Appl Microbiol Biotechnol (2016) 100:10193–10202
DOI 10.1007/s00253-016-7869-x
ENVIRONMENTAL BIOTECHNOLOGY
Biological treatment of actual petrochemical wastewater using
anaerobic/anoxic/oxic process and the microbial diversity analysis
Pengyuan Ding 1 & Libing Chu 1,2 & Jianlong Wang 1,2
Received: 31 May 2016 / Revised: 5 September 2016 / Accepted: 16 September 2016 / Published online: 5 October 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract A novel process integrating anaerobic hydrolysis-
acidification (HA) and anoxic/oxic (A/O) reactors was devel-
oped to treat the actual petrochemical wastewater, which was
operated for more than 8 months, the removal efficiency of
COD and NH4+-N was monitored, and the microbial commu-
nity was analyzed. The results showed that the effluent con-
centrations were maintained at around 99 and 1.3 mg/L, with
the removal efficiency of 70.6 and 95.4 %, respectively at a
total hydraulic retention time (HRT) of 20 h. The major pol-
lutants in the influent were identified as hydrocarbons, alde-
hydes, heterocyclic matters, amines, alcohols, phenols, ke-
tones, etc. by GC-MS analysis, while only heterocyclic com-
pounds, ketones, and esters were detected in the effluent after
HA-A/O treatment. Bacteria belonging to phyla Chloroflexi,
Proteobacteria, and Bacteroidetes were highly enriched in the
system. The predominant genera in HA, anoxic, and oxic
tanks were Anaerolineaceae uncultured and Desulfobacter,
B l a s t o c a t e l l a a n d A n a e ro l i n e a c e a e u n c u l t u re d ,
Saprospiraceae uncultured and Nitrosomonadaceae
uncultured, respectively. The sulfate-reducing bacteria
Desulfobacter, Desulfofustis and Desulfomicrobium were de-
tected only in HA reactor. The ammonium-oxidizing bacteria
Nitrosomonadaceae and Nitrosomonas and nitrite-oxidizing
bacteria Nitrospira were highly enriched in A/O reactor,
which is consistent with the good nitrification performance.
* Jianlong Wang
wangjl@tsinghua.edu.cn
1
Collaborative Innovation Center for Advanced Nuclear Energy
Technology, INET, Tsinghua University, Beijing 100084, People’s
Republic of China
2
Beijing Key Laboratory of Radioactive Waste Treatment, Tsinghua
University, Beijing 100084, People’s Republic of China
Keywords Hydrolysis . Acidification . Anoxic/oxic reactor .
Petrochemical wastewater . Pyrosequencing
Introduction
The petrochemical industry is a pillar industry and plays an
important role in national economy of China. The wastewater
generated in the processes of petroleum refining and manu-
facture of numerous organic chemicals and raw materials has
become one of the most serious issues (Liu et al. 2014). The
petrochemical wastewater contains many priority organic pol-
lutants involving phenols, cyanide, phthalate esters, chlorinat-
ed aliphatic compounds, and polycyclic aromatic hydrocar-
bons, which are characterized by complex composition, high
toxicity and poor biodegradability (Estrada-Arriaga et al.
2012).
Different technologies have been used for petrochemical
wastewater treatment, including pre-treatment such as dis-
solved air flotation (Hami et al. 2007), coagulation and floc-
culation (Verma et al. 2010), advanced oxidation process (Lin
et al. 2001), and anaerobic hydrolysis-acidification to improve
the biodegradation and decrease toxicity, followed by biolog-
ical processes which play the primary role in degrading the
organic pollutants (Guo et al. 2014). Hami et al. (2007) inves-
tigated the effects of activated carbon on the performance of
dissolved air flotation (DAF) unit for treating the refinery
wastewater. The results showed that biochemical oxygen
demand (BOD) removal efficiency increased from 27 to 70
and 76 to 94 %, chemical oxygen demand (COD) from 16 to
64 and 72 to 92.5 % when influent values were 45–95 and
110–200 mg/l for BOD and COD, respectively. Verma et al.
(2010) used coagulation-flocculation as a pretreatment pro-
cess for treating purified terephthalic acid (PTA) wastewater.
The results indicated that the wastewater was best treated
10194
when 3000 mg/l of ferric chloride was dosed at pH 5.6. The
removal efficiency of COD reached 75.5 %. Lin et al. (2001)
investigated the treatment of phenol, benzoic acid,
aminobenzoic acid, and petrochemical industry wastewater
which contains acrylonitrile butadiene styrene (ABS) using
pre-ozonation and lifted moving bed biological activated car-
bon. After pre-ozonation of 30 min, BOD/COD ratio of influ-
ent increased apparently from 20 to 35 % approximately. COD
removal efficiency was 85–95 % at organic loading rate of
3.2–6.3 kg COD/(m3/day). Guo et al. (2014) studied oil-field
wastewater treatment by installing airlift tubes and adding an
activated carbon bioreactor (ACB) to form a combined hydro-
lysis acidification and bio-contact oxidation system. The re-
sults showed that an average reduction of COD was 60 %, oil
and grease were 62 %, total suspended solid was 75 %, and
