Article
https://doi.org/10.1038/s41467-024-52364-9
Strategy to mitigate substrate inhibition in
wastewater treatment systems
Received: 19 September 2023
Accepted: 3 September 2024
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Beiying Li1, Conghe Liu1, Jingjing Bai1, Yikun Huang1, Run Su1, Yan Wei2 &
Bin Ma 1
Global urbanization requires more stable and sustainable wastewater treatment to reduce the burden on the water environment. To address the problem
of substrate inhibition of microorganisms during wastewater treatment, which
leads to unstable wastewater discharge, this study proposes an approach to
enhance the tolerance of bacterial community by artificially setting up a nonlethal high substrate environment. And the feasibility of this approach was
explored by taking the inhibition of anammox process by nitrite as an example.
It was shown that the non-lethal high substrate environment could enhance
the nitrite tolerance of anammox bacterial community, as the specific anammox activity increasing up to 24.71 times at high nitrite concentrations.
Moreover, the system composed of anammox bacterial community with high
nitrite tolerance also showed greater resistance (two-fold) in response to
nitrite shock. The antifragility of the system was enhanced without affecting
the operation of the main reactor, and the non-lethal high nitrite environment
changed the dominant anammox genera to Candidatus Jettenia. This approach
to enhance tolerance of bacterial community in a non-lethal high substrate
environment not only allows the anammox system to operate stably, but also
promises to be a potential strategy for achieving stable biological wastewater
treatment processes to comply with standards.
By 2050, over two thirds of the global population will live in cities1,2.
Urbanization has led to the pollution of water environments with a
variety of pollutants from human sources, including nutrients and
pathogens3. Eutrophication, caused by nutrients such as nitrogen (N)
and phosphorus (P), is widespread in many regions worldwide, particularly in developing countries4. Rapid global urbanization is placing
an unprecedented burden on the water environment5–7. More effective
wastewater treatment strategies are required to address rising pollutant levels while minimizing energy emissions to meet peak carbon and
carbon-neutral targets8,9.
Microbiological processes are essential to wastewater treatment
strategies10 as they remove pollutant elements such as carbon, nitrogen, phosphorus and sulfur9,11–14. Taking biological nitrogen removal as
an example, the nitrogen removal pathways in wastewater treatment
include nitrification and denitrification reactions15. The process of
nitrification involves the oxidation of ammonia to nitrate by ammoniaoxidizing bacteria, ammonia oxidizing archaea and nitrite-oxidizing
bacteria16,17. Denitrification, on the other hand, completes the nitrogen
cycle by converting nitrate into nitrogen gas18,19. Anaerobic ammonia
oxidation (Anammox) is an emerging microbial processes to reduce
energy consumption and enhance the efficiency of nitrogen
removal20,21.
Substrates are necessary for microbial growth in wastewater
treatment processes. However, high substrate inhibition is usually
triggered when the concentration exceeds a certain threshold22,23. For
instance, high concentrations of ammonia, nitrite and hydrogen
1
Key Laboratory of Agro-Forestry Environmental Processes and Ecological Regulation of Hainan Province, School of Environmental Science and Engineering,
Hainan University, Haikou 570228, China. 2State Key Laboratory of Marine Resources Utilization in the South China Sea, Hainan University, Haikou 570228,
e-mail: mabin@hainanu.edu.cn
China.
Nature Communications | (2024)15:7920
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Article
sulphide can be toxic to wastewater treatment microorganisms24,25.
Increased substrate concentrations can reduce microbial activity,
hindering the conversion of the substrate. The decreased conversion
can cause substrate accumulation around the microorganisms,
resulting in stronger toxic effects26. Substrate inhibition can disturb
the balance of the system through negative feedback, thereby reducing the efficiency of the wastewater treatment process27,28.
The existing strategies for dealing with substrate inhibition
revolve around avoiding or decreasing inhibition and recovery after
inhibition in the wastewater, involve reducing the concentration of
substances and adjusting operating conditions29–32. The main principle
of these measures is to decrease the contact between the microbial
community and the high substrate, making the community more vulnerable to inhibitory substrates33. However, substrate fluctuations are
a global situation in wastewater treatment and vary over time due to
factors such as industrial discharges, seasonal variations and population fluctuations34. The field survey revealed that high-substrate is
unavoidable35,36.
When substrate fluctuations cannot be controlled, another
potential strategy involves enhancing resistance of microbial community. The increased resistance has the potential to maintain or even
increase the system treatment efficiency and stability in the presence
of substrate stress. This mechanism is analogous to antifragility37. To
achieve the antifragility wastewater treatment system by enhance
tolerance of microbial community, a feasible hypothesis was proposed
to create a non-lethal high substrate inhibition environment.
