Accepted Manuscript

Autotrophic and heterotrophic nitrification-anoxic denitrification dominated the

anoxic/oxic sewage treatment process during optimization for higher loading
rate and energy savings

Xueyu Zhang, Shaokui Zheng, Hangyu Zhang, Shoupeng Duan

PII: S0960-8524(18)30636-9
DOI: https://doi.org/10.1016/j.biortech.2018.04.113
Reference: BITE 19893

To appear in: Bioresource Technology

Received Date: 29 March 2018

Revised Date: 22 April 2018
Accepted Date: 27 April 2018

Please cite this article as: Zhang, X., Zheng, S., Zhang, H., Duan, S., Autotrophic and heterotrophic nitrification-
anoxic denitrification dominated the anoxic/oxic sewage treatment process during optimization for higher loading
rate and energy savings, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.04.113

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Autotrophic and heterotrophic nitrification-anoxic denitrification
dominated the anoxic/oxic sewage treatment process during
optimization for higher loading rate and energy savings

Xueyu Zhang, Shaokui Zheng*, Hangyu Zhang, Shoupeng Duan

School of Environment, MOE Key Laboratory of Water and Sediment Sciences/State Key Lab

of Water Environment Simulation, Beijing Normal University, Beijing 100875, China

* Corresponding author.
E-mail address: zsk@bnu.edu.cn (S. Zheng).

1
ABSTRACT

This study clarified the dominant nitrogen (N)-transformation pathway and the key

ammonia-oxidizing microbial species at three loading levels during optimization of the

anoxic/oxic (A/O) process for sewage treatment. Comprehensive N-transformation activity

analysis showed that ammonia oxidization was performed predominantly by aerobic

chemolithotrophic and heterotrophic ammonia oxidization, whereas N2 production was

performed primarily by anoxic denitrification in the anoxic unit. The abundances of

ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria, and anaerobic AOB in

activated sludge reflected their activities on the basis of high-throughput sequencing data.

AOB amoA gene clone libraries revealed that the predominant AOB species in sludge samples

shifted from Nitrosomonas europaea (61% at the normal loading level) to Nitrosomonas

oligotropha (58% and 81% at the two higher loading levels). Following isolation and

sequencing, the predominant culturable heterotrophic AOB in sludge shifted from

Agrobacterium tumefaciens (42% at the normal loading level) to Acinetobacter johnsonii

(52% at the highest loading level).

Keywords: Nitrogen-transformation pathway; Anoxic/oxic (A/O) process; Domestic

wastewater; Nitrification startup; Heterotrophic nitrification

2
1. Introduction

Conventional biological nitrogen removal (BNR) through the anoxic/oxic (A/O) process is

widely employed in large-scale municipal wastewater treatment plants (WWTPs) worldwide

as a means to control eutrophication of natural water bodies (Guo et al., 2010; Gao et al., 2013;

Zhang et al., 2017a). In most cases, the maximum chemical oxygen demand (COD) and

NH4+-N loading rate (CODLR and NH4+-NLR) of the A/O process for municipal wastewater are

just ~1.0 kg COD·m-3·d−1 and ~0.2 kg NH4+-N·m-3 ·d−1, respectively (Guo et al., 2010), which

are far lower than those of other sewage treatment processes (e.g., ~3.0 kg COD·m-3·d−1 and ~

0.3 kg NH4+-N·m-3·d−1 for membrane bioreactors (Khan et al., 2013) and ~3.4 kg

COD·m-3·d−1 and ~0.4 kg NH4+-N·m-3·d−1 for a circulating fluidized bed bioreactor (Patel et

al., 2006). Furthermore, aeration often represents 45~75% of the total energy expenditure in

municipal WWTPs (Rosso & Stenstrom, 2006). Therefore, low loading rate and high energy

consumption often limit the widespread application of the A/O process in municipal WWTPs.

Based on 258 days of continuous operation of a lab-scale A/O system for sewage treatment,

which was reported before (Zhang et al., 2017a), with a decrease in hydraulic retention time

(HRT) for the A/O reactor from 8 to 2 h，the optimized A/O process could operate stably at far

higher CODLR and NH4+-NLR levels (~3.7 kg COD·m−3·d−1 and ~0.6 kg NH4-N·m−3·d−1,

respectively), with COD, NH4+-N, total nitrogen (TN), and total phosphorus (TP) removals

that remained stable at 95%, 98%, 79% and 74%, respectively.

Optimized A/O offers numerous advantages over the conventional process, including

lower energy consumption (Zhang et al., 2017a). These findings are helpful for the future

development of A/O process designs for sewage treatment.