sulfides were 77 %.
Among the biological treatment processes available involv-
ing activated sludge process, the anoxic/oxic (A/O) process,
membrane bioreactor, fluidized bed reactor, etc., the A/O pro-
cess which developed from the conventional activated sludge
process has become a promising alternative because both or-
ganics, nitrogen, and phosphorus compounds could be re-
moved effectively with low operational costs (Fu et al. 2009).
The anaerobic hydrolysis-acidification process is to control
the anaerobic digester in the first two stages. In this process,
the macromolecular organic matters such as polycyclic aro-
matic hydrocarbons and long-chain hydrocarbons and partic-
ular substances could be broken down into smaller molecular
organics which were further converted into volatile fatty acids
(VFAs) by acidogenic bacteria. The anaerobic hydrolysis-
acidification process has become a prevailing choice for pre-
treating the refractory industry wastewater owing to the low
operation cost and easy management. The bioreactors used for
anaerobic hydrolysis-acidification involve continuous stirring
tank, up-flow sludge blanket, hybrid sludge blanket, anaerobic
baffled reactor, and anaerobic contact reactor (Lei et al. 2010;
Lin et al. 2012; Qiu et al. 2013; Wang et al. 2014). In many
cases, uneven water distribution and short circuit occurred,
resulting in bad mixing of sludge and wastewater.
In recent years, a hydrolysis-acidification (HA) reactor
with pulse water allocation was developed to enhance the
mass transfer (Bai et al. 2013). The HA reactor consisted of
a pulse water distribution tank and an up-flow hydrolysis-
acidification reactor. The pulse tank works in the following
two circular stages: (1) filling stage in which influent goes to
the pulse tank continuously in about 5–6 min and (2) pulsing
stage in which all wastewater accumulated was evacuated
rapidly to the hydrolysis-acidification reactor in 30–40 s. As
a result, the sludge in the reactor was stirred severely and
mixed adequately with wastewater. The process is cost-
effective in practice since the capital and running cost for
submerged agitators was not needed. Bai et al. (2013) has
studied the treatment of combined domestic and industrial
Appl Microbiol Biotechnol (2016) 100:10193–10202
wastewater using a hybrid hydrolysis-acidification reactor
with periodic water allocating. Results showed that the peri-
odic water allocating could keep the sludge bed in periodic
expansion-sedimentation-expansion state. COD removal effi-
ciency was 22–42 % when hydraulic retention time (HRT)
was 6.5–16 h.
In the HA-A/O processes treating petrochemical wastewa-
ter, the microbial composition and activity depend on the char-
acteristics of petroleum hydrocarbons, nutrient composition,
and operational conditions such as temperature, dissolved ox-
ygen (DO), mixing system and configuration, etc. For a better
understanding the degradation mechanism of the organic pol-
lutants, further study on the microbial community of sludge in
the system is necessary. Through comparing the removal effi-
ciency of pollutants and the existence of the special microor-
ganism in the system, a relationship between the treatment
efficiency and the major microorganisms can be established.
In the present study, a combined HA and A/O process was
developed to treat the real petrochemical wastewater from a
large petrochemical company in northeast China.
The objectives of this study were to (1) evaluate the per-
formance of the combined processes in terms of the removal
of organics, nitrogen, and phosphorus compounds as well as
analysis of the pollutant composition in influent and effluent
by GC-MS and (2) analyze the microbial community of
sludge in each tank by pyrosequencing.
Materials and methods
Experimental setup
As shown in Fig. 1, the experimental apparatus was composed
of a pulse tank, an up-flow hydrolysis-acidification reactor, an
A/O reactor, and a secondary sedimentation tank with an ef-
fective volume of 0.75, 150, 150, and 25 L, respectively. The
A/O reactor consisted of six identical compartments. The first
compartment served as anoxic tank which was equipped with
an agitator for mixing. Air was supplied into the bottom of the
following five compartments through aquarium-type diffusers
with a flow rate of 0.2 m3/h, and DO concentration in the oxic
tank was maintained at 4–6 mg/L by adjusting the input aer-
ation flow rate. The DO concentration in the anoxic tank was
less than 0.5 mg/L. The sludge from the settling tank was
recycled into the anoxic tank with a recirculation ratio of
100 %.
Wastewater characteristics
The experimental setup was built in an Integrated
Petrochemical Wastewater Treatment Plant (IPWWTP) locat-
ed in northeastern China. The feeding wastewater was the
same as the influent of the IPWWTP. It consisted of
Appl Microbiol Biotechnol (2016) 100:10193–10202 10195

