Environmental Pollution 238 (2018) 783e791

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Biodegradation of nonylphenol during aerobic composting of sewage

sludge under two intermittent aeration treatments in a full-scale plant
Guodi Zheng a, d, *, Tieyu Wang b, d, Mingjie Niu a, d, Xijuan Chen c, d, Changli Liu a, d,
Yuewei Wang a, d, Tongbin Chen a, d
a
Center for Environmental Remediation, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing, 100101,
China
b
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
c
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, 110016, China
d
College of Resources and Environment, University of Chinese Academy of Sciences, Beijing, 100049, China

a r t i c l e i n f o a b s t r a c t

Article history: The urbanization and industrialization of cities around the coastal region of the Bohai Sea have produced
Received 1 December 2017 large amounts of sewage sludge from sewage treatment plants. Research on the biodegradation of
Received in revised form nonylphenol (NP) and the influencing factors of such biodegradation during sewage sludge composting is
22 March 2018
important to control pollution caused by land application of sewage sludge. The present study investi-
Accepted 31 March 2018
gated the effect of aeration on NP biodegradation and the microbe community during aerobic com-
Available online 4 April 2018
posting under two intermittent aeration treatments in a full-scale plant of sewage sludge, sawdust, and
returned compost at a ratio of 6:3:1. The results showed that 65% of NP was biodegraded and that Bacillus
Keywords:
Nonylphenol
was the dominant bacterial species in the mesophilic phase. The amount of NP biodegraded in the
Biodegradation mesophilic phase was 68.3%, which accounted for 64.6% of the total amount of biodegraded NP. The
Sewage sludge amount of NP biodegraded under high-volume aeration was 19.6% higher than that under low-volume
Compost aeration. Bacillus was dominant for 60.9% of the composting period under high-volume aeration,
Aeration compared to 22.7% dominance under low-volume aeration. In the thermophilic phase, high-volume
aeration promoted the biodegradation of NP and Bacillus remained the dominant bacterial species. In
the cooling and stable phases, the contents of NP underwent insignificant change while different
dominant bacteria were observed in the two treatments. NP was mostly biodegraded by Bacillus, and the
rate of biodegradation was significantly correlated with the abundance of Bacillus (r ¼ 0.63, p < 0.05).
Under aeration, Bacillus remained the dominant bacteria, especially in the thermal phase; this phe-
nomenon possibly increased the biodegradation efficiency of NP. High-volume aeration accelerated the
activity and prolonged the survival of Bacillus. The risk of organic pollution could be decreased prior to
sewage sludge reuse in soil by adjusting the ventilation strategies of aerobic compost measurements.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction
The Bohai Sea, also known as the Bohai Gulf, is the innermost
gulf of the Yellow Sea and Korea Bay on the coast of Northeastern
and North China. It is approximately 78,000 km2 in area, and its
proximity to Beijing, the capital of China, makes it one of the busiest
seaways in the world. Some of the rivers entering the gulf include
* Corresponding author. Center for Environmental Remediation, Institute of
Geographic Sciences and Natural Resources Research, Chinese Academy of Science,
Beijing, 100101, China.
E-mail address: zhenggd@igsnrr.ac.cn (G. Zheng).
the Yellow, Hai, Liao, and Luan Rivers. There are also important oil
reserves in the vicinity of the gulf, including the Shengli Field and
Liaohe Oilfield. The coastal region of the Bohai Sea is the largest
pool of modern industry and is the heavy and chemical industrial
base of China. The coastal region of the Bohai Sea shows large
developmental potential as the center of the international economy
shifts to the Asia-Pacific region; however, a large amount of
wastewater, exhaust gas, and waste residue was released without
treatment and reached the national standard beginning in the
1980s. Based on reporting from Economic Information Daily on
August 10, 2015, annually, approximately 28  104 t waste water
and 70  104 t solid waste flowed into the Bohai Sea through the 57

