Environ Sci Pollut Res (2016) 23:4919–4930

DOI 10.1007/s11356-015-5721-y

RESEARCH ARTICLE

A field pilot-scale study of biological treatment of heavy

oil-produced water by biological filter with airlift aeration
and hydrolytic acidification system
Min Zhang 1 & Junming Wang 2 & Zhongzhi Zhang 1 & Zhaozheng Song 3 &
Zhenjia Zhang 4 & Beiyu Zhang 2 & Guangqing Zhang 5 & Wei-Min Wu 6

Received: 21 July 2015 / Accepted: 30 October 2015 / Published online: 9 November 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract Heavy oil-produced water (HOPW) is a by-product
during heavy oil exploitation and can cause serious environ-
mental pollution if discharged without adequate treatment.
Commercial biochemical treatment units are important parts
of HOPW treatment processes, but many are not in stable
operation because of the toxic and refractory substances, salt,
present. Therefore, pilot-scale experiments were conducted to
evaluate the performance of hydrolytic acidification-
biological filter with airlift aeration (HA-BFAA), a novel
HOPW treatment system. Four strains isolated from oily
sludge were used for bioaugmentation to enhance the biodeg-
radation of organic pollutants. The isolated bacteria were eval-
uated using 3-day biochemical oxygen demand, oil, dodecyl
benzene sulfonic acid, and chemical oxygen demand (COD)
removals as evaluation indices. Bioaugmentation enhanced
the COD removal by 43.5 mg/L under a volume load of
0.249 kg COD/m3 day and hydraulic retention time of
33.6 h. The effluent COD was 70.9 mg/L and the correspond-
ing COD removal was 75.0 %. The optimum volumetric air-
to-water ratio was below 10. The removal ratios of the total
extractable organic pollutants, alkanes, and poly-aromatic hy-
drocarbons were 71.1, 94.4, and 94.0 %, respectively. Results
demonstrated that HA-BFAA was an excellent HOPW treat-
ment system.
Keywords Heavy oil-produced water . Biological filter with
airlift aeration . Refractory organic matter . Bioaugmentation .
Biodegradation . Removal of total nitrogen

Responsible editor: Philippe Garrigues

Introduction
* Zhongzhi Zhang
bjzzzhang@163.com
With the decline of conventional oil reserves, the heavy oil
industry is becoming more important in the global economy
1
State Key Laboratory of Heavy Oil Processing, College of Chemical (Pei et al. 2012). Large amounts of heavy oil-produced water
Engineering, China University of Petroleum, Beijing 102249,
People’s Republic of China
(HOPW) have been produced, which amounts to as much as
2
10–20 times the extracted oil (Shpiner et al. 2009). Besides
Dalian Design Branch, China Petroleum Engineering & Construction
Corporation, Dalian 116011, China
conventional pollutants such as n-alkanes, benzene deriva-
3
tives, phenolic compounds, and polycyclic aromatic hydrocar-
State Key Laboratory of Heavy Oil Processing, Faculty of Sciences,
China University of Petroleum, Beijing 102249, People’s Republic of
bons (PAHs), various kinds of non-biodegradable oilfield
China chemicals, such as polymers, emulsifiers, demulsifiers, and
4
School of Environmental Science and Engineering, Shanghai Jiao
bactericides, are also present in HOPW. These pollutants are
Tong University, Shanghai 200240, China caused by the extensive application of heavy oil recovery
5
School of Mechanical, Materials & Mechatronic Engineering,
techniques, resulting in low ratio of biochemical oxygen de-
University of Wollongong, Wollongong NSW2522, Australia mand (BOD) to chemical oxygen demand (COD). The emul-
6
Department of Civil & Environmental Engineering, Center for
sification, mineralization, viscosity, and temperature of
Sustainable Development & Global Competitiveness, Stanford HOPW are much higher than conventional oilfield-produced
University, Stanford, CA 94305-4020, USA water (OPW) (Fakhru’l-Razi et al. 2009). Effectively treating
4920 Environ Sci Pollut Res (2016) 23:4919–4930

