Science of the Total Environment 636 (2018) 968–974

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Bioavailability of weathered hydrocarbons in engine oil-contaminated

soil: Impact of bioaugmentation mediated by Pseudomonas spp.
on bioremediation
Kavitha Ramadass a,c, Mallavarapu Megharaj b,⁎, Kadiyala Venkateswarlu d, Ravi Naidu b
a
Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia
b
Global Centre for Environmental Remediation, Faculty of Science, CRC CARE, University of Newcastle, Callaghan NSW2308, Australia
c
Research and Innovation Division, University of Newcastle, Callaghan NSW2308, Australia
d
Formerly Professor of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India

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

• Weathered TPHs in engine oil-

contaminated soil were bioaugmented
by Pseudomonas spp.
• Combination of biostimulation and bio-
augmentation inhibited bioremediation
of weathered TPHs.
• Soil dehydrogenase activity is in confor-
mity with bioremediation of weathered
TPHs.
• Soil extraction with cyclodextrin is suit-
able for assessing bioavailability of
weathered TPHs.
• Pyrosequencing data clearly corroborate
the results of weathered TPHs
bioremediation.

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

Article history: Heavier fraction hydrocarbons (C15-C36) formed in soil after biotic and abiotic weatherings of engine oil are the
Received 4 April 2018 continuing constraints in the bioremediation strategy, and their bioavailability remains a poorly quantified reg-
Received in revised form 27 April 2018 ulatory factor. In a microcosm study, we used two strains of Pseudomonas, P. putida TPHK-1 and P. aeruginosa
Accepted 27 April 2018
TPHK-4, in strategies of bioremediation, viz., natural attenuation, biostimulation and bioaugmentation, for re-
Available online xxxx
moval of weathered total petroleum hydrocarbons (TPHs) in soil contaminated long-term with high concentra-
Editor: Frederic Coulon tions of engine oil (39,000–41,000 mg TPHs kg−1 soil). Both the bacterial strains exhibited a great potential in
remediating weathered hydrocarbons of engine oil. Addition of inorganic fertilizers (NPK), at recommended
Keywords: levels for bioremediation, resulted in significant inhibition in biostimulation/enhanced natural attenuation as
Weathered TPHs well as bioaugmentation. The data on dehydrogenase activity clearly confirmed those of bioremediation strate-
Pseudomonas spp. gies used, indicating that this enzyme assay could serve as an indicator of bioremediation potential of oil-
Bioaugmentation contaminated soil. Extraction of TPHs from engine oil-contaminated soil with hydroxypropyl-β-cyclodextrin
Bioremediation (HPCD), but not 1-butanol, was found reliable in predicting the bioavailability of weathered hydrocarbons.
Pyrosequencing
Also, 454 pyrosequencing data were in accordance with those of bioremediation strategies used in the present
microcosm study, suggesting the possible use of pyrosequencing in designing approaches for bioremediation.
© 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Global Centre for Environmental Remediation (GCER), Cooperative Research Centre for Contamination Assessment and Remediation of Environment (CRC
CARE), Faculty of Science, The University of Newcastle, ATC Building, University Drive, Callaghan NSW2308, Australia.
E-mail address: megh.mallavarapu@newcastle.edu.au (M. Megharaj).