Compared with COD removal, complete N removal from wastewater is often a greater
3
challenge for the A/O process (Wells et al., 2009). Traditionally, N removal by the A/O

process is predominantly achieved through a combination of oxic ammonia oxidation to

nitrite and nitrate (i.e., nitrification) by aerobic chemolithoautotrophic microorganisms,

including ammonia-oxidizing bacteria (AOB) or archaea (AOA) and nitrite-oxidizing bacteria

(NOB), and anoxic denitrification to reduce the nitrification products to nitrogen gas (N2) by a

large diversity of anoxic denitrification bacteria (Hallin et al., 2005). In the last two decades,

novel microbial N-transformation mechanisms, including aerobic heterotrophic ammonium

oxidation by heterotrophic AOB, chemolithoautotrophic anaerobic ammonia oxidation

(anammox) by anaerobic AOB, and aerobic denitrification by denitrifying microorganisms,

have been frequently identified in various BNR processes (Zhang et al., 2017b). For example,

Fitzgerald et al. (2015) observed that the nitrification was achieved by aerobic heterotrophic

ammonium oxidation in a microaerobic nitrification reactor when no significant population of

AOA, AOB or anaerobic AOB was detected. Many heterotrophic AOB were isolated from

activated sludge and simultaneously achieved aerobic heterotrophic ammonium oxidation and

aerobic denitrification (Chen et al., 2015; Padhi et al., 2017). Other researchers (Chen et al.,

2009; Wen et al., 2017) demonstrated that aerobic chemolithotrophic ammonia oxidation,

anammox, and denitrification were actively achieved in a microaerobic rotating biological

contact reactor. Furthermore, the dominant species in specific ammonia-oxidizing microbial

communities, such as AOB, often varies based on differences in environmental or operational

conditions (Gao et al., 2013). Thus, from a practical perspective, a fundamental understanding

of the dominant N-transformation pathway and the key ammonia-oxidizing microbial species

in the optimized A/O process and the tracking of changes during process optimization are

essential for accelerating nitrification startup and ensuring stable N-removal.

4
 As two key steps in N removal from wastewater, ammonia removal is mainly achieved

through three pathways including aerobic chemolithotrophic ammonia oxidation, aerobic

heterotrophic ammonia oxidation and anammox, while N2 production is also achieved by

three main pathways including anammox, anoxic denitrification and aerobic denitrification

(Zhang et al., 2017b). Their relative contribution to ammonia removal (or N2 production) can

be determined and compared through comprehensive N-transformation activities (Zhang et al.,

2017b). In this manner, the dominant N-transformation pathways for ammonia removal and

N2 production can be elucidated. Furthermore, the rapid development of modern molecular

biological techniques, such as quantitative polymerase chain reaction (qPCR), clone libraries,

and high-throughput pyrosequencing in recent decades have enabled better determination of

the presence, distribution, and population dynamics of AOB (Hallin et al., 2005), NOB (Ge et

al., 2014), AOA (You et al., 2009), and anaerobic AOB (Schmidt et al., 2003) in BNR

systems. Based on these methods, the primary objectives of this study were as follows: (i) to

determine the dominant N-transformation pathway in the optimized A/O process and track its

changes during process optimization from the normal loading rate to the highest loading rate;

and (ii) to elucidate the key ammonia-oxidizing microbial species responsible for the

dominant ammonia oxidization pathway (i.e., the rate-limiting step of the BNR process) at

three loading levels.

2. Materials and methods

2.1 Lab-scale A/O system and its treatment performance

The detailed information on the lab-scale A/O system, its operation and treatment

performance during 258 days of continuous operation has been described in our previous

study (Zhang et al., 2017a). Briefly, the lab-scale lucite A/O system includes an anoxic zone
5
and an oxic zone (effective volumes: 1.45 and 4.35 L, respectively) in a rectangular A/O

reactor, as well as a clarifier. The dissolved oxygen (DO) level of the aerobic zone was

maintained at 2.0~3.5 mg L-1. The synthetic domestic wastewater with COD, NH4+-N, and

TP levels of 200~300, 45, and 7 mg L-1, respectively, was prepared by dissolving food-grade

glucose, KH2PO4, NH4Cl, and NaHCO3 in tap water. A quasi-steady-state was achieved

when effluent pollutant levels varied by < 10% over three consecutive samplings. The

average values and standard deviations of consecutive measurement sets collected under

quasi-steady-state conditions were considered to represent the corresponding treatments.

Table 1 summarizes the operational conditions and treatment performance of the A/O system

during quasi-steady-state operation at three loading rate levels: 0.9 (Day 31~98, Phase A),

2.4 (Day 133~178, Phase B), and 3.3 (Day 179~258, Phase C) kg COD·m-3·d−1, respectively.

During each quasi-steady-state period, activated sludge samples were collected from the

aeration column every 10 days to immediately evaluated using the comprehensive

N-transformation activities. When these N-transformation activities remained stable during

continuous operation, activated sludge samples were collected from the aeration column

every 10 days for qPCR analysis and high-throughput pyrosequencing during 1~2 months.

Additionally, a fresh sludge sample collected at the end of the pseudo-steady-state period

was immediately used for analysis of AOB diversity with a clone library and for isolation of

heterotrophic AOB on agar plates. Subsequently, the A/O system was prepared for the next

phase at a higher loading level.