In the wastewater treatment, anammox is the most promising lowenergy wastewater treatment technology38,39. It convert nitrite and
ammonia simultaneously into nitrogen under anaerobic conditions
without organic carbon source40,41, which can save up to 60% of energy
consumption for aeration and eliminate the requirement for external
organic carbon sources42. The promotion of anammox is consistent
with the objectives of peak carbon and carbon neutrality43,44. Anammox was a process driven by autotrophic bacteria, including four
genera (Kuenenia, Brocadia, Anammoxoglobus and Jettenia) commonly
found in activated sludge45. The enrichment and physiology work
demonstrated that nitrite is an important substrate for anammox
bacteria, but nitrite with a high level can also act as an inhibitor46,47.
Despite taking measures such as reducing nitrite concentration and
adjusting pH to minimize nitrite inhibition, half of the wastewater
treatment plants (WWTPs) still exhibited high levels of nitrite accumulation during the field survey48.
In this study, nitrite inhibition of anammox bacterial community
was used as an example, developing a sidestream nitrite treatment unit
located outside the anammox systems (Fig. 1). A portion of the
https://doi.org/10.1038/s41467-024-52364-9
anammox sludge was transported from the system to this sidestream
unit with a high nitrite concentration, and then returned to the anammox system. To verify the feasibility of improving the nitrite tolerance of anammox bacterial community in a non-lethal high nitrite
environment, firstly, the response to different nitrite levels of anammox bacterial community was investigated to evaluate the variation
in nitrite resistance. Secondly, the operational stability of the anammox system during substrate fluctuations was examined by analyzing the nitrogen removal performance under nitrite shock.
Furthermore, the nitrogen conversion rate of the anammox system
was measured to evaluate the effect of sidestream nitrite treatment on
the anammox system. Finally, the effect of a high nitrite environment
on the community was explored through 16S rRNA and metagenomics
sequencing. This study contributes to the understanding of the resistance of anammox bacterial community to nitrite and further proposed a potential strategy to cope with substrate inhibition in
wastewater treatment engineering, promoting the stability of wastewater treatment systems under substrate fluctuations.
Results
Improved the tolerance of anammox bacterial community
through high-nitrite exposure
To explore the nitrite tolerance of anammox bacterial community, the
SAA was detected in different nitrite levels after exposure to highnitrite environment. The SAA of anammox bacterial community from
UASB1 and UASB2 were expressed by SAA1 and SAA2, respectively.
Trends in SAA1 and SAA2 at different nitrite concentrations were
shown in Fig. 2, and the MLSS were 7.28 g·L−1 and 5.35 g·L−1, respectively.
The results showed that exposure to high nitrite concentrations,
but not lethal, enhanced the tolerance of anammox bacterial community. The SAA1 reached its peak when the nitrite-to-ammonia ratio
was 1.0 (with a nitrite concentration of 10 mg·L−1). When the ratio
increased to 3.0 (with a nitrite concentration of 30 mg·L−1), SAA1
decreases by over 69%. And SAA1 decreases by 80.81% when the ratio
reaches 4.0.
Comparing the results, SAA2 was much higher than SAA1
throughout all five nitrite concentrations. Anammox bacterial community that had been previously exposed to high-nitrite were able to
maintain their activity while the nitrite-to-ammonia ratio increased
to 3.0. Furthermore, even when the nitrite concentration was up to
40 mg·L−1, SAA2 was 24.71 times higher than that of SAA1, and the
nitrite removal rate of anammox bacterial community in UASB2 was
20.15 times higher than that of UASB1.
These results indicate that anammox bacterial community in
UASB2 were more tolerant to high nitrite levels. Anammox bacterial
Fig. 1 | Device details of two UASB reactors. UASB1 was the control reactor, UASB2 was equipped with a sidestream unit separately.
Nature Communications | (2024)15:7920
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https://doi.org/10.1038/s41467-024-52364-9
TN removal efficiency
5.0
150%
120
4.0
120%
90
3.0
60
2.0
30
1.0
30%
0.0
0%
5.0
150%
120
4.0
120%
90
3.0
60
2.0
30
1.0
30%
0.0
0%
UASB1
0
0
1
2
3
4
NRR (g-N·L-1·d-1)
Nitrite in effluent (mg·L-1)
a
NRR
90%
60%
TN removal efficiency
Nitrite in effluent
150
5
150
UASB2
0
0
1
2
3
4
90%
60%
TN removal efficiency
Nitrite in effluent (mg·L-1)
b
NRR (g-N·L-1·d-1)
Time (h)
5
Time (h)
Fig. 2 | The nitrite tolerance of anammox bacterial community was explored by
detecting the nitrite removal rate and SAA in different nitrite levels. (a) was
UASB1 reactor and (b) was UASB2 reactor.