https://doi.org/10.1016/j.scitotenv.2018.04.379
0048-9697/© 2018 Elsevier B.V. All rights reserved.
 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974
1. Introduction
Lubricant or engine oil production, transportation, usage, and dis-
posal have several environmental impacts. A large percentage of used
oil is discharged into the ecosystems without any treatment even in de-
veloped countries. Engine oil persists for longer than six years in some
ecosystems, leading to chronic problems for the biota. Even under con-
trolled conditions, oil cannot be quickly and completely metabolized by
microorganisms, and it takes weeks to months for engine oils to degrade
(Bidoia et al., 2010). Thus, lubricant discharge in nature is of a continu-
ous and great concern due to its non-quantified impact and potential
chronic damage to the ecosystem (Bakke et al., 2013). Weathering is
an action where the physical, chemical and biological processes affect
the type of hydrocarbons remaining in a soil (Loehr et al., 2001). More-
over, weathering enhances sorption of the contaminants into soil solid
matrix and their diffusion into soil pores with increased contact time
leading to decreased bioavailability of pollutants to microorganisms
(Gao, 2009). Aging is thus an important natural process of pollutants
in soil environment that affects their bioavailability, biological toxicity
and biodegradability. Long-term oil-contaminated soil consists mostly
of recalcitrant compounds of high molecular weight hydrocarbons
(NC25 compounds) that are harder to degrade by indigenous microor-
ganisms. Weathered petroleum hydrocarbons that are less or non-
bioavailable complicates the performance of remediation technologies.
Furthermore, weathered hydrocarbons have significant qualitative and
quantitative differences compared to fresh petroleum products, and
these are not yet considered in most risk assessments (Jiang et al.,
2016). Hence, there is a necessity to assess the efficiency of
hydrocarbon-degrading microorganisms in the context of weathered
hydrocarbons in oil-contaminated soil.
Soil and sediment contamination with petroleum hydrocarbons is a
serious ecological problem, warranting remediation of petroleum-
contaminated sites. Bioremediation, among the remediation tech-
niques, is feasible because petroleum compounds including both
branched and unbranched chain aliphatic and aromatic compounds
are amenable to biodegradation process (Chatterjee et al., 2008; Smith
et al., 2015). However, bioremediation of an oil-contaminated soil can
be much influenced by the nature of hydrocarbons and duration of con-
tamination. Moreover, soil microbes are unlikely to respond in the same
way to fresh and weathered motor oils (Loehr et al., 2001). Hence, the
primary objective of this study was to investigate the efficiency of differ-
ent strategies of bioremediation of soil collected from a site that has
been contaminated for a long time with engine oils and other petroleum
products from automobiles or through any unintentional disposal. In
our most recent study, two strains of Pseudomonas sp., P. putida TPHK-
1 and P. aeruginosa TPHK-4, capable of growing on petroleum hydrocar-
bons, were isolated from oil-contaminated sites, and their great poten-
tial in degrading diesel and n-alkanes has been well established
(Ramadass et al., 2016). Bioaugmentation using efficient bacterial
strains is an effective approach for bioremediation of soils polluted
with organics (Megharaj et al., 2011; Kuppusamy et al., 2016a,
2016b). Therefore, these strains were further used in the present micro-
cosm study for their efficiency in bioremediation of engine oil-
contaminated soil following bioaugmentation.
The efficiency of microbes in biodegradation of hydrocarbons can be
better evaluated when bioavailability/bioaccessibility of these com-
pounds is clearly known. Since bioavailability is the tendency of individ-
ual oil components to be taken up by microorganisms, it should be
considered as a priority research objective in the field of bioremediation
(Semple et al., 2003; Riding et al., 2013; Naidu et al., 2015; Ren et al.,
2018a, 2018b). However, it is a poorly quantified regulatory factor for
the biodegradation of oil and other organic compounds in contaminated
soils (Naidu et al., 2008; Harmsen and Naidu, 2013). Most of the bio-
availability studies published to date have focused on individual com-
pounds or representatives for a group of compounds such as
polycyclic aromatic hydrocarbons (PAHs) and polychlorinated
969
biphenyls (PCBs) (Liste and Alexander, 2002; Latawiec and Reid,
2010). Only very meagre information is now available on bioavailability
of complex chemical mixtures in soils and sediments (Muijs and Jonker,
2011; Ortega-Calvo et al., 2015). Hence, another objective of the present
study was to evaluate the bioavailability of a complex mixture of weath-
ered hydrocarbons in oil-contaminated soil. To predict the bioavailabil-
ity of complex petroleum hydrocarbon mixtures from engine oil-
contaminated soil, most frequently followed chemical analytical
methods involving cyclodextrin (McAllister and Semple, 2010; Lal
et al., 2015) or butanol (Liste and Alexander, 2002) extraction were
used in the present investigation. Also, validity of the data from different
approaches of bioremediation followed in the present investigation was
verified using the recently developed assay of 16S rRNA-based pyrose-
quencing for assessing microbial diversity in soils contaminated with
environmental pollutants (Ramadass et al., 2015a; Kuppusamy et al.,
2016a, 2016b).
2. Materials and methods
2.1. Soil sample and bacterial isolates
Engine oil-contaminated soil was collected from an automobile ser-
vice and disposal yard in South Australia. The soil sample was homoge-
