Chemosphere 224 (2019) 265e271

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Enrichment of the soil microbial community in the bioremediation of

a petroleum-contaminated soil amended with rice straw or sawdust
Yongjie Huang a, Huan Pan a, Qingling Wang b, Yanyan Ge b, Wuxing Liu b, *,
Peter Christie b
a
College of Life Sciences, Anhui Normal University, Wuhu, 241000, China
b
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, 210008, China

h i g h l i g h t s

Rice straw and sawdust significantly enhance the TPHs and PAHs removal respectively.

Petroleum removal was irrelative with microbial community diversity.

Certain petroleum degraders play a vital role in petroleum removal.

a r t i c l e i n f o a b s t r a c t

Article history: Two common organic wastes from agriculture (rice straw) and forestry (sawdust) were applied to a
Received 30 May 2018 petroleum-contaminated soil to estimate their effectiveness in the removal of total petroleum hydro-
Received in revised form carbons (TPHs) and polycyclic aromatic hydrocarbons (PAHs). Rice straw was the more effective
3 February 2019
amendment than the other treatments in reducing TPH contents and addition of sawdust resulted in a
Accepted 22 February 2019
Available online 23 February 2019
significant decrease in PAH removal, particularly high-molecular-weight (5e6 ring) PAHs. Principal co-
ordinates analysis (PCoA) indicates that rice straw treatment separated only the bacterial community but
Handling Editor: Chang-Ping Yu sawdust greatly affected both the soil bacterial and fungal communities. Moreover, the abundance of
some petroleum degraders such as the bacteria Sphingomonas, Idiomarina and Phenylobacterium and the
Keywords: fungi Humicola, Wallemia and Graphium was promoted by inputs of the two agricultural and forestry
Agricultural and forestry wastes wastes. These results highlight the potential of waste applications in accelerating hydrocarbon biodeg-
Bioremediation radation which may be attributed to the enrichment of keystone taxa that show strong positive asso-
Petroleum degraders ciations with hydrocarbon degradation.
Petroleum hydrocarbons
© 2019 Published by Elsevier Ltd.

1. Introduction implementation of innovative technologies for the clean-up of

Rapid development of the oil industry has been accompanied by
increasing concern over the environmental consequences of pe-
troleum contamination (Oleszczuk et al., 2017). The remediation of
soils contaminated with petroleum hydrocarbons has been widely
studied due to their toxic and carcinogenic components such as
polycyclic aromatic hydrocarbons (PAHs) that threaten the envi-
ronment and human health (Johnston et al., 2015; Zeng et al., 2016).
There has been increasing interest in the development and
* Corresponding author. Key Laboratory of Soil Environment and Pollution
Remediation, Institute of Soil Science, Chinese Academy of Sciences, Address: 71
East Beijing Road, Nanjing, Jiangsu Province 210008, China.
E-mail address: liuwuxin@issas.ac.cn (W. Liu).
contaminated soils (de Boer et al., 2016). Bioremediation methods
such as biosparging, biostimulation and bioaugmentation are
receiving increased attention compared with physical and chemical
remediation technologies owing to their relatively low cost and
limited site disturbance (Liu et al., 2013; Lu et al., 2014).
Key factors in microbial bioremediation are soil nutrient con-
tent, oxygen status and moisture content (Galdames et al., 2017).
However, soils contaminated with petroleum residues often have
low porosity and water-holding capacity and this affects the effi-
ciency of microbial degradation. Numerous recent studies report
that agricultural and forestry wastes may help to maintain soil
quality and achieve high efficiency of removal of hydrocarbon
compounds (Shahsavari et al., 2013; Zhang et al., 2015). For
example, Callaham et al. (2002) have attributed enhanced