HOPW to meet strict environmental standards is a serious wastewater treatment systems specifically for the biodegrada-
challenge. tion of refractory pollutants.
Physical, chemical, and biological technologies, such as This paper reports a pilot-scale study that evaluates the
ultrafiltration, nanofiltration, reverse osmosis, photocatalytic ability of immobilized microorganisms to degrade oil and
oxidation, fenton oxidation, electrochemical technology, oilfield chemicals using a hydrolytic acidification-biological
room temperature ionic liquids, membrane bioreactor, anaer- filter with airlift aeration (HA-BFAA). The performance of the
obic bioreactor, and moving bed biofilm (Li et al. 2006, 2010; system operated at outdoor temperatures was evaluated with
Mondal and Wickramasinghe 2008; Murray-Gulde et al. regard to COD removal, ability to resist shock loading, and
2003; Bessa et al. 2001; Moraes et al. 2004; Ramalho et al. amount of scale formed on the biofilms.
2010; McFarlane et al. 2005; Scholz and Fuchs 2000; Dong
et al. 2011), have been investigated for the treatment of OPW.
However, these technologies are still in the stage of laboratory
Materials and methods
studies. In oilfields, onsite treatment technologies such as
American Petroleum Institute separators, tilted plate intercep-
Isolation and screening of bacteria from oily sludge
tor separators, and dissolved air flotation units are used to
pretreat produced water, after which wastewater is further
The bacteria used for bioaugmentation in this study were iso-
treated by an activated sludge process (Chavan and
lated and screened from oily sludge collected from the bottom
Mukherji 2008).
of an oil tank in Henan Oilfield. The bacteria that are capable
The conventional activated sludge (CAS) process is used
of degrading oil or are resistant to surfactants (i.e., bacteria
worldwide as an inexpensive and efficient wastewater
that can grow in surfactant-containing wastewater) were iso-
biotreatment. However, given that HOPW is low in nitrogen
lated as follows. Dodecyl benzene sulfonic acid (DBSA), a
and phosphorus compounds but rich in toxic and refractory
surfactant widely used in oilfields, was added in the media
substances, the CAS process cannot operate efficiently over a
used for screening surfactant-resistant bacteria. All the media
long term, as bulking and foaming tend to occur (Tellez et al.
used in this investigation contain the following nutrients
2002). Fixed film biological reactors can prevent some of
(per liter): 1.0 g NaCl, 0.5 g CaCl2, 0.5 g KH2PO4, 0.5 g
these problems (Rahimi et al. 2011). During the past decades,
K2HPO4, 0.02 g FeCl3, and 1.0 g NH4NO3. Approximately
various biofilm technologies have been developed to treat
5.0 g of the oily sludge was added to 200 mL of the medium
OPW.
containing 10.0 g/L crude oil or 1.0 g/L DBSA. The mixture
Zhao et al. (2006) used a biological aerated filter to remove
was cultivated for 3 days on a shaking table (150 r/min,
pollutants from OPW and achieved 78 and 94 % in TOC and
30 °C). Afterward, 2 mL of the mixture was transferred into
oil removal, respectively. However, the system may be prone
a fresh medium. This process was repeated three times, and
to blockages in the aeration and water distribution systems
the resulting mixture was isolated using the streak plate meth-
caused by suspended solids and mineral salts (Moore et al.
od with a gelated medium with 2 % agar containing 2.0 g/L
2001). Lu et al. (2009) studied the performance of hydrolysis
crude oil or 0.5 g/L DBSA. The culture dishes were kept in an
acidification/bio-contact oxidation system to remove COD
incubator at 30 °C for 24 h. Each colony was inoculated into
from OPW. An efficiency of 63.5 % in COD removal was
200 mL of liquid medium containing 2.0 g/L crude oil or
achieved with initial mean COD of 350 mg/L.
0.5 g/L DBSA. After cultivation for 3 days on a shaking table
However, almost all biofilm processes are associated with
(150 r/min, 30 °C), two strains of petroleum hydrocarbon-
numerous operational problems. For instance, the salts in high
degrading bacteria and two strains of DBSA-resistant bacteria
concentrations in the OPW combine with carbon dioxide in
were chosen by comparing the removal rates of oil and DBSA,
the aeration air to form carbonates on biofilm surfaces. This
respectively.
phenomenon reduces the efficiency of biodegradation by hin-
dering the contact between microorganisms in the biofilms
and the OPW. To reduce the formation of carbonate scaling, Inoculum preparation
this paper investigates a novel process named biological filter
with airlift aeration (BFAA), which is a combination of In a BioFlo 110 Fermentor (NBS, USA) with Lysogeny broth,
biological aeration filtration and bio-contact oxidation further cultivation of the isolated and screened bacteria in
techniques. large amounts was completed for augmentation in the pilot
Bioaugmentation has been successfully demonstrated as an plant investigation. The dissolved oxygen (DO) in the fermen-
effective measure to increase the efficiency of pollutant re- tation broth was kept at about 3 mg/L, and pH was maintained
moval in a laboratory wastewater treatment system and in at 7.2 using hydrochloric acid and sodium hydroxide. The
the oilfield. Aboriginal microorganisms, exogenous microor- microorganisms were cultured for 3 days, which generated a
ganisms, and genetic engineering strains are introduced into dry cell concentration of about 1.09±0.21 g/L.
Environ Sci Pollut Res (2016) 23:4919–4930 4921