https://doi.org/10.1016/j.envpol.2018.03.112
0269-7491/© 2018 Elsevier Ltd. All rights reserved.
784 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791
rivers, which accounts for 50% of China's total marine pollutants
(Bai, 2015). Pollutants have made environmental pollution partic-
ularly serious in the Bohai Sea, which has consequently harmed the
development of the Bohai economic circle (Chen et al., 2017; Zhang
et al., 2017; Jiang et al., 2018).
In recent years, the wastewater treatment industry has devel-
oped rapidly in the coastal region of the Bohai Sea. By 2016, the rate
of municipal wastewater treatment (MWT) was 90.58%, 92.08%,
93.61%, 95.37% and 96.21% in Beijing, Tianjin, Liaoning, Hebei and
Shandong, respectively. Sewage sludge is the end product of
sewage treatment. The improvement in sewage treatment capacity
has gradually increased the production of sludge. Therefore, prob-
lems regarding the disposal of sewage sludge must be urgently
addressed. Despite the risks of heavy metals, pathogens, organic
pollutants, and other pollutants that sewage sludge brings, this
method can be applied similarly to soil fertilizer after proper
treatment because it can provide plant nutrients and humus. In the
EU-27 countries, 53% of urban sludge is treated through compost-
ing (Kelessidis and Stasinakis, 2012). Thus, the principle of harmless
and resource-oriented treatment and disposal of sewage sludge can
and should be followed. Composting is a suitable method for
sewage sludge treatment. The heat produced during composting
can kill most of the pathogenic bacteria, and sewage sludge can be
effectively stabilized after composting. Compost can also be used to
produce organic fertilizer.
Sewage sludge composting is not designed to treat organic
pollutants, as evidenced by the lack of early warning and moni-
toring mechanisms for organic pollutants in sewage sludge treat-
ment plants; thus, the presence of organic pollutants in sludge may
pose a risk to the environment (Luo et al., 2014). Nonylphenol (NP)
is an estrogen and mainly originates from the biodegradation of NP
ethoxylates (NPEs), which are widely used in industrial products
(Bai et al., 2017; Jambor et al., 2017). During sewage treatment,
NPEs easily biodegrade into NP, which then contaminates sewage
sludge (Samaras et al., 2013; Esteban et al., 2016; Murdoch and
Sanin, 2016). NP can be detected in sewage sludge at a concentra-
tion ranging from 1  103 mg kg1 to 1  106 mg kg1 (Stasinakis,
2012). Meng et al. (2016) conducted a systematic review and a
meta-analysis of organic contaminants in Chinese sewage sludge
based on the literature published over the past 30 years and found
the amount of 4-nonylphenol to be 3.14  103 mg kg1. NP is also an
endocrine-disrupting chemical that may enter the human body
through the food chain and affect the liver and reproductive organs
once released into the environment (Dur
an and Beiras, 2016;
Esteban et al., 2016). Therefore, the NP content of sewage sludge
must be controlled.
Multiple studies on the transport, sorption, and biodegradation
of NP in soil and river sediments have been conducted (Chen et al.,
2011; Mao et al., 2012; Liu et al., 2014; Lu and Gan, 2014; Lyons
et al., 2014; Wang et al., 2014, 2015; Zhang et al., 2016), whereas
studies on the biodegradation of NP during sewage sludge com-
posting are limited. A few studies have found that NP in soil and
river sediments can be biodegraded by various microorganisms,
such as Pseudomonas (Watanabe et al., 2012; Xie et al., 2015),
Sphingomonas (Weert et al., 2010; Collado et al., 2013), Acidovorax
(Watanabe et al., 2012), and Bacillus (Hsu et al., 2013). The oxygen
content, temperature, and organic matter content of windrow
during sewage sludge composting differ, however, from those of
soil and river sediments. The effect of these microorganisms on the
sewage sludge composting process remains unclear. Hao et al.
(2008) studied the bio-degradation pathways of NP in wastewater