nized and passed through a 2-mm sieve. Some of the physico-chemical
characteristics of the soil measured, following standard methods, were:
pH, 7.6; sand, 24%; silt, 60%; clay, 16%; electrical conductivity, 890
μS cm−1; cation-exchange capacity, 2.1 meq 100 g−1; N0− 3 nitrogen,
90 mg L−1; NH+ 3 nitrogen, 3.3 mg L
−1
; total petroleum hydrocarbons,
39,000–41,000 mg kg−1; Cu, 798 mg kg−1; Pb, 355 mg kg−1; Cd,
0.7 mg kg−1; Zn, 604 mg kg−1; Cr, 56 mg kg−1 and Ni, 86 mg kg−1.
The pH and electrical conductivity were measured in 1:5 (soil:water;
w/v) slurry using Smart CHEM-Lab (TPS Pty Ltd., Brisbane, Australia).
Soil particle size distribution was determined using the micropipette
method (Miller and Miller, 1987), and the soil organic carbon (OC) con-
tent by the wet oxidation method (Walkley and Black, 1934). The con-
centration of nitrate and ammonia in the soil-water extract was
determined using ion chromatography (IC) with a Dionex ISC-2000
Ion Analyzer fitted with an AS18 column (Dionex). Heavy metal analysis
was conducted using ICP-MS Agilent 7500c (Inductively Coupled
Plasma Mass Spectrometer (ICP-MS Agilent Technologies, Tokyo,
Japan)) after extraction in a micro-wave accelerated reaction system
(CEM- MARS X®, CEM Corporation, Matthews, NC, U.S.A) (Ramadass
et al., 2015a, 2015b). P. putida TPHK-1 and P. aeruginosa TPHK-4, iso-
lated earlier from petroleum-contaminated soil that exhibited a great
potential in degrading high concentrations of hydrocarbons
(Ramadass et al., 2016), were used for bioaugmentation together with
biostimulation. Bacterial strains were grown up to a late log phase
using 500 mL of minimal salts medium (MSM). The cells were har-
vested, washed in sterile Bushnell-Hass medium (BHM), and pellets
were resuspended in sterile BHM to obtain an optical density at
600 nm of 1.0 (5 × 1010 cells mL−1). The cell suspension was further di-
luted in MQ water for inoculation to provide a final soil moisture level at
60% water-holding capacity (WHC).
2.2. Soil microcosm study
Soil microcosms consisted of portions (400 g) of engine oil-
contaminated soil, placed in 1 L glass jars, maintained at 60% WHC,
and incubated for 30 weeks at room temperature (25 ± 2 °C). Abiotic
controls (T1) were prepared by mixing soil samples with 2% HgCl2
(Cerniglia, 1992). Ammonium nitrate and potassium dihydrogen phos-
phate were used as nutrient amendments to provide a molar ratio
100:10:1:2.5 of carbon (C), nitrogen (N), phosphorus (P) and potassium
(K), respectively, as recommended for bioremediation studies
(Alexander, 1999). The details of different treatments (T2–T7) included
in microcosms like natural attenuation, bioaugmentation using two
970 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974
Table 1
Details of treatments used for bioremediation of engine oil-contaminated soil (OCS).
Treatment Treatment details
T1 Sterilized (2% HgCl2) OCS
T2 OCS
T3 OCS + inorganic NPK
T4 OCS + P. putida TPHK-1
T5 OCS + P. aeruginosa TPHK-4
T6 OCS + P. putida TPHK-1 + inorganic NPK
T7 OCS + P. aeruginosa TPHK-4 + inorganic NPK
bacterial strains, and biostimulation with the application of inorganic
fertilizers (NPK) are presented in Table 1. Each treatment was replicated
in three microcosms. Twice a week, each microcosm was aerated and
moisture level maintained using deionized water.
2.3. Analysis of soil samples for total hydrocarbons
To determine TPHs concentrations, microcosm soil samples were
withdrawn at 0, 7, 15, 30, 60, 90, 150 and 210 days for extraction with
a mixture of dichloromethane (DCM) and acetone (1:1). The solvent ex-
tracts were analyzed for total hydrocarbons using a gas chromatograph
fitted with a flame ionization detector (GC-FID Agilent model 6890).
Chromatography was performed on a fused-silica capillary column
BPX-5 from SGE (15 m × 0.32 mm internal diameter) coated with HP-
5 (0.10-μm film thickness). Helium was used as the carrier gas at
2.5 mL min−1, and the FID detector temperature was kept at 300 °C.
Split-less injection with a sample volume of 1 μL was applied. The
oven temperature was increased from 50 to 300 °C at a gradient of 25
°C min−1, and held at this temperature for 5 min. The total run time
was 19.6 min (Risdon et al., 2008).
2.4. Analysis for bioavailable concentrations of hydrocarbons
A mild extraction technique for estimating bioavailable fractions of
hydrocarbons in microcosm soil samples using aqueous
hydroxypropyl-β-cyclodextrin (HPCD) was adopted as described ear-
lier (Reid et al., 2000). Triplicates of dried and sieved soil samples
(1.25 g) from each microcosm were taken into 40 mL Teflon centrifuge
tubes, and 25 mL of 50 mM aqueous solution of HPCD was added to each
tube. Extract of HPCD from an empty tube served as an analytical blank.
The tubes were sealed and placed on an orbital shaker (Fisher Scientific,
Australia), set at 100 rpm, for 20 h prior to centrifugation at 27,000 ×g
for 30 min (Beckman JA21/2 centrifuge, USA). The supernatant was
liquid–liquid extracted using 10 mL of DCM. Organic phases were
dehydrated by passing through anhydrous Na2SO4, combined, and con-
centrated to 1.0 mL under gentle stream of nitrogen. The method of Liste
and Alexander (2002) was also adopted for mild extraction of hydrocar-
bons using n-butanol. Briefly, 15 mL of n-butanol was added to 5 g por-
tions of moist soil, and then suspended in a vortex mixer for 2 min. The
suspension was centrifuged, and the supernatant was separated from
the soil portion.
2.5. Soil dehydrogenase assay
To determine the influence of hydrocarbon pollution on microbial
activity in engine oil-contaminated soil, dehydrogenase activity was
measured at periodic intervals in microcosm soil samples, and
expressed in terms of triphenyl formazan (TPF) formed from triphenyl
