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Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
69
ABSTRACT: This study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing
batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and
bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high-
throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/Llevel
after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled
reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial
community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and
sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing
batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi
and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon
degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen
demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing
bacteria, the performance of these bioreactors had no significant difference between summer and winter.
KEYWORDS: Anaerobic baffled reactor (ABR); Denaturing gradient gel electrophoresis (DGGE); High throughput
sequencing; Microbial community; Seasonal variations; Sequencing batch reactor (SBR); Oilfield produced
water; Sulfide reducing bacteria (SRB); Wastewater treatment plant (WWTP)
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
DOI: 10.7508/gjesm.2016.01.008
*Corresponding Author Email: shaoyuanbai@126.com
Tel.: +86 13978389750; Fax: +86 13978389750
Note. Discussion period for this manuscript open until March
1, 2016 on GJESM website at the “Show Article”.
Seasonal variations of microbial community in a full scale oil field
produced water treatment plant
Q. Xie1
, S. Bai1,*
, Y. Li1
, L. Liu2
, S. Wang1
, J. Xi3
1
Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China
2
Hezhou University, Hezhou 542800, China
3
State Environmental Protection Key Laboratory of Microorganism Application and Risk Control,
Tsinghua University, Beijing 100084, China
INTRODUCTION
Oilfield produced water consists of toxic, aromatic,
and contaminated hypersaline water, which normally
are complex organic compounds, such as phenolics
and aromatic hydrocarbons, benzene, toluene,
ethylbenzene, xylene, naphthalene, phenanthrene, and
dibenzothiophene. The discharges of oilfield-produced
water without proper treatment can contaminate soil
and water bodies due to the existence of the toxic
organic compounds. Conventional treatment for
oilfield-produced water mainly relies on physical-
chemical methods, including gravity separation,
dissolved air flotation, hydrogen peroxide treatment,
photocatalytic degradation, coagulation and
flocculation . Since some halophilic bacteria, yeast and
fungi can grow well with crude oil as a source of carbon
and energy, some of them were utilized for
bioremediation to purify petroleum-produced water
because of the low cost. Sequencing batch reactor
(SBR) has been widely used in petroleum-produced
Received 24 September 2015; revised 29 October 2015; accepted 10 November 2015; available online 1 December 2015
ORIGINAL RESEARCH PAPER
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
70
Q. Xie et al.
water treatment, in which wastewater is initially
hydrolyzed under anoxic conditions and then treated
under aerobic conditions, by doing this, most of the
chemical oxygen demand (COD) can be removed.
Ghorbanian et al., (2014) showed that the average
petroleum hydrocarbon removal rates in an SBR were
99.9%, 99.6%, and 93.7% at initial concentrations of
950, 1450, and 2500 mg/L. Moreover, the removal rates
of COD, total organic carbon (TOC), and oil and grease
removed were 86.2%, 90.8%, and 90% respectively in a
MBR, with organic loading rate of 1.124 kg of COD/m3
d and hydraulic residence time (HRT) of 48h. Generally,
an anaerobic baffled reactor (ABR) is also used to
enhance the COD removal efficiency as a pretreatment.
Therefore, anABR and three SBRs in parallel integrated
process (Fig. 1) were employed in the oilfield-produced
wastewater treatment plant.
Microbial communities contribute to the
biodegradation of hydrocarbons and COD have been
investigated in previous molecular studies using sludge
obtained from oilfield produced water treatment plants.
Molecule microbial analytical methods such as
denaturing gradient gel electrophoresis (DGGE) profile
and high throughput sequencing, revealed that the
bacteria related to organic matter degradation belong
to different taxonomic groups, including
Methylobacterium sp., Rhodococcus aetherovorans,
Achromobacter xylosoxidans, Alcaligenes sp.,
Aquamicrobium defluvium, Rhizobium, and
Stenotrophomonas spp. . Moreover, temperature plays
a major role in biodegradation ofhydrocarbon. Changes
of the operating temperature in wastewater treatment
plants are due to seasonal variations. As removal
efficiency of hydrocarbon in a full-scale treatment plant
is different in summer and winter, the microbial
communities responsible for hydrocarbon degradation
during summer and winter are different. While the
microbial communities refer to oilfield-produced water
is scarce, especially with consideration of seasonal
effect on the microbial communities.
The studies aimed at assessment of the effects of
temperature and treatment units on microorganisms
targetingoilfield-producedwaterare still unclear.Abetter
understanding of the microbial organisms can lead to a
better understanding of the contribution ofthe microbial
organisms in the degradation of hydrocarbons in oilfield-
produced water. The aims of the present study were (i)
to assess the pollutant removal efficiency of COD in a
treatment plant; (ii) to analyze the compositions of
bacterial and fungi in the treatment plant; (iii) to select
the dominant species in the process of degrading
hydrocarbon in oilfield produced water.
Thisstudyhasbeen performed inBeihaiCity, Guangxi
ZhuangAutonomous region during 2012 to 2013.
MATERIALS AND METHODS
Study site description
The wastewater treatment plant (WWTP) was
established in 2006 to treat 1000 tons of wastewater per
day from Weizhou Island Oil Production Plant (China
National Offshore Oil Corporation). The influent
wastewater composition is illustrated in Table 1. The
plant is located in the southeast of Beihai City, Guangxi
Zhuang Autonomous Region in China. During the
system, anABR was followed bythree sequencing batch
reactors. The ABR (21 m long, 16.6 m wide and 6.5 m
high)andthreeSBRs(20.0 mlong,12.0 mwide,and5.8m
high) were connected to enhance the performance of the
WWTP. The effective volumes of the ABR and three
sulfide reducing bacteria (SRBs) were 1500, 360, 360
and 360 m3
respectively.All of the SBRs were operated
according to the following strategies: filling (1.0 h),
reaction (aeration, 8.0 h), settling (2.0 h), extraction (1
h), and idle.