treatment, and the results showed that the long branches in nonyl
of NP are first degraded into short methyl branches, which creates a
series of short chain alkyl phenols with different branch structures.
Given that the biodegradation of NP always starts from the oxygen-
adding open loop of benzene, the amount of oxygen exerts a limited
effect on such biodegradation. Studies have shown that NP is
completely biodegraded under the effect of aeration; in contrast,
the rate of biodegradation in the absence of aeration is only 60%
under laboratory scale (Abargues et al., 2013; Lu et al., 2015). The
biodegradation efficiencies of laboratory scale are insufficient to
satisfy the demand of environmental governance (Bai et al., 2017).
During sewage sludge composting, forced aeration can increase the
oxygen content of a material and the duration of aerobic conditions.
In the absence of forced aeration or under a low amount of aeration,
the oxygen content of a material is depleted completely, thereby
resulting in an anaerobic environment (Shen et al., 2011). There-
fore, aeration may be an important affecting factor of the biodeg-
radation of NP during sludge composting (Zheng et al., 2015).
Research on the succession of the microbial community struc-
ture and the biodegradation kinetics of NP in sludge compost can
reveal which microorganisms are involved in such biodegradation.
Measures can then be taken to promote the biodegradation of NP
during sewage sludge composting and consequently reduce the
risks posed by the land application of sludge compost products.
Full-scale composting for aeration optimization and development,
particularly an aeration method for sewage sludge composting
with improved efficiency for the biodegradation of NP, should be
developed. Thus, the objective of this work is twofold. First, the
dynamic changes in the biodegradation of NP during aerobic
composting of sewage sludge under two intermittent aeration
treatments in a full-scale plant are assessed. The abundance of
microorganisms is studied by high-throughput sequencing in
addition to the composting process. Second, the relationship be-
tween the microbe community and NP biodegradation is investi-
gated by comparing the effect on the biodegradation of NP and the
abundance of microorganisms by using different aeration
treatments.
2. Materials and methods
2.1. Instruments and reagents
Dichloromethane, methanol, and acetone (chromatographically
pure) were purchased from SigmaeAldrich (St. Louis, USA). Ethanol
and acetone (analytically pure) were purchased from the China
National Pharmaceutical Group (Beijing, China). Standard NP so-
lution (2500 mg/L, dissolved in methanol:acetone ¼ 1:1 mixture)
was purchased from AccuStandard (New Haven, USA). DNA
extraction kits (PowerSoil@ DNA Isolation Kit) were purchased
from MO BIO Laboratories (Carlsbad, Canada). Ultrapure water,
10  reaction buffer, PCR primers (50 and 30 primers), dNTPs, and
pfuTaq enzyme were used in DNA PCR amplification. A solid-phase
extraction column (Waters Sep-Pak tC18, 6 mL, 500 mg, 37e55 mm)
and high-performance liquid chromatography (HPLC) column (C18
symmetry, 5 mm, 4.6 mm  250 mm) were purchased from Waters
(Milford, USA).
2.2. Sludge, conditioner, and returning compost
Sludge was collected from the Qinhuangdao Sludge Treatment
Plant in Hebei Province, China. The water content of the sludge was
approximately 80% after mechanical dewatering. The sawdust was
obtained from a local timber processing plant; the equivalent
diameter of the sawdust was approximately 1.0 cm, and the mois-
ture content was 4.0e5.0%. Returning compost was obtained from
matured compost and then directly transported from the com-
posting workshop to the mixing workshop. The initial moisture
content of the windrow was approximately 60%, and the C/N ratio
was approximately 25:1. On the basis of the moisture content and
 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791 785