tetrazolium chloride (μg TPF g−1 soil h−1) as described previously
(Ramadass et al., 2015b).
2.6. Amplification of 16S rRNA genes and pyrosequencing
The DNA from total microbial community was isolated from 1.0 g
soil sample of each treatment using MoBio soil DNA extraction kit.
Purpose
Abiotic control
Natural attenuation
Enhanced natural attenuation (biostimulation)
Bioaugmentation
Bioaugmentation
Bioaugmentation + biostimulation
Bioaugmentation + biostimulation
Amplification of 16S rRNA genes and pyrosequencing of all the ex-
tracted soil DNA was performed at the Australian Genomic Research
Centre (AGRF), Brisbane, Australia. PCR amplicons were generated
using primers, AmpliTaq Gold 360 mastermix (Life Technologies,
Australia), and conditions as outlined previously (Ramadass et al.,
2015a). The resulting amplicons were measured by fluorometry, nor-
malized and measured by qPCR, normalized again second time, and
then pooled in equimolar ratios. This amplicon pool was then run on
the GS-FLX platform using XLR70 chemistry (Roche, Australia). Since
Roche 454 GS FLX pyrosequencing can provide one million sequence
reads of 500 million base pairs of sequence information in a single run
at relatively low cost (Voelkerding et al., 2009), this strategy has been
adopted for environmental microbial diversity (Ramadass et al.,
2015a; Kuppusamy et al., 2016a, 2016b). We also followed 454 pyrose-
quencing here for microbial diversity profiling before and after biore-
mediation of soil contaminated long-term with weathered
hydrocarbons.
2.7. Statistical analysis
All statistical tests were conducted using SPSS version 17.0. Duncan's
multiple-range (DMR) test was applied to determine the statistical sig-
nificance of mean values (n = 4) at P ≤ 0.05 (Ramadass et al., 2015b).
3. Results and discussion
3.1. Bioremediation of soil contaminated with weathered hydrocarbons in
engine oil
The engine oil-contaminated soil was silty loam type with neutral
pH. The total organic carbon content was 4.7%, while NO3-N, available
phosphorus (Olsen-P) and K were 90, 70 and 375 mg kg−1, respectively.
Since a ratio 100:10:1:2.5 of C, N, P and K, respectively, was recom-
mended for bioremediation of organic contaminants (Alexander,
1999), the nutrient status of soil used was adjusted to this ratio by ap-
plying inorganic fertilizer compounds, ammonium nitrate and potas-
sium dihydrogen phosphate. Also, these ratios of C:N and C:P were
used as they are known to provide the required quantity of N and P to
convert 100% petroleum C to cellular biomass (Dibble and Bartha,
1979). Among heavy metals, Cu concentration (798 mg kg−1) in oil-
contaminated soil was found to be higher, followed by Zn, Pb, Ni and Cr.
The range in recovery of different fractions of hydrocarbons from
treatments T1-T7 of engine oil-contaminated soil at 0-day following
DCM + acetone extraction was 860–1102, 24,500–27,174 and
12,904–14,832 mg kg−1 soil for C8-C14, C15-C28 and C29-C36, respec-
tively. Very limited presence of short chain aliphatic hydrocarbons
(bC14) in soil samples indicates the larger extent of weathering already
occurred in engine oil-contaminated soil. Also, the solvent extracts ob-
tained on day 0 revealed that the concentrations of TPHs in all the treat-
ments (T1–T7) were nearly the same. In all the treatments of
microcosm study, there was a rapid increase in biodegradation of
TPHs until only 90 days of incubation, and then followed by a slow
rate of degradation in soil samples contaminated with engine oil
(Fig. 1). The extent of bioremediation in any of the treatments
used was not N74% although soil samples were incubated for a total
 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974
100
T1 T2 T3
T4 T5 T6
T7
80
TPHs degradation (%)
60
40
20
0
0 30 60 90 120 150 180
Sampling time (days)
Fig. 1. Bioremediation of weathered hydrocarbons in engine oil-contaminated soil
samples. Data are the means of four replicates.
period of 210 days. The reason for incomplete bioremediation could be
due to the presence of appreciably higher concentrations
(39,000–41,000 mg kg−1) of TPHs in the selected oil-contaminated
soil sample since it was established that bioremediation of soils polluted
with TPHs at 50,000 mg kg−1 is very difficult to occur (Admon et al.,
2001; US EPA, 2004). Abiotic control samples (T1) exhibited only 4% de-
crease in hydrocarbons, possibly accounting for chemical degradation
during this period. Natural attenuation (T2), mediated by indigenous
hydrocarbon-degrading soil microorganisms resulted in 41% degrada-
tion of TPHs. Joo et al. (2008) opined that oil-contaminated soils exhibit
enhanced microbial hydrocarbon-degrading capacity (natural attenua-
tion) due to prior exposure to hydrocarbons. Surprisingly, soil amend-
ments with NPK (T3 and T4) employed for enhanced natural
attenuation resulted in pronounced decrease (17%) in hydrocarbon
degradation as compared to natural attenuation. Although NPK fertil-
izers were used for selective enhanced microbial growth followed by
biodegradation of TPHs in oil-contaminated soil, there was no biostim-
ulation in bioremediation.
The negative impact of nutrient amendments on TPHs biodegrada-
tion is not uncommon. For instance, Akbari and Ghoshal (2014) re-
ported inhibition in degradation of oil with the application of N
content. Braddock et al. (1997) correlated the negative impact of N ap-
plication for hydrocarbon remediation to salinity increase due to
partitioning of fertilizer salts into the pore water. Similarly, Wang
et al. (2012) noticed a negative impact of nutrient addition on popula-
tions of TPH degraders in soil. Although the purpose of nutrient amend-
ments for contaminant removal is to stimulate metabolic activities in