The activated sludge used for initial inoculation in the pilot which allows the microorganisms to adhere to the carrier.
plant test was collected from a wastewater treatment plant in a HOPW is pumped from a storage tank into the regulating
petroleum refinery of Henan Oilfield. The mixed liquor reservoir and passes through the HA reactor, primary sedi-
suspended solids (MLSS) and mixed liquor volatile mentation tank, the BFAA reactors, and the sedimentation
suspended solids (MLVSS) of the activated sludge were tank in sequence. The flow rate of influent water is controlled
6740 and 4956 mg/L, respectively. via a ball valve and recorded using an electric flowmeter.
The startup procedure of the pilot HA-BFAA wastewater
treatment plant was as follows. A total of 200 L of seed sludge
Pilot-scale wastewater treatment plant and operation each was injected into the HA reactor and BFAA reactors for
inoculating. The plant was filled with HOPW until the over-
The investigation was carried out in a pilot-scale HA-BFAA flow port level of the sedimentation tank. Then, 7.37 kg glu-
wastewater treatment plant in Sinopec Henan Oilfield, China. cose, 0.71 kg NH4NO3, 0.22 kg KH2PO4, 33.6 g FeSO4,
Before entering the biological system, the HOPW was 134.4 g NaMoO4, and 84.0 g MnCl2 were added to promote
pretreated by flocculation and air flotation. The characteristics microorganism growth. A diving mixer in the HA reactor was
of the HOPW are listed in Table 1. Figure 1 shows the sche- used to mix the sludge and HOPW homogeneously. The airlift
matic of the pilot plant, which consists of a regulating reser- aeration system of the BFAA reactors was switched on to
voir, a hydrolytic acidification (HA) reactor, two BFAA reac- supply oxygen to the microorganisms and to accelerate the
tors, and two sedimentation tanks. The total effective volume attachment of microorganisms on the carriers. Continuous
of the plant is 16.8 m3. flow of wastewater was started after 3 days, with an initial
The HA reactor is a fixed biofilm type reactor with 4 m3 flow rate of 6 m3/day. The flow rate was increased to 12 m3/
bulk volume of biofilm carrier consisting of hydroformylated day with a 2-m3/day increment every 15 days. After the flow
polyvinyl alcohol fibers and polyethylene rings with double rate reached 12 m3/day and held for 30 days, a steady state
circles. Specially, hydroformylated polyvinyl alcohol fibers operation was established. The steady state operation took a
are pressed on the circles to keep a uniform distribution total of 75 days to establish since a continuous flow was
around the polyethylene rings. Two BFAA reactors have elas- started.
tic packings with 6 m3 bulk volume and an effective waste- The isolated special bacteria were applied to enhance the
water capacity of 8.4 m3. Four PVC tubes surrounded by biodegradation of refractory organic pollutants in the HOPW.
elastic packing made of polyolefin are vertically installed in The influent was stopped and four strains (100 L) of special
each BFAA reactor for internal circulation of wastewater driv- bacteria for bioaugmentation were added into the BFAA reac-
en by airlift aeration. Specifically, a large amount of polyolefin tors. Then, 3.67 kg glucose, 0.36 kg NH4NO3, 0.11 kg
filaments 0.4 mm in diameter and 150 mm in length is fixed KH2PO4, 16.8 g FeSO4, 67.2 g NaMoO4, and 42.0 g MnCl2
on a rope to form a unit. The aerators are located inside the were added, and aeration of BFAA reactors was implemented.
tubes; thus, water flows upwards through the action of Continuous flow was started after 3 days with an initial flow
uplifting air and is distributed onto the packing from the top rate of 12 m3/day. The flow rate was then increased stepwise
of the tubes. Given this aeration pattern, the scouring force of to 18 m3/day with a 2-m3/day increment every 15 days. The
airflow to the packing/microorganism carriers decreases, effect of air-to-water ratio (AWR) on the COD removal was
evaluated from day 151 until day 195. The AWR was changed
Table 1 Characteristics of HOPW to 15, 10, and 5, each being run for 15 days while the water
flow rate was maintained at 16 m3/day. The temperatures of
Parameter Concentration Parameter Concentration the HA and BFAA reactors during the testing were 45 to 50
(mg/L) (mg/L)
and 35 to 45 °C, respectively.
COD 283.2±5.2 Na+ 932±4.2
BOD5 9.2±2.2 Mg2+ 5.17±0.2 Characterization of bacteria
NH3–N 12.1±2.1 Al3+ 7.81±0.3
SS 18.4±6.4 K+ 23.8±2.1 COD, oil, and DBSA removal, as well as the BOD in 3 days
Oil 32.1±2.2 Ca2+ 55.0±1.6 (BOD3), were used to evaluate the activities of the bacteria
Total nitrogen 23.2±4.1 Cl− 1155±4.2 isolated. BOD3 was selected rather than BOD5 because the
Total phosphorus 0.62±0.13 Fe2++Fe3+ 0.59±0.1 former is more similar to the actual hydraulic retention time
TOC 74.9±3.4 Br− 0.69±0.3 (HRT) in the HOPW treatment plant. The respirometric BOD
NPOC 56.4±2.1 Sr 1.38±0.2
Oxitop method was used for BOD 3 determination
pH 7.08∼7.54 Si 70.1±0.1
(Kuokkanen et al. 2004). Briefly, 10 mL of bacterial culture
Total salts 3075±6.3 S2− 21.5±0.6
was centrifuged, the supernatant fluid was discarded, and the
thallus was washed twice with a phosphate buffer. The thallus
4922 Environ Sci Pollut Res (2016) 23:4919–4930