2.2 Comprehensive N-transformation activity test

The comprehensive N-transformation activity test has been fully described in previous

study (Zhang et al., 2017b). All N-transformation activity tests were conducted in triplicate.

6
The potential AOB (PAO), anaerobic AOB (PAnammox), and heterotrophic AOB (PHAO)

activities were individually calculated based on the decrease in NH4+–N concentration, while

the potential NOB (PNO) activity was calculated based on the decrease in NO2––N

concentration. The potential aerobic denitrification (PAeD) and anoxic denitrification (PAnD)

activities were individually calculated based on the decrease in NO3––N concentration. The

relative contribution of the anammox reaction to N2 production was determined as two times

the PAnammox activity value (Zhang et al., 2017b).

2.3 DNA extraction

Total genomic DNA was extracted from individual sludge samples using the MP

FastDNA SPIN Kit (MP Biomedicals LLC, Solon, OH, USA) following the manufacturer’s

instructions. DNA extracts were quantified with a NanoDrop 1000 Spectrophotometer

(NanoDrop Technologies, Wilmington, DE, USA). The quality of the DNA extracts was

verified by electrophoresis on a 1% agarose gel, extracts were then stored at -20 °C for

subsequent molecular analyses.

2.4 Bacterial community structure analysis of sludge samples via high-throughput

sequencing

High-throughput pyrosequencing of bacterial 16S rRNA genes in sludge samples was

utilized to compare the overall bacterial communities in nine sludge samples (three samples

from each loading level) as described in previous studies (Bhattacharjee et al., 2017) and to

investigate the abundances of AOB, NOB, anaerobic AOB, and heterotrophic AOB. Briefly,

DNA was PCR-amplified using the primer set PRK338F/PRK806R targeting the V3 and V4

regions of the bacterial 16S rRNA gene (Liu et al., 2016). PCR products were purified using

the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA) and then sent to

7
Allwegene Technology (Beijing, China) for pyrosequencing with an Illumina MiSeq PE300

sequencer (Illumina, San Diego, CA, USA). The reads obtained were processed using

Quantitative Insights into Microbial Ecology (QIIME) version 1.8.0. Paired-end MiSeq reads

were first assembled using FLASH version 1.2.11 with the default parameters. Sequences

with a quality score of less than 20 and ambiguous N bases were removed. Subsequently,

chimeric sequences were identified using QIIME with USEARCH and also removed.

Non-chimeric 16S rRNA gene sequences were clustered into operational taxonomic units

(OTUs) using UPARSE (version 7.1) at the 97% nucleotide similarity level. Representative

sequences from each cluster were queried against the SILVA 119 rRNA database. Singleton

OTUs (those represented by only one read) were omitted from downstream analysis to reduce

overprediction of rare OTUs. Prior to analysis, datasets were normalized to the total number

of reads per sample. Diversity indices (Chao 1, Coverage, Shannon, and Simpson) were

calculated in mothur using RDP reference taxonomy based on the normalized OTUs.

2.5 Analysis of AOB diversity in sludge samples via cloning and sequencing

Following methods from previous studies (Gao et al., 2014; Cabrol et al., 2016), three

clone libraries were constructed to individually determine AOB diversity in three sludge

samples collected during the three phases. The ammonia monooxygenase subunit A (amoA)

gene fragment of AOB was PCR-amplified from genomic DNA using the primer set

amoA-1F/amoA-2R (Cabrol et al., 2016), providing longer sequence reads than those

produced by pyrosequencing. The PCR products were pooled and subjected to 2% agarose gel

electrophoresis to identify the presence of the target gene. Then, these products were purified

using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). The

purified products were ligated into plasmids using pGEM®-T Easy Vectors (Promega, USA).

8
The recombinant plasmids were transformed into Escherichia coli JM109 competent cells

(Takara, Dalian, China) to construct clone libraries. In total, 50 AOB amoA clones in each

library were randomly selected for sequencing using a capillary sequencer (ABI 3730 XL).

All sequences and those of their relatives obtained from the National Center for

Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) were

aligned using Clustal X1.83 program. Sequences with 99% similarity were grouped into the

same OTU using DOTUR software with the furthest neighbor approach. The sequences of

these OTUs were compared to those in the NCBI database to identify closely related bacterial

sequences. Biodiversity indicators (Shannon and Chao 1) were also calculated with DOTUR

software.

2.6 Analysis of culturable heterotrophic AOB diversity in sludge samples via isolation,

sequencing and phylogenetic analysis

Following the isolation from three sludge samples on agar plates (0.47 g·L-1 (NH4)2SO4,

5.49 g·L-1 sodium succinate, 50 mL trace element solution, pH 7.5, 2% agar), ~50 colonies

from each agar plate were randomly selected for further purification by repeated streaking, as

described in Ren et al. (2014). Subsequently, genomic DNA from each pure culture was

extracted, purified, PCR-amplified using the universal primer set 27F/1492R for the bacterial

16S rRNA gene (Yang et al., 2011), and finally sequenced by Majorbio Bio-pharm

Technology Co., Ltd. (Beijing, China). The resulting nucleotide sequences (1260~1510 bp)

were aligned using MEGA 6.0 software and then divided into OTUs.