C/N ratio of the sludge, sawdust, and returning compost, the
compost material was adjusted at a sludge:sawdust:returning
compost ratio of 6:3:1. The total mass of windrow was approxi-
mately 200 t. Table 1 shows the NP content and other properties of
the three substances.
2.3. Composting trail
The compost experiment was conducted in a full-scale plant
with a processing capacity of 200 t d1. The plant possessed 21
fermentation tanks, and the volume of each fermentation tank was
200 t. Intermittent aeration was performed for the sewage sludge
composting in the plant. The aeration duration was 6 min in a 40-
min ventilation cycle during the entire composting process. The
aeration system was compatible with computer controls for air
volume. Two aeration treatments were performed, and the amount
of aeration was adjusted by changing the aeration frequency. The
aeration frequency for the low aeration treatment was 35 Hz over
the entire composting process, whereas the aeration frequency for
the high aeration treatment was increased from 35 Hz to 45 Hz 3 d
later.
The composting period lasted for 20 d, and the windrow tem-
perature was measured daily at 9:00 and 21:00. The oxygen content
of a ventilation cycle was automatically monitored with a real time
on-line system during the mesophilic, thermophilic, cooling and
stable phases of composting. The probe measured the oxygen of the
compost pile in real time. The air sample is pulled through the
probe stem using an electric pump.
Samples were collected at d 0, 1, 3, 5, 8, 11, 14, 17, and 20, and
they were stored in a portable refrigerator (4  C) during transport
to the laboratory. A portion of each sample was used for extracting
microorganism for high-throughput sequencing, and the remaining
portion was freeze dried to complete dryness. The samples were
preserved in a freezer (20  C) for NP determination.
2.4. Determination methods
2.4.1. Determination of the NP concentration
The collected solid samples were placed in a low-pressure
freezer (Labconco Freezone, Kansas, USA) and completely dried
under 1 mba at 4  C. The dried sample was homogenized in a mill
(IKA A11 BASIC, Staufen, Germany) to a fine powder. NP was
extracted by accelerated solvent extractor (ASE 350, Dionex, Sun-
nyvale, USA) by dichloromethane:acetone (1:1). A sample (5.0 g)
was weighed and placed in an extraction pool containing 34 mL of
the extraction solvent. The sample was extracted twice at 75  C and
1500 psi. After extraction, the liquid extract was transferred into a
round-bottom flask and then concentrated to 1 mL using a rotary
evaporator (IKA@ rv10 basic, Staufen, Germany) at 50  C and 70
mba.
To avoid the effect of any impurity on the detection results, the
C18 column for the reversed-phase solid-phase extraction was used
to purify the liquid extract, which was conditioned by a methanol
and dichloromethane mixture (1:4; 5 mL), activated methanol
(5 mL), and dichloromethane (5 mL) at a flow rate of 1 mL min1.
The extraction liquid was subsequently allowed to flow through the
column at a flow rate of 0.5 mL min1, after which 5 mL of ultrapure
water was leached through the column. After completely drying in
a drying shed, the column was eluted two times with 5 mL of
methanol and dichloromethane (1:4) mixture at flow rate of
0.5 mL min1. The elution was transferred into a round-bottom
flask, concentrated to 1 mL using a rotary evaporator at 50  C and
70 mba, and then dried to nearly dryness under nitrogen blowing
conditions. Methanol (5 mL) was added to the elution prior to
determination.
The HPLC column and fluorescence spectrum analyzer (Agilent
1260 Infinity LC) were used in NP detection and were conditioned
as follows: sample volume: 50 mL; mobile phase: methanol; ultra-
pure water: 85:15; flow rate: 1 mL min1; testing time: 15 min;
detection temperature: 30  C; excitation wavelength: 280 nm; and
emission wavelength: 310 nm. The detection limit for NP in the
study was 2.5 ng g1, and the recovery rate was 80.0%.
2.4.2. Determination of microorganisms
1)DNA extraction
DNA was extracted from the samples using the Soil Power® DNA
extraction kit (MO BIO Laboratories, Carlsbad, Canada) in accor-
dance with the manufacturer's instructions.
2)PCR amplification
The PCR amplification system consisted of the following com-
ponents: DNA: 100 ng template; 10  Buffer (Mg2þ): 5 L; 5ʹ primer:
1 L; 3ʹ primer: 1 L; DNTPs: 0.6 mL; Taq PFU enzyme: 1.6 mL (4U); and
DdH2O: used to reach 50 L. The PCR amplification conditions were
as follows: 94  C for 4 min, followed by 25 cycles each at 94  C
(30 s), 54  C (30 s), 72  C (90 s), and 72  C (10 min). The product was
approximately 1.5 kb. After PCR amplification, the sample was used
for third-generation high-throughput sequencing using the RS II
PacBio sequencing platform. The Mothur software package was
used to analyze the 16S rDNA (Schloss et al., 2009).
2.5. Calculation of the biodegradation rate of NP
The ash content was determined by dry combustion in a muffle
furnace at 550 ± 10  C. Given that part of the organic matter in
sewage sludge could be decomposed into CO2 and H2O during
composting, the weight of the dry substance decreased, whereas
the ash quantity of sewage sludge did not change. The concentra-
tion of NP would be increased if the NP could not be biodegraded