https://doi.org/10.1016/j.chemosphere.2019.02.148
0045-6535/© 2019 Published by Elsevier Ltd.
266 Y. Huang et al. / Chemosphere 224 (2019) 265e271
biological activity in response to the application of wheat straw to
an increase in soil porosity which facilitates oxygen transfer.
Moreover, agricultural and forestry wastes have some capacity to
hold water and thus maintain the metabolism of indigenous mi-
croorganisms (Dong et al., 2013). All these measures are used to
provide a suitable habitat for microbes to enhance biodegradation
activity.
Straw and sawdust have been reported to increase soil porosity
and water holding capacity and the large amounts of cellulose and
lignin they contain can also serve as substrates for the growth of
hydrocarbon-utilization microorganisms (Ali et al., 2011; Dashti
et al., 2017). These microorganisms usually have an extracellular
enzymatic system that oxidizes lignin efficiently by producing en-
zymes such as laccase, lignin peroxidase, manganese peroxidase
and versatile peroxidase (Mathieu et al., 2013). Agricultural and
forestry wastes may therefore enhance the activity of specific en-
zymes to increase the efficiency of hydrocarbon biodegradation.
Although numerous studies have found that both materials
remediate petroleum contaminated soils individually, few studies
have focused on the different components of the two materials that
may exert different effects on the microbial community, thereby
resulting in distinct efficiencies and residues of petroleum degra-
dation. Further investigations on the changes in the abundance and
community structure of microorganism in petroleum degradation
are required to better understand how the two additives influence
the biodegradation of pollutants in soils. Indigenous microbial
community structure is considered to be a key factor for successful
bioremediation (Varjani et al., 2015). Most studies report variation
in the abundance and community structure of petroleum degraders
during petroleum degradation monitored by DGGE (Koshlaf et al.,
2016) or T-RFLP (Giebler et al., 2014) analysis. However, these
molecular methods usually give low taxonomic resolution of soil
microbial communities responding to the biodegradation of
organic contaminants. High-throughput sequencing has recently
become a popular technique because it provides the maximum
amount of data with high resolution at low cost (Hou et al., 2015).
A pot experiment using two common agricultural and forestry
wastes (rice straw and sawdust) was conducted with an aged
petroleum-contaminated soil to further elucidate the mechanisms
by which agricultural and forestry wastes may enhance petroleum
bioremediation, and particularly the relationships between bacte-
rial and fungal communities and petroleum hydrocarbons. The
removal efficiency of total petroleum hydrocarbons (TPHs) and
PAHs by fertilization alone and fertilization with rice straw or
sawdust was studied using FT-IR and GC-MS. Illumina MiSeq
sequencing was used to monitor the soil bacterial and fungal
communities to help elucidate the relationships between the two
additives and the biodegradation process.
2. Materials and methods
2.1. Soil samples and agricultural and forestry wastes
Weathered petroleum-contaminated soil was collected near an
oil production well over 20 years at Daqing oilfield, Heilongjiang
province, northeast China. Soil samples were air-dried and passed
through a 2-mm sieve. The soil samples were then mixed thor-
oughly and stored at 4  C until use. Physical and chemical proper-
ties of the original soil were: pH 8.29, available N 126 mg kg1,
available P 10 mg kg1, available K 13.2 mg kg1; and organic mat-
ter content 55.0 mg kg1. Rice straw was obtained from agricultural
land in Hunan province and sawdust was purchased from a local
market (Shown in Fig. 1). The rice straw was chopped into 2-cm-
long pieces.
Fig. 1. The two additives mentioned in the Materials and methods: (a) rice straw (b)
sawdust.
2.2. Pot experiment
The experimental design consisted of three treatments with
three replicates. The treatments were (1) fertilizer only (Control),
(2) rice straw þ fertilizer (RF); and (3) sawdust þ fertilizer (SF).
Each pot contained 1 kg of air-dried soil and the soil water content
was maintained at about 60% field capacity during the experi-
mental period. Aeration was conducted every two weeks in all the
treatments. Waste addition treatments comprised rice straw or