Fig. 3 | The nitrogen removal performance of effluent nitrite concentrations,
NRR and TN removal efficiency under nitrite shock. (a) was UASB1 reactor and
(b) was UASB2 reactor.
community had higher activity after exposure to nitrite, even when the
nitrite concentration exceeded the theoretical anammox stoichiometric ratio. The tolerance of microbial community to inhibitory
substrates was enhanced by a non-lethal inhibitory environment.
was significantly slower. This can be attributed to the anammox bacterial community in UASB2 being able to maintain greater activity at
high nitrite concentrations. It enabled the simultaneous removal of
ammonia and nitrite, providing the anammox system a more stable
nitrogen removal performance. In conclusion, exposing the microorganisms to high substrate concentrations in the sidestream unit can
improve the stability of the system to substrate fluctuations.
Increased the shock resistance of anammox systems through
high-nitrite exposure
In order to investigate the resistance of the anammox system to high
nitrite concentrations, a sudden increase of influent nitrite concentration from 80 mg·L−1 to 200 mg·L−1 to achieve the nitrite shock.
The nitrogen removal performances, such as effluent nitrite concentration, NRR and TN removal efficiency was shown in Fig. 3.
Anammox systems with the sidestream nitrite treatment unit
exhibited greater operational stability during the nitrite shock test.
Within 5 hours, NRR1 was decreased by 57.35%, while NRR2 was only
decreased by 28.85%. The trend of TN removal efficiency was consistent with the NRR. In UASB1, the decrease in TN removal efficiency
was more than double that of UASB2. The slower decline in nitrogen
removal performance indicated that the anammox system was more
antifragile to high nitrite concentrations, suggesting that UASB2
exhibiting twice the level of antifragility compared to UASB1.
Nitrite concentrations in UASB1 was increased from 7.92 mg·L−1 to
108.51 mg·L−1 over time was caused by the lower nitrite tolerance of
anammox bacterial community. As the nitrite concentration increased
within the reactor, it diminished the activity of anammox bacterial
community and the ability to remove nitrite, consequently leading to
further nitrite accumulation and stronger nitrite inhibition. This
negative feedback mechanism caused NRR1 to decline significantly
faster than NRR2. The increase rate of nitrite concentration in UASB2
Nature Communications | (2024)15:7920
Enhanced antifragility of anammox system through high-nitrite
exposure
In order to evaluate the effect of high nitrite treatment on main system,
the nitrogen removal performance of UASB2 reactor was investigated
during the operation of the sidestream unit. The analyses showed that
there was an effect of the high nitrite treatment on the anammox
system, as evidenced by fluctuations in the nitrogen removal performance. However, this effect did not reappear during the second high
nitrite treatment. The nitrogen removal performance of the UASB1
reactor was shown in Supplementary Fig. 1.
A portion of the anammox sludge was transported from the system to the sidestream unit for exposure to high nitrite on day 34
(phase II). After one day of operation, the effluent nitrite concentration
of UASB2 immediately increased from 5.44 mg·L−1 to 30.47 mg·L−1
(Fig. 4). During ten days of operating the sidestream nitrite treatment
unit, the average effluent nitrite concentration in UASB2 was
28.03 mg·L−1. Consequently, there was a significant decrease in the
NRR of UASB2, amounting to approximately 56.04%. These results
emphasize that the first operation of the sidestream nitrite treatment
unit had an impact on the nitrogen removal performance of the
reactor.
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Phase II
Influent TN (mg·L-1 )
240
4.0
Influent TN
NLR
180
3.0
120
2.0
60
1.0
0
0.0
NLR (g N·L-1·d-1 )
a
b
First
exposure
Second
exposure
120
Third
exposure
120
90
Effluent TN (mg·L-1 )
NRR (g N·L-1·d-1 )
NRR
Nitrite exposure
Effluent TN
Nitrite exposure (mg·L-1 )
3.2
2.4
90
1.6
60
0.8
30
0.0
0
0
2.0
125%
60
30
Nitrite in eff
effluent
ffluent
TN removal
eff
fficiency
efficiency
FNA
100%
1.5
)
45
-1
Nitrite in effluent (mg·L-1 )
60
30
75%
1.0
50%
15
0.5
0
0.0
0
33
44
67
78
116
127 144
TN removal efficiency
c
25%
0%
180
Days
Fig. 4 | Reactor performances of the UASB2 reactor. a Influent TN concentrations and NLR. b Effluent TN concentrations, nitrite exposure concentrations and NRR.
c Effluent nitrite concentrations, FNA and TN removal efficiency.