Fig. 1 The schematic diagram of the experimental setup

petrochemical industrial wastewater and domestic wastewater
at a ratio of around 3:1. The petrochemical wastewater was
from over 70 various sets of petrochemical production instal-
lations, including acrylonitrile butadiene styrene plants, calci-
um carbide plant, oil refinery plant, and so on. The industrial
wastewater was pre-treated in each plant and then discharged
to the IPWWTP. The domestic wastewater came from the
living and commercial areas near the IPWWTP. The influent
was characterized with severe fluctuation in COD and sulfate
concentration, high levels of toxic compounds such as volatile
phenolic compounds (VPCs), benzene compounds (BCs), and
cyanides. The characteristics of the influent wastewater (the
unit is mg/L, except pH) is shown in Table 1.
Experimental procedures
The HA and A/O reactors were started by inoculating the
recycled sludge from the recirculation line and the activated
sludge from the aerobic tank of the IPWWTP, respectively.
The concentration of the mixed liquor suspended solids
(MLSS) of the seeds was 10 and 5 g/L, respectively. After
48 h of batch operation, the reactors were run continuously.
The HRT was decreased stepwise and stabilized at 20 h after
3 weeks of operation. The sludge retention time were 60 and
40 days in HA and A/O reactor, respectively. The temperature
of the system was controlled at 21–28 °C. The average
oxidation-reduction potential (ORP) in the HA reactor was
−388 mV which facilitated the growth of the anaerobic and
facultative bacteria.
During steady state of operation, the samples of the influent
and effluent from both HA and A/O reactors were taken peri-
odically to detect the following parameters: COD, BOD5, TN,
NH4+-N, nitrate (NO3-N), nitrite (NO2-N), TP, and UV254.
The concentrations of VFA, SO42−, and sulfide (S2−) in the
influent and HA effluent were evaluated. All the samples were
settled and filtered using 0.45-μm syringe filters before anal-
ysis. The distribution of COD and NH4+-N at each tank of the
system and the concentrations of total organic carbon (TOC),
BOD5, total suspended solids (TSS), and volatile suspended
solids (VSS) were detected 2–3 times every month. On the
day 200, the sludge in the HA, anoxic, and oxic tanks were
taken for microbial community analysis.

