Chemosphere 302 (2022) 134870

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Chemosphere
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Enhanced remediation of fracturing flowback fluids by the combined

application of a bioflocculant/biosurfactant-producing Bacillus sp. SS15 and
its metabolites
Feng Zeng a, b, Hanghai Zhou b, Xiaoyun Lin b, Yanhong Li a, 1, Yanpeng Liang a, Qinglin Xie a,
Edidiong Okokon Atakpa b, Chaofeng Shen c, Chunfang Zhang b, *, 1
a
College of Environmental Science and Engineering, Guilin University of Technology, Guilin, 541006, China
b
Ocean College, Zhejiang University, Zhoushan, 316021, Zhejiang, China
c
College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Bacillus sp. SS15 is capable of producing

both bioflocculant and biosurfactant.
• Both bioflocculant and biosurfactant
showed excellent activity and stability.
• Bioflocculant and biosurfactant were
employed in treating fracturing flow­
back fluids.
• 86% of chroma, 94% of SS, 85% of COD,
and more than 50% of hydrocarbons
were removed.
• SS15 genome contains abundant genes
related to BF/BS synthesis.

A R T I C L E I N F O A B S T R A C T

Handling Editor: Chang-Ping Yu Fracturing flowback fluids (FFFs), which is generated from the process of oil and gas exploitation, is one of the
major environmental concerns. In this study, a bacterial strain, Bacillus sp. SS15, capable of producing both
Keywords: bioflocculant (BF) and biosurfactant (BS), was isolated from oil-contaminated mudflat sediment. The BS pro­
Bioflocculant duced by SS15 was identified as lipopeptide, which could reduce the surface tension of water from 74.2 mN/m to
Biosurfactant
36.6 mN/m with a critical micelle concentration of 44.4 mg/L. It also exhibited strong tolerance against a wide
Fracturing flowback fluids
range of pH (2–12), temperature (4–60 ◦ C), and salinity (0–100 g/L). Meanwhile, the BF produced by SS15
Flocculation
Biodegradation exhibited high flocculating activity (84.9%) for kaolin suspension, and was confirmed to be thermostable, salt-
tolerant, and alkaliphilic. The combined treatment of bioremediation (introducing SS15 and BS) followed by
flocculation (introducing BF) greatly promoted the removal of chroma (85.7% reduction), suspended solids
(94.4% reduction), chemical oxygen demand (84.9% reduction), n-alkanes (50.0% reduction), and polycyclic
aromatic hydrocarbons (66.5% reduction), respectively. The genome analysis showed that strain SS15 possessed
abundant genes related to the synthesis of carbohydrate, protein, and lipid, which might play an important role
in BF and BS synthesis. The findings in this study demonstrated that Bacillus sp. SS15 has promising prospect in
the remediation of FFFs.

* Corresponding author.
E-mail addresses: lyh1685@163.com (Y. Li), zhangcf@zju.edu.cn (C. Zhang).
1
These authors contributed equally to this article and are joint corresponding authors.

https://doi.org/10.1016/j.chemosphere.2022.134870
Received 21 October 2021; Received in revised form 23 March 2022; Accepted 4 May 2022
Available online 7 May 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
F. Zeng et al.
1. Introduction
A large amount of fracturing flowback wastewater would be pro­
duced when hydraulic fracturing is used as a recovery technology in
unconventional oil and gas fields. Fracturing flowback fluids (FFFs) is a
complex mixture, which contains various substances such as naturally
occurring radioactive materials, inorganic substances, metals, hydro­
carbons and chemical additives (Ferrer and Thurman, 2015; Fajfer et al.,
2022). Thus, it generally characterized by high chemical oxygen de­
mand (COD), total suspended solids (TSS), total dissolved solids (TDS),
stability and toxicity. Untreated FFFs could cause harmful impacts on
ecosystems, such as contaminating drinking water, and endangering
human health (Rish and Pfau, 2018).
Up to date, various treatment methods have been proposed for
remediating FFFs, such as active carbon adsorption, coagulation and
flocculation, chemical oxidation, biological treatment, evaporation with
mechanical vapor compression, and membrane filtration (Estrada and
Bhamidimarri, 2016; Abramowska et al., 2017; Freedman et al., 2017;
Conrad et al., 2020; Ma et al., 2020). Among these methods, flocculation
and biological treatment are cost-effective and energy efficient for the
removal of SS and petroleum hydrocarbons. Nevertheless, the treatment
process selected for remediating FFFs is dependent on their composi­
tions as well as the disposal requirement. For instance, a lot of chemicals
such as guar gum and hydrolyzed polyacrylamide are added to frac­
turing fluid in order to prevent precipitation of SS and clogging of small
fissures formed during fracturing the rock, which would make the
flocculation process very ineffective (Hasan and Abdel-Raouf, 2018; Liu
et al., 2020a). Meanwhile, high salinity, nutrient deficiencies, and bio­
cides would inhibit microbial growth (Stringfellow et al., 2014).
Flocculation, as an efficient strategy for SS removal, is widely used in
wastewater treatment (Abu Tawila et al., 2019; Abbas et al., 2020).