Fig. 1 Schematic of the HOPW

treatment pilot plant

1 Regulating reservoir, effective volume 1.05 m3

2 Hydrolytic acidification reactor, effective volume 5.25 m3

3 Primary sedimentation tank, effective volume 1.05 m3

4, 5 Biological filter with airlift aeration reactors, effective volume 4.2 m3×2

6 Final sedimentation tank, effective volume 1.05 m3

and wastewater were loaded in a closed bottle of a WTW
BOD Oxitop instrument and stored for 3 days at 20 °C to
obtain BOD3.
Polymerase chain reaction-denaturing gradient gel electro-
phoresis (PCR-DGGE) was employed to analyze the microbi-
al community of the wastewater treatment. The samples ana-
lyzed include microorganisms from biofilms in the HA and
BFAA reactors.
The genomic DNA of the microorganisms was extracted
using TIANamp Bacteria DNA Kit (Tiangen Biotech, China).
The variable V3 regions of eubacterial 16S rRNA genes of the
microorganism samples were amplified by PCR using the
bacterial universal primers 341F-GC (5′-CGC CCG GGG
CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG
GCC TAC GGG AGG CAG CAG-3′) and 518R (5′-
ATTACCGCGGCTGCTGG-3′). The PCR mixture contained
3 μL DNA extract, 0.5 μL of each primer, 21 μL deionized
water, and 25 μL MIX. The samples were first incubated for
4 min at 96 °C, then for 35 cycles of 94 °C for 30 s, 55 °C for
40 s, and 72 °C for 40 s. The final extension step was main-
taining at 72 °C for 30 min prior to cooling to 4 °C. Agarose
gel electrophoresis was carried out to detect whether the PCR
product existed before DGGE.
The DGGE of the PCR product was performed in a poly-
acrylamide gel (8w/v%), which contained 40 to 80 % dena-
turing gradients (urea and formamide) using a JY-TD331A
DGGE system (Junyi Electrophoresis, China).
Electrophoresis was conducted using a 1×TAE buffer at
160 V and 60 °C for 8 h. After electrophoresis, the gel was
stained with ethidium bromide for 15 min and rinsed with
Milli-Q water. The gel was scanned using the TEP-M/WL
gel imaging system (Vilber Lourmat, France) to obtain the
DGGE band image, which were cut off afterward. The DNA
was recovered from bands using a TIANgel Midi Purification
Kit (Tiangen Biotech, China) and then cloned and sequenced
with assistance from Sangon Biotech, China.
GC–MS analysis
The organic components in wastewater were quantitatively
analyzed using a gas chromatograph–mass spectrometer
(GC–MS). In brief, wastewater samples were extracted using
dichloromethane three times at pH levels of 2, 7, and 12 each.
The three extracts were combined and condensed to 1 mL in a
rotary evaporator (Lu and Wei 2011). Organic species were
obtained by evaporating the solvent from the condensate in a
sample bottle using nitrogen purging. Dibenzothiophene-d8
(D-DBT) was added in a ratio of 20 μg D-DBT per 3 mg of
organic species as internal standard. The GC-MS (Thermo
Fisher DSQ) was equipped with an Agilent HP-5MS fused
with silica capillary column (60 m×0.25 mm×0.25 μm).
Gas chromatography was conducted with 99.999 % pure
helium as gas carrier operating at 37 kPa and 1 mL/min. The
temperatures of the sampling port and transmission line were
270 and 250 °C, respectively, and the sample injection volume
was 1 μL. The temperature program consisted of three sec-
tions, namely, an initial isothermal section at 50 °C for 5 min,
ramping the temperature to 250 °C at a constant rate of
5 °C/min, and maintaining isothermal conditions for 25 min.
Mass spectrometer conditions included electron impact and
energy of 70 eV, filament current at 100 mA, multiplier volt-
age at 1047 V, and full-range scanning.
Environ Sci Pollut Res (2016) 23:4919–4930
Other characterization methods
The concentrations of COD, NH3–N, total nitrogen (TN), total
phosphorus (TP), MLSS, MLVSS, suspended solids, and S2−
were measured following the standard methods (State
Environmental Protection Administration of China, 2002).
Mercury sulfate was added to remove chlorine from the water
samples. Temperature, pH, and DO were measured with a
portable water quality analyzer (model Multi 3420, WTW
Laborprodukte, Germany), whereas TOC and non-purgeable
organic carbon of water samples were measured by a TOC
analyzer (Shimadzu Co., Japan). The concentrations of metal-
lic elements were measured by analyzing the residue after
water was vaporized using an X-ray fluorescence spectrome-
ter (model ZSX Primus II, Japan).
Results and discussion
Laboratory evaluation of the chosen strains
Four strains of special bacteria that targeted various refractory
organic pollutants were isolated from the oily sludge. Two
strains of petroleum hydrocarbon-degrading bacteria, namely,
B-4-9 and I, and two strains of surfactant-resistant bacteria, B-
3 and B-4, were isolated. Using 16S rDNA gene sequencing
analysis, the bacteria strains were identified as Bacillus
thuringiensis, Bacillus licheniformis, Citrobacter freundii,
and Pseudomonas aeruginosa.
In the screening experiments for effective oil-biodegrading
bacteria and surfactant-resistant bacteria, 70.9, 81.3, 69.6, and
65.7 % of the 2.0 g/L oil and 58.4, 53.2, 67.2, and 72.1 % of
the 0.5 g/L DBSA were degraded by Bacillus thuringiensis,
Bacillus licheniformis, Citrobacter freundii, and
Pseudomonas aeruginosa, respectively. All four strains of
bacteria were effective in the degradation of oil and DBSA.
In general, wastewater with BOD5/COD ratios greater than
0.5 such as brewery wastewater is easy to biodegrade (Rao
et al. 2007). Wastewater with BOD5/COD below 0.3, such as
municipal sewage (Lin and Chang 2000), contains toxic com-
ponents and requires special types of microorganisms to bio-
degrade (Lu et al. 2009). As shown in Table 1, the BOD5/
COD ratio of the HOPW used in this investigation was ap-
proximately 0.04, which was slightly less than that of HOPW
(0.07–0.11) (Liu et al. 2013; Ji et al. 2002, 2007) and smaller
than ordinary OPW (0.43–0.51) (Zhao et al. 2006; Campos
et al. 2002). The oilfield chemicals added in the oil extraction
process, such as viscosity breakers, emulsion splitters, antisep-
tics, and surface active agents, had an inhibitory effect on the
biodegradation of pollutants in HOPW. The performance of
the isolated special bacteria in wastewater treatment was eval-
uated using BOD3 of the wastewater, with the addition of
individual bacteria and removal of COD after 3 days of
4923
biodegradation. Table 2 shows the enhancement effects of
the chosen bacterial strains compared with a blank test without
the chosen strain. The BOD3/COD ratio of the original
HOPW was 0.04 and increased to 0.26–0.32 after the addition
of individual bacterial strains. The COD removal by 3 days of
degradation was increased from 17.8 % in the blank test to
about 50 % after the individual bacterial strains were added.
All four bacterial strains were effective in wastewater
treatment.
The surfactants in wastewater affect the microbiological
degradation of organic pollutants in two ways. On the one
hand, surfactants can enhance microbiological degradation
by increasing the solubility of petroleum hydrocarbons in
wastewater and promoting the diffusion of petroleum hydro-
carbons to the cells of microorganisms (Bury and Miller
1993). On the other hand, surfactants can inhibit microbial
growth or they can become toxic to microorganisms for two
reasons (Laha and Luthy 1991). First, the interaction of sur-
factants and lipid materials in cells of microorganisms de-
stroys the cell membrane. Second, surfactants react with and
deactivate the enzymes or functional proteins in the cells
(Volkering et al. 1995). In comparison, the surfactant-
resistant bacteria performed better than the petroleum
hydrocarbon-degrading bacteria in COD and DBSA removal.
The surfactants present in the wastewater may have sup-
pressed the growth of the petroleum hydrocarbon-degrading
bacteria to some extent, whereas the surfactant-resistant bac-
teria were less affected by this effect. The two strains of
surfactant-resistant bacteria were isolated from the medium
containing DBSA, making them less prone to the suppression
of the surfactant than the petroleum hydrocarbon-degrading
bacteria.
Performance of HOPW treatment plant
The HOPW treatment plant was run for 75 days without bio-
augmentation and 75 days with bioaugmentation. The COD
level of the HOPW influent fluctuated within 260 to 300 mg/
L, with an average of 283.2 mg/L during the whole period.
The effects of HRT and AWR on COD removal were
Table 2 Enhancement of BOD3/COD ratio and COD removal by four
chosen bacterial strains
Bacteria BOD3 BOD3/COD COD after BOD3 COD removal
(mg/L) test (mg/L) (%)
Blank 10.2 0.04 233 17.8
B-4-9 82.2 0.29 156 48.5
I 90.7 0.32 160 47.2
B-3 73.7 0.26 146 51.8
B-4 76.5 0.27 137 54.8
4924
examined during the pilot plant test. Figure 2 shows the COD
levels in the influent and effluents of the HA and BFAA
reactors.
Effect of bioaugmentation
From day 46 until day 75, operation without bioaugmentation
was stable, with HRT at 33.6 h. The average effluent COD,
COD removal, and volume loading of the HA reactor were
218.5 mg/L, 22.8 %, and 0.647 kg COD/m3 day, respectively;
whereas the corresponding values of the BFAA reactors were
114.5 mg/L, 47.6 %, and 0.312 kg COD/m3 day, respectively.
The COD removal of the BFAA reactors was relative to the
COD of the BFAA influent. The overall COD removal of the
plant was 59.6 %.
The COD of the effluent was significantly decreased after
bioaugmentation, which was attributed to the enhancement of
biodegradation by the BFAA reactors. As shown in Figure 2,
the performance of the HA reactor did not show significant
changes after bioaugmentation as long as the HRT was not
changed. This result was expected because the bioaugmenta-
tion was for the BFAA reactors only. From day 76 until day
105, the average effluent COD and COD removal of the HA
reactor were 208.3 mg/L and 26.4 %, respectively, which are
similar to the values without bioaugmentation. The BFAA
reactors had a slightly lower volume load of 0.298 kg COD/
m3 day, showing a 10-day transitional period when the efflu-
ent COD steadily decreased. Thereafter, the average effluent
COD and COD removal of the BFAA reactors were 70.9 mg/
L and 66.0 %, respectively. Overall, bioaugmentation de-
creased the average effluent COD by 43.5 mg/L, enhancing
the COD removal by 15.4 % and the overall COD removal to
75.0 %. The COD removal effect using the present HA-BAFF
system is more predominant than are other biotechniques used
Without bioaugmentation
300
250
200
COD (mg/l)
150
100
50
HRT=67.2 h HRT=50.4 h HRT=40.3 h
0
0 25 50
Fig. 2 COD concentration and removal rate with and without bioaugmentation
Environ Sci Pollut Res (2016) 23:4919–4930
to treat real OPW in previous studies, such as sequence batch
reactor, anaerobic baffled reactor, CAS process coupled with
immobilized biological filter, and airlift reactor (Campos et al.
2002; Freire et al. 2001; Ji et al. 2009; Tong et al. 2013). The
results demonstrated that the isolated bacteria effectively
biodegraded the refractory organic pollutants in the HOPW
and that bioaugmentation was an efficient measure to improve
the capacity of the wastewater treatment system.
Ability to resist shock loading
The practical operation of a wastewater treatment plant is sub-
ject to perturbation in influent conditions, such as flow rate,
pollutant composition, and temperature (Vymazal 2009;
Knoop and Kunst 1998). The shock loading experiments were
carried out to evaluate the ability of the wastewater treatment
system to resist shock loading from day 106 to day 150. This
experiment was conducted by increasing the influent flow rate
from 12 to 18 m3/day, which increased the volume load of the
bioaugmentation system from 0.249 to 0.373 kg COD/m3 day.
The HRT of the HOPW also decreased in the system from 33.6
to 22.4 h, which was unfavorable to the biodegradation of the
wastewater.
As shown in Figure 2, the COD of the effluent from the
BFAA reactors marginally increased from 70.9 to 73.9 mg/L,
when the wastewater flow rate was increased from 12 to
14 m3/day. This increment was smaller than that of the HA
reactor effluent. More marked changes were observed when
the water flow was further increased to 16 and 18 m3/day,
where the COD of the effluent from the BFAA reactors was
81.6 and 98.7 mg/L, respectively. Along with the increase of
water flow from 12, 14, 16, and 18 m3/day, the overall COD
removal was decreased from 75.0, 73.9, 71.2, and 65.1 %,
respectively. Notably, the decrease in the overall COD
Influent Effluent of HA Effluent
Bioaugmentation
HRT=25.2 h
AWR=15 AWR=10 AWR=5
HRT=33.6 h HRT=33.6 h HRT=28.8 h HRT=25.2 h HRT=22.4 h
75 100 125 150 175 200
Time (d)
Environ Sci Pollut Res (2016) 23:4919–4930 4925