The second nitrite exposure operation in the sidestream unit was
started on day 68. The nitrite effluent concentration of UASB2
remained below 10 mg·L−1 throughout the second operation, and the
NRR of UASB2 was consistent that before nitrite exposure. Compared
to the first operation, the nitrite exposure from the sidestream unit had
a significantly lower impact on the UASB2 reactor.
The sidestream nitrite treatment unit then began its third operation on day 117. Remarkably, during this operation, the TN removal
efficiency was maintained at 88.5% despite the nitrite exposing concentration in the sidestream unit gradually increased from 30 mg·L−1 to
100 mg·L−1. Anammox bacterial community were exposed to a higher
nitrite concentration during the third operation, the NRR of UASB2
increased significantly by 33.3%. The anammox system are not only
able to withstand nitrite stress, but also exhibit antifragility by displaying high nitrogen removal efficiency in the presence of stress.
The exposure of the anammox bacterial community to high nitrite
concentrations in the sidestream unit played a crucial role in enhancing their nitrite tolerance. Upon returning to the normally operating
UASB2 reactor, the anammox bacterial community had the
Nature Communications | (2024)15:7920
opportunity to recover under low nitrite concentrations and simultaneous presence of ammonia. This non-lethal exposure and timely
recovery of anammox bacterial community likely acquired a heightened resistance and improved nitrite tolerance, which potentially
enhanced the ability to withstand and adapt to varying nitrite levels.
High-nitrite environments alter population diversity
The Ace and Chao indices were used to measure species richness.
Lower values in these estimators correspond to decreased species
richness. Concurrently, the Simpson and Shannon indices were
employed to assess species evenness, where a lower Simpson index
and a higher Shannon index indicate higher species evenness49,50. The
microbial diversity in seed sludge and UASB1 was higher than UASB2.
Higher diversity means that there are more complementary units
adapted to nitrite stress, which could work together to cope with
stresses51. In this way, the anammox bacterial community were able to
adjust and repair more quickly when exposed to high nitrite concentrations, enhancing the adaptation of the community. There was a
slight decrease in microbial richness and evenness in UASB2, and this
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https://doi.org/10.1038/s41467-024-52364-9
a
Ace
b
Chao
800
550
6.0
700
Shannon
Simpson
0.12
4.5
0.10
3.0
0.08
1.5
0.06
500
Simpson
Shannon
600
Chao
Ace
525
500
100
0
475
Seed
UASB1
0.0
UASB2
0.04
Seed
UASB1
UASB2
c
Stochastic
Drift
Homogenizing Dispersal
Dispersal Limitation
UASB1
Deterministic
Homogeneous Selection
Heterogeneous Selection
UASB2
0.0
0.2
0.4
0.6
0.8
1.0
Relative abundance
Fig. 5 | Characteristics of population diversity. a Species richness expressed via Ace and Chao estimator. b Species evenness expressed via the Shannon and Simpson
indices. c The summary of assembly processes.
a
b
Abundence
Relative abundance at the phylum level
Distance
0.3
Hydrogenophaga
0.2
norank_f__A4b
0.1
norank_c__WWE3
0.0
Thermoanaerobaculum
100%
Limnobacter
other
Latescibacterota
Armatimonadota
Calditrichota
Patescibacteria
Actinobacteriota
Acidobacteriota
Chloroflexi
Proteobacteria
Bacteroidota
Planctomycetota
75%
50%
25%
0.01
0.1
0.2
0.3
OLB13
norank__o__SJA-15
norank_c__OLB14
norank_o__SBR1031
norank_c__SJA-28
Denitratisoma
Candidatus Brocadia
Candidatus Kuenenia
Candidatus Jettenia
0%
d
See
UA
SB
1
UA
SB
2
d
See
UA
SB
1
SB
UA
2
Fig. 6 | The structure of microbial communities. a Hierarchical clustering tree and distribution of the bacterial community on phylum level. b Analysis of bacterial
community abundance at the genus level.
decrease was a response of the microbial community to high nitrite
stress (Fig. 5).