Inorganic and organic polymer flocculants are the most widely used
chemical flocculants in water treatment. However, the massive appli­
cation of chemical flocculants could cause severe secondary pollution
due to their toxicity and low biodegradability (Bolto and Gregory, 2007;
Lofrano et al., 2013). Bioflocculant (BF) is a group of polymer metabo­
lites produced by microorganisms with the advantages of high floccu­
lating efficiency, good biodegradability, and environmental friendliness
(Macczak et al., 2020). Previous studies showed that BF could effectively
remove chroma (Ma et al., 2020), dyes (Pu et al., 2020), heavy metals
(Ayangbenro et al., 2019), soot (Liu et al., 2016), and straw ash (Liu
et al., 2020b) from wastewater, indicating that BF has a wide application
prospect in the water treatment field.
Biosurfactant (BS) is a group of metabolites produced by microor­
ganisms, containing both hydrophobic and hydrophilic domains (Shuai
et al., 2018; Huang et al., 2020b; Zhou et al., 2020, 2021). BS can reduce
the surface tension (ST) and enhance the solubility and bioavailability of
hydrophobic hydrocarbon pollutants, thus improving the removal effi­
ciency of pollutants (Huang et al., 2020a). Compared to synthetic sur­
factants, BS exhibits excellent environmental compatibility, making it
more suitable for wastewater treatment applications (Sun et al., 2019).
Previous study showed that the introduction of BS or BS-producing
microorganisms could enhance the FFFs bioremediation. For example,
Huang et al. (2020b) reported that the inoculation of Serratia marcescens
ZCF25 effectively promoted the removal of petroleum hydrocarbons.
Also, Huang et al. (2020a) reported the feasibility of applying BS with
sub-CMC dose to remediate FFFs.
Among the remediation technology of FFFs, combined treatment of
flocculation and biodegradation is considered as a promising approach
(Huang et al., 2020b). However, the excessive addition of chemicals in
the flocculation process could cause negative impacts on the environ­
ment. Therefore, the application of BF is a promising alternative for
reducing the use of chemicals. Meanwhile, the presence of hydrophobic
organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and
chemical additives would inhibit the removal performance. Herein, the
introduction of BS and BS-producing bacteria for enhancing the removal
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Chemosphere 302 (2022) 134870
of organic pollutants is considered. Nevertheless, there is limited
knowledge regarding the treatment of FFFs using a combined strategy
consisting of flocculation and biodegradation, not to mention the
application of BF and BS from the same bacterium.
The application of BF and BS was limited by their low yield.
Recently, the mechanisms of metabolites synthesis could be analyzed
based on the genomic information, which provides guidance to increase
the yield of metabolites. For instance, a hypothetical biosynthesis
pathway of polysaccharide-based BF produced by Agrobacterium tume­
faciens F2 was proposed based on the genomic and transcriptomic
analysis (Pi et al., 2020). Meanwhile, genomic and transcriptomic ana­
lyses were employed to investigate the effect on surfactin synthesis by
adding L-Leu and D-Leu into the culture medium (Zhou et al., 2019).
Considering that both BF and BS can be synthesized in SS15, the analysis
of the corresponding genes involved in the synthesis of the two metab­
olites could provide novel approaches in regulating the yield.
Therefore, the objectives of this study were to: 1) isolate the bacteria
capable of producing both BS and BF; 2) characterize the physico­
chemical features of the BS and BF produced by a single bacterium; 3)
investigate the treatment performance of chroma, SS, COD, and petro­
leum hydrocarbons in FFFs through the combined biological treatment;
4) investigate the potential genes involved in the production of BF and
BS. It is worth noting that this study is the first attempt to employ BF and
BS from a single strain to treat FFFs through a combined treatment.
2. Material and methods
2.1. Isolation and identification of bacteria capable of producing BF and
BS
The bacteria strains capable of producing BF and BS were isolated
from oil-contaminated mudflat sediment collected from Zhoushan,
Zhejiang, China (122◦ 11′ 24′′ N, 29◦ 59′ 26′′ E). The medium for the
screening test of BF-producing bacteria is composed of (g/L): glucose 10,
yeast extract 3.5, urea 0.5, K2HPO4 5, KH2PO4 2, NaCl 0.1, and MgSO4
0.5. Firstly, the BF-producing bacteria were isolated as previously re­
ported (Ayangbenro et al., 2019). The flocculating activity of the isolates
was measured using kaolin suspension. Briefly the individual isolate was
incubated in a rotary shaker (30 ◦ C, 180 rpm) for 3 days, then 1 mL of
culture broth and 1 mL of CaCl2 (1%, w/v) were mixed with kaolin
suspension (5 g/L, 23 mL). The mixture was subsequently vortexed for 3
min and settled for 10 min, and measured as previously reported
(Ayangbenro et al., 2019). The bacterial strains exhibiting excellent
performance in flocculating kaolin suspension were selected for the
subsequent examination of the BS-producing ability. To evaluate the
surface activity, the selected strains were inoculated into the medium
with olive oil 1% (w/v) as carbon source, and incubated at 30 ◦ C and
180 rpm for 3 days. Morphological analysis and Gram stain reaction as
well as molecular identification were employed to identify the strain.