5.0
removal actually occurred in the HA reactor. The average BFAA 1
4.5 Distance from reactor bottom
COD reduction by the BFAA reactors marginally increased,
0.1 m 0.5 m 0.9 m 1.3 m 1.7 m
with 137.4, 144.4, 148.0, and 144.4 mg/L for the water flow 4.0
of 12, 14, 16, and 18 m3/day, respectively. 3.5

Dissolved oxygen (mg/L)

The 17.1 mg/L increase in the effluent COD during the 3.0
flow rate increase from 16 to 18 m3/day and the HRT decrease
2.5
from 25.2 to 22.4 h had a significant impact on the treatment
2.0
system. An HRT at 25.2 h can be considered the most appro-
priate for the HOPW treatment system with the conditions 1.5

concerned. Under these conditions, the COD removal of the 1.0

HA and BFAA reactors was 18.9 and 64.5 %, respectively, 0.5
with overall COD removal of 71.2 %. The volume loading of 0.0
HA and BFAAwas 0.863 and 0.437 kg COD/m3 day, showing
0.0 0.5 1.0 1.5 2.0
that the plant has high potential in increasing the volume load Horizontal distance along water flow direction (m)
from its designed operation capacity. This result indicated that
5.0
the BFAA can excellently withstand wastewater influent dis- Distance from reactor bottom BFAA 2
turbances in flow rate and organic pollutant content. 4.5
0.1 m 0.5 m 0.9 m 1.3 m 1.7 m
4.0