Table 1 Characteristics of the

influent wastewater COD BOD5 NH+4-N TN (mg/ TP (mg/ SO42− Cyanide VPCs BCs
(mg/L) (mg/L) (mg/L) L) L) (mg/L) (mg/L) (mg/L) (mg/L)

205–561 67–218 16.9–38.2 20.5–60.0 0.44–2.57 294–802 0.01–0.02 10–15 13–20

VPCs volatile phenolic compounds, BCs benzene compounds

10196 Appl Microbiol Biotechnol (2016) 100:10193–10202

Analytical methods
The concentrations of COD, BOD5, NH4+-N, NO3−-N, NO2−-
N, SO42−, S2−, and TSS were analyzed using the Chinese stan-
dard methods (S.E.P.A. 2002). VFAwas determined by titration
method (Lutzhoft et al. 2014). TOC and TN were detected by
TOC analyzer (TOC-VCPH, Shimadzu). DO and ORP were
monitored by a multi-parameter analyzer (WTW Multi 3420).
pH was detected by a portable pH meter (SX721).
Molecular weight distribution of influent and effluent was
determined by filtering the water sample by ultrafiltration
membrane with different pore size (1, 3, 5, 10, 30, and
100 kDa, respectively) using ultrafiltration cup (Models
8400, Millipore, USA) which was driven by 0.1 MPa nitrogen
gas. The content of TOC of each permeate was detected. A
GC/MS analyzer (Agilent 7890A/5975C) was used to quali-
tatively analyze composition of the dissolved organic matters.
The water samples were acidified firstly using sulfuric acid,
and the organic substances were extracted from the aqueous
phase using dichloromethane. The extracts were evaporated to
1 mL before analysis. For GC-MS analysis, an HP-5 capillary
column (50 m × 0.25 mm × 0.25 μm) was used and the split
ratio was 5:1. The sample loop volume was 1.0 μL. Helium
was used as the carrier gas with a flow rate of 1.0 mL/min. The
oven temperature program was followed by starting at 40 °C
for 3 min, increasing the temperature to 300 °C at a rate of
5 °C/min and holding 300 °C for 3 min. The temperature of
GC injection and MS ion source was 280 and 230 °C, respec-
tively. The MS was performed in scan mode with the electron-
ic impact ionization energy of 70 eV, and scanning ranged
from 29 to 350 m/z.
The sludge samples were taken from HA, anoxic, and oxic
tanks and used for microbial community by Illumina MiSeq
sequencing according to the following steps (Yang et al.
2015): (1) The genomic DNA was extracted from sample by
DNA extraction kit (Omega, Japan) and examined by 1 %
agarose gel electrophoresis; (2) the specific primers with
barcode was synthesized in specified sequencing regions,
and SRX1715657 for activated sludge in HA, anoxic, and
oxic tanks, respectively.
Results
Performances of the HA-A/O system
The variation of COD concentrations in influent and effluent
with operation time is shown in Fig. 2. Although the influent
COD fluctuated between 206 and 566 mg/L, the effluent COD
maintained at stable levels of 99 ± 18 mg/L, indicating the
HA-A/O reactor was good at withstanding the fluctuation in
influent organic load. The total COD removal efficiency
ranged 65–90 %, among them HA reactor contributed to
15–30 % of COD removal.
The major role of the HA was to increase the biodegradabil-
ity of petrochemical wastewater, facilitating the following A/O
treatment. The BOD5/COD ratio increased from 0.39 ± 0.06 in
influent to 0.42 ± 0.08 in HA effluent and decreased remark-
ably to 0.04 in A/O effluent, indicating that the biodegradability
of the petrochemical wastewater was slightly improved by HA
treatment and almost all the biodegradable substances were
removed by A/O processes. Because the influent of the HA
tank was the mixture of the petrochemical wastewater and do-
mestic wastewater at a ratio of around 3:1, its biodegradability
was not poor, so the improvement by HA treatment was not
significant. However, HA treatment was still important because
it could change the structures of some recalcitrant organic pol-
lutants existed in petrochemical wastewater. The content of
UV254, which represents the aromatic compounds in wastewa-
ter (Wang et al. 2008), decreased from 1.81 ± 0.60 cm−1 in
influent to 1.62 ± 0.44 and 0.79 ± 0.25 cm−1 in effluent of
700
Influent
600
HA effluent
500 A/O effluent
COD (mg/L)