2.7 Accession numbers

The raw sequences generated by high-throughput pyrosequencing of bacterial 16S rRNA

genes in this study have been submitted to the NCBI Sequence Read Archive under accession

9
number SRP129929. The sequences of the AOB amoA genes obtained in this study are

deposited in the GenBank database under accession numbers MG846213~MG846350 and

MG831305~MG831306. All 16S rRNA gene sequences from heterotrophic AOB obtained in

this study were deposited in the GenBank database under accession numbers MF156926 ~

MF156973 and MG757534 ~ MG757556.

3. Results and discussion

3.1 Dominant N-transformation pathway during optimization of the A/O process

The changes of AOB and AOA amoA gene abundances in activated sludge were

quantified by qPCR and the AOB/AOA ratios during three pseudo-steady-state periods of the

A/O system at three loading levels were investigated. It appears that AOB amoA gene

abundance in all sludge samples varied over the wide range of 2.5×109 ± 8.7×108~8.6×1012 ±

7.9×1010 copies g-1 volatile suspended solids (VSS), whereas the AOA amoA gene abundance

varied within a narrow range of 4.7×106 ± 7.8×105~5.2×107 ± 1.5×107 copies g-1 VSS. AOB

amoA genes remarkably outnumbered AOA amoA genes, with the AOB/AOA ratio varying

over a wide range of 6.0×102~2.8×105, indicating that AOB are much more competitive than

AOA at oxic chemolithoautotrophic ammonia oxidization in the A/O process during three

phases. In fact, AOB have frequently been identified at abundances more than two (Gao et al.,

2014), three (Wells et al., 2009), or five (Zhang et al., 2015) orders of magnitude greater than

AOA in many industrial or municipal WWTPs worldwide. Under these conditions, the

potential oxic chemolithoautotrophic ammonia oxidization activity in activated sludge was

predominantly attributable to AOB rather than AOA, which is a prerequisite for the

comprehensive N-transformation activity test (Zhang et al., 2017b).

Figure 1 summarizes the average values and standard deviations of various

10
N-transformation activities of sludge samples collected during the three experimental phases.

It has been demonstrated that the optimized ratios of initial substrate to biomass concentration

(S0/X0) and culture times effectively reduced the determination errors in N-transformation

activity data caused by biomass production (Zhang et al., 2017b). Furthermore, 10 mg L–1

allylthiourea (ATU) can effectively inhibit AOB activity when it shows extremely low or even

no inhibition of heterotrophic AOB activity (Zhang et al., 2017b). Under the conditions, when

the contribution of AOA to chemolithoautotrophic ammonia oxidization was negligible (e.g.,

in this study), the PHAO activity determined in this study should be predominantly

contributed by aerobic heterotrophic ammonium oxidation.

In this study, the mean PAO, PNO, and PAnD activities during three phases varied within

narrow ranges of 5.0~6.0 mg NH4+-N g–1 MLSS h–1, 2.8~5.8 mg NO2 --N g–1 MLSS h–1, and

8.1~9.4 mg NO3--N g-1 MLSS h-1, respectively, which agree with previous reports in activated

sludge (e.g., 4~7 mg NOx--N · g–1 VSS · h–1 (Hallin et al., 2005) or 2 mg NOx--N · g–1 VSS ·

h–1 (Tanaka et al., 2003) for PAO activity, 4~6 mg NOx--N · g–1 VSS · h–1 for PNO activity

(Hallin et al., 2005), and 3~12 mg NOx--N · g–1 VSS · h–1 for PAnD activity

(Morgan-Sagastume et al., 2008). In contrast, the mean PAnammox activity during three

phases remained at low levels (i.e., 0.4~1.3 mg NH4+-N g–1 MLSS h–1 or 6-23% of PAO

activity), indicating that the anammox process always plays a minor role in ammonia removal

during the three phases. At the same time, the mean PHAO activity during three phases

(3.3~4.0 mg NH4+-N g–1 MLSS h–1) amounted to 66~67% of the PAO activity in this study. It

appears that ammonia removal during the three phases of the A/O process was achieved

predominantly by both chemolithotrophic and heterotrophic ammonia oxidization.

In this study, besides ammonia removal, it seems that the anammox process also plays a

11
minor role in N2 production (approximately 9~30% of PAnD activity) during all three phases.

