Bioremediation of Oily Sludge Using Solid Complex Bacterial Agent

Ecotoxicology and Environmental Safety 208 (2021) 111673
Available online 21 November 2020
0147-6513/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Bioremediation of oily sludge by solid complex bacterial agent with a
combined two-step process
Cong-Yu Ke a
, Fang-Ling Qin a
, Zhi-Gang Yang b,c
, Jun Sha a
, Wu-Juan Sun a
, Jun-Feng Hui d
,
Qun-Zheng Zhang a
, Xun-Li Zhang a,*
a
Shaanxi Province Key Laboratory of Environmental Pollution Control and Reservoir Protection Technology of Oilfields, Xi’an Shiyou University, Xi’an 710065, China
b
Shaanxi Key Laboratory of Lacustrine Shale Gas Accumulation and Exploitation, Xi’an 710065, China
c
Research Institute of Yanchang Petroleum (Group) Company Limited, Xi’an 710065, China
d
School of Chemical Engineering, Northwest University, Xi’an 710069, Shaanxi, China
A R T I C L E I N F O
Edited by Dr. G. Liu
Keywords:
Bioremediation
Oily sludge
Solid complex bacterial agent
Two-step combined treatment
A B S T R A C T
In the present research, a bioremediation process was developed using solid complex bacterial agents (SCBA)
through a combined two-step biodegradation process. Four isolated strains showed high efficiency for the
degradation of total petroleum hydrocarbons (TPH) and the reduction of COD of the oily sludge, at 96.6% and
92.6%, respectively. The mixed strains together with bran prepared in form of SCBA exhibited improved per
formance compared to individual strains, all of which had an optimal temperature of around 35 ◦
C. The use of
SCBA provided advantages over commonly used liquid media for storage and transportation. The two-step
process, consisting of firstly biosurfactant-assisted oil recovery and secondly biodegradation of the remaining
TPH with SCBA, demonstrated the capability for treating oily sludge with high TPH content (> 10 wt%) and
short process period (60 days). The large-scale (5 tons oily sludge) field test, achieving a TPH removal efficiency
of 93.8% and COD reduction of 91.5%, respectively, confirmed the feasibility and superiority of the technology
for industrial applications.
1. Introduction
Along with the petroleum products consumed all over the word,
large amounts of petroleum hydrocarbons are released into the envi
ronment during the process of oil exploitation, storage, transportation,
refining and oily sewage treatment (Nwankwegu et al., 2016; Varjani,
2017). In China, for example, about one million tons of oily sludge are
produced every year. That has consequently caused significant envi
ronmental pollution problems (Rodrigo et al., 2014). As a highly haz
ardous solid waste, it is composed of crude oil, water, heavy metals and
sludge (Wang and Wang, 2018). The total petroleum hydrocarbon (TPH)
in oily sludge accounts for 5–86 wt% (da Silva et al., 2012; Hu et al.,
2013). The heavy metals in oily sludge generally include nickel, chro
mium, zinc, lead, manganese and copper, and the concentrations are
higher than that in general soil (Asgari et al., 2017; Hu et al., 2013;
Koolivand et al., 2017). In addition, there are a large number of other
toxic substances, such as benzene series, phenols, anthracene, and pyr
ene, which are presented in oily sludge from different sources (Aguel
mous et al., 2019; Ozyurek and Bilkay, 2020).
It has been understood that not only the large number of com
pounds/chemicals exist in oily sludge, but also the interaction between
them, makes the treatment more challenging, which is however essen
tial before disposal to the environment (da Silva et al., 2012; Pinheiro
and Holanda, 2009). For example, the bridge structures between various
organic pollutants and inorganic solids in oily sludge enhance their
stability. The co-existence of oil and water together with other potential
surfactants results in emulsions. These issues in turn increase difficulty,
both technically and economically, for the development of effective
processes for oily sludge treatment (Ozyurek and Bilkay, 2020).
At present, there are a range of oily sludge treatment approaches,
including physical and chemical methods. However, the effective
treatment of oily sludge has still remained a worldwide unmet need,
mainly due to its complexity and the stability. Also, there are different
technical issues and limitations associated with the commonly used
chemical and physical methods such as the use of large amount of
chemical solvents, harsh operating conditions, and high energy con
sumption (Sayara et al., 2010; Varjani, 2017). Recently, there has been
increasing interest in developing biological processes for oily sludge
* Correspondence to: College of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an 710065, China.
E-mail address: xlzhang@xsyu.edu.cn (X.-L. Zhang).
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
https://doi.org/10.1016/j.ecoenv.2020.111673
Received 2 September 2020; Received in revised form 3 November 2020; Accepted 15 November 2020

2
treatment (Aguelmous et al., 2019; Johnson and Affam, 2019; Varjani
et al., 2020; Wang and Wang, 2018). It is mainly due the potential ad
vantages over conventional physical and chemical treatments, such as
low cost, environment-friendly, mild operating conditions, and simple
operation (Bento et al., 2005; Patowary et al., 2016). By selecting spe
cific microorganisms it has demonstrated strong ability to degrade the
TPH and organic matters in oily sludge, which can eventually be
transformed into harmless materials e.g. CO2 and H2O (Mishra et al.,
2001; Silva-Castro et al., 2013). Different processes have also been
explored, e.g. composting and bio-augmentation technology (Dzondo-
Gadet et al., 2005; Lazar et al., 1999; Namkoong et al., 2002).