Dissolved oxygen (mg/L)

Effect of AWR on COD removal 3.5
3.0
The aeration system of an aerobic unit that supplies oxygen
2.5
for the metabolism of microorganisms is the major energy-
2.0
consuming unit in a wastewater treatment plant. AWR is the
determinant parameter of energy consumption in the plant. 1.5

The effect of AWR on COD removal was examined at a 1.0

wastewater flow rate of 16 m3/day by changing AWR from
15 to 10 and then to 5 (v/v ratio).
The COD removal by the HA reactor returned to the same
level as that from day 121 to day 135, when the water flow
was retained after a short transitional period caused by the
reduction of water flow from 18 to 16 m3/day (Fig. 2).
The change in AWR of aeration in the BFAA reactors did
not affect the operation of the HA reactor. The stabilized COD
of the BFAA effluent was slightly lower than that achieved
during day 121 to day 135, showing that the biodegradation
activity of the bacteria did not decay in the testing period. A
decrease in the AWR from 15 to 10 did not affect the COD
removal. The average effluent COD was about 72.2 mg/L, and
COD removal was kept at 74.5 %. Further decrease of AWR
to 5, however, significantly deteriorated wastewater treatment
performance. The COD of the effluent increased to about
100 mg/L. From the experimental data, the optimum AWR
was between 5 and 10. Air-to-COD-removal ratio (ACRR),
defined as air needed per 1 kg COD removal, was introduced
to compare the AWR in this research with the values of some
OPW treatment process. The ACRR of BFAA was 33.78 to
67.57 m3 air/kg CODremoval, which was below the activated
sludge process of 92.84 m3 air/kg CODremoval (Tellez et al.
2002).
The distribution of DO in the BFAA reactors was detected
at a wastewater flow of 16 m3/day and AWR of 10 (Fig. 3). In
each BFAA reactor, the highest DO content was that closest to
the water surface, which decreased with the increase in the
0.5
0.0
0.0 0.5 1.0 1.5 2.0
Horizontal distance along water flow direction (m)
Fig. 3 Distribution of dissolved oxygen in BFAA reactors
depth in the reactors from 3.7 mg/L to nearly 0 in the first
BFAA reactor. The DO level was slightly higher in the second
BFAA reactor compared with the equivalent locations of the
two reactors. Therefore, most of the areas in the BFAA reac-
tors were in aerobic conditions where COD removal and ni-
trification were enhanced. However, the bottoms of the reac-
tors were anaerobic, which was beneficial to denitrification.
The TN and ammonia concentrations of BFAA1 influent and
BFAA2 effluent were detected to investigate the TN and
(NH3–N) removal rates of the BFAA reactors. As shown in
Fig. 4, the TN and NH3–N concentrations were reduced from
23.21 to 8.16 mg/L and from 12.36 to 5.73 mg/L. Meanwhile,
the TN and the NH 3 –N removal rates were 64.84 and
53.64 %, respectively. Thus, the BFAA reactors functioned
to remove COD and TN, simultaneously.
Fouling on biofilm surfaces
Formation of inorganic scales on biofilm surfaces is a com-
mon problem in biofilm systems treating salt-rich wastewater.
As shown in Table 1, the concentrations of Ca2+, Al3+, and
4926
35
Total nitrogen concentration of BFAA1 influent
Total nitrogen concentration of BFAA2 effluent
30 Ammonia concentration of BFAA1 influent
Ammonia concentration of BFAA2 effluent
25
Concentration (mg/L)
20
15
10
5 pollutants during wastewater treatment, the organic pollutant
164 168 172 176 180
Time (d)
Fig. 4 Total nitrogen and ammonia concentrations of BFAA1 influent
and BFAA2 effluent
Mg2+ in the HOPW were 55.0, 7.81, and 5.17 mg/L, respec-
tively. These inorganic ions reacted with carbon dioxide in the
aeration air to form solid carbonates on the surface of the
biofilms. This reaction prevented the microorganisms in the
biofilms from interacting with wastewater, reducing the effi-
ciency of wastewater treatment. The MLVSS/MLSS ratio of
the biofilms is used to evaluate the amount of scale formed on
the biofilms. As the amount of inorganic substances within the
microbial cells is steady, a higher MLVSS/MLSS ratio means
that the amount of inorganic substances in the biofilms is
smaller and the scaling is less severe.
After the pilot plant was operated continuously for
12 months under the optimized conditions, the MLVSS/
MLSS ratio of the biofilms on the elastic packing was 0.59
and the COD removal was 64.5 %. In comparison, a commer-
cial biological wastewater treatment plant having similar
flowsheets and operation conditions had a MLVSS/MLSS ra-
tio of 0.39 and COD removal of 41.3 %. This commercial
plant contained fixed biofilm carriers consisting of soft fiber
packing and plastic frames but had no airlift tubes installed.
The amount of scale on the biofilms in the BFAA was lesser
than that in the commercial plant, whereas the COD removal
was higher than the latter. These results indicated that the
BFAA system reduced the formation of scales effectively be-
cause of the installation of airlift tubes.
Treatment energy costs
Treatment economics in environmental protection has always
been a major concern and priority. Researchers worldwide are
continually in search of new treatment and development op-
portunities that will effectively decrease environmental
Environ Sci Pollut Res (2016) 23:4919–4930
treatment costs without compromising profit. Therefore, the
economic energy cost of the HA-BFAA system was calculat-
ed. Actual design considerations were as follows: design treat-
ment capacity=100 m3/h, energy usage (lift pump and air
blower)=7.5+7.5=15 kW, and energy cost=$ 0.13/kW h.
Thus, the actual field treatment energy cost is US$0.195/m3,
which is much cheaper than that of the activated sludge sys-
tem (Tellez et al. 2002).
Change in pollutant composition during biodegradation
To better understand the biodegradation of different organic
components were separated from wastewater at different
stages of biodegradation through liquid–liquid extraction
using methylene dichloride as extractant. After biodegradation
via the HA and BFAA reactors, the amount of total organic
pollutants in the wastewater was reduced by 71.1 %, from
62.0 to 17.9 mg/L. The degradation of organic pollutants by
the HA and BFAA reactors was 19.6 and 51.5 %, respectively,
which was relative to the amount of pollutants in the plant
influent. This result agrees with amounts in COD removal.
The HOPW contained a very complex mixture of dissolved
and dispersed oil compounds, antiseptics, emulsion splitters,
and surfactants. Up to 64 organic compounds were identified
in the influent, which can be classified into three categories,
namely, alkanes, PAHs, and others that mainly consist of
oilfield chemicals including antiseptics, emulsion splitters,
and surfactants. Table 3 lists the compounds and their
changes.
The total alkane content in the influent was 28.3 mg/L and
occupied 45.7 % of the total organic pollutants, which was
decreased to 1.60 mg/L after biodegradation. The removal of
the total alkanes by the HA and BFAA reactors was 43.5 and
50.9 %, respectively. This finding disagreed with the COD
removal results because the alkanes were more biodegradable
by HA than were other pollutants in the HOPW. GC–MS
analysis showed that the alkanes included C12 to C38 n-al-
kanes, alkyl cyclohexane, pristine, and phytane. The concen-
trations of C18 to C26 n-alkanes were high, each produced
above 1 mg/L water. Dodecane had a significantly lower re-
moval (69.6 %) than did other alkanes. This finding is in
contradiction with that observed by Olivera et al. (1997),
who explained that the high volatility of dodecane resulted
in its higher removal. The low removal of dodecane can be
attributed to the initial abundance of dodecane and the rate and
extent of degradation of other components in the complex
mixture (Mohanty and Mukherji 2008). Formation of mole-
cules smaller than dodecane cannot be excluded; however,
they were not detected in the treated wastewater due to their
high volatility. For C15 to C22 alkanes, the biodegradation
Environ Sci Pollut Res (2016) 23:4919–4930 4927