sawdust (0.2% on a dry weight basis). Inorganic nutrients in the
form of (NH4)2SO4 and K2HPO4 were added to all treatments to give
final rates of 250 mg N kg1 and 100 mg P kg1. The pot experiment
was conducted in a sunlit greenhouse without supplementary
illumination for five months.
At the end of the pot experiment, part of each soil sample was
freeze-dried in a vacuum freeze drier to detect the concentrations
of TPHs and PAHs. The remainder was stored at 20  C for soil
microbial analysis.
2.3. Quantification of TPHs and PAHs
Lyophilized soil samples were passed through a 0.25-mm sieve
before TPH and PAH analysis. TPHs extracted from the soil samples
were determined by Fourier transform-infrared analysis (FT-IR). In
brief, 5-g aliquots of soil were extracted using 25 mL of tetra-
chloromethane by ultrasonic extraction for 30 min and then the
extracts were centrifuged. The supernatants were filtered through
filter paper and made up to 50 mL in glass centrifuge tubes. The soil
samples were then extracted ultrasonically using 10 mL of tetra-
chloromethane and filtered again. The extracts were made up to a
constant volume of 50 mL and homogenized. The hydrocarbon
extracts were passed through 20-cm columns containing anhy-
drous sodium sulfate and florisil. The first 5 mL of the eluates were
discarded. The remainder of the eluates was used for analysis using
a Model F2000-ⅡK FT-IR oil spectrometer (Ou Yier, Jilin, China). The
setup of external standard calibrations and quantity analysis fol-
lowed the HJ 637e2012 method (China Standards Press, 2012).
PAHs were determined by gas chromatography-mass spec-
trometry (GC-MS). Aliquots of 3 g of the soil sample were mixed
with 3 g of anhydrous sodium sulfate and extracted using 70 mL
dichloromethane in a Soxhlet extractor for 24 h. The extracts were
rotary-evaporated and the residues dissolved in 2 mL cyclohexane.
An aliquot of 0.5 mL of the cyclohexane solution was passed
through a 0.5 cm (i.d.)  20 cm column containing 1 g activated
silica gel (200e325 mesh size) from top to bottom. The polycyclic
aromatic hydrocarbon fraction was eluted in 3 mL of a 1:1 mixture
of dichloromethane/hexane from the column. The first 0.5 mL of
elute was discarded because it contained non-polar saturated
 Y. Huang et al. / Chemosphere 224 (2019) 265e271
hydrocarbons and was less retained than PAHs by the silica gel. The
eluates were concentrated to 2 mL for GCeMS analysis.
GCeMS analysis was performed using a Model 7890 GC-5975
MSD (Agilent Technologies, Santa Clara, CA) fitted with a DB-5 ms
capillary column (Restek Corporation, Bellefonte, PA),
30 m  0.25 mm i.d., 0.25 mm. The carrier gas was He at
1.4 mL min1; the injection temperature was 280  C; and the tem-
perature program was: 50  C (held for 3 min) to 40  C at 2  C min1
and then to 300  C (held for 5 min) at 25  C min1. The setup of
external standard calibrations and quantitative analysis of different
fractions were followed the WSDE method (Washington State
Department of Ecology, 1997).
2.4. Bacterial and fungal analysis
The nine samples were sent to Genewiz, Inc. (Suzhou, east
China) for analysis of bacteria and fungi in the different treatments.
DNA was extracted from soil sub-samples using a TIANamp Soil
DNA Kit (Tiangen Biotech, Beijing, China). DNA samples were
quantified using a Qubit 2.0 fluorometer (Invitrogen, Waltham,
MA). For the bacterial community, 30e50 ng DNA was used to
generate amplicons using a MetaVx™ Library Preparation kit. V3
and V4 hypervariable regions of prokaryotic 16S rDNA were
selected to generate amplicons and subsequent taxonomic analysis.
Genewiz designed a panel of proprietary primers aimed at rela-
tively conserved regions bordering the V3 and V4 hypervariable
regions of bacterial and archaeal16S rDNA. The V3 and V4 regions
were amplified using forward primers containing the sequence
“CCTACGGRRBGCASCAGKVRVGAAT” and reverse primers contain-
ing the sequence “GGACTACNVGGGTWTCTAATCC”. First round PCR
products were used as templates for second round amplicon
enrichment PCR. Indexed adapters were simultaneously added to
the ends of the 16S rDNA amplicons to generate indexed libraries
ready for downstream NGS sequencing on the Illumina Miseq
platform.
For the fungal community, 50e100 ng DNA was used to generate
amplicons using a panel of primers designed by Genewiz. Oligo-
nucleotide primers were designed to anneal to the relatively
conserved sequences spanning fungal ITS regions. ITS2 region was
