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.

1 INTRODUCTION

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

https://orcid.org/0000-0003-4124-1874

https://orcid.org/0000-0002-3574-2154

https://orcid.org/0000-0002-8753-8254

https://orcid.org/0000-0002-1024-8275

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

867

2 CHARACTERISTICS AND

INFLUENCING FACTORS OF

DPAOs

2.1 The Metabolic Mechanism of

DPAOs

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

condition.

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

868

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.

3 RECENT TECHNOLOGIES OF

DPAOs

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

869

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

WWTPs.

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..

4 CONCLUSIONS

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

870

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