Sampling and analysis
Water samples were collected from the influent and
effluent of the ABR, and three parallel SBRs effluent
(1#SBR, 2#SBR, and 3#SBR) between 9:00 and 10:00
during a day in August 2012 and February 2013
respectively. COD was measured according to standard
method. The temperature of the samples was also
determined. Statistics analysis was employed to
investigate the relationship between temperature and
COD removal efficiency/microbial diveristies using the
one-way ANOVA test in the SPSS 17.0 software and
difference were considered to be significant when
p<0.05.
Table 1: Composition of oilfield produced wastewater
Parameter Range
Cl-
/mg/L 14000–15000
CODCr/mg/L 384–584
BOD5/mg/L 50–127
Total petroleum hydrocarbon /mg/L 11.5–15
S2-
/mg/L 11.8–20.1
TN/mg/L 9–13
TP/mg/L 7–12
SS/mg/L 140–610
pH 7.8–8.2
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
71
Sludge samples was collected in August and
February from theABR (denoted as SAin summer and
WAin winter) and three parallel SBRs (denoted as SS1,
SS2, and SS3 for samples collected in summer and WS1,
WS2, and WS3 for samples collected in winter) for
molecular and biological analyses. Six samples at
different sites were collected from each reactor, and
then fixed and stored at “20 C before analysis.
Micobial community analysis
DNA extraction, PCR amplification, DGGE and
sequencing
The total genomic DNA was extracted from sludge
samples using Power Soil DNA isolation kit (Mo Bio
Laboratories, Inc., Carlsbad, CA, USA). DNA was
purified using Universal DNA purification kit
(TIANGEN Biotech Co., Ltd., Beijing, China) and
quantified using Nano-Drop UV-3000
spectrophotometer (Nanodrop Technologies Inc.,
Delaware, USA) according to the manufacturer’s
instructions. Isolated DNA was amplified through PCR
using primers specific for fungi (ITS1F
CTTGGTCATTTAGAGGAAGTAA and ITS4
TCCTCCGCTTATTGATATGC), targeting the ITS1
region to analyse sludge fungal communities. PCR
amplifications of the ITS1 region from genomic DNA
were performed in 25 μL mixture containing 5 μL of 5×
PrimeSTAR®
 buffer with MgCl2
, 2 μL of deoxyri-
bonucleotide triphosphate mixture, 0.5 μLofeachprimer,
0.25 μL of PrimeSTAR®  
HS DNA polymerase, 1 μL of
DNA, and 15.75 μL of molecular-grade water. PCR
reactions were performed as follows: 95 °C for 5 min
(one cycle); 95 °C for 30 s, 59 °C for 30 s, and 72 °C for
50 s (30 cycles); and 72 °C for 7 min (one cycle). The
amplified products were subjected to electrophoresis
on 0.8% agarose gels.All PCR amplification reactions
were performed in an S1000TM
BioRad model iCycler.
The purified PCR products were analysed on 8%
polyacryamide gels containing gradients of 31%–53%
denaturant. DGGE was performed in aDCode Universal
Mutation Detection System (Bio-Rad) at a constant
voltage of 80 V and temperature of 60 °C for 16 h in
1×TAE runningbuffer.The gels werestained with SYBR
Gold nucleic acid gel stain (Molecular Probes, Eugene,
OR) and then imaged with the FluoroImager System
Model 595 (Molecular Dynamics, Sunnyvale, CA). Gel
images were analyzed with Gelcompar II v5.0 (Applied
Maths, Sint-Martens-Latem, Belgium) to generate
dendrogram profiles.
The individual bands of the DGGE gels were excised
and eluted with 30 μL of dH2
O for 48 h at 4 °C before re-
amplification using the same set of primers. Sequencing
was performed on a 3730 DNA Analyzer using
BigDyeqR
Terminator v3.1 cycle sequencing kit
(Applied Biosystems, United States) according to the
manufacturer’s instructions. A phylogenetic tree was
constructed through neighbor-joining method using
the MEGA 4.1 with sequences and bootstrapped for
1000 iterations.
High throughput sequencing analysis
The purified totalcommunity DNA samples prepared
in 2.2.2 were submitted to Novogene Bioinformatics
Institute (Beijing, China) for high-throughput
Fig. 1: Simplified system of the Weizhou wastewater treatment plan
(pumps were not shown)
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
72
Microbial community changes in oil field produced water treatment plant
sequencing using Miseq Illumina. Primers for
sequencing were 515F (5’ -GTG CCAGCM GCC GCG
GTAA-3’) and 806R (5’-GGACTACHV GGG TWT
CTAAT-3’), with different barcodes for the V4 region
of the 16S rRNA gene. Reads with incorrect barcodes,
incorrect primer sequences, average quality scores of
< 25, homopolymers of > 6 and read lengths < 200 bp
were excluded from further analysis. High-quality
sequences were processed through CD-HIT to generate
operational taxonomic units (OTUs) with 97% sequence
similarity threshold. Representative sequences from
each OTU were aligned using PyNAST. Average data
were calculated for each sludge sample before analyzing
the unique and shared OTUs/genera.
RESULTS AND DISCUSSION
Effect of temperature on COD removal in the full scale
ABR-SBR
The average temperature in summer (August, 2012)
and winter (February, 2013) were 29 °C and 18 °C. The
influent concentration of COD was 535±49 mg/L in
summer and 418±34 mg/L in winter (Fig. 2). Statistical
analyses of COD removal in ABR system showed
significantly(P<0.05)lowerefficiencyinwinter(23±4%)
than in summer (30±3%), even though it received lower
influent concentration. The corresponding of low
temperature and low COD removal inABR may cause
by lower functional microbial activity appearance in
winter. However, the removal efficiencies of COD in
both season achieved 90±2% in summer and 88±4% in
winter, both with effluent concentration lower than 50
mg/L after full-scale ABR-SBR system, which proves
the high application performance of ABR-SBR
technology for oilfield produced wastewater.