amplified using forward primer containing the sequence
“GTGAATCATCGARTC” and reverse primer containing the sequence
“TCCTCCGCTTATTGAT”. In addition to the ITS target-specific se-
quences, the primers also contained adaptor sequences allowing
uniform amplification of the library with high complexity ready for
downstream NGS sequencing on the Illumina Miseq platform.
DNA libraries were validated with an Agilent 2100 bioanalyzer
(Agilent Technologies, Palo Alto, CA), and quantified with a Qubit
2.0 fluorometer. DNA libraries were multiplexed and loaded on an
Illumina MiSeq instrument according to the manufacturer's in-
structions (Illumina, San Diego, CA). Sequencing was performed
using a 2  300 paired-end (PE) configuration. Image analysis and
base calling were conducted with the MiSeq control software (MCS)
embedded in the MiSeq instrument.
2.5. Data analysis
The QIIME data analysis package was used for 16S rRNA and ITS
rRNA data analysis. The forward and reverse reads were joined and
assigned to samples based on barcode and truncated by cutting off
the barcode and primer sequence. Quality filtering on joined se-
quences was performed and sequences which did not fulfil the
following criteria were discarded: sequence length <200 bp, no
ambiguous bases, mean quality score 20. Then the sequences
were compared with the reference database (RDP Gold database)
using the UCHIME algorithm to detect chimeric sequences and then
267
the chimeric sequences were removed. The effective sequences
were used in the final analysis. Sequences were grouped into
operational taxonomic units (OTUs) using the clustering program
VSEARCH (1.9.6) against the Silva 119 database and the UNITE ITS
database pre-clustered at 97% sequence identity. The ribosomal
database program (RDP) classifier was used to assign a taxonomic
category to all OTUs at a confidence threshold of 0.8. The RDP
classifier uses the UNITE ITS database which has taxonomic cate-
gories predicted to the species level. Sequences were rarefied prior
to calculation of alpha and beta diversity statistics. The alpha di-
versity indices Ace, Chao, Shannon and Simpson were calculated
using QIIME. The richness indices Ace and Chao were analyzed
according to Chao et al. (1993) and Chao (1984), respectively. The
diversity indices Shannon and Simpson were analyzed according to
Shannon (1948) and Simpson (1949), respectively. Beta diversity
was calculated using weighted and unweighted UniFrac and prin-
cipal coordinates analysis (PCoA) was conducted.
2.6. Statistical analysis
Significant differences among the treatments were analyzed
using Duncan's multiple range test at p<0.05. All data analysis was
conducted using the SPSS for Windows version 19 statistical soft-
ware package.
3. Results
3.1. Removal of TPHs and PAHs
TPHs and PAHs in soil samples collected from the three treat-
ments were monitored after five months. The concentration of
TPHs in the original contaminated soil was 2278 mg L1. The TPH
removal efficiencies were 23.9, 45.2 and 27.5% in the Control, RF and
SF treatments, respectively. The final concentration of TPHs was
lower in RF but there was no significant difference between the
control and SF (Fig. 2 a). The content of PAHs in the original
contaminated soil was 13.0 mg kg1. The PAHs decreased by 26.9,
30.3 and 66.3% in the Control, RF and SF treatments, respectively.
The final PAH concentration was significantly lower (P < 0.05) in SF
than in the control or RF (Fig. 2 b). The 16 PAH fingerprint species
were divided into five groups based on the numbers of aromatic
rings. Fig. 3 shows the concentrations of different ring numbers of
PAHs in the different treatments and all levels of PAHs in the
sawdust treatment were significantly lower than in the control.
Moreover, the final concentrations of PAHs with 5e6 rings
decreased more than those with 2, 3, or 4 rings in the SF treatment
(43.9, 49.1 and 54.0%, respectively), decreasing by 60.6 and 64.8%,
respectively, compared with the control.
Fig. 2. The residual contents of (a) TPHs and (b) PAHs in the different treatments.
Original, untreated soil; Control, fertilizer only; RF, rice straw þ fertilizer; and SF,
sawdust þ fertilizer. Error bars show the standard deviation (n ¼ 3). Mean values
followed by the same letter are not significantly different by Duncan's multiple range
test at the 5% level.
268 Y. Huang et al. / Chemosphere 224 (2019) 265e271