Table 1
Basic properties of the composting material components.

Windrow components NP content (ng/g) pH Moisture (%) C/N Volatile solid (%)

Low-volume aeration Sludge 1379.7 7.1 80.6 7.0 73.6

Conditioner 494.0 6.6 6.2 e 98.4
Returning compost 457.7 6.8 54.1 20.7 83.8
Initial compost 1021.8 6.9 66.1 19.4 85.2
High-volume aeration Sludge 1374.9 7.1 79.5 6.9 73.6
Conditioner 474.6 6.5 41.6 e 98.4
Returning compost 414.3 6.7 54.4 22.2 83.8
Initial compost 1008.8 6.7 63.0 19.2 85.2

Note: “-” represents lack of data.

786 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791
during composting. Therefore, the decomposition of organic matter
should be considered when calculating the biodegradation rate of
NP. The biodegradation rate of NP was calculated as follows:
ðAi  C0 Þ  ðA0  Ci Þ
Biodegradation Rate ð%Þ ¼  100%
Ai  C0
where A0 is the ash content of the original material of compost, Ai is
the ash content of the compost on day i, C0 is the NP content of the
original material of compost, and Ci is the NP content of the
compost on day i.
2.6. Data processing
SPSS Statistics V22.0 IBM software was used for correlation and
comparative analyses of the results. The software packages used for
microbial sequencing were Analysis (2.3.0.140936. p4.150482),
Mothur (v.1.34.3), Silva database (Release 123), and rDnaTools1
(SMRT).
3. Results and discussion
3.1. Changes in pile temperature and oxygen during composting
3.1.1. Dynamics of the pile temperature
The temperature of windrow under the two aeration treatments
increased first and then decreased to room temperature. On the
basis of the changes in the temperature of the composting pile
(Fig. 1), the composting period can be divided into four stages as
follows: mesophilic phase (0e3 d), thermophilic phase (3e10 d),
cooling phase (10e16 d), and stable phase (16e20 d). In the mes-
ophilic phase, the trend of the temperature change of the two
aeration treatments was similar. After the third day, the compost
entered into the thermophilic phase. The temperature of windrow
under high-volume aeration decreased to 65  C within half a day,
and the reduction continued until d 10. In the low-volume aeration
windrow, the temperature of windrow remained higher than 70  C
until d 10. After d 10, the temperature of windrow in both treat-
ments decreased. At the end of composting, the temperature of
windrow under low-volume aeration was slightly higher than the
Fig. 1. Temperature of piles during composting under two intermittent aeration
treatments. Note: M. Mesophilic phase. T. Thermophilic phase. C. Cooling phase. S.
Stable phase.
ambient temperature. During composting, high-volume aeration
dissipated the increased amount of heat, thereby resulting in a
significantly lower temperature than that under low-volume
aeration.
During sewage sludge composting, microorganisms break down
(1)
organic matter and produce heat, carbon dioxide, water, and hu-
mus. Different communities of microorganisms predominate dur-
ing different composting phases (Khalil et al., 2011). Initial
decomposition in the current study was carried out by mesophilic
microorganisms, which rapidly broke down the soluble, readily
degradable compounds. The heat they produced increased the
compost temperature rapidly. As the temperature rose above
approximately 55  C, the mesophilic microorganisms became less
competitive and were replaced by other thermophilic microor-
ganisms. Given that temperatures over nearly 65  C can kill many
forms of microbes and limit the rate of decomposition, compost
managers usually use aeration and mixing to keep the temperature
below this point. The aeration rate is considered an important
influencing factor of successful composting because it can supply
oxygen and remove moisture and heat (Yuan et al., 2016). During
the thermophilic phase in the present study, high temperatures
accelerated the breakdown of organic matter in sewage sludge and
sawdust. As the high-energy compounds became exhausted, the
temperature of compost decreased gradually.
3.1.2. Dynamics of the pile oxygen
Oxygen is one of the most important indicators of compost pile
health, and it can deplete rapidly if a pile is not aerated (Zeng et al.,
2016; Oazana et al., 2018). During a composting ventilation cycle
(40 min), the oxygen content increased dramatically when the
blower was open, peaking after a few minutes (Fig. 2). The oxygen
content began to decline when the blower was off and then
decreased to the lowest point until the next ventilation cycle began.
There were many differences in the oxygen parameters during the
mesophilic stage, thermophilic phase, cooling and the stable phase
of composting, however, such as the speed of increase, speed of
decrease, peak value, and the lowest point of oxygen content. Dif-
ferences also existed between the two aeration treatments. The