The variation in the community assembly process was further
investigated by comparing the βNTI index, and the results of the βNTI
analyses were used to calculate the proportions of deterministic and
stochastic processes (Fig. 5c). The results showed that heterogeneous
Nature Communications | (2024)15:7920
selection dominated for microbial communities in UASB2, with basic
abiotic parameters as the driving factors52. Nitrite concentration was
probably the main driver in this study. Hierarchical clustering analysis
were used to compare bacterial communities from different samples,
as shown in Fig. 6a. The analysis revealed that the UASB2 sludge
samples were dissimilar from the other samples based on phylogeny.
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UASB1
100%
UASB2
Candidatus Brocadia
Candidatus Jettenia
Candidatus Kuenenia
others
Contribution
75%
50%
25%
0%
hzs
hdh
hao
hzs
hdh
hao
Fig. 7 | The contribution of anammox bacterial community to key functional genes. Left panel: Control group without nitrite addition (UASB1). Right panel: Treatment
group with nitrite addition (UASB2).
The observed dissimilarity in UASB2 may be attributed to microbial responding environmental stressors, such as nitrite exposure,
prompted a deeper investigation into the microbial community
structure.
High-nitrite environments shift the structure of microbial
communities
The study found that the abundance of three phyla, Planctomycetes,
Chloroflexi and Bacteroidetes, increased in UASB2 as compared to
UASB1, while there was a significant decrease in the abundance of
Proteobacteria (Fig. 6a). Planctomycetes phylum contained three types
anammox genera, Candidatus Kuenenia, Candidatus Brocadia and
Candidatus Jettenia (Fig. 6b). Among these, Candidatus Kuenenia was
the dominant genera in the seed sludge and UASB1 sludge, the relative
abundance was 9.93% and 12.62%, respectively. The dominance of
Candidatus Kuenenia can be attributed to its k-strategist characteristics, making it more competitive under conditions of low nitrogen
concentrations53–55.
After nitrite exposure, the main anammox bacterial genera were
Candidatus Jettenia, accounting for 28.26% in UASB2, while the
remaining two genera accounted for less than 0.5%. Candidatus Kuenenia was decreased abruptly due to their susceptibility to fluctuating
substrate conditions34. This transformation in response to nitrite shock
potentially contributed to the antifragility of UASB2 reactor. Since
Candidatus Jettenia may possess superior adaptive advantages in high
nitrite concentrations compared to other anammox genera, it was able
to adapt well to influent fluctuation. The variation of anammox bacterial community in UASB2 was consistent with the diversity. The
adapted groups grew and multiplied, and the non-adapted groups
withered and died, changing the proportions of different genera in the
anammox bacterial community.
Denitratisoma and norank_c__SJA-28 are the two genera in the
UASB2 system with relatively significant abundance variations, except
anammox genera. Both Denitratisoma and norank_c__SJA-28 have the
ability to participate in denitrification reactions56–58, which was
important in anammox systems59,60. Nevertheless, Denitratisoma
decreased in abundance at high nitrite concentrations61. In some cases,
organisms in the SJA-28 lineage encoded an ammonia-forming cytochrome c nitrite reductase of the nrfAH-type involved in nitrite
reduction62. That may allow SJA-28 survive higher nitrite concentrations, which may explain the increased abundance in UASB2.
More analyses of the different bacterial communities in UASB1
and UASB2 in the Supplementary Information. Differences of bacterial
communities in UASB2 may be due to different responses to nitriteinduced stress. Further exploration of microbial functions could provide insights into changes in community distribution.
Nature Communications | (2024)15:7920
High-nitrite environment transformed the community contribution to functional genes
The anammox process involves the conversion of nitrite and ammonia
to N2 gas through three possible pathways. This conversion process
relies on the synergistic action of three key functional enzymes:
hydrazine synthase (HZS), hydrazine dehydrogenase (HDH), and
hydroxylamine oxidoreductase (HAO)63. HZS catalyses the conversion
of NO from nitrite to hydrazine (N2H4) with NH4+. The N2H4 generated
is then oxidized by HDH and eventually converted to N264. HAO was
believed to reconvert any hydroxylamine (NH2OH) that escaped from
the HZS complex back into NO, thereby detoxifying this inhibitor
NH2OH and ensuring the normal progression of the anammox
process65,66.
The genes producing these enzymes were detected by metagenomic analysis to investigate the contribution of anammox bacterial
community to key functional genes. It was found that the functional
genes hzs, hdh and hao, encoded by Candidatus Kuenenia in the
UASB1, were highly abundant, contributing 91.76%, 92.37%, and
80.82%, respectively (Fig. 7). After the nitrite treatment, the abundance
of functional genes encoded by Candidatus Jettenia increased significantly and became the largest contributing anammox genus. It
indicated that the anammox process was dominated by Candidatus
Jettenia bacteria in UASB2, which was consistent with the change in
community structure.