Composting involves microbial strains that feed on waste while
transforming organic substances into stable and humified products
(Antizar-Ladislao et al., 2005), normally by mixing oily sludge with a
bulking agent (Aguelmous et al., 2019). As a one-step process for direct
biodegradation, research has shown its advantageous performance with
relatively low capital and maintenance costs, simple design and opera
tion (Ouyang et al., 2005). However, the microbial strains used in this
process are normally not screened and domesticated, therefore the
degradation efficiency of oily sludge is limited. In order to enhance the
process efficiency, bio-augmentation technology has been examined,
where on the basis of composting method, screened and optimized
bacteria with high-efficiency degradation are used to enhance the
treatment effect while effectively shorten the treatment time.
At present, a variety of microorganisms have been reported capable
of using petroleum hydrocarbons as the sole carbon source and energy,
such as Pseudomonas, Achromobacter, Candida digboiensis, Micro
monospora, Bacillus subtilis, and Leuconostoc mesenteroides (Cerqueira
et al., 2012; Gojgic-Cvijovic et al., 2012; Joo et al., 2008; Ozyurek and
Bilkay, 2020; Sood et al., 2010). Furthermore, bio-augmentation
involving microbial communities composed of different functional
bacteria has showed improved performance compared to the individual
strain for oily sludge treatment (Guerra et al., 2018; Simon et al., 2004;
Bento et al., 2005; Ouyang et al., 2005). However, in either composting
or microbial enhancement method, the one-step treatment process
generally adopted for bioremediation of oily sludge with high content of
TPHs needs long treatment time and is not conducive to oil recovery
(Gogoi et al., 2003; Pannu et al., 2004; Simon et al., 2004). In addition,
the commonly used microbial agents in liquid form is associated with a
number of limitations in industrial application such as storage, trans
portation and cost.
The aim of the present study was to develop a two-step oily sludge
bioremediation process with improved efficiency and reduced time for
high TPH content, by screening and isolating bacteria with higher
degradation capability, followed by the development of solid complex
bacterial agent (SCBA). The first step was designed to remove most of
the TPH in the oily sludge by using oil recovery method with bio
surfactant, that was followed by the second step of biodegradation of the
remaining TPH with SCBA. The bioremediation performance was char
acterized by the reduction of the total petroleum hydrocarbon (TPH),
chemical oxygen demand (COD) and heavy metals in the oily sludge.
The key operating parameters were investigated and optimized,
including temperature, initial TPH concentration and water content.
The process was then evaluated through field tests in Xinjiang Oilfield
under industrial relevant conditions towards large-scale applications.
2. Materials and methods
2.1. Materials
All chemicals and reagents were obtained commercially and used as
received. The biochemical reagent kits were purchased from Beijing
Leadman Biochemical Limited, China. Glucose, peptone, yeast extract,
urea, ammonium sulfate, potassium dihydrogen phosphate, dipotassium
hydrogen phosphate, ammonium nitrate, magnesium sulfate, calcium
chloride, sodium citrate and sodium chloride were purchased from
Tianjin Tian Da Chemical Factory, China. Bran purchased from Hunan
Huinong Technology Co., Ltd. Other reagents used in this study were
analytical grade, and the water was deionized.
The strains Luteimonas huabeiensis sp. Nov (GenBank accession
number: JX658136) and Chelatococcus daeguensis (GenBank accession
number: JX658135) used in this study were isolated from the production
fluid of Baolige Oilfield. The strains Pseudomonas aeruginosa (GenBank
accession number: CP053110.1) and Bacillus subtilis (GenBank accession
number: GCA_006088795.1) were isolated from oily sludge in Xinjiang
Oilfield.
Instrument and apparatus used in this study included GC-MS (Agilent
6890N-5975, Agilent), automatic infrared oil detector (FLY6800, Bei
jing Fly Seth Technology Co., Ltd), COD analyzer (LH-T725, Lohand
Biological), Constant temperature shaking table (TY-70B, Suzhou
Jiangdong Precision Instrument Co., Ltd), Biochemical incubator (SPX-
250BIII, Tianjin Taisite), Automatic fermentor (RZY-SJB-50L, Naijing
Runzhe Biology Engineering Facility Co., Ltd.) and digital pH meter
(PHSJ-3F, Rex Electric Chemical).
Oily sludge was collected with plastic containers (1 L capacity) from
No. 1 Combining Station in No.2 Oil Production Plant of Xinjiang Oil
field, and analyzed before experiments. The oily sludge samples were
milled to pass through a 2-mm sieve and kept at 4 ◦
C for within 48 h
before starting the experiments.
2.2. Methods
2.2.1. Bacterial isolation and screening
10.0 g of each collected sample from oily sludge in Xinjiang Oilfield
was weighed into 500 mL flasks containing 100 mL broth medium
(Peptone 10.0 g/L, beef extract 5.0 g/L, NaCl 5.0 g/L, pH 7.2–7.4.
Sterilized at 121 ◦
C for 20 min). After 48 h enrichment culture at 30 ◦
C
and 120 r/min, a series of diluents (10− 2
, 10− 3
, 10− 4
, 10− 5
, 10− 6
) were
prepared by gradient dilution method, and 150 µL diluents were coated
on nutrient agar medium, respectively. The inoculated plates were
incubated at 30 ◦
C and 180 r/min under aerobic conditions for 14 days.
Bacterial colonies with rapid growth and large diameter were picked
and purified (Gao et al., 2017).