The bacteria were incubated at 30 ◦ C for 12 h for morphological
observation. Gram stain reaction was conducted using a Gram strain Kit
(Hangzhou Tianhe Micro-organism Reagent) according to the manu­
facturer’s instructions (Ye et al., 2020). The molecular identification was
conducted using the protocol reported by Huang et al. (2020a). The
sequence alignment was conducted on NCBI using Basic Local Align­
ment Search Tool (BLAST). The phylogenetic tree was constructed with
the MEGA 5.0 software using neighbor-joining method. Bootstrap
analysis (1000 replicates) was employed to evaluate the topology of the
tree (Ye et al., 2020).
2.2. Extraction and characterization of BS and BF produced by Bacillus
sp. SS15
2.2.1. Extraction of BS and BF
The BS extraction was performed according to the methods as pre­
viously reported with slight modifications (Zhou et al., 2020). After
F. Zeng et al.
cultivation at 30 ◦ C and 180 rpm for 5 days, the fermentation broth was
centrifuged at 9000 rpm for 5 min to collect the supernatant, which was
then extracted with ethyl acetate. Ultimately, the extracts were
concentrated with a rotary evaporator.
The BF extraction was conducted based on the method as previously
reported with modifications (Pu et al., 2020). In brief, the strain SS15
was cultivated at 30 ◦ C and 180 rpm for 24 h, followed by centrifugation
at 9000 rpm and 4 ◦ C for 5 min. Subsequently, the supernatant was
mixed with pre-cooled (4 ◦ C) anhydrous ethanol (1:2, v/v) and stood
overnight at 4 ◦ C. After precipitation, the mixture was centrifuged at
9000 rpm and 4 ◦ C for 30 min to collect the precipitate. The precipitate
was washed with distilled water three times and freeze-dried.
2.2.2. Surface activity of the BS
The determination of critical micelle concentration (CMC) was
conducted by preparing BS solutions with a concentration gradient
ranging from 0 to 500 mg/L. The ST of each solution was measured until
a constant value was reached. Meanwhile, oil-spreading test, drop
collapse assay, and emulsification index (E24) determination were car­
ried out based on previous description (Huang et al., 2020b).
2.2.3. Stability evaluation of the BS and BF
BS solutions were prepared at the CMC while BF solutions were
prepared at 3.0 g/L, and they were evaluated under different pH (i.e., 2,
4, 6, 8, 10, and 12) adjusted by HCl and NaOH, temperature (i.e., 4, 20,
40, 60, 80, and 100 ◦ C), and salinities (i.e., 0, 20, 40, 60, 80, and 100 g/
L). In each case, the ST and flocculating activity were measured,
respectively (Xia et al., 2019).
2.2.4. Chemical characteristics of the BS and BF
Functional groups of the BS and BF were analyzed by Fourier
transform infrared (FTIR) spectroscopy. Thin-layer chromatography
(TLC) was used to analyze the chemical properties of BS per the method
of Shuai et al. (2018). For the constitution analysis of BF, the Bradford
and the phenol-sulfuric acid method were employed (Ma et al., 2019).
The NMR spectral analysis for the structural information of the BF was
performed as previously reported (Sivasankar et al., 2020). About 30 mg
of the BF was dissolved in 0.5 mL of D2O and the spectrum was detected
at 70 ◦ C using a JNM-ECZ600R/S1 spectrometer (JEOL, Japan) at 600
MHz. The predictions of polysaccharides based on the chemical shifts δ
were conducted by CASPER program (Ronnols et al., 2013). Further­
more, gas chromatography-mass spectrometry (GC-MS) was used to
detect the fatty acid of BS (Zhou et al., 2020). To explore the flocculation
mechanism of the BF, zeta potential analysis and scanning electron
microscopy (SEM) observation were employed (Aljuboori et al., 2015).
2.3. Remediation of FFFs by Bacillus sp. SS15 and its metabolites
The strain SS15 and its metabolites (BS and BF) were applied to treat
the FFFs through biodegradation and flocculation. According to the
preliminary experiments (Fig. S5), the best flocculation performance
was achieved by adding 0.6 g/L of BF and 4 g/L of AlCl3, adjusting the
pH of FFFs to 5, and settling for 30 min. The optimization experiments
for degradation showed that the addition of either yeast extract (YE), BS,
or strain SS15 significantly (p < 0.01) promoted the removal of COD,
and the best COD removal was achieved by adding YE, BS, and SS15 into
FFFs (Fig. S6). The above optimum condition of flocculation and
biodegradation were applied in the following research.
Different treatments including flocculation, biodegradation, and
combined treatments consist of flocculation and biodegradation were
conducted to investigate the remediation performance. The BF and BS
used in all the treatments were prepared beforehand using the fermen­
tation medium. Briefly, five treatments were carried out: 1) the positive
control was provided by supplementing 4 mL of sterilized Milli-Q water,
this is the treatment using indigenous microorganisms present in the
FFFs, designated as control (degradation); 2) the flocculation treatment
3
Chemosphere 302 (2022) 134870
was carried out by amending 0.6 g/L of BF and 4 g/L of AlCl3, and
adjusting the pH of FFFs to 5; 3) the degradation treatment was provided
by inoculating 3 mL of SS15 and 1 mL of BS solution (50 mg/L); 4)
combined process of flocculation followed by degradation designated
the flocculation + degradation treatment; 5) combined process of
degradation followed by flocculation, designated the degradation +
flocculation treatment. All the experimental treatments were prepared
in triplicate and the treatments involving biodegradation were incu­
bated in a rotary shaker at 30 ◦ C and 180 rpm for 7 days. The condition
for the flocculation and degradation treatment was the same as the
solely flocculation and degradation treatments mentioned earlier.