microorganisms, Sarkar et al. (2005) did not notice any benefit in TPH
degradation rate with the addition of nutrients because of the toxicity
of ammonia at high levels. Also, very little change in population of
TPH degraders was observed in soil amended with nutrients by
Coulon et al. (2010). Our present observation of clear inhibition in
TPH biodegradation in oil-contaminated soil samples that received
NPK fertilizers clearly suggests that the optimal level of soil N content
necessary for efficient bioremediation following natural attenuation is
around 90 mg kg−1 soil which is the level of N present in originally col-
lected soil, and any additional N would inhibit biodegradation of TPHs.
Expectedly, in soil samples (T4 and T5) bioaugmented with bacterial
strains of Pseudomonas sp. biodegradation of hydrocarbons was greatly
significant when compared to natural attenuation (Fig. 1). In particular,
bioaugmentation of engine oil-contaminated soil samples with
P. aeruginosa TPHK-4 resulted in 22% more degradation of hydrocarbons
than that occurred in naturally-attenuated samples within 90 days. On
the other hand, P. putida TPHK-1 also performed better by effecting
18% more degradation of hydrocarbons when compared with natural
attenuation. Again, a combination of treatments for the purpose of
both bioaugmentation and biostimulation inhibited hydrocarbon
971
degradation in oil-contaminated soil samples (T6 and T7), possibly by
exerting significant toxicity towards bacteria introduced for bioaug-
mentation. Thus, the degradation of hydrocarbons in soil samples that
received P. aeruginosa TPHK-4 or P. putida TPHK-1 along with NPK fertil-
izers was only 31% as against to 41% biodegradation in naturally-
attenuated samples during the corresponding period of 90 days. How-
ever, very little effect of biostimulation and bioaugmentation on popu-
lation counts of TPH degraders as well as TPH degradation in oil-
contaminated soil was reported by Bento et al. (2005).
Of the hydrocarbon-degrading bacteria, species of Pseudomonas such
as P. aeruginosa, P. putida and P. chlororaphis are the best known for hy-
drocarbon utilization as sources of carbon and energy, and for
210 biosurfactants (glycolipid type) production (Das and Chandran, 2011).
The striking capability of the selected strains of Pseudomonas sp. in bio-
remediation of engine oil-contaminated soil observed in the present
study could be due to their ability in producing biosurfactants impli-
cated in biodegradation of hydrophobic petroleum hydrocarbons. Gen-
erally, bacterial isolates are considered to be biosurfactant producers or
emulsifiers if they are hydrophobic, and able to reduce surface tension
(Franzetti et al., 2006). Our preliminary studies indicated that the pres-
ent strains, TPHK-1 and TPHK-4, showed high emulsification activity
(80–82%), and also exhibited surface tension values b 35 mN·m−1 be-
sides high cell surface hydrophobicity (79–82% adherence of cells to hy-
drocarbons) for waste engine oil. Thus, the biosurfactants that may have
been produced by these introduced bacterial strains could facilitate the
solubilization and uptake of insoluble long-chain hydrocarbons from
soil samples contaminated with weathered oil.
3.2. Bioavailability of weathered hydrocarbons in soil contaminated with
engine oil
Recently, there has been a shift in certain regulations of contami-
nated land and assessment policies since the current regulatory proce-
dures tend to overestimate the truly biodegradable fraction of a
contaminant in the ecosystem (Naidu et al., 2008; Harmsen and
Naidu, 2013). Thus, the total measured hydrocarbon concentration in
a soil following exhaustive solvent extraction will not provide an insight
into its potentially biodegradable fraction. Hence, predicting contami-
nants' ‘bioavailable or bioaccessible fraction’ would be useful for pre-
suming “actual” exposure limits in risk assessment (Naidu et al., 2015;
Ren et al., 2018a, 2018b). Non-exhaustive extraction of contaminated
soil using mild organic extractants has been proposed for predicting
the bioaccessibility of PAHs and pesticides for biodegradation (Liste
and Alexander, 2002; Latawiec and Reid, 2010). Majority of bioavailabil-
ity studies dealt with individual aromatic hydrocarbons or PAH com-
pounds, yet very little has been understood on the bioavailability of
aliphatic weathered hydrocarbons in contaminated soils (Stroud et al.,
2007). Hence, this study was focused on evaluating the feasibility of
using mild organic extractants like HPCD (Semple et al., 2003; Riding
et al., 2013; Diplock et al., 2009) and 1-butanol (Liste and Alexander,
2002) to assess the bioavailable and potentially degradable fraction of
weathered hydrocarbons from engine oil-contaminated soils.
A comparison of measured per cent degradation of weathered hy-
drocarbon fractions in different treatments after 210 days following ex-
haustive extraction with a mixture of DCM and acetone was made with
predicted percentage of degradation obtained following either HPCD or
butanol extraction at 0-day in order to determine the extent of bioavail-
able portions of TPHs for bioremediation of engine oil-contaminated
soil. In general, butanol extraction predicted higher TPHs degradation
when compared with HPCD extraction in all the treatments (Table 2).
Thus, the predicted per cent degradation values for butanol and HPCD
extractions were in the range of 53–61 and 36–41, respectively. Except-
ing in soil treatments used for bioaugmentation (T4 and T5), the pre-
dicted degradation of TPHs following butanol extraction was more
than the measured degradation in all other approaches of bioremedia-
tion. For instance, the predicted degradation values for HPCD extraction
972 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974