After activation, the purified strains were inoculated into 500 mL
flasks containing 200 mL inorganic salt medium (including oil sludge 20
g/L(with 221.3 g/kg TPH), K2HPO4 5.0 g/L, MgSO4 0.25 g/L, NH4NO3
2.0 g/L, NaCl 5.0 g/L, KH2PO4 1.0 g/L, MgSO4⋅7H2O 2 g/L, CaCl2⋅2H2O
1 g/L, pH 7.0–7.2, and autoclaved at 121 ◦
C for 30 min before use), and
incubated at 30 ◦
C and 180 r/min under aerobic conditions for 14 days.
Bacterial growth, COD and oil content were determined and recorded
during incubation. Two strains with better removal of TPH were
screened out and identified by 16S rRNA sequence (Wu et al., 2013) as
Pseudomonas aeruginosa and Bacillus subtilis.
The other two strains Luteimonas huabeiensis sp. Nov and Chelato
coccus daeguensis were isolated from the production fluid of Baolige
Oilfield by our group in the previous work (Ke et al., 2019, 2018b).
2.2.2. Shake cultivation
The four strains, Pseudomonas aeruginosa, Bacillus subtilis, Luteimonas
huabeiensis sp. Nov and Chelatococcus daeguensis, were inoculated into
seed medium (Glucose 6 g/L, ammonium sulfate 1 g/L, yeast extract 0.1
g/L, KH2PO4 1.5 g/L, MgSO4⋅7H2O 2 g/L, CaCl2⋅2H2O 1 g/L, sodium
citrate 0.5 g/L, and the pH 6–8), respectively. The above four types of
bacteria were then cultured at 30 ◦
C for 48 h under shake cultivation,
before obtaining seed liquid for further use. This seed medium was used
to rapidly propagate the of bacteria activation process, as it had a
relatively rich and complete nutrient composition including nitrogen
source and vitamin.
2.2.3. Preparation of solid complex bacterial agent (SCBA)
The wheat bran (with the size of 1–5 mm) was used as the culture
medium. The seed liquid of the four strains was cultured by
C.-Y. Ke et al.

3
amplification fermentation, and it was fully mixed with culture medium.
The thickness of the storage on the ground was about 30–40 cm. The
fermentation was carried out at a constant temperature of 30 ◦
C for
about 48 h. The number of viable bacteria in the solid culture was
monitored with plate counting during the fermentation process. When
the effective number of viable bacteria became greater than 108
CFU/
mL, the fermentation was stopped. The obtained fermentation products
were then dried in a fluidized bed to obtain solid bacterial agent of
different strains. Finally, the above four solid bacterial agents were
mixed in equal proportion to obtain the SCBA.
2.2.4. Preparation of biosurfactant solution
Based on the previous findings that bacteria-produce biosurfactants
played a crucial role for oil degradation (Ke et al., 2018b), biosurfactants
were prepared with selected bacteria. Luteimonas huabeiensis sp. nov was
cultured in slant (30 ◦
C, 48 h) and shake flask (30 ◦
C, 180 r/min, 48 h),
respectively, and then inoculated to 50 L automatic fermentation tank
for automatic fermentation. The medium used was glucose 2%, peptone
0.05%, yeast 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium
dihydrogen phosphate 0.5%, magnesium sulfate (7H2O) 0.02%, sodium
chloride 0.01%. Through the dynamic detection and analysis of the
fermentation process, the fast consumption and utilization of the sub
strate were supplemented timely and appropriately. The pH of fermen
tation broth was automatically adjusted to 7.0–7.5 in the fermentation
process. After fermentation at 30 ◦
C for 7 days, the biosurfactant cyclic
lipopeptide in fermentation broth was obtained (Ke et al., 2018b). The
biosurfactant metabolized by strain Luteimonas huabeiensis sp. Nov was
cyclic lipopeptide which has been well studied in our previous studies
(Ke et al., 2018b). It was isolated by centrifuged and acid precipitation
method, then it was identified by thin-layer chromatography (TLC) and
Fourier transform infrared (FTIR) spectroscopy.
2.2.5. Determination of bacterial density in oily sludge
The four isolated strains grown on broth medium were centrifuged
and suspended in physiological saline to 106
CFU/mL. 0.5 mL of each
culture was employed to inoculate in 500 mL erlenmeyer flasks con
taining 200 mL marine mineral culture (10 g oily sludge on a dry basis,
0.2 g ammonium nitrate, 0.1 g potassium dihydrogen phosphate, 0.3 g of
disodium hydrogen phosphate, 0.2 g sodium chloride and 0.05 g/L of
magnesium sulfate heptahydrate, pH adjusted to 7, and sterilized at
121 ◦
C before use). Being incubated at different temperature of 10, 20,
30, 40 or 50 ◦
C for 48 h, the growth curves were determined by the
method of a serial dilution (10− 2
, 10− 3
, 10− 4
, 10− 5
, 10− 6
, 10− 7
, 10− 8
) on
agar plates (Baron et al., 2006). Each dilution was plated in triplicate on
a nutrient agar plate and incubated at 37 ◦
C for 24 h. The number of CFU
at each dilution rate was counted after incubation and the average
CFU/mL was determined. The temperature range of 10–50 ◦
C was
selected by taking into account of the outdoor temperature range on the
ground for oil sludge treatment.
2.2.6. Processing oily sludge with SCBA and indigenous microorganisms
500 g original oily sludge (TPH 221.3 g/kg) and selected content of
distilled water were added into a 2000 mL beaker, and urea and po
tassium dihydrogen phosphate were added to adjust the C:N:P = 90:3:1
with water content of 25%. The selected ratio of C:N:P was based on the
preliminary study as it can be affected by the types of bacteria and the
properties of oily sludge. The oily sludge samples were sterilized at high
temperature (121 ◦
C for 30 min) and then 5% of the 4 types of solid
individual bacterial agent and SCBA were added, respectively. The
fermentation was performed at 30 ◦
C for 14 weeks. The oily sludge
without high temperature sterilization was treated by indigenous
microorganism, with only 5% bran and distilled water added, while
other conditions were kept identical.