Variations in fungal community composition
The DGGE fingerprints of the fungal community of
sludge sampled from theABR and SBRs during summer
and winter were analyzed and 28 different bands were
detected (Fig. 3). Phylogenetic analysis indicated that
the majority of these 28 fungal clones belonged to the
Ascomycota, which consisted of the Pezizomycotina
and the Saccharomycotina. The Pezizomycotina was
the dominant taxon and further divided into four
distinct groups, including the Fusarium, Bionectria,
Stachybotrys, and Aspergillus (Fig. 4). The
Mucoromycotina was also observed in the fungal
clones (Fig. 4). Fig. 5 shows proportion of the major
fungi in the ABR and SBRs collected in summer and
winter, with the Saccharomycotina, Fusarium, and
Aspergillus were detected in all the samples and the
rest fungi were detected conditionally with
consideration of season and treatment units.
It’s apparent to see that the Stachybotrys was
shaped by seasons and treatment units, while the
Saccharomycotina, Fusarium, and Aspergillus were
ubiquitous in all sludge sampled from ABR and SBRs
during summer and winter, although their abundance
differed. Previous studies suggested that the
filamentous fungi can produce extracellular enzymes
and degrade polycyclic aromatic hydrocarbons (PAHs).
In the present study, significant difference in the
Fig. 2: COD concentration and removal efficiencies of the ABR influent
and effluent and three parallel SBRs. Data Bars followed by different
letters are significantly different (p< 0.05)
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
73
removal efficiency of COD were observed between
seasons and treatment units, which may due to the
difference of microorganisms communities existed in
the sludge samples. However, the proportion of the
fungi in theABR and SBRs during summer and winter
were not corresponded to the COD removal efficiency,
which may imply that fungal community will not
influence the COD removal in the present study.
Variation in microbial community compositions
The bacterial community diversities of the sludge
used in batch tests were analyzed by lllumina
sequencing. The number of bacterial species can be
estimated using the number of OTUs (Fig. 6). Fig. 6
shows the number of OUTs in summer was higher than
in winter, indicating that the bacterial diversity was
relatively higher in summer than that in winter in terms
of eitherABR or SBRs.
Microbial community in SRBs
In aerobic sludge, the number of OTUs were 4535
(genus level) and 9203 (species level) in SS and 3647
(genus level) and 7254 (species level) in WS. In
agreement with previous reports, the present study
showed that changes in environmental conditions,
particularly temperature shock, directly influenced
microbial communities. Temperature plays a vital role
in biodegradation, and the microbial diversity in the
wastewater treatment system reduced as the reactor
progressively recovered from low-temperature shock.
Extreme temperatures may confer irreversible damages
and even death in some bacteria .
Fig. 7 shows that the majority of the bacteria (39.3%)
in the treatment plant for oilfield-produced water
belonged to the phylum Proteobacteria, followed by
the phyla Firmicutes and Chloroflexi, which accounted
for 19.6% and 13.5% of the total population,
respectively. The phyla Bacteroidetes, Actinobacteria,
and Planctomycetes accounted for 5%, 4.7%, and 2%
of the total population, respectively. Similarly, previous
research showed that bacterial communities at different
operating temperatures consisted mostly of the phyla
Proteobacteria, Bacteroidetes, and Firmicutes, which
can effectively degrade organic compounds . Analysis
of MBR and conventional sludge produced from PAH-
contaminated wastewater showed that Proteobacteria
could be the potential organism for remediating
degraded petroleum.
SS1 SS2 SS3 SA WS1 WS2 WS3 WA 0
Fig. 3. DGGE analysis of the 16S rRNA gene fragment of fungal
population from the ABR (SA for summer and WA for
winter) and three parallel SBRs (SS for summer and WS
for winter). Sequenced bands are marked on the gel.
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
74
Q. Xie et al.
The majority of the OTUs contained only one tag,
and tag numbers were counted at different levels to
determine the optimal taxonomic level for relevant
comparisons . The proportions of assignable tags for
the SBR andABR samples were higher than 70% at the
order level, whereas lower than 50% at the family level.
Therefore, the order level was selected as the optimal
taxonomic level for comparison.
The results demonstrate that bacterial diversity
was relatively higher in summer than that in winter
(Fig. 7). In SBRs, namely, SS samples, the most
abundant order was DRC31 from the phylum
Chloroflexi (18.16%), followed by Lactobacillales
(11.7%) and Bacillales (6.9%) from the phylum
Firmicutes. The relative abundance of Rhodospirillales
(phylum Proteobacteria), and Gammaproteobacteria
Fig. 4: Phylogenetic tree of the 16S rRNA sequences derived from the DGGE bands
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
75
Fig. 5: Relative abundance of the major fungi in each sludge samples from the ABR and three
parallel SBRs collected in summer and winter
No.ofOTUs
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
SS WS SA WA
Sludge Samples
Species
Genus
Fig. 6: Analysis of OTUs from sludge sampled from the ABR and SBRs in
summer and winter
and Rhizobiales (phylum Proteobacteria) were
comparable, which were 4.6%, 4.3%, and 4.2%,
respectively. By contrast, WS samples harbored high
relative abundance of Pseudomonadales (35.6%,
phylum Proteobacteria), Clostridiales (12.2%, phylum
Firmicutes), and Lactobacillales (8.9%). Furthermore,
Ignavibacteriales (6.5%), and Sphingobacteriales
(7.4%) were detected in SS with low abundance and
were absent in WS (Fig. 8). The members of
Pseudomonadales, Lactobacillales, and Bacillales
participate in the biogeochemical transformation of
petroleum. Silva et al., (2013) has indicated on the
diversityofmicrobialcommunities in petroleumsamples
fromBrazilian oil fields showed that eight different phyla
(Actinobacteria, Bacteroidetes, Deferribacteres,
Spirochaetes, Firmicutes, Proteobacteria,
Thermotogae, and Synergistetes) were the dominant
groups involved in hydrocarbon degradation.According
to the performance of SBRs, the COD removal
efficiencies were similar (88.5% to 82.2%) when the
influent COD concentration were 375.5 and 320.9 mg/L
in thesummer and winter, respectively(Fig. 2).Although
the microbial communities significantly varied between
summer and winter, the relative abundance of the
dominant hydrocarbon degrading bacteria still remained
at a high level (see above metioned). Therefore, the
changes ofbacterial communities from summerto winter
did not affect the performance of SRBs.