increase speed and peak value of oxygen content can be improved
by increasing the aeration quantity.
3.2. Variation in the content of NP in compost
Fig. 3 shows that the content of NP decreased significantly
during composting in both treatments. Moreover, the rate of
reduction was higher in the earlier stage and relatively lower in the
later stage. Under high-volume aeration, the initial NP content of
windrow was 1008.8 ng g1 and then decreased rapidly to
590.3 ng g1 in the mesophilic phase. At the end of the thermo-
philic phase, the content of NP decreased slowly to 361.3 ng g1 and
then became nearly stable in the cooling phase. At the end of
composting, the NP content was 392.4 ng g1. Under low-volume
aeration, the initial NP content of windrow was 1021.8 ng g1 and
then decreased rapidly to 652.3 ng g1 in the mesophilic phase. At
the end of the thermophilic phase, the content of NP decreased
slowly to 480.5 ng g1 and became nearly stable in the cooling
phase. At the end of composting, the NP content was 471.9 ng g1.
The content of NP decreased mainly in the mesophilic and ther-
mophilic phases. Moreover, the reduction rate of NP content was
significantly higher under high-volume aeration than under low-
volume aeration. This trend was more evident in the thermo-
philic phase than in other phases. The decline in content indicated
the biodegradation of NP during composting, and the difference in
the rates of reduction between the two treatments indicated that
the biodegradation of NP was related to the composting conditions.
 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791
Fig. 2. Changes in oxygen contents in a ventilation cycle during the composting process under two intermittent aeration treatments. Note: M. Mesophilic phase. T. Thermophilic
phase. C. Cooling phase. S. Stable phase.
Fig. 3. Changes in the content of NP in the composting material and the biodegra-
dation rate of NP during composting under two intermittent aeration treatments.
Aeration control is the key to the aerated static aerobic composting
system (Zhu, 2006). In the current study, the difference in aeration
treatments affected the temperature of the compost pile, and the
substances in sewage sludge changed differently. The high-volume
aeration treatment could remove heat and provide oxygen for
microbial growth. Therefore, the temperature of the compost pile
under high-volume aeration differed from that of the compost pile
under low-volume aeration. Given the difference in microbial
growth and activity in the composting material, the biodegradation
of NP in sludge differed during composting. Therefore, the
biodegradation of NP in sewage sludge was influenced by the mi-
crobial oxygen uptake.
The biodegradation rate of NP was calculated on the basis of the
787
dynamic changes in the content of NP. Fig. 3 shows that the
biodegradation rate of NP could reach up to 40% within 3 d. At the
end of the thermophilic phase, the biodegradation rates of NP were
64.2% and 53.0% under high- and low-volume aeration treatments,
respectively. NP was mainly biodegraded in the mesophilic phase in
which no significant difference was observed between the two
treatments. The amount of NP that was biodegraded in the meso-
philic phase was 68.3%, which accounted for 64.6% of the total
amount of biodegraded NP. The amount of NP biodegraded under
high-volume aeration was 19.6% higher than that under low-
volume aeration, and the difference was mainly attributed to the
temperature difference in the thermophilic phase.
3.3. Relationship between the microbe community and NP
biodegradation
High-throughput sequencing results showed that microorgan-
isms in the samples included Firmicutes (49.7%), Proteobacteria
(24.9%), Bacteroidetes (12.4%), and Actinobacteria (4.7%). The cor-
responding abundance of these bacterial species (operational
taxonomic units, OTUs) changed with the duration of the com-
posting period (Table 2).
Under high-volume aeration, the net biomass changed with the
duration of the composting period. The abundance of Firmicutes
peaked at d 1, 5, 8, and 14, whereas the abundance of Proteobacteria,
Bacteroidetes, and Actinobacteria peaked at d 8 and 17. The abun-
dance of these microorganisms was significantly lower under low-
volume aeration than under high-volume aeration.
The structure of the microbial community and the NP content in
the composting material changed with the duration of the com-
posting period. The biodegradation of organic pollutants during
composting relied on microbial activity. Thus, the change in the
content of NP during composting directly reflected the effect of
microorganisms on the biodegradation of NP. The microorganisms
associated with the biodegradation of NP could be identified on the
basis of the changes in the structure of the microbial community
788 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791