2.4. Evaluation indicators analysis
Chroma, SS, COD, n-alkanes, and PAHs were analyzed to evaluate
the treatment performance. Chroma was measured with the spectro­
photometer at 380 nm (Ma et al., 2020). SS was determined by the
gravimetric method (Ma et al., 2020). COD was determined by the po­
tassium dichromate titration method (Zhou et al., 2020). It should be
noted that the calculated COD would be higher than the actual value due
to the presence of various ions such as chlorine, nitrogen, sulfur, and
phosphorus in the FFFs. The residual n-alkanes and PAHs after reme­
diation in all treatments were analyzed by GC-MS using the protocol
reported by Zhou et al. (2020).
2.5. Genome sequencing and analysis
The genomic DNA was extracted with an Easypure® Genomic DNA
kit following the manufacturer’s instructions (Transgene, Beijing,
China). A-tailed ligated to paired-end adaptors and PCR amplified with a
350 bp insert was used for the library construction at the Beijing
Novogene Bioinformatics Technology Co., Ltd (Qi et al., 2019). All good
quality paired reads in clean data which comes from raw data were
assembled by using the SOAP denovo into several scaffolds. All assem­
bled genes were annotated by several public databases, including NR
(Non-Redundant Protein Database databases), KEGG (Kyoto Encyclo­
pedia of Genes and Genomes), CAZy (Carbohydrate-Active Enzymes)
and GO (Gene Ontology) database. Meanwhile, secondary metabolite
biosynthetic gene clusters of strain SS15 were predicted by antiSMASH
website (version 6.0.1).
2.6. Data analysis
All data were mean values of three replicates. Statistical analysis was
performed in SPSS 19.0 software (IBM Corp., USA). To test the signifi­
cant difference (p value < 0.05) of results, one-way analysis of variance
(ANOVA) was employed.
3. Results and discussion
3.1. Isolation and characterization of BF and BS-producing bacteria
In total, twenty-five strains with flocculation activity were isolated
from oil-contaminated mudflat sediment (Fig. S1). Among these strains,
seven isolates showed high flocculating efficiency for kaolin suspension
and were selected for the evaluation of BS-producing ability. The results
suggested that strain 15 exhibited the highest flocculation activity
(78.0%) for kaolin suspension and the best BS-producing capacity (the
fermentation broth showed a low ST of 26.8 mN/m). Therefore, this
isolate was selected for further study (Fig. S2). Strain SS15 formed cir­
cular, convex, smooth, milky white in color colonies after 12 h of in­
cubation on the fermentation medium at 30 ◦ C (Fig. 1a). Gram stain
reaction showed that the strain SS15 was Gram positive (Fig. 1b). The
BLAST result showed that strain SS15 was closest to Bacillus velezensis
strain MFYC11X (99.97% similarity, accession number OK17640.1).
According to the phylogenetic tree, strain 15 is within the genus Bacillus
F. Zeng et al.
(Fig. 1c), and it was named Bacillus sp. SS15. The 16S rRNA sequence
was deposited in the GeneBank database with an accession number of
MW881545. Although the Bacillus has been reported for its ability to
produce either BF (Elkady et al., 2011; Hua et al., 2020) or BS (Zhou
et al., 2015; Parthipan et al., 2017), the ability to produce both has not
been reported.
3.2. Properties of BS produced by SS15
3.2.1. Surface activity of BS
As shown in Fig. 2a, Bacillus sp. SS15 had a CMC of 44.4 mg/L, and
the corresponding ST value was 36.6 mN/m. Compared with the CMC of
the BSs reported by Sun et al. (2019) (96.5 mg/L) and Huang et al.
(2020b) (220 mg/L), the BS produced by SS15 has a lower CMC, indi­
cating that it has better surface activity. In addition, oil-spreading
diameter (Fig. S3a), E24 index (Fig. S3b), and drop-collapse width
(Fig. S3c) of BS was 13.6 cm, 52% (with olive oil), and 0.7 cm (the
control was 0.5 cm), respectively. These results indicated the excellent
surface activity of the BS produced by SS15.
3.2.2. Effects of pH, temperature, and salinity on BS stability
As shown in Fig. 2b, the BS exhibited a lower surface activity under
acidic conditions (2–4) as compared to that of weak acid and alkaline
conditions (6–12). However, it generally maintained a low ST (<40 mN/
m), suggesting that it has good acid and alkali resistance. The ST of the
BS maintained a low value (35.5–36.6 mN/m) at different temperatures
(4–60 ◦ C), while the ST was 40.4 mN/m and 46.6 mN/m at 80 ◦ C and
100 ◦ C, respectively, indicating that a higher temperature (80–100 ◦ C)
hindered the performance of the BS. Different salinities (0–100 g/L) had
a negligible effect on the surface activity of BS (29.7–33.7 mN/m),
indicating that the BS had an excellent salinity tolerance. These results
indicate that the BS has strong environmental tolerance and harbors
Fig. 1. Morphology (a), gram stain (b), and phylogenetic tree (c) of strain SS15.