Diversity of microbial community in ABR
The OTUs were higher in SA and reached 1787 at
the genus level and 3540 at the species level. By
contrast, the number of OTUs in WA was 1555 at the
genus level and 3057 at the species level in theABR. In
anaerobic sludge, SA samples harbored abundant
populations of Rhizobiales (13.2%), Thermotogales
(phylum Thermotogae, 10.7%), and Actinomycetales
(phylum Actinobacteria, 10.4%). Rhodospirillales and
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
76
Microbial community changes in oil field produced water treatment plant
Fig. 7: Microorganism distribution at the phylum level in sludge sampled from
three parallel SBRs and ABR in summer and winter
Fig. 8: Microorganism distribution at the order level in sludge sampled from three
parallel SBRs and ABR in summer and winter
Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016
77
Rhodobacterales accounted for 8.5% and 4.7% of the
population, respectively (Fig. 7). Rhodospirillales
showed the largest proportion (19.1%) of the bacterial
population in WA samples, followed by the order
Actinomycetales (18.4%). DRC31 and
Gammaproteobacteria unclassified were also
abundant and accounted for 9.7% and 8.1%,
respectively (Fig. 7). The mean proportion of DRC31
in SS was 18.2% and decreased to 5.7% in WS. High
temperatures seemed to be more beneficial in DRC31
survival under aerobic conditions than low
temperatures. However, under anaerobic conditions,
DRC31 accounted for higher proportion (9.7%) in the
WA samples than that in the SA samples (2.3%).
Although information about the role of DRC31 in
petroleum hydrocarbon degradation remains limited,
DRC31 is speculated to positively affect high COD
degradation during summer.
Actinobactera, Thermotogales and Rhizobiales are
mainly isolated from petroleum hydrocarbon-
contaminated environments, such as marine sediment,
beach, and groundwater; and genera within these
families contain members with active roles in
biogeochemical transformation of petroleum. The
relative abundance of Rhizobiales and Thermotogales
were decreased from13.15% and 10.72% in the summer
period to 5.3% and 0.4% in the winter time respectively.
However, the relative relative abundance of
Actinomycetales increased by 8.1%. The results reveal
that the total amount of the dominant hydrocarbon
degrading bacteria was decreased in the winter period,
compared to summer time.Additionally, around 159.1
and 97.4 mg/L of COD, on average, were removed in
the ABR in summer and winter respectively (Fig. 2).
Therefore, the removed COD was declined resulting
from the dominant hydrocarbon degrading bacteria
decreasing.
CONCLUSION
COD removal rates afterABR treatment system were
significantly higher in summer than in winter, which
conformed to the microbial community diversity.
Moreover, COD effluent concentration achieved lower
than 50 mg/L level after full-scale ABR-SBR system,
which proves the high performance of the technology
for oilfield-produced wastewater. The fungal
communities inABR and SBR were shaped by seasons
and treatment units, while there was no correlation
between abundance of fungi and COD removal rates.
Different seasons affected the microbial community
both inABR and in SRBs. The performance of theABR
was slightly decreased in terms of COD removal.
However, the COD removal efficiencies in the three
parallel SRBs kept stable between summer and winter.
ACKNOWLEDGEMENT
The work was funded by the National Natural
Science Foundation of China (No. 51168012), the
State Environmental Protection Key Laboratory of 
Microorganism Application and Risk Control
(No. SMARC2013D 009), and supported by the project
of high level innovation team and outstanding scholar
in Guangxi Colleges and Universities.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of
interest regarding the publication of this manuscript.
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structure in two anaerobic systems to treat bagasse spraying
wastewater with and without addition of molasses alcohol
wastewater. Bioresource Tech., 131: 333–340 (8 pages).
Silva, T.R.; Verde, L.C.L.; Neto, E.V.S.; Oliveira, V.M., (2013).
Diversity analyses of microbial communities in petroleum
samples from Brazilian oil fields. Int. Biodeterior. Biodegr.,
81: 57-70 (14 pages).
Táncsics, A.; Szabó, I.; Baka, E.; Szoboszlay, S.; Kukolya, J.;
Kriszt, B.; Márialigeti, K., (2010). Investigation of catechol
2, 3-dioxygenase and 16S rRNA gene diversity in hypoxic,
petroleum hydrocarbon contaminated groundwater. Syst.
Appl. Microbiol., 33: 398–406 (6 pages).
Verma, V.; Raju, S.C.; Kapley, A.; Kalia, V.C.; Daginawala, H.F.;
Purohit, H.J., (2010). Evaluation of genetic and functional
diversity of Stenotrophomonas isolates from diverse
effluent treatment plants. Bioresource Tech., 101: 7744–
7753 (10 pages).
Wiszniowski, J.; Ziembinska, A.; Ciesielski, S., (2011). Removal
of petroleum pollutants and monitoring of bacterial
community structure in a membrane bioreactor.
Chemosphere, 83: 49–56 (8 pages).
Yeung, C.W.; Stempvoort, D.R.V.; Spoelstra, J.; Bickerton, G.;
Voralek, J.; Greer, C.W., (2013). Bacterial community
evidence for anaerobic degradation of petroleum
hydrocarbons in cold climate groundwater. Cold Reg. Sci.
Technol., 86: 55-68 )14 pages).
Zhang, X.; Gao, J.; Zhao, F.; Zhao, Y.; Li, Z., (2014).
Characterization of a salt-tolerant bacterium Bacillus sp.
from a membrane bioreactor for saline wastewater treatment.
J. Environ. Sci., 26: 1369-1374 (6 pages).
AUTHOR (S) BIOSKETCHES
Xie, Q., Ph.D., Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China. Email:
xqinglin@hotmail.com
Bai, S., Ph.D., Associate Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China.
Email: shaoyuanbai@126.com
Li, Y., Ph.D., Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China.
Email: 331842901@qq.com
Liu, L., Ph.D., Professor, Hezhou University, Hezhou 542800, China. Email: 1315099645@qq.com
Wang, S., M.D., Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China.