Table 2
Bioavailability (based on predicted and measured degradation) of weathered hydrocarbon fractions in engine oil-contaminated soil.

Treatment Measured degradation (%) following DCM + Predicted degradation (%) following HPCD Predicted degradation (%) following butanol
acetone extraction (210 days) extraction (0-day) extraction (0-day)

C8-C14 C15-C28 C29-C36 Total C8-C14 C15-C28 C29-C36 Total C8-C14 C15-C28 C29-C36 Total
b a a a a b a a a b ab
T1 57 3 2 4 100 51 40 42 100 59 60 61b
T2 100a 51c 51d 52d 100a 49ab 41a 40a 100a 54a 64b 59ab
T3 100a 32b 29b 33b 100a 47ab 39a 37a 100a 53a 50a 53a
T4 100a 73d 65e 71e 100a 44a 38a 35a 100a 56ab 61ab 59ab
T5 100a 78d 65e 74e 100a 51b 37a 37a 100a 56ab 44a 54a
T6 100a 33b 40c 37c 100a 50ab 38a 39a 100a 55a 47a 53a
T7 100a 33b 42c 38c 100a 49ab 39a 38a 100a 60b 57ab 60b

Refer to Table 1 for the details of different treatments (T1–T7) used. Means (n = 4) in a column sharing the same superscript letter are not significantly different (P ≤ 0.05) according to
DMR test.