Table 3 Contents of organic matters in the HOWP before and after treatment (μg/L)

Organic matter Influent Effluent Effluent Organic matter Influent Effluent Effluent
of HA of BFAA of HA of BFAA

Dodecane 21.83 23.64 6.56 Naphthalene 2.20 1.45 0.53

Tridecane 33.53 26.32 4.59 Acenaphthylene 124.9 60.60 31.64
Tetradecane 18.07 14.40 2.05 Acenaphthene 105.4 18.74 0.90
Pentadecane 59.73 36.39 2.58 Fluorene 32.69 32.51 5.34
Cetane 293.2 76.97 4.90 C-3 phenanthrene 381.5 22.82 0.97
Heptadecane 670.8 132.9 11.72 Phenanthrene 134.4 86.99 14.12
Pristane 1140 226.3 11.11 Anthracene 199.9 29.06 0.00
Octadecane 1571 294.6 16.62 Fluoranthene 39.57 23.76 0.74
Phytane 1713 546.2 41.95 Pyrene 2.86 2.17 0.31
Nonadecane 1996 1043 133.1 Benzofluorene 44.22 29.98 4.77
Eicosane 2367 1422 223.1 4-Methylpyrene 36.43 22.95 5.23
C13-alkyl cyclohexane 69.10 24.00 6.63 Benzanthracene 6.58 4.54 1.03
Heneicosane 2478 1560 262.7 Chrysene 6.05 4.12 0.18
C14-alkyl cyclohexane 51.59 28.23 8.25 Triaromatic steroid 9.84 5.39 0.20
Docosane 2504 1663 278.9 Benzo(k)fluoranthene 4.05 3.75 0.29
C15-alkyl cyclohexane 119.1 185.7 8.69 Benzo(g,hi)perylene 19.74 13.51 2.06
Tricosane 2075 1502 1.84 Indeno(1,2,3-cd)pyrene 16.57 7.53 0.72
Tetracosane 1787 1268 171.7 Benzo(b)fluoranthene 3.76 2.27 0.35
Pentacosane 1740 1184 87.73 Benzo(a)pyrene 2.28 1.29 0.42
Hexacosane 1722 1037 28.75 Dibenz(a,h)anthracene 3.05 1.60 0.20
Heptacosane 1323 930.5 76.89 Diethylene glycol ether 393.6 100.5 9.05
Octacosane 1141 779.0 54.86 Ethyl thiocyanate 427.5 294.8 43.84
Nonacosane 1059 682.4 52.15 Azelaic acid 560.8 402.1 77.85
Triacontane 1047 637.0 45.77 Methyl oleate 180.4 80.80 38.89
Hentriacontane 617.3 270.3 33.31 Stearyl alcohol 198.7 112.4 6.92
Dotriacontane 355.7 301.6 8.28 Dinonyl phthalate 237.3 194.4 18.00
Tritriacontane 137.3 147.9 8.33 Dodecylbenzenesulfonic acid 471.0 292.3 114.5
Tetratriacontane 152.8 94.45 1.02 Diisopropylidene acetone 351.2 59.29 16.77
Pentatriacontane 27.86 22.03 3.22 2,4-Diaminoanisole dihydrochloride 340.4 168.2 14.97
Hexatriacontane 20.73 9.99 1.81 2,4-Dichloronitrobenzene 174.7 85.34 7.71
Heptatriaconetane 16.29 3.91 0.53 2,6-Di-tert-butylphenol 358.0 246.5 17.91
Octatriacontane 6.57 3.94 0.27 Myristyl alcohol 242.9 39.72 3.33