Email: 410178957@qq.com
Xi, J., MSc. Student, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, Tsinghua University,
Beijing 100084, China. Email: 56362159@qq.com
DOI: 10.7508/gjesm.2016.01.008
URL: http://gjesm.net/article_14865_1931.html
How to cite this article:
Xie, Q.; Bai, Y.; Li, Y.; Liu, L.; Wang, S.; Xi, J., (2016). Seasonal variations of microbial community in a full
scale oil field produced water treatment plant. Global J. Environ. Sci. Manage. 2 (1): 69-78.

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Seasonal Variations of Microbial Communities in an Oil Field Produced Water Treatment Plant

  • 1. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 69 ABSTRACT: This study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high- throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/Llevel after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing bacteria, the performance of these bioreactors had no significant difference between summer and winter. KEYWORDS: Anaerobic baffled reactor (ABR); Denaturing gradient gel electrophoresis (DGGE); High throughput sequencing; Microbial community; Seasonal variations; Sequencing batch reactor (SBR); Oilfield produced water; Sulfide reducing bacteria (SRB); Wastewater treatment plant (WWTP) Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 DOI: 10.7508/gjesm.2016.01.008 *Corresponding Author Email: shaoyuanbai@126.com Tel.: +86 13978389750; Fax: +86 13978389750 Note. Discussion period for this manuscript open until March 1, 2016 on GJESM website at the “Show Article”. Seasonal variations of microbial community in a full scale oil field produced water treatment plant Q. Xie1 , S. Bai1,* , Y. Li1 , L. Liu2 , S. Wang1 , J. Xi3 1 Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China 2 Hezhou University, Hezhou 542800, China 3 State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, Tsinghua University, Beijing 100084, China INTRODUCTION Oilfield produced water consists of toxic, aromatic, and contaminated hypersaline water, which normally are complex organic compounds, such as phenolics and aromatic hydrocarbons, benzene, toluene, ethylbenzene, xylene, naphthalene, phenanthrene, and dibenzothiophene. The discharges of oilfield-produced water without proper treatment can contaminate soil and water bodies due to the existence of the toxic organic compounds. Conventional treatment for oilfield-produced water mainly relies on physical- chemical methods, including gravity separation, dissolved air flotation, hydrogen peroxide treatment, photocatalytic degradation, coagulation and flocculation . Since some halophilic bacteria, yeast and fungi can grow well with crude oil as a source of carbon and energy, some of them were utilized for bioremediation to purify petroleum-produced water because of the low cost. Sequencing batch reactor (SBR) has been widely used in petroleum-produced Received 24 September 2015; revised 29 October 2015; accepted 10 November 2015; available online 1 December 2015 ORIGINAL RESEARCH PAPER
  • 2. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 70 Q. Xie et al. water treatment, in which wastewater is initially hydrolyzed under anoxic conditions and then treated under aerobic conditions, by doing this, most of the chemical oxygen demand (COD) can be removed. Ghorbanian et al., (2014) showed that the average petroleum hydrocarbon removal rates in an SBR were 99.9%, 99.6%, and 93.7% at initial concentrations of 950, 1450, and 2500 mg/L. Moreover, the removal rates of COD, total organic carbon (TOC), and oil and grease removed were 86.2%, 90.8%, and 90% respectively in a MBR, with organic loading rate of 1.124 kg of COD/m3 d and hydraulic residence time (HRT) of 48h. Generally, an anaerobic baffled reactor (ABR) is also used to enhance the COD removal efficiency as a pretreatment. Therefore, anABR and three SBRs in parallel integrated process (Fig. 1) were employed in the oilfield-produced wastewater treatment plant. Microbial communities contribute to the biodegradation of hydrocarbons and COD have been investigated in previous molecular studies using sludge obtained from oilfield produced water treatment plants. Molecule microbial analytical methods such as denaturing gradient gel electrophoresis (DGGE) profile and high throughput sequencing, revealed that the bacteria related to organic matter degradation belong to different taxonomic groups, including Methylobacterium sp., Rhodococcus aetherovorans, Achromobacter xylosoxidans, Alcaligenes sp., Aquamicrobium defluvium, Rhizobium, and Stenotrophomonas spp. . Moreover, temperature plays a major role in biodegradation ofhydrocarbon. Changes of the operating temperature in wastewater treatment plants are due to seasonal variations. As removal efficiency of hydrocarbon in a full-scale treatment plant is different in summer and winter, the microbial communities responsible for hydrocarbon degradation during summer and winter are different. While the microbial communities refer to oilfield-produced water is scarce, especially with consideration of seasonal effect on the microbial communities. The studies aimed at assessment of the effects of temperature and treatment units on microorganisms targetingoilfield-producedwaterare still unclear.Abetter understanding of the microbial organisms can lead to a better understanding of the contribution ofthe microbial organisms in the degradation of hydrocarbons in oilfield- produced water. The aims of the present study were (i) to assess the pollutant removal efficiency of COD in a treatment plant; (ii) to analyze the compositions of bacterial and fungi in the treatment plant; (iii) to select the dominant species in the process of degrading hydrocarbon in oilfield produced water. Thisstudyhasbeen performed inBeihaiCity, Guangxi ZhuangAutonomous region during 2012 to 2013. MATERIALS AND METHODS Study site description The wastewater treatment plant (WWTP) was established in 2006 to treat 1000 tons of wastewater per day from Weizhou Island Oil Production Plant (China National Offshore Oil Corporation). The influent wastewater composition is illustrated in Table 1. The plant is located in the southeast of Beihai City, Guangxi Zhuang Autonomous Region in China. During the system, anABR was followed bythree sequencing batch reactors. The ABR (21 m long, 16.6 m wide and 6.5 m high)andthreeSBRs(20.0 mlong,12.0 mwide,and5.8m high) were connected to enhance the performance of the WWTP. The effective volumes of the ABR and three sulfide reducing bacteria (SRBs) were 