and the recycled PCR product by gel extraction kit (Axygen, 400
USA) was eluted by Tris_HCL, and then examined by 2 % 300
agarose gel electrophoresis; (3) the former PCR product was 200
quantified using QuantiFluor™—ST blue fluorescence quan- 100
titative system (Promega) and mixed at a corresponding pro-
portion according to the amount of sequencing of each sam- 80
COD removal (%)

ple; (4) after quantification and homogenization of the mixed 60

Total system
PCR product by TBS-380 fluorimeter, the amplicons were HA
40
sequenced by Ilumina Miseq PE250 (Shanghai Majorbio
Inc.); (5) all effective operational taxonomic units (OUTs) 20
were processed to obtain the statistics information about mi- 0
crobial community diversity and composition in each biology 0 40 80 120 160 200 240
classification level. Time (d)
The raw data of sequencing have uploaded to NCBI SRA Fig. 2 Variation of COD concentration in influent and effluent and the
database. The run numbers are SRX1715646, SRX1715653, removal efficiency
Appl Microbiol Biotechnol (2016) 100:10193–10202
HA and A/O reactors, respectively. The average removal effi-
ciency of UV254 in HA and A/O reactors was 10.4 and 51.3 %,
respectively.
In the case of ammonium removal, it was much higher than
COD removal (Fig. 3). The influent NH4+-N concentration
varied in the range of 16.9–38.3 mg/L, while the effluent
NH4+-N concentration maintained at 1.3 ± 0.6 mg/L. The
ammonium removal efficiency reached 95.4 ± 2.0 %. This
indicated that activity of ammonium oxidation bacteria in
A/O reactor was high during the whole operational period.
The increased NH4-N concentration in the HA effluent might
be owing to anaerobic ammonification of organic nitrogen
compounds in the influent, which were identified by the fol-
lowing GC/MS analysis.
Figure 4 depicts the distribution of TN, ammonium, nitrate,
nitrite, and TP in each part of the A/O reactor. Ammonium
was nitrified in oxic tank and converted to nitrate. Nitrate was
the major nitrogen compounds, and the nitrite concentration
was low (below 0.05 mg/L) in the effluent, indicating that
nitrification took place completely. The reduced ammonium
concentration in anoxic tank was ascribed to 100 % recycling
of sludge. Each part of A/O reactor contributed to TP degra-
dation. The TP and TN removal in A/O reactor reached 44.7
and 51.7 %, respectively.
Regarding the performance of the HA reactor, the VFA/
COD ratio increased remarkably from 0.36 ± 0.19 in influent
to 0.66 ± 0.27 in HA effluent, indicating the good efficiency of
acidogenic fermentation. The concentrations of sulfate de-
creased from 498.5 ± 155.8 mg/L in influent to
400.2 ± 134.1 mg/L in effluent, while the sulfide increased
from 0.17 ± 0.11 to 4.55 ± 2.89 mg/L because sulfate was
converted to sulfide by sulfate reduction bacteria during an-
aerobic digestion.
The characteristics of influent and effluent
The variations of molecular weight (MW) in influent and ef-
fluent were determined. The percentage of substances with
Fig. 3 Variation of NH4+-N
concentration in influent and
effluent and the removal
efficiency
NH 4-N (mg/L)
+
10197
MW <1 kDa accounted for a large portion in the wastewater,
which increased from 80.6 to 85.8 % after HA treatment and
declined to 63 % in the final effluent, which indicated that
some macromolecular organic pollutants were decomposed
into micro-molecular substances in HA process, which then
were degraded in A/O process. In addition, the organics with
MW 1–10 kDa accounted for 30 % in the A/O effluent, which
might be due to the production of soluble microbial products
(SMP). It was reported that the dissolved organic substances
of MW >1.8 kDa in the biologically treated effluent accounted
for above 50 % due to SMP production (Parkin and McCarty
1981).
Table 2 shows the variation of organic pollutants in influent
and effluent analyzed from GC-MS. The influent consisted of
a diversity of organic pollutants involving hydrocarbons, ke-
tone, heterocyclic matters, ethanol, esters, amides, nitriles,
phenols, etc. The variety and abundance of organic pollutants
were decreased by HA treatment at certain extent and reduced
significantly by following A/O treatment. It is worthwhile to
note that the number and abundance of some toxic organic
pollutants such as aldehydes, nitriles, and esters decreased,
while the content of hydrocarbons increased remarkably by
HA process. This proved that HA reactor could decompose
the refractory organics in the petrochemical wastewater at
some extent. Most of the contaminants were degraded in
A/O reactor. There are still some heterocyclic compounds,
ketones, and esters in the effluent. An advanced treatment to
polish the effluent seems to be needed.
Microbial community of sludge in each tank
The sludge concentration in HA and A/O reactor ranged
6600–8460 mg TSS/L and 4860–5130 mg TSS/L, with
VSS/TSS ratio of around 43.8 and 48.1 %, respectively, under
steady-state conditions. After screening and optimizing of the
raw reads, a total of 39,947 effective sequences were obtained.
The numbers of OTUs (3 % dissimilarity) of samples are listed
in Table 3. The higher the Ace and Chao1 indexes, the more
60 100
50
80
NH 4-N removal (%)
40
60
30
40
20
+
Influent
HA effluent 20
10
A/O effluent
0 0
0 40 80 120 160 200 240
Time (d)
10198 Appl Microbiol Biotechnol (2016) 100:10193–10202