Variations in other genes related to nitrogen metabolism were
shown in Supplementary Fig. 4. and Supplementary Fig. 5.
Discussion
The non-lethal high substrate environment could enhance the nitrite
tolerance of anammox bacterial community, as demonstrated in the
following two aspects: (1) Anammox bacterial community exhibited
24.71 times greater tolerance to nitrite, as shown by the higher SAA at
nitrite concentration was 40 mg·L−1; (2) Anammox systems displayed
greater resistance during sudden nitrite shock from 80 mg·L−1 to
200 mg·L−1.
The enhancing antifragility of the anammox system was attributed to the non-lethal exposure of nitrite in the sidestream unit, this
operation resulted in shifted the dominant genera of the anammox
bacterial community. Microbial communities have developed a variety
of strategies to survive in toxic environments, and the shift of nitrite
tolerance within the anammox dominant genus was a protective
mechanism. Candidatus Jettenia has been identified as the dominant
anammox population in response to increased substrate load, as a high
nitrogen concentration may be more favorable for Candidatus
Jettenia67,68. A study has found that Candidatus Jettenia was detected as
the influent substrate concentration increased, and its abundance
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increased by approximately 0.51% within 13 days69. In this experiment,
through nitrite exposing in the sidestream unit repeatedly and at a
maximum nitrite concentration up to 100 mg·L−1, the proportion of
Candidatus Jettenia increased and became the dominant anammox
genera. Candidatus Jettenia might possess a higher tolerance to nitrite.
High nitrite concentrations in the sidestream unit inhibited the growth
of other anammox genera such as Candidatus Brocadia. Meanwhile,
Candidatus Jettenia was in a better position to survive than other
anammox genera, increasing abundance.
During practical wastewater treatment, it has been observed that
the nitrogen removal efficiency of the anammox system was still
reduced following repetitive occurrences of high nitrite
concentrations27,28. This may be because reducing nitrite concentration immediately was the primary operation when high nitrite concentration appeared70,71. This operation is not sufficient to select for
another anammox genus into one that is more adapted to high nitrite
concentrations, and also making the anammox community more vulnerable towards fluctuations in nitrite concentrations.
Combined with the results of this study, a sidestream unit for
nitrite treatment could be added in the biological nitrogen removal via
anammox in wastewater treatment system. A portion of the anammox
sludge were channel into this unit, and then returned to system after
nitrite treatment, contributing to increase the antifragility of the anammox system to nitrite. The nitrite used in the sidestream unit can be
produced from anaerobic digester liquor of wastewater treatment
plants72,73, and nitrite as a green and renewable chemical can be treated
in the subsequent anammox reactor before its final discharge74,75.
However, the addition of a backflow device for nitrite treatment will
increase operating costs. Additions can be made based on the process
and operational goals of the actual WWTPs. Depending on local conditions, they may vary from region to region and country to country.
Creating non-lethal high substrate inhibition environment could
be used to improve resistance not only to nitrite, but also has the
potential to be used for other substrates, such as ammonia and sulfur
are present in wastewater treatment processes76,77. Variations in
microbial tolerance to different substrate inhibition in wastewater
treatment have been investigated extensively. For instance, Nitrosomonas cluster 7 has a stronger tolerance to high ammonia than other
ammonia oxidizing bacteria78, sulphur-oxidising bacteria Chromatiaceae are more tolerant of sulfur (thiosulphate) than Thiobacillus79, Ca.
Accumulibacter clades IID involved in denitrifying phosphorus
removal via nitrite pathway showed strong tolerance to nitrite than
clades IIC80. Consequently, the non-lethal high substrate could use to
increase tolerance of functional microbial communities. The creation
of a non-lethal high substrate environment in this study was achieved
using the sidestream treatment unit. This may be a feasible strategy to
enhance the antifragility of the system by improving the tolerance of
the microbial community without affecting normal operation. It is a
potential approach to ensure that the performance of the wastewater
treatment system remains stable even under fluctuating substrate
concentrations.
Methods
Experimental facilities
Two UASB reactors were operated in the experiment. The first UASB
reactor, UASB1, was used as the control reactor, which operated
without a sidestream unit. Another UASB reactors, UASB2, connected
with a sidestream nitrite treatment unit, as shown in Fig. 1.