In a previous study, 15N-isotope tracing technology has revealed that anammox contributes

1.71~7.26% to N2 production in traditional municipal WWTPs (Wang et al., 2015). Likewise,

the mean PAeD activities during three phases remained low (i.e., 0.2~0.5 mg NO3 --N g–1

MLSS h–1 or 3~6% of the PAnD activity). The considerable differences among the

PAnammox, PAeD, and PAnD activities demonstrated that N2 production during all three

phases was predominantly through the anoxic denitrification reaction in the anoxic zone of the

A/O process. Under such conditions, optimizing the design of the anoxic zone and the

recycling ratio are necessary to further improve N2 production and TN removal, as influent

ammonia was completely removed by the A/O process. Many previous experiments have

demonstrated that denitrifying enzymes are active under oxic and anoxic conditions (Patureau

et al., 2000). It has been reported that most aerobic denitrification bacteria can also undertake

anoxic denitrification reaction (Huang & Tseng, 2001) and that the PAnD activity of an

aerobic denitrification bacterial species was at least twice as high as its PAeD activity (Zhang

et al., 2017b). Therefore, it is difficult to differentiate the aerobic denitrification bacterial

community from the anoxic denitrification bacterial community due to the complex activities

of denitrifying enzymes. Under the conditions, no further investigation of the anoxic

denitrification bacterial community was conducted in this study.

3.2 Response of the overall bacterial community in activated sludge to varied loading levels

A total of 83407-128482 sequence reads per sample were obtained for each sludge sample

after low-quality, ambiguous, chimeric, singleton, chloroplastic, and mitochondrial reads were

removed from the database. After subsampling the database to the lowest read number, the

remaining reads were grouped into 534~761 OTUs per sample. High coverage estimates

12
(>0.998), as well as rarefaction curves and Shannon–Wiener curves that tended to plateau,

verified that the bacterial community in these sludge samples was represented well by the

collected gene sequences. The relative abundances of various taxonomic assignments (i.e.,

genus levels) in the three bacterial communities are shown in Fig. 2. The main three phyla

detected in the three bacterial communities (> 80% relative abundance in total) included

Proteobacteria (34~47% of the total reads in each group), Bacteroidetes (16~37%), and

Saccharibacteria (13~28%). The top four classes detected in the three groups (> 80% relative

abundance in total) included Betaproteobacteria (15~24%), Sphingobacteriia (12~34%),

Gammaproteobacteria (12~17%), and Alphaproteobacteria (5~9%). These taxa have been

reported to be dominant in both activated sludge and aerobic granules (He et al., 2017),

suggesting their ubiquity in BNR processes. Regarding genus-level abundances in the three

bacterial communities (Fig. 2), the majority of OTUs in all three groups (50~63%) were

unidentified (i.e., sequences could not be taxonomically classified at or below the genus level),

demonstrating that environmental microbes are still notably under-characterized (Che et al.,

2017; Mondav et al., 2017). Among all genera detected in the three groups, increased loading

level resulted in sharp increases in the abundances of Azospira (from 5% to 10%), Terrimonas

(from 3% to 10%), and Candidatus Competibacter (from 2% to 10%) as well as in a sharp

decrease in the abundance of Hydrogenophaga (from 6% to 0.4%). The Venn diagram of

three sludge microbial communities (Fig. 2d) illustrated that 65%, 60%, and 70% of the total

OTUs in the three samples were shared among all three, whereas the OTUs identified solely

in one group amounted to 12%, 6%, and 6%, respectively, of total OTUs. Fig. 3 summarizes

the Shannon, Chao1, and phylogenetic diversity (PD) whole-tree indices of the three bacterial

communities. Based on these three indices, differences in the alpha diversity were further

13
evaluated using the Tukey test for multiple comparisons, and no significant difference (P >

0.14) was observed between groups in most cases.

3.3 Abundance-activity relationship of specific ammonia-oxidizing bacteria

Many previous qPCR studies have demonstrated that the abundances of AOB and NOB in

activated sludge are correlated with their potential activities (Cebron et al., 2003; Kurola et al.,

2005; Ke et al., 2013), meaning the PAO and PNO activities can be used as indices of the

sizes of the active AOB and NOB populations in activated sludge (Ke et al., 2013). Recently,

high-throughput sequencing data have been used to determine the abundance of AOB

(Leyva-Diaz et al., 2015; He et al., 2017; Szabo et al., 2017), NOB (Leyva-Diaz et al., 2015;

He et al., 2017; Kinnunen et al., 2017), or anaerobic AOB (Szabo et al., 2017) in various

sludge samples based on phylogenetic classification of the functional microbial community at

the genus level. In this study, high-throughput sequencing data for AOB, NOB or anaerobic

AOB were tentatively used to correlate their abundances and activities (Fig. 4). It seems that

the relative abundances of AOB, NOB and anaerobic AOB among total bacteria in the sludge

samples were in the range of 0.05~0.17%, 0.42~1.83%, and 0.01~0.16%, respectively. These

values are similar to those reported in previous studies by high-throughput sequencing (e.g.,

0.12~1.00% for AOB and 1.92~3.68% for NOB (He et al., 2017) and ~0.3% for anaerobic