4
Chemosphere 302 (2022) 134870
great potential to be used in hostile environments.
3.2.3. Chemical characteristics of BS
TLC results of the BS were shown in Fig. 3a. Rf values of the two spots
were 0.25 and 0.59, respectively. Iodine fumigation and ninhydrin re­
agent staining test both showed positive results, indicating that the BS
contains lipid and peptide and may belong to lipopeptide (Shuai et al.,
2018).
For FTIR analysis (Fig. 3b), the peaks at 2921 cm− 1, 2852 cm− 1, and
1464–1160 cm− 1 were characteristics of stretching vibration of aliphatic
chain (-CH3 and –CH2-) (Zhou et al., 2015, 2020). The strong absorption
peak at 1743 cm− 1 indicates that it has carbonyl group (Sun et al.,
2018). The weak absorption peak at 1464 cm− 1 is caused by the bending
vibration of C–H, indicating the existence of a carbon chain structure,
and the absorption peaks observed at 800–500 cm− 1 may be caused by
the methylene shear vibration of bacterial protein (Zhou et al., 2020).
The hydrolyzed products of BS were methylated and analyzed by GC-
MS. As shown in Fig. 3c, three major adsorption peaks occurred. The
similarity search results showed that the three main peaks were closest
to the methyl hexadecanote (C17:0), methyl decadecanote (C19:0), and
methyl tetracosanote (C24:0), respectively. Therefore, heptadecanoic
acid (C17:0), decadecanoic acid (C19:0), and tetracosanoic acid (C24:0)
may be the main fatty acid components of the BS.
3.3. Properties of BF produced by SS15
3.3.1. Chemical characteristics of BF
The constitution analysis showed that the BF contains 6.0% of car­
bohydrates and 8.3% of protein, which is responsible for the flocculating
activity (Ma et al., 2020). As shown in Fig. 4a, the FTIR results of BF
showed weak CH tensile vibration bands at 2826.2 cm− 1, which corre­
sponded to carbohydrate derivatives (Rahman et al., 2014). The strong
F. Zeng et al.
Fig. 2. (a) CMC determination; (b) stability analysis of the BS produced by
SS15 in different pH, temperature, and NaCl concentration. The error bars
represent the standard deviations. ST indicates surface tension (mN/m).
Fig. 3. TLC (a), FTIR (b), and GC–MS (c) analysis results of extracted BS. (a): left was developed with iodine vapor to detect lipids, lemony yellow is positive; the
right was developed with a 0.25% ninhydrin solution to detect peptides, light purple is positive. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
5
Chemosphere 302 (2022) 134870
absorption peak at 1033 cm− 1 was caused by C–O in the polysaccharides
(Abbas et al., 2020), suggesting that the BF contains polysaccharides. As
shown in NMR spectrum, the signals at 4.8–5.3 ppm indicate the pres­
ence of four α-configuration monosaccharides in BF, while the signals at
4.4–4.8 ppm indicate the presence of two β-configuration mono­
saccharides (Fig. 4c). Based on the chemical shift signal of h-spectrum
and the prediction of CASPER, the signals at δ4.94, 4.07, 4.01, 3.83, 3.81
and 3.79 ppm corresponded to α-D-Gal, the signal at δ4.16, 4.01, 3.91,
3.79, 3.76 and 3.70 ppm corresponded to α-D-Glc, the signal at δ4.72,
4.04, 3.93, 3.76, and 3.63 ppm corresponded to β-D-Man, the signal at
δ3.97, 3.86, 3.85, 3.83, and 3.68 ppm corresponded to α-D-Arap-OMe,
and the signal at δ4.72, 3.91, 3.86, 3.60, 3.43 and 3.33 corresponded to
β-D-Glcp3NAC (Ronnols et al., 2013; Sivasankar et al., 2020). Among
these monosaccharides, α-D-Gal, α-D-Glc, and β-D-Man were the
repeating units of bioflocculant ABF which was produced by marine
actinobacterium Streptomyces sp. (Sivasankar et al., 2020).
3.3.2. Effects of pH, temperature, and salinity on BF stability
As shown in Fig. 4b, the flocculating activity of the BF at acidic
condition (pH 2–6) was lower than at alkaline condition (pH 8–12),
which is contrary to the result reported by some researchers that the
flocculating activity of BF decreased at alkaline conditions (Yin et al.,
2014). In general, the BF maintained good flocculating activity at a wide
pH range (pH 2–12). The flocculating activity of the BF showed little
variance at different temperature (4–100 ◦ C). This is different from other
researchers’ description that high temperature may reduce the floccu­
lating activity of BF (Elkady et al., 2011), indicating that the BF has a
better thermal resistance. The flocculating activity of the BF dropped
with the increase of NaCl concentration from 0 to 60 g/L, which might
be attributable to the decrease of binding sites caused by a large amount
of Na+ binding to the BF (Zhang et al., 2021). With the increase of
salinity (60–100 g/L) of the BF solution, the flocculating activity of the
BF gradually recovered. This can be interpreted as a constantly positive
correlation between the positive charge of the BF and the salinity of BF
solution. Overall, the BF produced by strain SS15 has strong environ­
mental tolerance.