Fig. 4 Distribution of nitrogen 60 2.5

compounds and TP in different Other N-compounds
part of the system NO3-N
50

Nitrogen compounds (mg/L)

NH4-N 2.0
TP
40

TP (mg/L)
1.5
30
1.0
20

0.5
10

0 0.0
Influent HA effluent A-tank A/O effluent
Stages

the community richness exists. The Shannon index is positive-
ly correlated with community diversity and the high Good’s
coverage reflects the reliability of the sequencing analysis re-
sults. The results showed that anoxic sludge has the highest
community richness and diversity, following the sludge in
oxic and HA tanks.
Figure 5 depicts the rarefactions curves of the three sludge
samples, which were also used to estimate the richness of
bacteria community. A flat rarefaction curve reflected that
the microbial sequencing could represent the accuracy of spe-
cies diversity (Liao et al. 2013). It is obvious that the diversity
level of anoxic sludge was richer than that of oxic and HA
sludge, which is consistent with the results of diversity indi-
ces. All of these results demonstrated that the library for each
sample was fully satisfactory to characterize bacterial
communities.
In the case of the abundance of bacteria, the major bacterial
community of the three sludge samples was different (Fig. 6).
At phylum level, the dominant bacteria were Chloroflexi
(25 %), Proteobacteria (22 %), Firmicutes (15 %), and
Bacteroidetes (12 %) for HA sludge; Proteobacteria (59 %),
Acidobacteria (9 %), Bacteroidetes (7 %), and Chloroflexi
(6 %) for anoxic sludge; and Proteobacteria (53 %),
Bacteroidetes (14 %), Actinobacteria (5 %), and
Planctomycetes (4 %) for oxic sludge, respectively.
There were 338, 292, and 287 genera identified in HA,
anoxic, and oxic samples, respectively. Figure 7 depicts the
heat map generated with major genera in sludge samples. The
most abundant genera were Anaerolineaceae uncultured and
Desulfobacter for HA sludge, B lastocatella and
Anaerolineaceae uncultured for anoxic sludge, and
Saprospiraceae uncultured and Nitrosomonadaceae

Table 2 The variation of organic

pollutants in influent and effluent Number of peaks Percentage (%)

Influent HA eff. A/O eff. Influent HA eff. A/O eff.

Hydrocarbons 17 21 ND 16.7 25.5 –

Aldehydes 11 6 ND 7.6 2.7 –
Heterocyclic matters 9 7 1 8.0 8.4 29.4
Amines 8 7 ND 5.7 11.0 –
Alcohols 7 6 ND 5.6 6.8 –
Phenols 6 5 ND 7.1 10.0 –
Ketones 5 8 2 1.4 4.9 35.1
Nitriles 2 3 ND 3.4 1.5 –
Esters 2 2 1 6.0 3.1 10.5
Others 12 22 1 12.7 26.4 25.1

Percentage means the ratio of peak areas and total peak areas. The organic matters with low concentration, namely
peak area percentage under 0.1 % were not listed in the table
ND not detected
Appl Microbiol Biotechnol (2016) 100:10193–10202 10199