Table 2
Dynamic abundance of microorganisms during composting under two intermittent aeration treatments.

Aeration strategy Microorganisms Operational taxonomic units (OTUs)

0d 1d 3d 5d 8d 11 d 14 d 17 d 20 d

High-volume aeration Firmicutes 13 21 6 20 19 5 22 21 7

Proteobacteria 1 3 2 2 36 1 6 26 0
Bacteroidetes 1 0 2 0 24 0 0 45 0
Actinobacteria 1 0 1 1 6 0 1 21 1
net 18 25 14 23 91 6 32 164 9
Low-volume aeration Firmicutes 7 10 3 21 5 7 11 9 5
Proteobacteria 5 4 7 4 7 2 11 10 5
Bacteroidetes 3 2 3 2 4 2 0 9 1
Actinobacteria 0 1 1 1 1 0 5 0 2
net 15 17 14 28 23 11 32 37 14

and the content of NP. Compared with the abundance of the three
other microorganisms, that of Firmicutes peaked in the main stage
(mesophilic phase) of NP biodegradation, thereby suggesting a
relationship between Firmicutes and NP biodegradation.
Firmicutes included two classes of microorganisms, namely
Bacilli (27.6%) and Clostridia (20.6%). The abundance of Bacilli
peaked at d 1 and 8, whereas that of Clostridia peaked at d 5 and 17.
The biodegradation of NP mainly occurred in the mesophilic phase
within the first 3 d. Therefore, Bacilli was the dominant microor-
ganism corresponding to the biodegradation of NP. The relationship
between NP biodegradation and Bacilli abundance was evaluated
using Pearson correlation analysis, and the result showed a sig-
nificant correlation between them (r ¼ 0.59, p < 0.05).
Bacillales (22.2%) accounted for more than 90% of the genus
Bacilli. Fig. 4 shows the dynamic change in the abundance of
Bacillales. During the composting period, the abundance of Bacil-
lales was higher under high-volume aeration than under low-
volume aeration. Consequently, the biodegradation rate of NP was
significantly higher under high-volume aeration than under low-
volume aeration. Moreover, the abundance of Bacillales was
higher under high-volume aeration than under low-volume aera-
tion in the thermophilic phase. This result further supports the
inference above. Pearson correlation analysis showed a significant
correlation between the NP biodegradation rate and Bacillus
abundance (r ¼ 0.63, p < 0.05).
Fig. 4. Changes in the abundance of Bacillus in the composting material during
composting.
The effect of Bacillus on the biodegradation of NP was further
explored by analyzing the succession of dominant microorganisms
during composting. The most abundant species in the sample was
the dominant microorganisms, which may have played a key role in
composting. Fig. 5 shows the succession of dominant microorgan-
isms in the two treatments. The structure of the microbial com-
munity in the windrow under the two treatments differed, and this
difference indicated that aeration exerted a certain effect on the
structure of the microbial community. Bacillus was dominant for
60.9% of the composting period under high-volume aeration but
only 22.7% dominant under low-volume aeration. The content of
oxygen in the compost was increased by high airflow rates, and this
increase promoted the growth and metabolic activity of Bacillus
and extended the survival time of Bacillus during composting.
In the series of treatments under high-volume aeration, the
succession of dominant microorganism was as follows: Bacillus e
Bacillus e Bacillus e Bacillus e Parapedobacter e Bacillus e Bacillus e
Paludibacter e Exiguobacterium. Several microorganisms, namely
Mobilitalea, Thermobacillus, Stenotrophomonas, and Parapedobacter,
were also detected during the process. NP was mainly biodegraded,
and Bacillus was the dominant microorganism in the mesophilic
phase. By contrast, Bacillus was not the dominant microorganism in
the cooling and stable phases, during which the biodegradation of
NP was not observed. Thus, Bacillus may have been the major factor
in the biodegradation of NP. Previous studies have reported that
Bacillus can degrade NP and polycyclic aromatic hydrocarbons
(PAHs) (Gioia et al., 2004; Chang et al., 2005). Additionally, Mobi-
litalea is a thermophilic cellulose-degrading bacterium
(Podosokorskaya et al., 2014; Koeck et al., 2015), and Thermobacillus
is a thermophilic polysaccharide-degrading bacterium isolated in
compost (Sakai et al., 2015). Parapedobacter is a mesothermal
bacterium that can degrade pyrene (Li et al., 2012), and Steno-
trophomonas is a cold-loving bacterium that can degrade NP as its
sole carbon source (Soares et al., 2003). Paludibacter is an anaerobic
bacterium that is dominant in the digestive system (Shen et al.,
2015), and Exiguobacterium is a PAH-degrading bacterium
(Jeswani and Mukherji, 2012). In the present study, Mobilitalea,
Thermobacilllus, and other microorganisms could biodegrade cel-
lulose and other polysaccharides to derive energy for microbial
activities. Several microorganisms could biodegrade NP in the
cooling and stable phases, but their biodegradation ability was
relatively poor.
Under low-volume aeration, the succession of dominant
microorganism was as follows: Bacillus e Bacillus e Parapedobacter
e Caldicoprobacter e Comamonas e Parapedobacter e Bacillus e
vadinBC27_wastewater e sludge_group e Caldicoprobacter e
Comamonas e Parapedobacter e Bacillus e vadinBC27_wastewater e
sludge_group e Caldicoprobacter. To the best of our knowledge,
Caldicoprobacter and Comamonas were two new species found in
 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791 789

Fig. 5. Succession of microbial community during composting under two intermittent aeration treatments. Note: a. Succession of the dominant microorganisms with the change in
composting cycle under different ventilation rates. b. Percentage of the dominant microorganisms during composting under high-volume aeration. c. Percentage of the dominant
microorganisms during composting under low-volume aeration.