2.2.7. Processing oily sludge with combined two-step treatment
In the first step, 500 g of sludge and 250 mL of 2 wt% fermentation
liquid were added into a 1000 mL beaker (the fermentation liquid pre
pared by 2.25 was diluted by 50 times), and stirred at 50 ◦
C for about 10
min. After a period of standing, the upper oil phase (crude oil) was
collected. Following that, in the second step 5 wt% SCBA was added to
the remaining oily sludge and fermentation was performed at 30 ◦
C for
selected period of time.
The oily sludge in the control experiment was not treated with
fermentation liquid or SCBA, with only 250 mL distilled water added,
and a small amount of floating oil on the surface was removed. It was
then left for 10 weeks and the relevant indicators were detected.
2.2.8. Large-scale field tests
In the large-scale field test involving the two-step process, 2 tons of 2
wt% fermentation liquid containing lipophthalein biosurfactant and 5
tons of oily sludge were added to the treatment tank for the first step oil
recovery. After fully mixed with agitator for 10 min, it was heated to
50 ◦
C and left for 1 h. When the oil and sludge were separated, the upper
floating oil was pumped to the oil-water separator for treatment where
the oil was recovered. The water phase liquid was returned to the
treatment tank for repeated use. This cycle was repeated for three times
until there was no large amount of liquid in the treatment tank.
Following that was the second step, 250 kg SCBA, 77 kg urea and 45
kg potassium dihydrogen phosphate were added to the remaining
sediment and mixed thoroughly, that was then removed from the
treatment tank for accumulation fermentation. Experiments lasted 60
days under the ambient temperature. The water content was controlled
at about 25%. Mixing and watering were repeated every week for oxy
gen exposure and water supplement. Oily sludge in the negative controls
were conduct at the same treatment process without fermentation liquid
and SCBA adding.
2.2.9. Chemical analyses
The contents of TPH and COD were measured weekly. The samples
were collected from the top, middle, and bottom of each sample first,
and then the subsamples were mixed to form one composite sample for
analysis. TPH and benzo[a]pyrene were extracted with n-pentane and
measured with gas chromatograph (GC) according to Texas Natural
Resource Conservation Commission (TNRCC, 2001). The water content,
P and N were measured based on American Public Health Association
(APHA, 2011). All tests were conducted in duplicate. COD was measured
according to the standard method (APHA, 2005). Heavy metal ions were
analyzed by atomic absorption spectrometry (AAS). TPH, benzo[a]
pyrene, heavy metal ions, P and N were calculated on a dry matter basis.
The TPH removal was calculated as a percentage change of the oil
content after the treatment. TPH removal = (oil content before treat
ment - oil content after treatment)/(oil content before treatment) ×
100%.
2.2.10. High-throughput sequencing
Generation sequencing library preparations and Illumina MiSeq
sequencing were conducted at Sangon Biotech Co., Ltd (Shanghai,
China). DNA was extracted in duplicate from the cell pellets using E.Z.N.
A™ Mag-Bind Soil DNA Kit (Omega Bio-Tek, Guangzhou, China). The
duplicate of each sample were pooled together for one high-throughput
sequencing. The qubit 3.0 DNA detection kit was used to quantify the
genomic DNA. A panel of proprietary primers was designed to anneal to
the relatively conserved regions bordering the V3, V4 hypervariable
regions. The V4 and V5 regions were amplified using forward primers
containing the sequence CCTACGGGNGGCWGCAG and reverse primers
containing the sequence GACTACHVGGGTATCTAATCC.
3. Results and discussion
3.1. Characterization of oily sludge
The chemical and microbiological properties of the oily sludge used
C.-Y. Ke et al.

4
were characterized before the bioremediation experiments. The results
are presented in Table S1 (Supplementary Information). It was found
that the oily sludge contained high concentrations of TPH, COD, and
some heavy metal ions. TPH content was 221.3 g/kg, including 43.03%
Saturated hydrocarbon, 21.35% aromatic hydrocarbon, and 30.42%
NSO and asphaltens. According to the local standards of Xinjiang Uygur
Autonomous Region for pollution control requirements for compre
hensive utilization of oil and gas field oily sludge, some important in
dicators were found to be over the standard limit, including the COD,
TPH, benzo[a]pyrene and some heavy metal ions. Therefore, these oily
sludges must be treated effectively before comprehensive utilization.
Both pH value (8.25) and water content (17.8%) were within the
suitable range needed for growing microorganisms (Koolivand et al.,
2017). However, the number of indigenous microorganisms in the oily
sludge was only 5 × 104
CFU/g due to the high concentration of con
taminants and the matrix effect in the sample (Gojgic-Cvijovic et al.,
2012). In addition, although the water content of the sample reached
17.8%, it was in the form of water in oil emulsion, which was difficult to
be utilized by microorganisms. Therefore, changing the properties of
oily sludge or controlling its free water content became necessary to
ensure the effective growth and reproduction of microorganisms.
The content of total nitrogen and total phosphate in the oily sludge
sample was very low relative to carbon content. It is known that the lack
of nitrogen (N) and phosphate (P) in nutrients can limit the TPH
biodegradation (Wu et al., 2017). The C/N value between 25 and 30 is
suitable for enhancing the biodegradation. By taking into account the
C/N/P ratio, the addition of N and P nutrients was also needed for
stimulating growth of exogenous and/or indigenous microorganisms
(Devlin, 2007; Ma et al., 2016).