Table 2
Richness and diversity of the fungal community in soil from the different
treatments.

Treatment Ace Chao Shannon Simpson

Control 117.05 ± 7.58a 118.63 ± 10.23a 2.42 ± 0.35a 0.63 ± 0.12a

RF 128.73 ± 3.07a 130.91 ± 1.38a 2.84 ± 0.99a 0.63 ± 0.21a
SF 117.89 ± 20.36a 117.50 ± 21.10a 2.92 ± 0.26a 0.80 ± 0.02a

The data are the mean ± standard deviation of three replicates, and mean values
followed by the same letter are not significantly different by Duncan's multiple
range test at the 5% protection level. Control, fertilizer only; RF, rice
straw þ fertilizer; and SF, sawdust þ fertilizer.

Fig. 3. The concentrations of individual groups of PAHs with different ring numbers in
the different treatments. Control, fertilizer only; RF, rice straw þ fertilizer; and SF,
sawdust þ fertilizer. Error bars show the standard deviation (n ¼ 3). Mean values
followed by the same letter are not significantly different by Duncan's multiple range
test at the 5% level.

3.2. Pyrosequencing and sequence analysis

A total of 384,438 bacterial and 463,077 fungal sequences were

obtained after the sequence optimization process with an average
number of 42,715 sequences per sample (ranging from 34,884 to
65,190) and 51,453 sequences per sample (ranging from 38,609 to
64,128), respectively.
The calculated bacterial community richness and diversity
indices indicate differences in the soil microbial community among
the different treatments (Table 1). The richness indices Ace and
Chao were significantly higher in the RF and SF treatments than in
the control. The Shannon index was significantly higher in SF than
in the control but the Simpson index showed no significant dif-
ference among treatments. The calculated fungal community
richness and diversity indices also showed no significant differ-
ences in ACE, Chao, Shannon and Simpson indices among the
different treatments (Table 2).
Principal coordinates analysis (PCoA) on Bray-Curtis dissimi-
larity matrices of OTUs at 97% cutoff was conducted using Rstudio
version 1.1.383 to reveal community level differences between the
different treatments and the control. As can be seen in Fig. 4, the
bacterial communities in rice straw and sawdust amended treat-
ments were separated from the control and samples within the
same treatment were clustered together. In the case of the fungal
communities, the sawdust treatment formed a separate group
away from the control and rice straw treatment which were posi-
tioned relatively close to each other.
Table 1
Richness and diversity of the bacterial communities in soil from the different
treatments.
Treatment Ace Chao Shannon
Control 506.92 ± 10.18b 517.81 ± 8.46b 6.74 ± 0.36b
RF 555.67 ± 10.64a 559.65 ± 6.65a 7.00 ± 0.18 ab
SF 572.50 ± 11.74a 575.33 ± 11.10a 7.39 ± 0.06a
The data are the mean ± standard deviation of three replicates, and mean values
followed by the same letter are not significantly different by Duncan's multiple
range test at the 5% protection level. Control, fertilizer only; RF, rice
straw þ fertilizer; and SF, sawdust þ fertilizer.
Fig. 4. Comparison of bacterial 16s rDNA and fungal ITs rDNA communities in the
different treatments using principal coordinates analysis. The percentages in paren-
theses indicate the proportions of variation by each ordination axis. Control, fertilizer
only; RF, rice straw þ fertilizer; and SF, sawdust þ fertilizer.
3.3. Taxonomic composition analysis
Fig. 5 shows that the relative abundance of petroleum-
degrading bacteria belonging to the genera Sphingomonas, Phenyl-
obacterium and Rhizobium increased in the SF treatments while the
genus Idiomarina increased in the rice straw treatment compared to
the control. The relative abundance of petroleum degrading fungi
also showed some changes and the relative abundance of Humicola
and Graphium increased significantly in the sawdust treatment.
Specifically, the abundance of Humicola was significantly higher in
SF than in the control or the RF treatment.
4. Discussion
Bioremediation is an effective method that has been widely
used to remediate soils contaminated with petroleum materials
including crude oil (Cai et al., 2016) and PAHs (Rostami et al., 2016).
The degradation results show that the fertilizer-only treatment
removed 23.9 and 26.9%, respectively, of TPHs and PAHs from the
contaminated soil. This may be partly due to the addition of
(NH4)2SO4 and KH2PO4 as supplementary nutrients. Agricultural
and forestry wastes have been widely used with great success in