Two UASB reactors were made of plexiglass. The reaction zone
was a cylinder with a height/diameter ratio of 10:1 and a reaction
volume of 1 L. The UASB were covered with silver paper and black tape
to avoid the growth of photosynthetic microorganisms. Influent was
controlled by an influent pump, and synthetic wastewater was prepared every day. To maintain an anaerobic environment, nitrogen gas
was used to reduce dissolved oxygen (DO) in synthetic wastewater.
Nature Communications | (2024)15:7920
https://doi.org/10.1038/s41467-024-52364-9
When the DO was reduced to less than 1 mg·L−1, the water surface was
covered with plastic film to avoid oxygen dissolving into the synthetic
wastewater. A small agitator, which connected the controller, was put
into the reactor for mixing synthetic wastewater and sludge. A sampling port was set on the side of the reactor so a syringe could be used
for quantitative sampling.
In the sidestream nitrite treatment unit, 100 ml mixture was taken
from the UASB2 reactor into 400 ml of water (DO < 0.3 mg·L−1), so the
total volume of the sidestream unit was 500 ml. The sidestream unit
was placed on a magnetic stirrer with a rotating speed of 100 rpm.
Black plastic was used to cover the liquid surface to avoid oxygen
dissolving during mixing, and DO and pH probes (WTW 3420, WTW
Company Germany) were put into the sidestream unit for monitoring.
Only nitrite was added to the sidestream unit. The maximum effluent
nitrite concentration (30 mg·L−1) of the UASB2 reactor was taken as the
initial inhibition concentration. After nitrite treatment, the sludge was
washed five times to ensure that the nitrite concentration was less than
1.0 mg·L−1, and pumped back to the UASB2 reactor.
Synthetic wastewater
NH4Cl and NaNO2 were used as substrates for anammox bacterial
community, and the ratio of nitrite to ammonia in UASB influent was
1.0. Other synthetic wastewater components include (per liter):
450 mg NaHCO3, 18 mg KH2PO4, 14 mg CaCl2·2H2O, 90 mg
MgSO4·7H2O, and 1.25 mL trace elements. Trace element solution A
contained (per liter): 5 g EDTA and 22 g FeSO4, the Fe2+ concentration
remained at 0.08 mM during the whole experiment81. Trace element
solution B contained (per liter): 15 g EDTA, 0.014 g H3BO4, 0.19 g
NiCl2·6H2O, 0.22 g NaMoO4·2H2O, 0.25 g CuSO4·5H2O, 0.99 g
MnCl2·4H2O, and 0.43 g ZnSO4·7H2O82.
Seed sludge
The seed sludge was 2.4 L of the floc anammox sludge which has been
enriched in the laboratory. The sludge was evenly mixed and divided
into two parts and inoculated into two UASB reactors. The mixed
liquor suspended solids (MLSS) of each reactor were 6.516 g·L−1. A
biological sample (50 mL) was reserved for analysis of community
structure.
Operation of UASB reactors and sidestream nitrite
treatment unit
The operation was divided into three phases.
In phase I (1–33 days), start-up of two anammox reactors. The
initial hydraulic retention time (HRT) of two UASB reactors was 1.2 h
for 23 days. Part of the sludge accumulated in the three-phase
separator, which may be due to the high up-flow rate. To prevent
sludge loss, the HRT of two reactors was adjusted to 2.4 h on day 24.
When the effluent nitrite concentration was below 15 mg·L−1, increasing
the influent total nitrogen (TN) concentration to gradually increase the
nitrogen loading rate (NLR) to meet the growing demands of anammox bacterial community.
In phase II (34–126 days), three nitrite exposure were performed.
One-tenth of the sludge from UASB2 was exposed to nitrite in the
sidestream unit. The sidestream unit was operated for ten days at a
time, with a total of three operations during the whole phase, on the
34th, 68th and 117th days (see Supplementary Methods and Supplementary Table 1 for more details of nitrite treatment concentrations).
UASB1 operated normally throughout this phase.
After phase II, to explore the adaptability of anammox bacterial
community to high nitrite concentrations, sludge was taken out from
the reactor to study the response of anammox bacterial community to
different nitrite levels through the specific anammox activity (SAA)
test70. The device used for the SAA test was the same as that of the
sidestream unit. The sludge in each reactor was divided into five sections, with five separate devices. The NH4+-N concentration was fixed at
7
Article
20 mg·L−1, while the NO2−-N concentrations were 10, 20, 30, 40,
50 mg·L−1, respectively. The medium was blown with N2 to remove DO,
and pH and temperature were measured. The MLSS was determined
and the SAA was expressed as g-N·g-SS−1·d−1. After the SAA test, the
sludge was starved at 4 °C for approximately two weeks.