AOB) (Szabo et al., 2017). Nitrosomonas was predominant in all sludge samples (89, 99.6,

and 100% of AOB during Phases A–C), whereas Nitrosococcus mobilis was detected only

during Phases A (11%) and B (0.4%). Among NOB, all detected sequence variants were

classified as Candidatus Nitrotoga (94% of NOB during Phase A) or Nitrospira (6% during

Phase A and 100% during Phases B and C). Compared with qPCR assays, high-throughput

sequencing offers a more efficient approach, as it provides more information and

14
simultaneously quantifies AOB, NOB and anaerobic AOB. Figure 4 illustrates the

relationships between the relative abundances of AOB, NOB, or anaerobic AOB and their

activities during the three phases. The correlation coefficient (R2) of the three linear

regression equations was 0.85~0.94, demonstrating a significant (P < 0.01) positive

correlation between the relative abundance and the activity of the three ammonia-oxidizing

bacterial groups. These results demonstrated that variation in ammonia-oxidizing activity of

each ammonia-oxidizing bacterial group (Fig. 1) reflected the corresponding variations in its

abundance in activated sludge.

3.4 AOB community structure in sludge samples at three loading levels

Three AOB amoA gene clone libraries were individually constructed for the three sludge

samples collected at three loading levels, and 140 AOB amoA gene sequences were retrieved,

generating 13 OTUs. The Good's coverage (93~98%) presented here reveals that the

sequenced clones covered the majority of the AOB amoA gene diversity in the sludge samples.

The majority of the clones collected were most closely related to Nitrosomonas europaea (4

OTUs, 45 clones, 32% of total clones) or Nitrosomonas oligotropha (2 OTUs, 64 clones, 46%

of total clones). The relative abundances and distributions of AOB species in the three clone

libraries, as well as their Chao1 and Shannon diversity indices, are depicted in Fig. 5. All

three AOB species detected during phase A (i.e., Nitrosomonas europaea/eutropha,

Nitrosomonas mobilis, and a Nitrosomonas-like AOB) gradually decreased and even

disappeared with increasing loading levels, whereas the abundance of Nitrosomonas

oligotropha in the AOB community increased remarkably, from 0% to 58% and then to 81%.

According to the Chao 1 estimator (Fig. 5b), the number of OTUs detected in Libraries A, B,

and C individually amounted to 100%, 83%, 100% of the number actually present in the three

15
sludge samples. However, the high-throughput sequencing data demonstrated that higher

AOB amoA gene diversity would be observed in Libraries A and B if more clones had been

sequenced. The Shannon index (Fig. 5b) showed a significant decrease in AOB diversity with

increased COD loading levels or shorter HRT. In previous research, Han et al. (2017) also

noted lower yeast diversity at higher loading levels due to shortened HRT in a yeast-dominant

activated sludge system.

The results presented here demonstrate that as loading levels increased, the predominant

AOB species in sludge samples shifted from Nitrosomonas europaea (61%) during Phase A

to Nitrosomonas oligotropha (58% and 81%) during Phases B and C, respectively. In a

previous investigation of eight WWTPs utilizing different sewage treatment processes,

Nitrosomonas europaea was the predominant AOB species (55~67%) in six sludge samples,

whereas Nitrosomonas oligotropha was predominant (48~97%) in four other samples (Gao et

al., 2013). Many previous researchers have shown that AOB communities in the activated

sludge of various sewage treatment systems do not exhibit seasonal variations (Limpiyakorn

et al., 2005; Kim, 2013). Therefore, seasonal variation is unlikely to have resulted in the

variations in the dominant AOB species observed in this study, as the water temperature in the

A/O reactor remained 25~28C throughout the 258-day experimental period. Previous

researchers have often attributed the predominance of these two AOB species during the

treatment of sewage-like wastewater to the difference in their ammonia (Koops &

Pommerening-Roser, 2001; Park & Noguera, 2007) or oxygen affinities (Park & Noguera,

2004; Park & Noguera, 2007). For example, Nitrosomonas europaea was frequently the

predominant AOB species in activated sludge processed for high ammonia levels (e.g., 30~50

mg L-1 (Park & Noguera, 2004), 45~58 mg L-1 (Gao et al., 2013), whereas Nitrosomonas

16
oligotropha was often dominant at low ammonia levels (e.g., 26 mg L-1 (Hallin et al., 2005),

13~30 mg L-1 (Limpiyakorn et al., 2005), 6~15 mg L-1 (Lydmark et al., 2007). However,

some findings on the effect of DO level on the predominance of these two AOB species have

been controversial. For example, researchers found that Nitrosomonas europaea frequently

became the dominant AOB species in activated sludge (Park & Noguera, 2004) or biofilms

(Wang et al., 2012) at high (e.g., 8.5 mg L-1) (Park & Noguera, 2004) and low (< 1 mg L-1)