recent years to remove organic pollutants from contaminated soils
(Wu et al., 2011; Alotaibi et al., 2018). In the present study, we found
Simpson
that two common agricultural and forestry waste amendments
0.98 ± 0.01a
enhanced the dissipation of TPHs and PAHs from soil microcosms.
0.98 ± 0.01a
0.99 ± 0.00a The addition of rice straw increased the removal efficiency of TPHs
compared to the control and SF (Fig. 2 a). A previous study reported
that the degradation of TPH in soil can be promoted by modifying
soil physicochemical properties (Akbari et al., 2015). Thus, we hy-
pothesized that the dissipation of the TPH in our study may be
 Y. Huang et al. / Chemosphere 224 (2019) 265e271
Fig. 5. The relative abundance of petroleum-degrading (a) bacterial and (b) fungal taxa
at genus level in the different treatments. Vertical bars show the standard deviation
(n ¼ 3). Columns with the same letter are not significantly different by Duncan's
multiple range test at the 5% level. Control, fertilizer only; RF, rice straw þ fertilizer;
and SF, sawdust þ fertilizer.
attributed to the hollow structure of the straw which may enable it
to increase soil aeration. This structure might make the amended
soil a more suitable habitat for the microorganisms that degrade
TPHs. Alotaibi et al. (2018) concluded that wheat straw when
applied to crude oil-contaminated soil has an important stimula-
tory effect on soil microbial respiration and organic C
mineralization.
PAHs are strongly hydrophobic and poorly water-soluble and
are therefore more resistant to degradation (Balachandran et al.,
2012). In our study there was a higher PAH degradation rate in
the sawdust treatment (Fig. 2 b). This may be due to differences in
the composition of the two waste materials. Straw and sawdust are
both lignocellulosic materials and previous studies show that the
lignin content of sawdust is 15% higher than that of common straw
(Sheng, 2006). Llado et al. (2013) reported that a decline in PAHs
was achieved by the addition of lignin-rich materials because lig-
ninolytic enzymes such as LiP, MnP and laccase have exceptional
capacities for PAH transformation and may contribute to soil PAH
dissipation through co-metabolic mechanisms. These proteins are
269
involved in the hydroxylation of aromatic rings, which contribute
to the oxidation of the resultant intermediates and are responsible
for the cleavage of the terminal ring structure (Haritash et al.,
2009). This is consistent with our study in which an increased
degradation rate of PAHs occurred in the sawdust treatment. The
compounds with the most complex structures, containing five or
more benzene rings in their molecules, i.e. high molecular weight
PAHs, are considered to be the most toxic, mutagenic and carci-
nogenic (Alagic et al., 2016). In our study a much larger decrease in
the 5e6 ring PAHs than in the 2e4 ring compounds was observed in
the SF soil (Fig. 3).
The dissipation of petroleum hydrocarbons is generally attrib-
uted to indigenous microbes (Wloka et al., 2017). Microbial com-
munity analysis was conducted using high-throughput sequencing
to reach a better understanding of the biological processes of pe-
troleum degradation after bioremediation. PCoA analysis (Fig. 4)
based on OTU composition shows that the bacterial community
was clearly differentiated between the control and the agricultural
and forestry waste amendments (RF and SF). Moreover, the rich-
ness and diversity indices of bacteria (Table 1) show that the Ace
and Chao indices and the Shannon index increased in RF and SF
compared with the control. However, only the rice straw addition
treatment showed a significant decrease in TPH degradation
compared with the addition of sawdust. We observed a switch
between different genera of bacteria and fungi and several groups
of bacteria and fungi appeared to be enriched in the rice straw
treatment. Fig. 5 shows the relative abundance of petroleum-
degrading bacterial and fungal taxa at genus level in the different
treatments. Lysobacter dominated the g-proteobacteria was
enriched in the rice straw treatment. This bacterium has been re-
ported to be a petroleum-hydrocarbon degrader (Cervantes-
Gonzalez et al., 2009). Some other genera belonging to the g-pro-
teobacteria such as Luteimonas, Arenimonas and Idiomarina are also
potential petroleum degraders (Kim et al., 2018; Ni et al., 2017;
Gomes et al., 2018). Idiomarina has been reported to show great
potential for both the biodegradation of hydrocarbons (Bayat et al.,
