ammonium
oxidizing bacteria (AOB) in a single SBR (Xu et al.,
2019). The main mechanism of SNADPR is shown in
Figure 3. The SNADPR could remove 89.15 ± 2.19%
of total nitrogen and 92.93 ± 0.60% of total
phosphorus. Interstingly, the SNADPR process used
a filler in the reactor and anammox mainly grew on
the filler while DPAOs and AOB were principally
located in suspended sludge. The strategy of adding
fillers solved the problem of sludge retention time
contradiction between anammox and DPAOs, which
allowed anammox, DPAOs and AOB to coexist in a
single SBR.
The technology of combining
activated sludge with carrier at the same time
may solve the problem of different microbial
sludge age conflict and provide the possibility of
mainstreaming anammox in sewage plants. But
in the meanwhile, this approach will also bring
about the carrier crushing and aging sludge
treatment efficiency decline.
Figure 3: The mechanism of SNADPR (The solid line
presents the metabolism in anaerobic condition, dotted line
presents the one in aerobic condition and chain-dotted line
presents the one in anoxic condition).
Although these technologies can achieve good
treatment effect, most of them are in the laboratory
stage, and the difficulty of sludge acclimation,
complexity of operation and process instability limit
their practical application. In addition, WWTPs tend
to operate in a continuous flow mode, and the SBR
model used in most studies also hinders the
application of these technologies in practice.
Therefore, future studies on these new processes
should pay more attention to their performance in
pilot tests, and the comprehensive evaluation should
be carried out in combination with the treatment
effect, economy and ease of operation..
Although the metabolic mechanism of DPAOs is
generally clear, there are still some confusions about
the utilization of nitrate and the classification of
DPAOs. In addition, few studies focused on the
interaction between DPAOs and other organisms,
such as DGAOs, which need to be studied in the
future. The combination of anammox technology and
DPR can effectively reduce the energy consumption
of wastewater treatment and the need for influent
organic matter, so it is also an important direction in
the future. In terms of process research, most used
SBR mode, and the future research should focus on
continuous flow process which is easier to be applied
in WWTPs.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
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Recent Advances in Characteristics and Technologies of Denitrifying Phosphorus-accumulating Organisms