1500, 360, 360 and 360 m3 respectively.All of the SBRs were operated according to the following strategies: filling (1.0 h), reaction (aeration, 8.0 h), settling (2.0 h), extraction (1 h), and idle. Sampling and analysis Water samples were collected from the influent and effluent of the ABR, and three parallel SBRs effluent (1#SBR, 2#SBR, and 3#SBR) between 9:00 and 10:00 during a day in August 2012 and February 2013 respectively. COD was measured according to standard method. The temperature of the samples was also determined. Statistics analysis was employed to investigate the relationship between temperature and COD removal efficiency/microbial diveristies using the one-way ANOVA test in the SPSS 17.0 software and difference were considered to be significant when p<0.05. Table 1: Composition of oilfield produced wastewater Parameter Range Cl- /mg/L 14000–15000 CODCr/mg/L 384–584 BOD5/mg/L 50–127 Total petroleum hydrocarbon /mg/L 11.5–15 S2- /mg/L 11.8–20.1 TN/mg/L 9–13 TP/mg/L 7–12 SS/mg/L 140–610 pH 7.8–8.2
  • 3. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 71 Sludge samples was collected in August and February from theABR (denoted as SAin summer and WAin winter) and three parallel SBRs (denoted as SS1, SS2, and SS3 for samples collected in summer and WS1, WS2, and WS3 for samples collected in winter) for molecular and biological analyses. Six samples at different sites were collected from each reactor, and then fixed and stored at “20 C before analysis. Micobial community analysis DNA extraction, PCR amplification, DGGE and sequencing The total genomic DNA was extracted from sludge samples using Power Soil DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). DNA was purified using Universal DNA purification kit (TIANGEN Biotech Co., Ltd., Beijing, China) and quantified using Nano-Drop UV-3000 spectrophotometer (Nanodrop Technologies Inc., Delaware, USA) according to the manufacturer’s instructions. Isolated DNA was amplified through PCR using primers specific for fungi (ITS1F CTTGGTCATTTAGAGGAAGTAA and ITS4 TCCTCCGCTTATTGATATGC), targeting the ITS1 region to analyse sludge fungal communities. PCR amplifications of the ITS1 region from genomic DNA were performed in 25 μL mixture containing 5 μL of 5× PrimeSTAR®  buffer with MgCl2 , 2 μL of deoxyri- bonucleotide triphosphate mixture, 0.5 μLofeachprimer, 0.25 μL of PrimeSTAR®   HS DNA polymerase, 1 μL of DNA, and 15.75 μL of molecular-grade water. PCR reactions were performed as follows: 95 °C for 5 min (one cycle); 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 50 s (30 cycles); and 72 °C for 7 min (one cycle). The amplified products were subjected to electrophoresis on 0.8% agarose gels.All PCR amplification reactions were performed in an S1000TM BioRad model iCycler. The purified PCR products were analysed on 8% polyacryamide gels containing gradients of 31%–53% denaturant. DGGE was performed in aDCode Universal Mutation Detection System (Bio-Rad) at a constant voltage of 80 V and temperature of 60 °C for 16 h in 1×TAE runningbuffer.The gels werestained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR) and then imaged with the FluoroImager System Model 595 (Molecular Dynamics, Sunnyvale, CA). Gel images were analyzed with Gelcompar II v5.0 (Applied Maths, Sint-Martens-Latem, Belgium) to generate dendrogram profiles. The individual bands of the DGGE gels were excised and eluted with 30 μL of dH2 O for 48 h at 4 °C before re- amplification using the same set of primers. Sequencing was performed on a 3730 DNA Analyzer using BigDyeqR Terminator v3.1 cycle sequencing kit (Applied Biosystems, United States) according to the manufacturer’s instructions. A phylogenetic tree was constructed through neighbor-joining method using the MEGA 4.1 with sequences and bootstrapped for 1000 iterations. High throughput sequencing analysis The purified totalcommunity DNA samples prepared in 2.2.2 were submitted to Novogene Bioinformatics Institute (Beijing, China) for high-throughput Fig. 1: Simplified system of the Weizhou wastewater treatment plan (pumps were not shown)
  • 4. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 72 Microbial community changes in oil field produced water treatment plant sequencing using Miseq Illumina. Primers for sequencing were 515F (5’ -GTG CCAGCM GCC GCG GTAA-3’) and 806R (5’-GGACTACHV GGG TWT CTAAT-3’), with different barcodes for the V4 region of the 16S rRNA gene. Reads with incorrect barcodes, incorrect primer sequences, average quality scores of < 25, homopolymers of > 6 and read lengths < 200 bp were excluded from further analysis. High-quality sequences were processed through CD-HIT to generate operational taxonomic units (OTUs) with 97% sequence similarity threshold. Representative sequences from each OTU were aligned using PyNAST. Average data were calculated for each sludge sample before analyzing the unique and shared OTUs/genera. RESULTS AND DISCUSSION Effect of temperature on COD removal in the full scale ABR-SBR The average temperature in summer (August, 2012) and winter (February, 2013) were 29 °C and 18 °C. The influent concentration of COD was 535±49 mg/L in summer and 418±34 mg/L in winter (Fig. 2). Statistical analyses of COD removal in ABR system showed significantly(P<0.05)lowerefficiencyinwinter(23±4%) than in summer (30±3%), even though it received lower influent concentration. The corresponding of low temperature and low COD removal inABR may cause by lower functional microbial activity appearance in winter. However, the removal efficiencies of COD in both season achieved 90±2% in summer and 88±4% in winter, both with effluent concentration lower than 50 mg/L after full-scale ABR-SBR system, which proves the high application performance of ABR-SBR technology for oilfield produced wastewater. Variations in fungal community composition The DGGE fingerprints of the fungal community of sludge sampled from theABR and SBRs during summer and winter were analyzed and 28 different bands were detected (Fig. 3). Phylogenetic analysis indicated that the majority of these 28 fungal clones belonged to the Ascomycota, which consisted of the Pezizomycotina and the Saccharomycotina. The Pezizomycotina was the dominant taxon and further divided into four distinct groups, including the Fusarium, Bionectria, Stachybotrys, and Aspergillus (Fig. 4). The Mucoromycotina was also observed in the fungal clones (Fig. 4). Fig. 5 shows proportion of the major fungi in the ABR and SBRs collected in summer and winter, with the Saccharomycotina, Fusarium, and Aspergillus were detected in all the samples and the rest fungi were detected conditionally with consideration of season and treatment units. It’s apparent to see that the Stachybotrys was shaped by seasons and treatment units, while the Saccharomycotina, Fusarium, and Aspergillus were ubiquitous in all sludge sampled from ABR and SBRs during summer and winter, although their abundance differed. Previous studies suggested that the filamentous fungi can produce extracellular enzymes and degrade polycyclic aromatic hydrocarbons (PAHs). In the present study, significant difference in the Fig. 2: COD concentration and removal efficiencies of the ABR influent and effluent and three parallel SBRs. Data Bars followed by different letters are significantly different (p< 0.05)