F. Zeng et al.
Fig. 4. FTIR spectrum (a), stability under different pH, temperature, and NaCl concentration (b), and the NMR 1H spectrum (c) of the BF produced by SS15. The error
bars represent the standard deviations.
3.3.3. The analysis results for zeta potential and SEM
The zeta potential of the BF solution, kaolin suspension, and super­
natant after flocculation treatment was − 0.4 mV, − 12.6 mV, and 0.3
mV, respectively. This was also evidenced by the change of zeta po­
tential of kaolin suspension from negative to positive after treatment. It
was reported that cations (i.e., Na+, Ca2+, Mg2+, and Al3+) could be used
as coagulant aids to neutralize the negative charge of kaolin suspension
(Mohammed and Dagang, 2019). It could also decrease the repulsive
interaction between flocculant and colloidal particles, thus promoting
flocculation treatment (Li et al., 2020). In general, divalent cations have
better promoting effect on flocculation activity than trivalent cations, as
the latter could change the charge of kaolin particle and cover the ab­
sorption sites (Gong et al., 2008). Charged and zero-charged kaolin are
formed by the electric neutralization with cations, and they are adsorbed
or bridged by BF to form large flocs and precipitate. Additionally, pH can
affect the flocculation activity of BF, since the formation of ionic bonds
in the floc could be influence by HCl (Shahadat et al., 2017). Based on
SEM images (Fig. S4), the rough and amorphous surface of the BF has a
large specific surface area, which provides a large number of functional
groups and attachment sites to adsorb and bridge suspended particles
(Fang et al., 2021). The functional groups bound to the suspended
particles may be the hydroxyl, and methoxy groups as reported in other
study (Li et al., 2009).
3.4. Remediation of FFFs by biodegradation and flocculation
In this study, strain SS15, BF, and BS were used to remediate FFFs
through flocculation and biodegradation treatment, and the remediation
performance of different treatments was evaluated regarding the
removal of chroma, SS, COD, and petroleum hydrocarbons.
As shown in Fig. 5a, the degradation + flocculation treatment
showed the best chroma removal performance (85.7%) among all the
treatments, while the removal rate in control (degradation),
6
Chemosphere 302 (2022) 134870
flocculation, degradation, and flocculation + degradation treatment was
− 12.2%, 79.7%, − 5.8%, and 55.2%, respectively.
The SS content in initial FFFs was 1090 mg/L, and it decreased to
816 mg/L (25.1% reduction), 275 mg/L (74.8% reduction), 554 mg/L
(49.2% reduction), 327 mg/L (70.0% reduction), and 61 mg/L (94.4%
reduction) in control (degradation), flocculation, degradation, floccu­
lation + degradation, and degradation + flocculation treatments
(Fig. 5b), respectively. Notably, significant (p < 0.05) SS removal was
achieved by flocculation, degradation, and flocculation + degradation
treatment while the latter had an extremely significant (p < 0.01)
removal performance.
The COD in initial FFFs was 8371 mg/L, and it decreased to 7219
mg/L (13.8% reduction), 3878 mg/L (53.7% reduction), 2419 mg/L
(71.1% reduction), 1459 mg/L (82.0% reduction), and 1267 mg/L
(84.9% reduction) in control (degradation), flocculation, degradation,
flocculation + degradation, and degradation + flocculation treatment,
respectively (Fig. 5c). Notably, significant (p < 0.01) COD removal
performance occurred in all the treatments except the control.
Compared with the treatment performance (85% chroma, 52% SS
and 7% COD) of the biological flocculant BW-P3 (1 g/L) used in FFFs
(Ma et al., 2020), the BF produced by SS15 showed better treatment
performance (79.7% chroma, 74.8% SS, and 53.7% COD reduction) with
a lower dosage (0.06 g/L), indicating that the BF has great remediation
performance in treating FFFs. Also, BF has comparable treatment per­
formance and low dosage compared with chemical flocculants. For
example, Zhao (Zhao et al., 2018) achieved the maximum removal rate
of oil and turbidity (87.5% and 92.0%) in oilfield wastewater by using
40 mg/L cationic polyacrylamide. This suggested that the BF has the
potential to be applied in wastewater treatment as an eco-friendly
alternative to chemical flocculants (Abu Bakar et al., 2021).
As shown in Fig. 6a, the degradation + flocculation treatment has the
best remediation performance compared with other treatments. To
better understand the removal effect and residual concentration of
F. Zeng et al. Chemosphere 302 (2022) 134870

Fig. 5. The chroma removal efficiency (a), the suspended solid content and corresponding removal efficiency (b), COD and corresponding removal efficiency in
different treatments(c). Asterisks indicate a significant difference (*P < 0.05) or extremely significant difference (**P < 0.01) between the original sample and the
other treatments. The error bars represent the standard deviations.

Fig. 6. The concentrations of total n-alkanes and n-alkanes of different chain lengths (C8~10, C11~20, and C21~40) in different treatments (a); the concentrations
of total PAHs in different treatments (b). The error bars represent the standard deviations.