Table 3 Relevant indexes of

bacterial richness and diversity Sludge Reads OTUs Average length Ace Chaol Shannon Good’s coverage

HA 12,574 583 435 680 701 5.03 0.9900

Anoxic 15,292 780 438 887 904 5.34 0.9899
Oxic 12,081 618 440 719 764 4.84 0.9888

uncultured for oxic sludge, respectively. Cluster analysis sug-
gested that the organisms in the anoxic and oxic zones showed
high similarity.
Discussion
The above results indicated that the combined HA with pulse
water allocation and A/O processes were promising to treat
petrochemical wastewater. Although the organic load of influ-
ent fluctuated, the content of effluent COD and NH4+-N main-
tained at around 99 and 1.3 mg/L, which met the first-grade
discharge standard (GB8978-1996) for integrated wastewater
in China. The treatment performance of the reactor depends
on the characteristics of influent, operational conditions, reac-
tor configuration, etc. The COD removal was 15–30 % by HA
treatment and reached 65–90 % for the total system, which is
comparable to that reported in the literature. The COD remov-
al efficiencies ranging 20–40 % were reported for treating the
industrial wastewater such as pharmaceutical, starch, and tan-
nery wastewater by the traditional HA processes (Bai et al.
2013; Wang et al. 2014), while 59–89 % of COD removal was
reported using A/O processes for treating petrochemical
wastewater (Shokrollahzadeh et al. 2008; Ma et al. 2009).
Our previous studies (Yang et al. 2015) for treating the petro-
chemical wastewater from the same plant demonstrated that
the average effluent COD maintained at 97 ± 19 mg/L by
using the microaerobic hydrolysis-acidification-A/O system
which was similar to this study. The microaerobic HA reactor
contributed to 33–42 % COD removal. The HA reactor with
pulse water allocation exhibited a good mixing of wastewater
and sludge, leading to a higher efficiency of acidogenic fer-
mentation and COD removal. Moreover, the strict anaerobic
condition in HA reactor ensures a stable and higher efficiency
of acidogenic fermentation. Regarding the operational cost of
the HA reactor with pulse water allocation, it is generated
mainly from the power consumption to elevate the influent.
The estimated cost was 0.04 CNY/m3 according to 0.55 CNY
per kilowatt-hour in China.
To find the relationships between the microbial community
and the performance of the reactors, the sequencing of sludge
in each tank was analyzed by pyrosequencing. Many
researchers revealed the correlation of microorganism
community and organic pollutants degradation. The
filamentous Chloroflexi are reported to be abundant not only
in anaerobic sludge but also in aerobic sludge in wastewater
treatment. Yamada et al. (2005) found that organisms affiliat-
ed with Chloroflexi phylum are ubiquitous microbial popula-
tions in mesophilic and thermophilic sludge granules, and
these populations might contribute to the degradation of car-
bohydrates and other cellular components, such as amino
acids in the bioreactors. The Gram-negative Proteobacteria
is a large group of bacteria, including a broad variety of aer-
obic, anaerobic, and facultative bacteria. They could degrade a
wide spectrum of organic pollutants and were commonly
identified in the wastewater treatment processes (Snaidr
et al. 1997). It is worthwhile to note that the major function
of the anaerobes Bacteroidetes in the fermentation system is to

800
100 Others
Verrucomicrobia
600 Nitrospirae
80
Gemmatimonadetes
Abundance (%)