the low-volume aeration treatment. Caldicoprobacter is a thermo-
philic anaerobic bacterium, and its presence indicates that an
anaerobic environment is formed under low-volume aeration
(Bouanane-Darenfed et al., 2011). Comamonas is a common deni-
trifying bacterium (Spain, 1995). During the composting process of
sewage sludge with sawdust and returning compost, the abun-
dance of Bacillus and the biodegradation rate of NP were lower
under low-volume aeration than under high-volume aeration,
which suggests that Bacillus was the main microbe working to
biodegrade NP during composting. The biodegradation of NP in
sewage sludge was influenced by the microbial growth, and the
abundance of Bacillus was higher under high-volume aeration than
under low-volume aeration. Given the difference in the abundance
of Bacillus between the two treatments, the biodegradation of NP in
sludge differed during composting.
Bacillus is a gram-positive bacterium. The most suitable ranges
of pH and temperature for this bacterium are 7.0e9.5 and 15e55  C,
respectively. Bacillus can resist many adverse environmental con-
ditions: it demonstrates a higher capacity to resist heat, salinity,
and anaerobic conditions than vegetative cells (Hecker et al., 1996).
This genus is widely distributed in the soil, plant surfaces, food, and
water. Table 3 lists some studies on the effect of the abundance of
Bacillus obtained from the environment on the biodegradation rate
of NP (Yang et al., 2011; Chang et al., 2012; Hsu et al., 2013; Wang

zalska
et al., 2014; Reddy et al., 2015; Ro _ et al., 2015). All of the re-
sults demonstrated the strong effect of the abundance of Bacillus on
the biodegradation rate of NP. Chang et al. (2012) isolated microbial
strains from urban sludge and found that these strains biodegrade
NP under aerobic conditions. The results showed that Bacillus
demonstrates the best effect. Moreover, Gioia et al. (2004) found
that the Bacillus strain VA160 can enhance the biodegradation
efficiency of NP and NPE. Bai et al. (2017) found that the biodeg-
radation efficiencies of many microorganisms are insufficient to
satisfy the demand of environmental governance. Moreover, most
pure microorganisms are effective in the biodegradation of only
limited species of NP at low concentrations. NP-M2, which may be
an excellent tool for the bioremediation of environments contam-
inated with NP for the consortium, is found to mainly comprise
members of the Sphingomonas, Pseudomonas, Alicycliphilus, and
Acidovorax genera. The biodegradation efficiency of the treatment
under high-volume aeration was detected in a full-scale compost-
ing plant in the present study. Thus, the effectiveness of Bacillus in
the biodegradation of NP during composting presents a good en-
gineering application value.
4. Conclusions
NP in sewage sludge was mainly biodegraded in the mesophilic
phase of composting, and the amount of NP biodegraded in this
stage accounted for 64.6%e68.3% of the total biodegraded NP. The
biodegradation rate of NP was significantly correlated with the
abundance of Bacillus (r ¼ 0.63, p < 0.05). High-volume aeration
promoted Bacillus activity, thereby improving the biodegradation
efficiency of NP. The abundance of Bacillus was higher under high-
volume aeration than under low-volume aeration. The biodegra-
dation efficiency of the treatment was higher under high-volume
aeration than under low-volume aeration for the sewage sludge
composting, and this disparity could be mainly attributed to the
differences observed in the thermophilic phase. Despite these
findings, whether Bacillus as a strain additive could enhance the
biodegradation of NP during composting remains unclear and re-
quires further study.

Table 3
Effect of the abundance of Bacillus on the biodegradation of NP.

Microorganism Degradation condition Degradation effect Reference

Bacillus sphaericus strain CT7 pH ¼ 7.0, 30  C, 2.0 mg/l 100% (Hsu et al., 2013)
Bacillus cereus pH ¼ 7.0, 50  C, 10.0 g/l 80% (Yang et al., 2011)
Bacillus pumilus pH ¼ 7.0, 30  C, 50 mg/g t1/2 ¼ 2.4 (Chang et al., 2012)
Bacillus sp. CYR1 100 mg/l 54% (Reddy et al., 2015)
Brevundimonas, Flavobacterium, Lysobacter and Rhodobacter 25  C, 150 mg/g 93.2% (Wang et al., 2014)
M. robertsii IM 6519 28  C, 50 mg/l 99.2%
zalska
(Ro _ et al., 2015)
790 G. Zheng et al. / Environmental Pollution 238 (2018) 783e791
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant numbers 41101463, 41371455).
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