3.2. Growth of different strains in different temperature and oily sludge
leaching solution
Some components in oily sludge are harmful to bacterial growth,
such as heavy metals, excessive salinity, polymers, chemical treatment
agents and other inorganic substances (Ozyurek and Bilkay, 2020). They
generally have toxic and inhibitory effects on cells, thus likely limiting
the growth and reproduction rate of cells themselves, and potentially
reducing the activity of microbial degradation of oil.
To evaluate the growth and reproduction of the four strains, the
bacterial activity was investigated by mixing the SCBA with the leaching
solution from oily sludge. The bacterial density was measured at
different temperature ranging from 10 ◦
C to 50 ◦
C under aerobic con
ditions. The results are illustrated in Fig. 1.
The results showed that all four strains were able to grow and
propagate well in the oily sludge leaching solution. This was indicative
that these microorganisms had good tolerance to oily sludge. In addi
tion, the growth activity as a function of temperature showed an optimal
temperature around 30–40 ◦
C, which is largely consistent with the
suitable temperature of the general bioremediation technology of oily
sludge (Aguelmous et al., 2019).
In principle, the bioremediation process involves the introduction of
selected microbial culture into oily sludges to facilitate biodegradation
through fermentation mostly at constant temperature. One major limi
tation of this process for industrial application is associated with the
requirement for storage and transportation of the large amount of bac
terial culture in liquid for fermentation especially at industrial scale. In
this regard, the solid based culture, i.e. SCBA was investigated in this
work. Moreover, the use of bran as solid media in the SCBA provided
unique advantages such as (a) being environmentally friendly by using
biomass application, (b) having economic benefit from using abundant
low-cost raw materials, and (c) its porous physical structure being
conducive to the aerobic fermentation of microorganisms.
3.3. Biodegradation with different bacteria
In the bioremediation process of oily sludge through biodegradation,
the type and activity of microbial strains are the key factors affecting the
overall performance for oily sludge treatment. At present, most of the
research has been focused on the indigenous microorganisms (Aguel
mous et al., 2019) by adding activator, bulking agents and adjusting the
carbon nitrogen ratio. At the same, the exogenous microbial strains
isolated from other sources have also been used for oily sludge treat
ment, but the treatment mechanism and performance can be different.
By using SCBAs developed as exogenous microbial strains, their
biodegradation performance for oily sludges was characterized with the
four microbial strains at different temperatures, by measuring TPH
removal and COD reduction in the oily sludge samples. The results are
depicted in Figs. 2 and 3.
As seen in Fig. 2, among the four individual strains Pseudomonas
aeruginosa showed the highest removal efficiency for TPH in the range of
18.0–55.4%. The highest TPH removal efficiency of this strain was also
observed to apply to all temperature levels investigated. An optimal
temperature of 35 ◦
C was observed for all four strains. Furthermore, the
performance of the mixture of these four strains was clearly improved,
reaching the maximal removal efficiency of 64.1% also at the optimal
temperature of 35 ◦
C, which was 8.7% higher than that of the best in
dividual stain. This improved was largely attributed to the mixed pop
ulations in the complex bacterial consortium equipped with broad
enzymatic capacities enabling increased rate and extent of hydrocarbons
biodegradation, as also observed previously by other researchers (Cer
queira et al., 2011; Patowary et al., 2016; Shen et al., 2015).
Compared to the performance of exogenous microbial strains as
discussed above, the degradation activity of indigenous microorganisms
for oily sludge was notably lower although it was also composed of a
large number of different bacterial groups, while the bacterial concen
tration detected reached a comparable level of 109
CFU/g (Fig. 2). This
was likely due to the fact that there were only a limited number of
functional bacteria within the indigenous microorganisms which were
able to degrade petroleum hydrocarbons. That was in contrast to the
four exogenous strains used, which have been well studied through
screening and evaluation. Specifically, all four strains of Luteimonas
huabeiensis sp. nov, Pseudomonas aeruginosa, Bacillus subtilis and Chela
tococcus daeguensis were previously demonstrated to use petroleum hy
drocarbon as their sole carbon source. At the same time, they produced
bioemulsifiers or biosurfactant, enabling demulsification of oily sludge
and/or emulsifying petroleum hydrocarbon, further promoting the
degradation of TPH (Amani et al., 2013; Ke et al., 2018a, 2019, 2018b;
Pathak and Keharia, 2014).
Fig. S1 (Supplementary Information) shows the reduction of COD of
the oily sludge samples by the four strains, their mixture and also the
indigenous microorganism at the temperature ranging from 15 ◦
C to
40 ◦
C. Overall, the trends of the COD reduction profiles followed that of
the TPH removal efficiency. Among the four individual strains, Pseu
domonas aeruginosa exhibited the highest activity for COD reduction
5
6
7
8
9
10
10 20 30 40 50
Bacterial
density
(log
10
CFU/mL)
Temperature(oC)
Luteimonas huabeiensis
Chelatococcus daeguensis
Pseudomonas aeruginosa
Bacillus subtilis
Fig. 1. Bacterial density of four strains in oily sludge at different temperature.
C.-Y. Ke et al.

5
(reaching 89.5%) at the optimal temperature of 35 ◦
C. Also, the mixed
culture (where the four types of bacteria were mixed in the ratio of
1:1:1:1) showed improved performance compared to the single strain, in
line with the previous observation (Cerqueira et al., 2011; Patowary
et al., 2016; Shen et al., 2015).