(Park & Noguera, 2004; Wang et al., 2012) DO levels because it has a higher oxygen affinity

than Nitrosomonas oligotropha. However, other researchers found that Nitrosomonas

oligotropha dominated in activated sludge under low-DO (0.5 mg L-1) and high-DO

conditions (1.5~3.5 mg L-1) (Bellucci et al., 2011) or in the deeper biofilm layers with low

DO levels (Bai et al., 2015). In this study, there were no significant changes in the DO and

influent ammonia levels, and the sole changes involved the HRT (from 8 h to 2 h in the A/O

reactor) and the resultant volumetric loading levels. It has been reported that, among five

yeast species seeded into a yeast-dominant activated sludge system, the only yeast species

was finally remained in the system at the end of the run due to its superior settleability rather

than its degradation capability or growth rate (Park & Noguera, 2007). These results

demonstrate that the shift between these two dominant AOB species during A/O process

optimization should be attributed to other factors (e.g., better settleability of Nitrosomonas

oligotropha) rather than to DO or influent ammonia levels.

3.5 Community structure of heterotrophic AOB in activated sludge at three loading levels

The broad phylogenetic range of heterotrophic AOB makes it difficult to develop a

genomic approach that can clarify their community structure and abundance in environmental

samples (Zhang et al., 2017b). To date, most information about novel HNB species has been

17
obtained through isolation, identification, and characterization (Ren et al., 2014; Li et al.,

2017). Moreover, succinate has been widely used as a carbon source for the isolation of the

most abundant heterotrophic AOB species in previous investigations (Ren et al., 2014; Li et

al., 2017). Therefore, this study tentatively compared the species composition and relative

abundances of culturable heterotrophic AOB from three sludge samples following their

isolation, sequencing, and phylogenetic analysis.

Three libraries were finally constructed for the three sludge samples collected at different

loading levels, and the relative abundances of these identified isolates in these three libraries

are depicted in Fig. 6. The predominant isolate was Agrobacterium tumefaciens (42%) during

Phase A, which shifted to Microbacterium lacus (36%) and Acinetobacter johnsonii (52%)

during Phases B and C, respectively. Among them, Agrobacterium tumefaciens and

Acinetobacter johnsonii are reported to be capable of both heterotrophic nitrification and

aerobic denitrification in previous investigations. Besides, other isolates identified in this

study (e.g., Pseudomonas putida, Enterobacter cloacae, and Pseudomonas fluorescens) have

also been reported to be capable of both heterotrophic nitrification and aerobic denitrification.

Based on high-throughput sequencing data, Pseudomonas putida was detected at relative read

abundances of 0.09 and 0.02% during Phases A and C, respectively. Furthermore, two genera

that include the isolated heterotrophic AOB species (i.e., Acinetobacter and Pseudomonas)

were detected in all sludge samples, with relative abundances of 0.17%, 0.09%, and 0.24%,

respectively, during the three phases. The relative abundances of possible heterotrophic AOB

in sludge samples approximated to those of AOB (i.e., 0.05~0.17%) in this study, although

their relative abundances may be overestimated due to inclusion of other species within these

two genera.

18
 The heterotrophic nitrification performances of these OTU-identified isolates from the

lab-scale A/O system during Phase C were further evaluated by the conventional procedure.

Results showed that, in most cases, cell growth reached the stationary phase within 24 h, as

the OD600 levels increased from <0.01 to >1.00 (or from <0.35 to >1.20), and during this time

~89 mg·L-1 NH4+-N was completely oxidized. The simultaneous formation of significant

amounts of NO3--N (>8.0 mg·L-1) was frequently observed in these batch cultures, while

nitrite was hardly detectable (<0.1 mg·L-1) throughout the culture period. No significant

heterotrophic nitrification by Pseudomonas fluorescens was observed possibly due to the

differences in carbon source, C/N ratio, or temperature, although this species was shown to be

capable of both heterotrophic nitrification and aerobic denitrification in previous reports. The

results presented here demonstrated that all isolates in this library exhibited typical

heterotrophic nitrification, either in this study or in previous reports, when succinate was used

as carbon source. All these results demonstrate the ubiquitous presence of heterotrophic AOB

in all sludge samples, which explains why a high PHAO activity was stably determined in this

study. Future research should devote more attention to the roles and functions of heterotrophic

AOB in various BNR processes.

4. Conclusions

In this study, the variation of these ammonia-oxidizing activities reflected variations in

their abundance in activated sludge. Besides aerobic chemolithotrophic ammonia oxidization,

the ubiquitous presence of heterotrophic AOB and a high PHAO activity indicated that

ammonia removal was also carried out through aerobic heterotrophic ammonia oxidization

during the optimization of an A/O process for sewage treatment. Future research should

devote more attention to the roles and functions of heterotrophic AOB in various BNR
19
processes. During optimization of the A/O process, the predominant AOB species in sludge

samples shifted from Nitrosomonas europaea to Nitrosomonas oligotropha, whereas the

predominant heterotrophic AOB species shifted from Agrobacterium tumefaciens to

Acinetobacter johnsonii. On the other hand, N2 production was predominantly through anoxic

denitrification processes at all three loading levels.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No.