and butanol extraction concerning natural attenuation (T2) were 40
and 59%, while the corresponding measured per cent degradation was
52. This could be due to the fact that butanol extraction, being some-
what exhaustive than HPCD extraction, overestimated the bioavailable
portions of TPHs in the treatments used. Our present microcosm study
thus suggests that mild extraction with HPCD can be more appropriate
while predicting the bioavailable concentrations of hydrocarbons pres-
ent even at the higher range of 39,000–41,000 mg kg−1 in weathered
oil-contaminated soils.
3.3. Soil dehydrogenase activity – an indicator of TPHs bioremediation
Pollutants cause serious stresses on microbial community structure,
diversity and enzymatic activities (Li et al., 2005). During remediation,
pollutants and various other substances continuously alter the micro-
bial communities. Soil microbial activities and community diversities
are critical indicators of the ecological health of soils, and hence it is im-
portant to evaluate the dynamics of the whole microbial community
rather than just pollutants-degrading microbes during bioremediation
(Bastida et al., 2008). In line with remediation efficiency, soil ecosystem
health also has to be considered in bioremediation of soil contaminated
with pollutants such as engine oil. Continuous monitoring of microbial
activity is therefore imperative for sustaining a sound soil ecosystem
as well as achieving successful bioremediation. Dehydrogenase is an ex-
clusive intracellular enzyme in a majority of soil microorganisms, and its
activity in soil is an estimation of total oxidative potential of the mi-
crobes which has been used as an index for monitoring microbial activ-
ity (Ramadass et al., 2015b; Alrumman et al., 2015; Richardson et al.,
2015). Likewise, the assay of dehydrogenase in diesel oil-polluted soils
was used as a simple method to examine the possible inhibitory effects
of contaminants on microbial population (Bento et al., 2005). We, there-
fore, wanted to verify if the response of dehydrogenase activity was in
line with the bioremediation potential of different treatments of engine
oil-contaminated soil.
weathered hydrocarbons present in engine oil suggests that the dehy-
drogenase assay could be considered as an indicator for evaluating bio-
remediation potential of a soil contaminated with oils.
3.4. Pyrosequencing – implication to TPHs bioremediation
A shift in the dynamics of microbial communities in soils results due
to selective forces exerted by environmental contaminants (Griebler
and Lueders, 2009). Linking contaminants' biodegradation to phyloge-
netic identity of active microbes has therefore become a main challenge
in bioremediation studies. The distribution, diversity and composition
of bacterial communities in different treatments of soil contaminated
with engine oil were explored at 0 and 210 days following high-
throughput 454 pyrosequencing as this would provide a detailed infor-
mation on taxonomy and metabolic potential of bacteria in bioremedi-
ation (Mason et al., 2012; Yergeau et al., 2012). Very recently,
Kuppusamy et al. (2016a, 2016b) for the first time implicated the 454
pyrosequencing assay in bioremediation of long-term PAHs- and
heavy metal-contaminated soils. In particular, our main objective in
using pyrosequencing was therefore to determine the extent of survival
and growth, in terms of population abundance, of introduced bacterial
strains in order to validate the data obtained on their potential in biore-
mediation of engine oil-contaminated soil.
In all, our results on RNA-based pyrosequencing grouped the se-
quences obtained from all the six soil treatments (T2–T7) into 13 differ-
ent bacterial phyla (Fig. 3). The 0-day results on the relative abundance
of bacterial communities in different treatments of soil contaminated
long-term with engine oil revealed the predominant occurrence of
Proteobacteria followed by other bacterial phylotypes consisting of
Gemmatimonadetes, Actinobacteria, Bacteriodetes and Chloroflexi,
whereas the minor communities included Acidobacteria, Chlorobi,
4.0
T1 T2 T3
T4 T5 T6
3.5
prot)

There was a gradual increase in activity of dehydrogenase in T7

naturally-attenuated soil sample (T2) until 30 days of incubation, and 3.0
1

was inhibited thereafter (Fig. 2). Addition of inorganic fertilizers to the

Dehydrogenase (U mg

oil-contaminated soil (T3) was greatly inhibitory to the enzyme activity. 2.5

Clearly, dehydrogenase activity correlated well with the bioaugmented 2.0

treatments (T4 and T5) that contained the introduced bacterial strains
up to 90 days, and the enzyme was significantly inhibited subsequently. 1.5
As with hydrocarbon biodegradation, P. aeruginosa TPHK-4 performed
1.0
better than P. putida TPHK-1 in expressing dehydrogenase during biore-
mediation of engine oil-contaminated soil. Thus, the bioaugmentation 0.5
mediated by this strain was 2.6-fold when compared with natural atten-
uation at the end of 90 days. Again, the addition of a bacterial strain to- 0.0
gether with NPK to soil samples (T6 and T7) containing weathered 0 7 15 30 90 150 210

hydrocarbons resulted in significant inhibition of bioremediation. In Sampling time (days)