7
F. Zeng et al.
different n-alkanes after treatment, total n-alkanes were divided into
low-chain (C8–C10), medium-chain (C11–C20), and long-chain
(C21–C40) (Zhou et al., 2020). In this study, the proportions of the
three different chain lengths from low to long in the initial samples of
total n-alkanes was 2.3%, 60.8%, and 37.0%, respectively, indicating
that the middle-chain and long-chain were the main components of the
total n-alkanes. The total n-alkane concentrations in initial FFFs were
860.7 mg/L, and it decreased to 645.6 mg/L (25.0% reduction), 850.1
mg/L (1.2% reduction), 475.0 mg/L (44.8% reduction), 477.0 mg/L
(44.6% reduction), and 430.8 mg/L (50.0% reduction) in control
(degradation), flocculation, degradation, flocculation + degradation,
and degradation + flocculation treatments, respectively. Moreover, the
main constituents degraded by treatments were middle- (C11–20) and
long-chain (C21–40) n-alkanes. Similar treatment performance has been
reported in previous studies (Huang et al., 2020b; Zhou et al., 2020),
which suggested that the addition of bacterial culture, BS, and YE can
effectively promote the biodegradation of total n-alkanes, especially the
middle- and long-chain.
PAHs are generally characterized as highly toxic, teratogenic, and
carcinogenic; the effective removal of which can reduce the harm of
FFFs to the ecological environment (Lee et al., 2018). The residual
concentrations of PAHs in each treatment are shown in Fig. 6b. The
PAHs concentration in initial FFFs was 1161.2 μg/L, and it decreased to
883.4 μg/L (24.0% reduction), 1003.7 μg/L (13.6% reduction), 421.2
μg/L (63.7% reduction), 379.6 μg/L (67.3% reduction) and 389.4 μg/L
(66.5% reduction) in control (degradation), flocculation, degradation,
flocculation + degradation, and degradation + flocculation treatments,
respectively.
In all treatments, biodegradation is the main step to remove total n-
alkanes and PAHs from FFFs, while flocculation plays a minor role. The
inoculation of BS-producing bacteria combined with the supplemented
BS enhanced bioremediation of hydrocarbons. It was considered that the
produced biosurfactant during the degradation process can emulsify and
solubilize the hydrophobic compounds, thereby increasing the remedi­
ation performance (Huang et al., 2020a). Notably, small addition of
carbon sources such as acetic acid could improve the biodegradation as
bacteria need some energy from decompositions of simpler compounds
in order to decomposes petrochemicals. For instance, the addition of YE
has been reported to promote the growth and activity of microorganisms
in wastewater (Zhou et al., 2020). Thus, significant petroleum hydro­
carbon removal performance was observed in treatments involving
biodegradation.
In general, flocculation treatment mediated by the BF can effectively
remove the chroma, SS, and COD, whereas biodegradation treatment
enhanced by BS, YE, and SS15 can effectively promote the removal of
COD and petroleum hydrocarbons in wastewater. The combined treat­
ments, regardless of the sequences, achieved better remediation per­
formance than single treatments. The degradation + flocculation
treatment achieved the best FFFs treatment performance and could be
expected to be applied in industrial treatment of FFFs.
3.5. Genome analysis and AntiSMASH prediction
The complete assembled chromosome of strain SS15 contained a
circular molecule of 3,899,660 bp with 47.22% G + C content (Fig. S7).
There were 4035 predicted genes, of which 3963 were protein-coding
genes, 52 were tRNA genes, 3 were rRNA genes, and 7 were sRNA
genes. CRISPR gene was not identified, whereas 133 TR genes, 8 GIs
genes, and 9 prophage genes were detected in the genome. The detailed
genomic information of strain SS15 was listed in Table S2. The genome
data was deposited in the Genebank database accession number of
JAJAJA000000000.
All genes were annotated using BLAST with several public databases,
including the NR, KEGG, and CAZy databases. The NR database was
applied to annotate non-redundant protein, since the annotation result
contains species information, it can be used for species classification.
8
Chemosphere 302 (2022) 134870
The genus with the highest matching number of the strain SS15 genes
was Bacillus amyloliquefaciens, which matched 1541 genes, indicating
that strain SS15 belongs to the genus Bacillus (Fig. S8). According to the
KEGG database, genes matched to carbohydrate, amino acid, and lipid
metabolism groups were 236, 203, and 75, respectively (Fig. 7). These
genes could be responsible for producing polysaccharides, proteins in BF
and lipid in BS. Amino acid components in the BS and BF probably come
from arginine, phenylalanine, tyrosine, tryptophan, valine, leucine, and
isoleucine, since their metabolism pathways were detected. Meanwhile,
peptidoglycan biosynthesis pathway has been annotated in the genome
and probably be applied to BF synthesis. Genes involved in the fatty acid
biosynthesis were also detected for producing components of BS. The
CAZy database was applied to comment genes to enzymes families
related to degradation, modification, and biosynthesis, such as Glyco­
side Hydrolases, Glycosyl Transferases, Polysaccharide Lyases, Carbo­
hydrate Esterases, Auxiliary Activities, and Carbohydrate-Binding
Modules. GTs are related to the biosynthesis of oligosaccharides, poly­
saccharides, and glycoconjugates through catalyzing the formation of
glycosidic linkages to form glycosides (Qi et al., 2019). In this study, 52
genes from the genome of SS15 were matched in this group and could be
applied to form glycosides then produce carbohydrate components of BF
(Fig. S9).