Temperature plays a vital role in the biodegradation as in other
biological processes, mostly with an optimal temperature range. It can
be seen in Figs. 2 and 3 that the degradation performance, in terms of
both TPH removal efficiency and COD reduction, of these different mi
croorganisms increased significantly with rising temperature before
reaching the maxima around 35 ◦
C. Along with the increase in metabolic
activity of microorganisms by rising temperature affects, it also caused
positive changes in the physical properties of the petroleum hydrocar
bons such as solubility, viscosity and interfacial tension, thus further
enhancing the degradation (Chandra et al., 2013; Gibb et al., 2001). On
the other hand, further increase in temperature over the maximum can
reduce the activity of microorganisms even potentially denature their
enzymes. Based on the experimental results, the appropriate operational
temperature range was selected to be 30–40 ◦
C.
3.4. Effects of initial TPH concentration on biodegradation
The bioactivity is generally dependent of the feed concentration.
Fig. 3 compares the profiles of THP removal efficiency at four levels of
initial TPH concentrations ranging from 2.5 wt% to 20.5 wt%. It shows
that a higher THP removal efficiency was obtained at a lower initial
concentration. In general, with a certain amount of microorganisms a
higher conversion percentage is expected for the lower feed concentra
tion. However, previous studies indicated that the biodegradation was
independent of the initial TPH concentration or quantity (Ramadass
et al., 2015), and the mechanism remained unclear.
The variation of TPH removal efficiency as a function of time showed
a similar trend for the four initial THP concentrations. It started initially
following a linear relationship with different slopes, before leveling off.
The higher initial THP concentration gave a relatively lower slope, thus
a lower level of final removal efficiency. When the initial concentration
of TPH was 20.5%, the final removal efficiency was only 30.2% in Week
12, whilst the initial concentration of 2.5% allowed a final removal ef
ficiency of 96.1%.
The period following the linear relationship was also dependent of
the initial THP concentration. It needed shorter time to reach the
levelling off stage at lower initial THP concentration. When the initial
concentration was 2.5%, it took 6 weeks to reach the plateau at a rate of
2.15% per day, whilst for the initial THP concentration of 20.5% it
levelling off stage was about to start at Week 11 at a much lower rate of
0.27% per day. This was an important observation for process design in
particular to decide the operation time associated with the TPH con
centration in the TPH. In general, the oily sludge with high TPH content
needed a longer time for degradation to a desired extend, and based on
previous studies (Huesemann, 2004) a suitable TPH concentration range
between 0.2% and 5.5% was recommended. In other studies, TPH
concentrations exceeding 16% were used while achieving TPH removal
efficiency above 90%. However, these processes generally took more
than 6 months even up to one year to complete (Kriipsalu et al., 2007;
Kriipsalu and Nammari, 2010; Sood et al., 2010). A recent study also
processed oily sludge with a TPH initial concentration of 13.1%
achieving a 95.2% TPH removal over 12 weeks, however, through a
two-stage composting process (Koolivand et al., 2017).
3.5. Combined two-step process for bioremediation of oily sludge with
high TPH content
The two-step process was conducted by firstly treating the oily sludge
containing high TPH content (initial concentration 22.1%) with the
microbial fermentation liquid (after sterilization) having functional
biosurfactant (cyclophthalide) to recover the majority of the petroleum
hydrocarbons, and secondly employing SCBA for biodegradation of the
remaining TPH. The results are presented in Table 1.
In the first step (less than 5 h, 85.2% of the TPH was removed, and a
further 11.4% was removed during the second step within 6 weeks,
reaching a 96.6% total TPH removal. However, for the same oily sludge
0
10
20
30
40
50
60
70
80
15 20 25 30 35 40
TPH
removal
(%)
Temperature (oC)
Luteimonas huabeiensis
Chelatococcus daeguensis
Pseudomonas aeruginosa
Bacillus subtilis
mixed culture
indigenous microorganism
Fig. 2. Removal efficiency of TPHs by different bacteria at temperature 15–40 ◦
C.
0
20
40
60
80
100
0 2 4 6 8 10 12 14
THP
removal
(%)
Time(week)
8.1%
15.2%
20.5%
2.5%
Fig. 3. Profiles of TPH removal efficiency with time at different initial THP
concentrations by SCBA.
C.-Y. Ke et al.

6
samples, when only one-step bioremediation (i.e. the second step)
method was adopted by treating the sample directly with SCBA for
biodegradation (while skipping the first step involving biosurfactants),
the TPH removal rate was found to be less than 20% over the same time
period (data not shown). This was clearly indicative that the process
would take longer time to reach the same removal rate compared to the
combined two-step process.
In contrast, the COD reduction in the first step was only 18.6% that
was noticeably increased to 74.0% in the second step. This was mainly
attributed to the addition of fermentation broth which had a high level
of COD with organics brought in. The majority of other components
were removed in the first step (82.8%–90.1%), with high total removal
efficiencies (94.5%–98.9%), including saturated and aromatic hydro
carbons and NSO/asphaltens which are generally more difficult to
degrade. Overall, the results were significantly higher removal effi
ciencies than that using the traditional one step direct composting
method (Aguelmous et al., 2019).
Although the removal efficiency was relatively high in the first step it
was believed necessary and important as an induction phase to enable
the second step for much higher efficiency. In our previous studies (Ke
et al., 2018b), it was demonstrated that strain Luteimonas huabeiensis sp.
Nov was able to effectively produce cyclophthalide as functional bio
surfactant (with a production rate of > 1 g/L) during fermentation. The
effects of this biosurfactant on water/oil interfacial property and
emulsification of crude oil were all beneficial for microbial fermentation
thus biodegradation of organics in the oily sludge (Calvo et al., 2009).