all, the results on dehydrogenase activity clearly support the data on
biodegradation of TPHs in different soil treatments until 90 days. Thus, Fig. 2. Soil dehydrogenase activity in different approaches of bioremediation of engine oil-
the observed response of the enzyme during biodegradation of contaminated soil samples. Data are the means of four replicates.
 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974
100
90
Realtive abundance (%)
80
70
60
50
40
30
20
10
0
T2 a T2 b T3 a
Other
Chlorobi
Gemmatimonadetes
WPS-2
Fig. 3. Changes in bacterial diversity in engine oil-contaminated soil samples during bioremediation. Letters a and b associated with the treatments of T2-T7 on X-axis indicate sampling
times, 0 and 210 days, respectively.
Verrucomicrobia, Firmicutes and WPS-2. Members of Cyanobacteria
and Nitrospirae were totally absent. Similarly, members of
Proteobacteria, Actinobacteria and Acidobacteria were among the bac-
terial phylogenetic groups observed earlier in hydrocarbon-
contaminated soils (Ramadass et al., 2015a; Prince, 2010). Also, Zhang
et al. (2012) reported the dominance of Proteobacteria followed by
Actinobacteria in soils contaminated by a leakage of heavy oil hydrocar-
bons. Thus, Proteobacteria which are known TPH degraders accounted
for nearly 35–37% of the total bacterial phyla in soil samples of T2a
and T3a. An increase of 15–18% in population of the bacterial phylum
Proteobacteria was observed in soil samples (T4a, T5a, T6a and T7a)
that received the inoculum of a TPHs degrader, P. putida TPHK-1 or
P. aeruginosa TPHK-4. This result is expected because the introduced
bacteria belong to Gammaproteobacteria which is one of the three
major classes of Proteobacteria.
Incubation of soil samples with different treatments for 210 days re-
sulted in a major change/shift in bacterial communities during bioreme-
diation. A 2-fold increase in bacterial populations belonging to
Proteobacteria in soil sample T2b is in clear conformity with the signif-
icant natural attenuation resulted (Fig. 1), and suggests that this group
can use TPHs for growth and proliferation. Also, an increase (N3-fold)
in bacteria of the phylum Acidobacteria suggested their possible role
in bioremediation of TPHs in engine oil. In particular, members of
Gemmatimonadetes were significantly decreased from 28 to 0.8%, indi-
cating the selective enrichment of TPH degraders (Proteobacteria and
Acidobacteria) to mediate natural attenuation. The growth of
Actinobacteria, which are typically Gram-positive soil bacteria that can
survive even in dry environment, was also not favored during bioreme-
diation. The striking 19% decline in Proteobacteria in soil sample treated
with inorganic fertilizers (T3b) is a true reflection of the observed inhi-
bition of TPHs bioremediation. Certainly, Actinobacteria, whose popula-
tion significantly increased (50%) possibly under the influence of
inorganic fertilizers, was not involved in TPHs degradation.
The significant increase in members of the phylum Proteobacteria in
engine oil-contaminated soil samples (23% in T4b, and 31% in T5b) that
received P. putida TPHK-1 or P. aeruginosa TPHK-4 clearly corroborates
bioaugmentation mediated by these introduced strains. Moreover,
973
T3 b T4 a T4 b T5 a T5 b T6 a T6 b T7 a T7 b
Soil treatments
Acidobacteria Actinobacteria Bacteroidetes
Chloroflexi Cyanobacteria Firmicutes
Nitrospirae Proteobacteria Verrucomicrobia
there was a significant decline in populations of Actinobacteria in both
these samples, confirming that this phylum is not implicated in TPHs
bioremediation. Again, of particular interest is the clear confirmation
of the results on significant inhibition in biodegradation of TPHs in oil-
contaminated soil samples that received simultaneously both the bacte-
rial strains and inorganic fertilizers. Thus, the pyrosequencing data
showed pronounced decrease (48% in T6b, and 35% in T7b) in bacterial
communities related to Proteobacteria, followed by a significant in-
crease (62% in T6b, and 58% in T7b) in population abundance of
Actinobacteria. Clearly, our study demonstrates that the introduced bac-
terial strains belonging to the phylum Proteobacteria can grow on
higher fractions TPHs present in engine oil-contaminated soil. Further-
more, the soil treatment used for biostimulation significantly inhibited
biodegradation of TPHs. The findings of the present microcosm study
suggest that pyrosequencing data could be used in designing suitable
methods for bioremediation of soils contaminated with environmental
pollutants such as weathered hydrocarbons.
4. Conclusions
In a microcosm study, we assessed the bioremediation potential of
soil long-term contaminated with engine oil following different ap-
proaches such as natural attenuation, biostimulation and bioaugmenta-
tion. Although the soil contained high concentrations of weathered
hydrocarbons, natural attenuation occurred appreciably; however, soil
treatment with NPK fertilizers at the recommended levels for biostimu-
lation resulted in significant inhibition of natural attenuation. Introduc-
tion of hydrocarbon-degrading bacterial strains, P. putida TPHK-1 or
P. aeruginosa TPHK-4 into oil-contaminated soil samples resulted in pro-
nounced bioaugmentation. However, an approach of combining both
bioaugmentation and biostimulation significantly inhibited bioremedi-
ation of weathered TPHs. These results on bioremediation were in ac-
cordance with the response of dehydrogenase activity in soil
contaminated with oil which suggests that the assay of this enzyme
could serve as an indicator for evaluating bioremediation potential of
soils contaminated with hydrocarbons. Extraction of oil-contaminated
soil for weathered hydrocarbons with HPCD, but not butanol, would
974 K. Ramadass et al. / Science of the Total Environment 636 (2018) 968–974

predict their availability better for bioremediation. The data on 16S Liste, H.H., Alexander, M., 2002. Butanol extraction to predict bioavailability of PAHs in
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rRNA pyrosequencing of soil samples with different treatments were Loehr, R.C., McMillen, S.J., Webster, M.T., 2001. Predictions of biotreatability and actual re-
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Megharaj, M., Ramakrishnan, B., Venkateswarlu, K., Sethunathan, N., Naidu, R., 2011. Bio-
This research was supported by the Australian Government, Univer- remediation approaches for organic pollutants: a critical perspective. Environ. Int. 37,
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sity of South Australia through an IPRS scholarship and CRC for Contam- Miller, W.P., Miller, D.M., 1987. A micro-pipette method for soil mechanical analysis.
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