51378066, 91547207 and 21677016). We also thank Professor Guibing Zhu and Dr. Shanyun

Wang, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, for

high-throughput sequencing and qPCR assays of sludge samples.

Appendix A. Supplementary data

E-supplementary data for this work can be found in e-version of this paper online

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List of Tables and Figures

Table 1. Operational conditions and treatment performance of the lab-scale A/O system

during three experimental phases.

Fig. 1. The average values and standard deviations of various N-transformation activities of

sludge samples collected during the three experimental phases. D: various potential

N-transformation activities; PAO: potential aerobic chemolithotrophic ammonia oxidation;

PHAO: potential aerobic heterotrophic ammonia oxidation; PAnammox: potential anammox;

PAnD: potential anoxic denitrification; PAeD: potential aerobic denitrification; PNO:

potential aerobic chemolithotrophic nitrite oxidation.

Fig. 2. Genus-level abundances in the three bacterial communities in sludge samples collected

during Phases A (a), B (b) and C (c), and the Venn diagram (d) of three sludge microbial

communities during Phases A (I), B (II) and C (III).

Fig. 3. Diversity, richness and evenness estimates of the total community in AS of OLR at

0.9(A), 2.4(B), 3.3(C) kg COD m-3 d-1, determined after normalizing the 16S rRNA libraries

to 75070 sequences.

Fig. 4. The relationships between the relative abundances of AOB, NOB, or anaerobic AOB

and their activities during the three phases.

Fig. 5. The species composition (a), shannon, and chao1 index (b) of the three AOB libraries.

Fig.6. The relative abundances and distributions of culturable heterotrophic AOB species in

the heterotrophic AOB libraries.

28
Table 1

Operational conditions and treatment performance of the lab-scale A/O system during three experimental phases.

Phase A Phase B Phase C

(0.9±0.1 kg COD m-3 d-1; (2.4±0.1 kg COD m-3 d-1; (3.3±0.5 kg COD m-3 d-1 ;
Item
0.14±0.01 kg NH4-N m-3 d-1; 0.34±0.02 kg NH4-N m-3 d-1; 0.48±0.05 kg NH4-N m-3 d-1;
3.8±1.1 g MLSS L-1) 7.7±1.1 g MLSS L-1) 10.3 ±1.1 g MLSS L-1)
Periods Day 31~98 Day 133~178 Day 179~258
Influent (mg L-1) 281±19 310±16 307±19
COD
Effluent (mg L-1) 15±10 18±12 15±10
Remvoval (%) 94±5 94±4 95±3
Periods Day 40~98 Day 133~178 Day 212~258
Influent (mg L-1) 48±2 45±2 44±2
NH4+-N
Effluent (mg L-1) 1.3±1 0.5±0.7 0.5±0.8
Remvoval (%) 97±2 99±2 99±2
Periods Day 40~98 Day 133~178 Day 179~258
Influent (mg L-1) 45±3 41±3 46±4
TN
Effluent (mg L-1) 11±1 6.8±3 10±3
Remvoval (%) 75±2 84±6 78±6

29
Fig.1. The average values and standard deviations of various N-transformation activities of

sludge samples collected during the three experimental phases. D: various potential

N-transformation activities; PAO: potential aerobic chemolithotrophic ammonia oxidation;

PHAO: potential aerobic heterotrophic ammonia oxidation; PANAMMOX: potential

anammox; PAnD: potential anoxic denitrification; PAeD:potential aerobic denitrification;

PNO: potential aerobic chemolithotrophic nitrite oxidation.

30
Fig.2. Genus-level abundances in the three bacterial communities in sludge samples collected

during Phases A (a), B (b) and C (c), and the Venn diagram (d) of three sludge microbial

communities during Phases A (I), B (II) and C (III).

31
Fig.3. Diversity, richness and evenness estimates of the total community in AS of OLR at

0.9(A), 2.4(B), 3.3(C) kg COD m-3 d-1, determined after normalizing the 16S rRNA libraries

to 75070 sequences.

32
Fig. 4. The relationships between the relative abundances of AOB, NOB, or anaerobic AOB

and their activities during the three phases.

33
Fig.5. The species composition (a), shannon, and chao1 index (b) of the three AOB libraries.

34
Fig.6. The relative abundances and distributions of culturable heterotrophic AOB species in

the heterotrophic AOB libraries.

35
36
 Highlights

 Dominant N-transformation pathway was identical during sewage A/O process

optimization.

 There is correlation between abundance and activity of AOB, anaerobic AOB or

heterotrophic AOB.

 Both autotrophic and heterotrophic nitrification dominated the sewage A/O process.

 The shift between two dominant AOB species should be attributed to HRT or loading

levels.

37