Furthermore, such biosurfactants were produced by microorganisms
and able to be further degraded by microorganisms, providing an
environmentally friendly and cost-effective approach for waste treat
ment in the oil industry.
3.6. Effects of water content on oily sludge biodegradation by SCBA
Water exists unavoidably in most of the oily sludge with the content
of 10%–40%, and its content has important effect on the performance of
biodegradation process. In the process of bioremediation of oily sludge,
the water content has a great influence on the removal of TPH and COD.
As shown in Fig. 4, for oily sludge with initial TPH concentration of 5%,
when the water content increases from 10% to 40%, a similar trend of
increasing first and then decreasing were seen in the removal of both
TPH and COD, and the treatment effect is the best when the water
content is 20%–30%. The growth, reproduction and metabolism of mi
croorganisms are inseparable from the presence of water, when the
water content is lower than 10%, the activity of microorganisms are
significantly reduced.
However, when the water content is more than 30%, too much water
will occupy the space volume and prevent the oxygen from entering,
which will lead to anoxic fermentation of microorganisms (Viñas et al.,
2005). Although the previous research shows that the four kinds of
bacteria used in this experiment are facultative anaerobes, but their
treatment effect under anaerobic condition is obviously inferior to that
under aerobic condition (Ke et al., 2018a, 2018b). In addition, consid
ering the practical application, too much water will make the SCBA and
oily sludge mixed to form mud, which is not conducive to later plowing.
At the same time, too much free water will penetrate into the ground,
thus causing pollution to the underground soil.
3.7. Effects of solid agents dosage and composition on biodegradation of
oily sludge
It is generally understood that aerobic biodegradation is the most
effective way for TPH degradation, where the presence of oxygen is key
to facilitating the process. The introduction of co-substrates such as soil,
sand or wood chips has been demonstrated to improve aeration thus
enhance the oily sludge biodegradation (Eftoda and McCartney, 2004;
Malińska and Zabochnicka-Świątek, 2013).
Fig. 5 illustrates the effect on biodegradation by adding wood chips
to different amount of bran-based SCBA, in terms of TPH removal effi
ciency and COD reduction over 6 weeks. In this system, the bran used in
the SCBA provided both the bulking substrate, and also the carbon
Table 1
The component removal in oily sludge by two-step combined process.
Component First step
removal (%)
Second step
removal (%)
Total removal
(%)
TPH 85.2 11.4 96.6
COD 18.6 74.0 92.6
Saturated
hydrocarbons
90.1 8.8 98.9
Aromatic
hydrocarbons
84.3 12.5 96.8
NSO + asphaltens 82.8 11.7 94.5
0
20
40
60
80
100
0 2 4 6 8 10 12 14
TPH
removal
(%)
Time(week)
10%
20%
30%
40%
0
20
40
60
80
100
0 2 4 6 8 10 12 14
COD
removal
(%)
Time (week)
10%
20%
30%
40%
Fig. 4. Effects of water content on the removal of TPH and COD.
0
20
40
60
80
100
A B C D E
Removal
rate
(%)
TPH
COD
Fig. 5. Effects of solid agents dosage and composition on biodegradation
(Conditions: Temperature, 30 ◦
C; processing time, 6 weeks; water content,
25%. Bioagent addition (wt%), A: 1.0% SCBA, B: 2.5% SCBA, C: 5.0% SCBA, D:
5.0% SCBA + 5.0% wood chips, E: 5.0% SCBA + 10.0% wood chips).
C.-Y. Ke et al.

7
source of microorganism metallization, while the added wood chips
were supposed to act as aeration promoter to accelerate the biodegra
dation as the second step in the two-step process.
By increasing SCBA dosage form 1.0–5.0%, the TPH removal effi
ciency increased correspondingly, from 70.5% to 95.2%. However, the
addition of wood chips gave just a slight further increase by 1.3%. This
might be associated with the role that wood chips played. On one hand,
the co-substrate wood chips may improve aeration. On the other hand,
the increased amount of carbon source including both bran and wood
chips themselves required microorganism for fermentation, thus
contributed insignificantly to the degradation of oily sludge. This was
also reflected on the trend of change in COD reduction which decreased
by adding wood chips.
In general, the addition of solid agents has been designed according
to the TPH content in the oily sludge, the higher of the TPH content, the
larger amount of solid agents added. In the previous studies, the applied
ratios of oily sludge/solid agents ranged widely from 4/1–1/10
(Aguelmous et al., 2019; Asgari et al., 2017; Koolivand et al., 2017;
Ouyang et al., 2005). It is true that increasing the amount of solid agents
can reduce the TPH content in the composting mixture. However,
exceeded amount of solid agents can also cause a significant increase in
the volume of solid waste. In this study, the use of only 5% solid bacteria
agents showed a higher TPH removal (95.2%) from the oily sludge,
which was a significantly lower dosage compared to the previously re
ported (Aguelmous et al., 2019; Asgari et al., 2017; Koolivand et al.,
2017; Ouyang et al., 2005), thus providing great potential for large-scale
industrial application.
3.8. Large-scale field tests
Based on the characterization and examination for the performance
of individual, mixed and SCBAs prepared in this study, a large-scale field
test was conducted in Xinjiang Oilfield for the combined two-step
bioremediation of 5 tons oily sludge over 60 days. Table 2 summarizes
the experimental results to compare the measured content of the main
components before and after bioremediation.