Chlorobi
OTUs

HA 60 Planctomycetes
400
Anoxic Cyanobacteria
Acidobacteria
Oxic
40 Actinobacteria
200 Synergistetes
Bacteroidetes
20 Firmicutes
Proteobacteria
0
Chloroflexi
0 3000 6000 9000 12000 15000 18000 0
Number of reads sampled
HA Anoxic Oxic
label: 0.97 Sludge samples
Fig. 5 Rarefaction curves of sludge samples at HA, anoxic, and oxic Fig. 6 Relative abundance of the dominant bacterial community in three
reactors reactors at phylum level
10200
Fig. 7 Heat map of the dominant
genera in three reactors (the color
bar indicates the range of the
percentage of a genus in a sample,
based on the color key at the right
bottom) (color figure online)
degrade macromolecular substances such as protein, starch,
cellulose, and fiber into small molecules (Nakasaki et al.
2009). Some species in the phylum Firmicutes are able to
excrete extracellular enzymes such as cellulose, lipase, and
protease (Hasan et al. 2006), which could contribute the hy-
drolysis of the refractory chemicals in petrochemical
wastewater.
At genus level, bacteria affil iated with genus
Anaerolineaceae (19.0 % in HA sludge) in the phylum
Chloroflexi are known as semi-syntrophic and fatty acid-
oxidizing bacteria, enabling to degrade carbohydrate couples
with hydrogenotrophic methanogens (Narihiro and Sekiguchi
2007). Members of genera Blastocatella (6.1 % in anoxic sam-
ple) belonging to phylum Acidobacteria were aerobic and
chemoheterotrophic bacteria (Foesel et al. 2013). It was detect-
ed from an oil-contaminated soil in an oilfield in northeast
China (Liao et al. 2015). Members of family Saprospiraceae
(11.5 % in oxic sample and 2.2 % in anoxic sample) affiliated
with phylum Bacteroidetes were able to hydrolyze carbon
polymers, particularly proteins, and specialized in the break-
down and utilization of complex carbon sources (McIlroy and
Nielsen 2014). The ammonium-oxidizing bacteria (AOB)
Nitrosomonadaceae were highly enriched in the oxic tank,
while AOB Nitrosomonas and nitrite-oxidizing bacteria
(NOB) Nitrospira have high abundance in the anoxic and oxic
sludge, which contributed the higher removal efficiency of am-
monium, that is to say, the existence of abundant AOB and
NOB in the microbial community of oxic tank is consistent
with the good nitrification performance of the reactor.
Appl Microbiol Biotechnol (2016) 100:10193–10202
It should be noted that the sulfate-reducing bacteria
Desulfobacter, Desulfofustis, and Desulfomicrobium, which
reduced sulfate to hydrogen sulfide by utilizing organic mat-
ters such as alcohols, fatty acids, lactate, and some aromatic
compounds as electron donor and carbon source under anaer-
obic conditions (Lens et al. 1998), were detected only in HA
reactor with a total abundance of 6.5 %. This might be the
reason why the high sulfide ions concentration was detected in
HA effluent.
Shokrollahzadeh et al. (2008) reported that the culturable
bacterial population isolated from the activated sludge in a
petrochemical wastewater treatment plant mainly belonged
to genera Pseudomonas, Flavobacterium, Comamonas,
Cytophaga, Sphingomonas, Acidovorax and Bacillus, which
were mostly detected in our system. Among the genera iden-
tified, the bacteria belonging to genus Pseudomonas were
reported to be able to degrade oil, alkanes, and aromatic sub-
stances (Wei et al. 2015) and capable of producing extracel-
lular proteases and depolymerases to improve the anaerobic
hydrolysis (Zheng et al. 2013). Some strains of genus
Xanthobacter enable to degrade halogenated short-chain hy-
drocarbons and halogenated carboxylic acids (Janssen et al.
1985). Some species of genus Bacillus were capable of
degrading refractory and high toxic pesticides, fungicide,
and polycyclic aromatic hydrocarbons, such as chlorpyrifos,
chlorothalonil, and naphthalene (Anwar et al. 2009; Lin et al.
2010; Zhang et al. 2007). All of these bacteria detected in the
HA-A/O system were favorable for degradation of the hazard-
ous chemicals in the petrochemical wastewater.
Appl Microbiol Biotechnol (2016) 100:10193–10202
Acknowledgments This research was supported by the National S&T
Major Project (2012ZX07201-005-06-01), the National Natural Science
Foundation of China (51338005), and the Program for Changjiang
Scholars and Innovative Research Team in University (IRT-13026).
Compliance with ethical standards
Human and animal rights This article does not contain any studies
with human participants or animals performed by any of the authors.
Conflict of interest The authors declare that they have no conflict of
interest.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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