In the genome of strain SS15, the peptidoglycan biosynthesis
pathway has been annotated. The UDP-GlcNAC produced in the pepti­
doglycan biosynthesis pathway can serve as a precursor for α-D-Glc and
β-D-Glc biosynthesis. In the glycolysis/gluconeogenesis pathway, the
glucosephosphomutase catalyzes the reversible conversion between Glc-
6P and Glc-1P. And Glc-6P are employed to produce energy for bacterial
growth, while Glc-1P is the donor of UDP-Glc (Barreto et al., 2005).
Meanwhile, UDP-Glc is the precursor for the biosynthesis of α-D-Glc in
the BF. In the galactose metabolism pathway, UDP-Glc can be conversed
to UDP-Gal by UDP-glucose-4-epomerase, then employed to biosyn­
thesis α-D-Gal in the BF. After biosynthesis of macromolecules by these
precursors, Carbohydrate-Binding Modules are in charge of forming
polysaccharides via the combination of macromolecules.
The biosynthetic gene cluster for secondary metabolite of strain SS15
was predicted by the antiSMASH website, and 15 clusters were pre­
dicted. The results showed that SS15 has multiple types of clusters such
as non-ribosomal peptide synthetase (NRPS), betalactone, type III pol­
yketide synthases (T3PKS), transAT-PKS, Linear azol(in)e-containing
peptides, terpene, and bacteriocin, suggesting that SS15 contain abun­
dant gene clusters for secondary metabolite synthesis. Herein, cluster 2
had 95% similarity to the gene cluster for surfactin synthesis in Bacillus
velezensis FZB42, suggesting that it might related to the synthesis of the
BS in Bacillus sp. SS15. Surfactin, a lipopeptide biosurfactant containing
a β-hydroxylated fatty acid chain and a circular heptapeptide, is syn­
thesized by a complex mechanism catalyzed with a NRPS encoded by the
srfA operon (Nakano et al., 1991; Hu et al., 2019). The srfA operon was
annotated in the NRPS metabolism pathway containing srfAA, srfAB,
and srfAC. This operon composes a linear array of seven modules
involving biosynthesis of seven amino acids (Hu et al., 2019). Also, the
srfAD was annotated in the pathway of quorum sensing and involved in
the cyclization and release of surfactin. In the fatty acid biosynthesis
pathway, the hexadecanoyl-ACP is synthesized from acetyl-CoA under
the regulation of FabD, FabF, FabG, FabZ, FabI, FabK, and FabL genes.
After synthesis of the fatty acyl precursors, these precursors are used to
form corresponding branched- and straight-chain fatty acid. At last, fatty
acids and cyclic amino acids are combined to form surfactin. The
detailed gene clusters information of strain SS15 are listed in Table S3.
4. Conclusion
In this study, a bacteria strain designated as Bacillus sp. SS15 which
could simultaneously produce BF and BS was isolated from the oil-
contaminated mudflat sediment. The lipopeptide BS showed good ST
with a CMC value of 44.4 mg/L. The BF contains carbohydrate and
F. Zeng et al.
Fig. 7. KEGG pathway annotation of assembled genes of Bacillus sp. SS15.
protein, and it exhibited high flocculating activity for kaolin suspension.
In addition, both BS and BF were thermostable, salt-tolerant, and alka­
liphilic. Electric neutralization and bridging occurred during the floc­
culation process through zeta potential and SEM observation suggested
that those activities may be associated with the flocculation of BF.
Further genomic analysis showed that many genes related to the syn­
thesis of carbohydrates, protein, and lipid existed in SS15 genome,
which might be related to BF and BS biosynthesis. The combined
treatment consisted of biodegradation and flocculation achieved excel­
lent remediation performance. The findings in this study suggested the
potential application of a BS and BF-producing bacteria in treating FFFs.
Credit author statement
Feng Zeng: Conceptualization, Methodology, Investigation, Formal
analysis, Data curation, Writing – original draft. Hanghai Zhou: Meth­
odology, Software, Investigation, Formal analysis, Data curation.
Xiaoyun Lin: Investigation, Methodology, Data curation. Yanhong Li:
Investigation, Data curation. Yanpeng Liang: Investigation, Data cura­
tion. Qinglin Xie: Investigation. Edidiong Okokon Atakpa: English pol­
ishing. Chaofeng Shen: English polishing, NMR analysis. Chunfang
Zhang: Validation, Writing – review & editing, Project administration.
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.
9
Chemosphere 302 (2022) 134870
Acknowledgements
This study was financially supported by Zhoushan Science and
Technology Department Project (2019C81056), the Fundamental
Research Funds for the Central Universities (2020QNA4045), the Sci­
entific and Technological Aid Project for Xinjiang (2021E02041), the
Guangxi Key Laboratory of Theory and Technology for Environmental
Pollution Control (No. 2001 K004), and the Major Science and Tech­
nology Project of Zhejiang Province (2021C02024).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2022.134870.
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