After 60 days of fermentation, the bacterial concentration in the oily
sludge increased from 5 × 104
to 8 × 109
CFU/g, indicating the signif
icant bacterial growth and reproduction. High-throughput sequencing
results indicated the presence of highly diverse microorganisms in the
oil sludge before and after SCBA treatment. As shown in Fig. S2 (avail
able as supplementary information), five main strains (Pseudomonas,
Thauera, Arcobacter, Fusibacter, Proteobacteria, Bacteroides) were detec
ted and accounted for 82.25% of all sequences on average in the oily
sludge before SCBA treatment, and a number of other genera only
appeared in low abundance. While 4 main strains (Luteimonas hua
beiensis sp. Nov, Chelatococcus daeguensis, Bacillus subtilis and Pseudo
monas aeruginosa) were detected and accounted for 87.99% of all
sequences on average in the oily sludge after SCBA treatment. Obvi
ously, after SBCA treatment, the amount of strains (Thauera, Arcobacter,
fusibacter, Proteobacteria, Bacteroides) in the original oily sludge
decreased significantly, while the amount of added strains (Luteimonas
huabeiensis sp. Nov, Chelatococcus daeguensis, Bacillus subtilis and Pseu
domonas aeruginosa) increased significantly, which indicated that the
four kinds of stains became the dominant flora after 60 days of
treatment.
At the same time, the levels of all main components of interest
showed noticeable decrease including TPHs, other organics and metals,
together with COD. The content of TPH and COD decreased by 93.8%
and 91.5%, respectively, indicating a significant biodegradation of oily
sludge. The content of heavy metals also decreased, with reduction be
tween 33.3% and 93.3%, showing the strong capacity of the microor
ganisms for heavy metal further removal. This is a very complex process
though there have been different mechanisms proposed for heavy metal
removal by microorganisms, such as biotransformation, bio
accumulation, biomineralization, bioadsorption, and bioleaching (Dixit
et al., 2015).
Fig. S3 (Supplementary Information) depicts the visible appearance
of the oily sludge before and after bioremediation, the SCBA used, and
the mixture of SCBA with oily sludge. As can be seen, the oily sludge
appeared to be dark and caking before treatment, which became cleaner
and more homogeneous. It was also interesting to observe the change in
the arability of the oily sludge for cultivation. Before bioremediation the
plant was unable to survive in the highly contaminated soil, and the
remediated soil supported its healthy growth. It is clearly evident of
capability of the bioremediation technology developed involving
application of SCBA and a combined two-step process for treating crude
oil contaminated soil, towards tackling the significant challenge in the
oil industry. Since the main objective of the large-scale field test as
presented was to demonstrate the feasibility of the process developed,
further studies are being carried out on the process implementation and
operation towards industrial applications.
4. Conclusions
By using solid complex bacterial agents (SCBA) through a combined
two-step biodegradation process, four screened strains showed high ef
ficiency for the degradation of total petroleum hydrocarbons (TPH) and
the reduction of COD of the oily sludge, at 96.6% and 92.6% respec
tively. The mixed strains together with bran prepared in form of SCBA
exhibited improved performance compared to individual strains. The
use of SCBA provided advantages over commonly used liquid media for
storage and transportation. The two-step process demonstrated the
capability for treating oily sludge with high TPH content and short
process period (60 days). The large-scale field test confirmed the feasi
bility and superiority of the technology for industrial applications.
To further improve the design and operation of the oily sludge
treatment process, an in-depth understanding of the process kinetics is
needed, e.g. to quantitatively characterize the key operating factors on
the degradation kinetics of oily sludge, including temperature, moisture
content, dosage of bacteria agent and oxygen exposure. In addition,
instead of investigating the individual factors as presented in this work,
more comprehensive statistical analysis, e.g. principal coordinates
analysis, is also valuable for evaluating the influence of these factors.
These are currently under active investigation in our laboratories.
CRediT authorship contribution statement
Cong-Yu Ke: Supervision, Conceptualization, Writing - original
draft, Writing - review & editing, Funding acquisition. Fang-Ling Qin:
Formal analysis, Funding acquisition. Zhi-Gang Yang: Investigation,
Validation, Data curation. Jun Sha: Data curation. Wu-Juan Sun:
Funding acquisition, Project administration. Jun-Feng Hui: Software,
Validation. Qun-Zheng Zhang: Writing - review & editing. Xun-Li
Zhang: Supervision, Resources, Writing - review & editing.
Table 2
Summary of field test results.
Parameter (unit) Negative control Experiment
TPH (g/kg) 235.2 14.7
COD (mg/L) 857.2 72.5
Copper (mg/kg) 27.41 11.14
Zinc (mg/kg) 39.85 10.12
Cadmium (mg/kg) 7.24 2.45
Lead (mg/kg) 61.33 4.11
Chromium (mg/kg) 21.28 5.50
Arsenic (mg/kg) 6.87 0.64
Mercury (mg/kg) 0.03 0.02
Benzo[a]pyrene (mg/kg) 1.29 0.59
Bacteria (CFU/g) 5 × 104
8 × 109
C.-Y. Ke et al.

8
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (21676215), Scientific Research Program Funded by Shaanxi
Provincial Education Department (20JY057), Open Project of Shaanxi
Key Laboratory of Lacustrine Shale Gas Accumulation and Exploitation
(YJSYZX19SKF0004) and Postgraduate Innovation and Practical Ability
Training Plan of Xi’an Shiyou University (YCS19211017). We
acknowledge the support of the Analysis and Test Center of Xi’an Shiyou
University and Collaborative Innovation Center for Unconventional Oil
and Gas Exploration and Development (17JF033).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2020.111673.
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