  • 5. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 73 removal efficiency of COD were observed between seasons and treatment units, which may due to the difference of microorganisms communities existed in the sludge samples. However, the proportion of the fungi in theABR and SBRs during summer and winter were not corresponded to the COD removal efficiency, which may imply that fungal community will not influence the COD removal in the present study. Variation in microbial community compositions The bacterial community diversities of the sludge used in batch tests were analyzed by lllumina sequencing. The number of bacterial species can be estimated using the number of OTUs (Fig. 6). Fig. 6 shows the number of OUTs in summer was higher than in winter, indicating that the bacterial diversity was relatively higher in summer than that in winter in terms of eitherABR or SBRs. Microbial community in SRBs In aerobic sludge, the number of OTUs were 4535 (genus level) and 9203 (species level) in SS and 3647 (genus level) and 7254 (species level) in WS. In agreement with previous reports, the present study showed that changes in environmental conditions, particularly temperature shock, directly influenced microbial communities. Temperature plays a vital role in biodegradation, and the microbial diversity in the wastewater treatment system reduced as the reactor progressively recovered from low-temperature shock. Extreme temperatures may confer irreversible damages and even death in some bacteria . Fig. 7 shows that the majority of the bacteria (39.3%) in the treatment plant for oilfield-produced water belonged to the phylum Proteobacteria, followed by the phyla Firmicutes and Chloroflexi, which accounted for 19.6% and 13.5% of the total population, respectively. The phyla Bacteroidetes, Actinobacteria, and Planctomycetes accounted for 5%, 4.7%, and 2% of the total population, respectively. Similarly, previous research showed that bacterial communities at different operating temperatures consisted mostly of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which can effectively degrade organic compounds . Analysis of MBR and conventional sludge produced from PAH- contaminated wastewater showed that Proteobacteria could be the potential organism for remediating degraded petroleum. SS1 SS2 SS3 SA WS1 WS2 WS3 WA 0 Fig. 3. DGGE analysis of the 16S rRNA gene fragment of fungal population from the ABR (SA for summer and WA for winter) and three parallel SBRs (SS for summer and WS for winter). Sequenced bands are marked on the gel.
  • 6. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 74 Q. Xie et al. The majority of the OTUs contained only one tag, and tag numbers were counted at different levels to determine the optimal taxonomic level for relevant comparisons . The proportions of assignable tags for the SBR andABR samples were higher than 70% at the order level, whereas lower than 50% at the family level. Therefore, the order level was selected as the optimal taxonomic level for comparison. The results demonstrate that bacterial diversity was relatively higher in summer than that in winter (Fig. 7). In SBRs, namely, SS samples, the most abundant order was DRC31 from the phylum Chloroflexi (18.16%), followed by Lactobacillales (11.7%) and Bacillales (6.9%) from the phylum Firmicutes. The relative abundance of Rhodospirillales (phylum Proteobacteria), and Gammaproteobacteria Fig. 4: Phylogenetic tree of the 16S rRNA sequences derived from the DGGE bands
  • 7. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 75 Fig. 5: Relative abundance of the major fungi in each sludge samples from the ABR and three parallel SBRs collected in summer and winter No.ofOTUs 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 SS WS SA WA Sludge Samples Species Genus Fig. 6: Analysis of OTUs from sludge sampled from the ABR and SBRs in summer and winter and Rhizobiales (phylum Proteobacteria) were comparable, which were 4.6%, 4.3%, and 4.2%, respectively. By contrast, WS samples harbored high relative abundance of Pseudomonadales (35.6%, phylum Proteobacteria), Clostridiales (12.2%, phylum Firmicutes), and Lactobacillales (8.9%). Furthermore, Ignavibacteriales (6.5%), and Sphingobacteriales (7.4%) were detected in SS with low abundance and were absent in WS (Fig. 8). The members of Pseudomonadales, Lactobacillales, and Bacillales participate in the biogeochemical transformation of petroleum. Silva et al., (2013) has indicated on the diversityofmicrobialcommunities in petroleumsamples fromBrazilian oil fields showed that eight different phyla (Actinobacteria, Bacteroidetes, Deferribacteres, Spirochaetes, Firmicutes, Proteobacteria, Thermotogae, and Synergistetes) were the dominant groups involved in hydrocarbon degradation.According to the performance of SBRs, the COD removal efficiencies were similar (88.5% to 82.2%) when the influent COD concentration were 375.5 and 320.9 mg/L in thesummer and winter, respectively(Fig. 2).Although the microbial communities significantly varied between summer and winter, the relative abundance of the dominant hydrocarbon degrading bacteria still remained at a high level (see above metioned). Therefore, the changes ofbacterial communities from summerto winter did not affect the performance of SRBs. Diversity of microbial community in ABR The OTUs were higher in SA and reached 1787 at the genus level and 3540 at the species level. By contrast, the number of OTUs in WA was 1555 at the genus level and 3057 at the species level in theABR. In anaerobic sludge, SA samples harbored abundant populations of Rhizobiales (13.2%), Thermotogales (phylum Thermotogae, 10.7%), and Actinomycetales (phylum Actinobacteria, 10.4%). Rhodospirillales and