2016) and the emulsification or reduction of surface tension
(Malavenda et al., 2015). Brevundimonas belongs to the a-proteo-
bacteria was involved in hydrocarbon degradation (Phillips et al.,
2008). The relative abundance of petroleum-degrading fungi
enriched in the RF treatment such as Penicillium, Wallemia and
Acremonium were reported to have the capacity to degrade hy-
drocarbons (Khan et al., 2016; Zheng et al., 2003; Ma et al., 2015).
Thus, our results highlight the importance of these microbial taxa
in petroleum decomposition while the soil community diversity
has no direct connection with hydrocarbon dissipation. This is in
agreement with Hou et al. (2015) who found that the removal ef-
ficiency was not related to bacterial diversity but certain microor-
ganisms were selectively stimulated and played an important role
in petroleum dissipation.
In the case of the fungi, the sawdust addition treatment was
significantly differentiated from the control and the rice straw
treatment but the fungal communities in the control and RF did not
diverge significantly (Fig. 4). The calculated fungal community
richness and diversity indices show no significant differences in
terms of the Ace, Chao, Shannon and Simpson indices in the
different treatments (Table 2). The sawdust treatment had the
highest degradation efficiency of PAHs (66.3%) that was signifi-
cantly higher than the control (26.9%) or the rice straw treatment
(30.3%). Analysis of community composition shows that several
petroleum-degrading microbes increased in the sawdust treatment
compared to the control and RF. For example, the bacteria Ther-
momonas, Sphingomonas, Gemmatimonas, Phenylobacterium and
Rhizobium have been reported to be associated with the decom-
position of petroleum (de la Cueva et al., 2016; Zhao et al., 2017;
270 Y. Huang et al. / Chemosphere 224 (2019) 265e271
Freire et al., 2017; Zhang et al., 2016; Ni et al., 2017). Moreover,
Sphingomonas and Phenylobacterium are strongly associated with
conditions exhibiting increased PAH removal (Bacosa et al., 2015;
Singleton et al., 2016). The fungi Humicola, Scedosporium, Tricho-
cladium and Graphium have been reported to be potential petro-
leum degraders (Cerniglia et al., 2010; Morales et al., 2017;
Alegbeleye et al., 2017; Chandra et al., 2013). The relative abun-
dance of Humicola in the Agaricomycetes was significantly higher in
the sawdust treatment with a maximum value of 18.59%. Humicola
is known to degrade PAHs with three or more rings (Cerniglia et al.,
2010). Notably, the sawdust treatment had a higher PAH removal
efficiency. The enriched abundance of Humicola may have been
associated with the decrease in PAH content. Moreover, Trichocla-
dium has been reported to produce ligninolytic enzymes (Silva
et al., 2010) which can transform PAHs through cometabolic
mechanisms.
5. Conclusions
One agricultural and one forestry waste were applied to reme-
diate a petroleum-contaminated soil in a pot experiment. The
addition of rice straw or sawdust resulted in a significant decline in
TPHs and PAHs, respectively. Moreover, soil PAHs clearly decreased
compared with the original soil. The PAH removal efficiencies were
26.9, 30.3 and 66.3% in the control and RF and SF treatments,
respectively. Principal coordinates analysis (PCoA) indicates that
the bacterial communities within the different wastes were distinct
and both treatments were clearly different from the control, while
the fungal community in the sawdust treatment was significantly
differentiated from the control and the rice straw treatment.
Removal efficiency was not related to bacterial diversity but rather
to the selective effect of specific microbial communities in the
bioremediation. The community composition analysis shows that
the abundance of petroleum-degrading taxa such as the bacteria
Sphingomonas, Phenylobacterium and the fungi Humicola and
Graphium increased after addition of rice straw and sawdust. Thus,
the current study highlights the potential of these microbial taxa in
petroleum dissipation to harness their potential for greater reme-
diation effectiveness under field conditions.
Acknowledgements
We thank the National Natural Science Foundation of China
(U1662110, 41671325), Jiangsu Provincial Natural Science Founda-
tion (BK20171521) and Foundation of the Provincial Key Laboratory
of the Conservation and Exploitation of Biological Resources in
Ecological Safety in Anhui Province for financial support.
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