removal in the HA stage was higher than in the BFAA stage,
which is similar to that reported by Liu et al. (2013). However,
the result of C23 to C35 alkanes behaved oppositely.
The total content of PAHs was 1176 μg/L in the HOPW,
which occupied only 1.9 % of the organic pollutants. Despite
their low content, PAHs are highly toxic (Cerniglia 1993). A
total of 20 PAHs were identified in the HOPW influent. C-3-
phenanthrene, anthracene, phenanthrene, acenaphthylene,
and acenaphthene were the dominant PAHs with concentra-
tions of 381.5, 199.9, 134.4, 124.9, and 105.4 μg/L, respec-
tively. The total content of the PAHs in the effluents of the
HA and BFAA reactors were 375 and 70 μg/L, respectively.
This result corresponded to the overall removal of the organic
pollutants by 68.1 and 25.9 % in the HA and BFAA stages,
respectively. PAHs were well degraded or converted through
the hydrolytic acidification and aerobic processes.
Naphthalene and acenaphthylene were more resistant to bio-
degradation, whose overall biodegradation was less than
80 %. Although 74.7 % of acenaphthylene was degraded,
its residual concentration was still as high as 31.6 μg/L after
treatment.
Twelve oilfield chemicals were found in the influent.
The oilfield chemicals were degraded well with removal
rates of more than 90 % except for DBSA and methyl
4928 Environ Sci Pollut Res (2016) 23:4919–4930

oleate, which were only degraded by 75.7 and 78.4 %, re- Table 4 16S rDNA gene sequence analysis of different reactor biofilms
bacterial communities
spectively. DBSA is an ionic component with a concentra-
tion of 471 μg/L in the HOPW and whose sodium salt is a Band number Genus/species HA BFAA1 BFAA2
popular anionic surfactant. The oilfield chemicals also in-
clude some alcohols, esters, and organic acids, which were 1 Flavobacteriaceae √ √ √
precursors or solvents of surfactants and some antiseptics 2 Rhodospirillaceae √ √ √
such as 2,4-dichloronitrobenzene. Some unresolved com- 3 Nitrospirae √ √
ponents formed an embossment in the mass spectrum of 4 Phenylobacterium √ √
organic pollutants in the influent. These components 5 Citrobacter freundii √ √
contained branched and cyclic compounds that cannot be 6 Enterobacter √ √
separated by capillary columns (Lu and Wei 2011). 7 Klebsiella √ √
8 Sphingomonas √ √
Bacterial community structure analysis 9 Alphaproteobacteria √ √
10 Coriobacteriaceae √ √
PCR-DGGE technology was used to assess the bacterial com- 11 Xanthobacter √ √
munity structure. The DGGE band profiles of the amplified 12 Pseudomonas nitroreducens √
16S rDNA fragments from the biofilms of the HA and BFAA
√ Detected in the biofilm of reactor
reactors are presented in Fig. 5. These band patterns are dif-
ferent in number, intensity, and position of the 16S rDNA
fragments, suggesting that the bacterial community structure previous studies (Janssen et al. 1985; Said et al. 2008).
in each reactor was different. The 16S rDNA sequences of all The microorganism species in the two BFAA reactors were
the DGGE bands were obtained by sequencing and compared generally similar; however, the primary microorganisms
with the bacterial sequences stored in the GenBank database were different. The result indicated that the function of the
to identify the corresponding bacteria. two BFAAs were different for water quality. The Citrobacter
As shown in Table 4, 12 bacterial bands yielded se- freundii and Pseudomonas aeruginosa used for bioaugmen-
quences that were analyzed using the BLAST program. In tation were observed in the biofilms of both BFAA reactors,
the HA reactor, only Flavobacteriaceae (band 1) and confirming the presence and efficiency of the bioaugmenting
Rhodospirillaceae (band 2) were detected, which have been bacteria in the wastewater treatment system.
previously documented in wastewater treatment systems
(Qiu and Ting 2013; HIRAISHI et al. 1991). A total of 10
bands were observed in the BFAA 1 reactor, among which
bands 3, 4, and 5 were predominant. The organisms related Conclusions
with Nitrospirae are usually observed in wastewater treat-
ment plants and laboratory reactors as primary nitrite bacte- This investigation shows that HA-BFAA is effective for the
ria (Lücker et al. 2010). Members of Phenylobacterium and removal of COD and organic pollutants. The system has a
Citrobacter freundii are known for the degradation of hydro- steady performance and low energy consumption compared
carbons and PAHs (Rodgers-Vieira et al. 2015; Ali et al. to the CAS treatment. The system is easily constructed com-
2012). The same bands were detected in the BFAA 2 reac- pared with the biological aerated filters with back flushing.
tor, but the organisms represented by bands 1, 2, 11, and 12 Moreover, bacterial performance can be reliably evaluated
have become more important. The Xanthobacter (band 11) using BOD3 and COD removal. After biodegradation treat-
have been reported as bacteria that degrade halogenated al- ment with bioaugmentation at a volume load of 0.249 kg
iphatic compounds, whereas Pseudomonas nitroreducens COD/m3.day and AWR of 10, the COD of the effluent was
(band 12) could degrade chlorobenzene and PAH in decreased to 70.9 mg/L.

Fig. 5 DGGE band profiles of

amplified 16S rDNA fragments of
the microorganisms from the
biofilms from HA and BFAA
reactors 1 and 2
Environ Sci Pollut Res (2016) 23:4919–4930
Acknowledgments This research was financially supported by the Na-
tional Key State Basic Research Development Program of China (973
Program, No. 2011CB200906), the National Natural Science Foundation
of China (Nos. 51474223 and 41172333), China Petroleum Science and
Technology Major Project of Low Carbon (No. 2011E-2408), and China
Petroleum Science and Technology Major Project of Development and
Industrial Applications of Sets of Technology in Ten Million Tons Large
Refinery (No. K1003-5).
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