  • 8. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 76 Microbial community changes in oil field produced water treatment plant Fig. 7: Microorganism distribution at the phylum level in sludge sampled from three parallel SBRs and ABR in summer and winter Fig. 8: Microorganism distribution at the order level in sludge sampled from three parallel SBRs and ABR in summer and winter
  • 9. Global J. Environ. Sci. Manage., 2(1): 69-78, Winter 2016 77 Rhodobacterales accounted for 8.5% and 4.7% of the population, respectively (Fig. 7). Rhodospirillales showed the largest proportion (19.1%) of the bacterial population in WA samples, followed by the order Actinomycetales (18.4%). DRC31 and Gammaproteobacteria unclassified were also abundant and accounted for 9.7% and 8.1%, respectively (Fig. 7). The mean proportion of DRC31 in SS was 18.2% and decreased to 5.7% in WS. High temperatures seemed to be more beneficial in DRC31 survival under aerobic conditions than low temperatures. However, under anaerobic conditions, DRC31 accounted for higher proportion (9.7%) in the WA samples than that in the SA samples (2.3%). Although information about the role of DRC31 in petroleum hydrocarbon degradation remains limited, DRC31 is speculated to positively affect high COD degradation during summer. Actinobactera, Thermotogales and Rhizobiales are mainly isolated from petroleum hydrocarbon- contaminated environments, such as marine sediment, beach, and groundwater; and genera within these families contain members with active roles in biogeochemical transformation of petroleum. The relative abundance of Rhizobiales and Thermotogales were decreased from13.15% and 10.72% in the summer period to 5.3% and 0.4% in the winter time respectively. However, the relative relative abundance of Actinomycetales increased by 8.1%. The results reveal that the total amount of the dominant hydrocarbon degrading bacteria was decreased in the winter period, compared to summer time.Additionally, around 159.1 and 97.4 mg/L of COD, on average, were removed in the ABR in summer and winter respectively (Fig. 2). Therefore, the removed COD was declined resulting from the dominant hydrocarbon degrading bacteria decreasing. CONCLUSION COD removal rates afterABR treatment system were significantly higher in summer than in winter, which conformed to the microbial community diversity. Moreover, COD effluent concentration achieved lower than 50 mg/L level after full-scale ABR-SBR system, which proves the high performance of the technology for oilfield-produced wastewater. The fungal communities inABR and SBR were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and COD removal rates. Different seasons affected the microbial community both inABR and in SRBs. The performance of theABR was slightly decreased in terms of COD removal. However, the COD removal efficiencies in the three parallel SRBs kept stable between summer and winter. ACKNOWLEDGEMENT The work was funded by the National Natural Science Foundation of China (No. 51168012), the State Environmental Protection Key Laboratory of  Microorganism Application and Risk Control (No. SMARC2013D 009), and supported by the project of high level innovation team and outstanding scholar in Guangxi Colleges and Universities. CONFLICT OF INTEREST The authors declare that there are no conflicts of interest regarding the publication of this manuscript. REFERENCES Ahmadun, F.R.; Pendashteh, A.; Abdullah, L.C.; Biak, D.R.A.; Madaeni, S.S.; Abidin, Z.Z., (2009). Review of technologies for oil and gas produced water treatment. J. Hazard. Mater., 170: 530-551 (22 pages). Bonfá, M.R.L.; Grossman, M.J.; Mellado, E.; Durrant, L.R., (2011). Biodegradation of aromatic hydrocarbons by haloarchaea and their use for the reduction of the chemical oxygen demand of hypersaline petroleum produced water. Chemosphere, 84: 1671–1676 (6 pages). Cho, H.U.; Kim, Y.M.; Choi, Y.-N.; Kim, H.G.; Park, J.M., (in press). Influence of temperature on volatile fatty acid production and microbial community structure during anaerobic fermentation of microalgae. Bioresource Tech. Availablt at: http://www.sciencedirect.com/science/article/ pii/S0960852415003120 Diya’uddeen, B.H.; Daud, W.M.A.W.; Aziz, A.R.A., (2011). Treatment technologies for petroleum refinery effluents: A review. Process. Saf. Environ., 89: 95-105 (11 pages). Ghorbanian, M.; Moussavi, G.; Farzadkia, M., (2014). Investigating the performance of an up-flow anoxic fixed- bed bioreactor and a sequencing anoxic batch reactor for the biodegradation of hydrocarbons in petroleum-contaminated saline water. Int. Biodeter. Biodegr., 90: 106-114 (9 pages). Isanta, E.; Bezerra, T.; Fernández, I.; Suárez-Ojeda, M.E.; Pérez, J.; Carrera, J., (2015). Microbial community shifts on an anammox reactor after a temperature shock using 454- pyrosequencing analysis. Bioresource. Tech., 181: 207-213 (7 pages). Lors, C.; Aldaya, M.M.; Salmon, S.; Ponge, J.-F., (2006). Use of an avoidance test for the assessment of microbial degradation of PAHs. Soil Biol. Biochem., 38(8): 2199- 2204 (6 pages). Lors, C.; Ponge, J.-F.; Aldaya, M.M.; Damidot, D., (2010). Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils. Environ. Pollut., 158(8): 2640-2647 (8 pages).
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Zhang, X.; Gao, J.; Zhao, F.; Zhao, Y.; Li, Z., (2014). Characterization of a salt-tolerant bacterium Bacillus sp. from a membrane bioreactor for saline wastewater treatment. J. Environ. Sci., 26: 1369-1374 (6 pages). AUTHOR (S) BIOSKETCHES Xie, Q., Ph.D., Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China. Email: xqinglin@hotmail.com Bai, S., Ph.D., Associate Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China. Email: shaoyuanbai@126.com Li, Y., Ph.D., Professor, Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China. Email: 331842901@qq.com Liu, L., Ph.D., Professor, Hezhou University, Hezhou 542800, China. Email: 1315099645@qq.com Wang, S., M.D., Center of Mining, Metallurgy and Environment, Guilin University of Technology, Guilin 541004, China. Email: 410178957@qq.com Xi, J., MSc. Student, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, Tsinghua University, Beijing 100084, China. Email: 56362159@qq.com DOI: 10.7508/gjesm.2016.01.008 URL: http://gjesm.net/article_14865_1931.html How to cite this article: Xie, Q.; Bai, Y.; Li, Y.; Liu, L.; Wang, S.; Xi, J., (2016). Seasonal variations of microbial community in a full scale oil field produced water treatment plant. Global J. Environ. Sci. Manage. 2 (1): 69-78.