In phase III (144–184 days), restart the two reactors. The sludge
was returned to each UASB reactor and the temperature was restored
to 31.0 ± 1.5 °C. To avoid substrate inhibition, influent TN concentration was increased gradually when the effluent nitrite concentration
was below 15 mg·L−1 to restore the nitrogen removal performance. On
day 185, the nitrite shock test was operated by suddenly increasing the
influent NO2−-N concentration from 80 mg·L−1 to 200 mg·L−1 to investigate the stability of two reactors, while the influent concentration of
NH4+-N was stable at 80 mg·L−1. The nitrite shock test was maintained
for 5 h, the concentration of NH4+-N, NO2−-N, and NO3−-N in the reactor
were detected every 30 min, and pH and temperature were monitored
simultaneously. The decline rate of NRR was used to characterize the
antifragility of the anammox reactor to high nitrite concentrations.
Chemical analysis and calculations
The mixed liquor samples were taken by syringes and filtered through
a 0.45 μm membrane immediately, and analysed for ammonium,
nitrite, and nitrate with an Automatic multi-parameter water quality
analyzer (DeChem-Tech Cleverchem Anna, Germany). The total
nitrogen (TN) and nitrogen removal rate (NRR) was calculated
accordingly. Free nitrous acid (FNA), the protonated form of nitrite,
was be determined through the nitrite concentration, pH and
temperature83.
Community structure analysis and visualization
Three sludge samples were collected. One samples from the initial
moments of two UASB reactors (day 1) were named Seed. Two samples
at the end of the two reactor operations (day 185) were named UASB1
and UASB2, respectively. Three replicates of amplicon sequencing
were performed for each sample. DNA extraction and purification were
conducted following the instruction of the E.Z.N.A.® soil DNA Kit
(Omega Bio-tek, Norcross, GA, U.S.), and the DNA concentration was
determined via using NanoDrop2000 (Thermo Scientific, Wilmington,
USA). The primer pair of 338 F (5’-ACTCCTACGGGAGGCAGCA-3’) and
806 R (5’- GGACTACHVGGGTWTCTAAT-3’) were selected for polymerase chain reaction (PCR) amplification, and paired-end sequenced
on the Illumina MiSeq PE300 platform (Illumina, San Diego, USA).
After quality filtering, a mean length ranging from 422 bp were
obtained for each sample. To avoid unequal sequencing depth the
number of valid sequences is 38223. These sequences were clustered
into operational taxonomic units (OTUs) at a 97% similarity level.
Based on the OTU clustering analysis, Mothur (v.1.30.2) software was
used to analyze the OTU diversity index (alpha diversity analysis within
samples)84. Beta diversity was calculated using QIIME, and hierarchical
clustering was performed based on the beta diversity distance
matrix85. To evaluate community assembly processes, the β-nearest
taxon index (βNTI) and Bray-Curtis-based Raup-Crick (RCBray) were
calculated through the package icamp (version 1.5.12)86.
https://doi.org/10.1038/s41467-024-52364-9
Paired-end sequencing was performed according to the manufacturer’s instructions. The raw data were trimmed (length< 50 bp or
with a quality value < 20 or having N bases) by fastp (https://github.
com/OpenGene/fastp, version 0.20.0). The size of metagenomic data
of each sample ranges from 7.3 Gb to 8.8 Gb after quality control. The
functional annotation was conducted using DIAMOND (version 0.8.35)
against the Kyoto Encyclopedia of Genes and Genomes database
(https://www.genome.jp/kegg/)87,88.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
All data generated in this study are provided in the article file, Supplementary Information, and Supplementary Data. The relevant source
data from each figure are provided in the Source Data files. The
amplicon sequence datasets generated during this study are available
with accession number PRJNA1102518. The metagenomic sequence
data could be found at BioProject PRJNA1094462. Source data are
provided with this paper.
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Acknowledgements
This research was financially supported by the National Natural Science
Foundation of China (52260003, U23A20675, to B.M.), the Hainan Provincial Natural Science Foundation of China (422QN274, to Y.W.) and the
Key Research and Development Plan of Hainan Province
(ZDYF2021SHFZ061, to B.M.).
Author contributions
B.M., B.L., C.L., and Y.W. conceived the study. B.L. and C.L. performed
the experiments, data analysis, and prepared the manuscript. B.M.
supervised the whole research and revised the manuscript. J.B.
performed the community assembly modeling. Y.H. visualized the
data. R.S. revised the title of the manuscript. All authors are involved
in the interpretation of the results and the preparation of this
manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-024-52364-9.
Correspondence and requests for materials should be addressed to
Bin Ma.
Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A
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