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Enhanced Removal of Hydrocarbons from Crude
Oil Sludge through Phytoremediation with
Biosurfactant-producing Rhizobacteria
To cite this article: Siti Shilatul Najwa Sharuddin
et al
2024
IOP Conf. Ser.: Earth Environ. Sci.
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International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
IOP Publishing
doi:10.1088/1755-1315/1307/1/012009
1
Enhanced Removal of Hydrocarbons from Crude Oil Sludge
through Phytoremediation with Biosurfactant-producing
Rhizobacteria
Siti Shilatul Najwa Sharuddin1*, Siti Rozaimah Sheikh Abdullah 1*, Hassimi Abu
Hasan1,2, Ahmad Razi Othman1, & Israa Abdulwahab Al-Baldawi3
1 Department of Chemical and Process Engineering, Faculty of Engineering and Built
Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia
2 Research Centre for Sustainable Process Technology (CESPRO), Faculty of
Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM
Bangi, Selangor, Malaysia
3Civil Engineering Department, College of Engineering, University of Baghdad,
Baghdad, Iraq
*Corresponding author: Email: sitishilatulnajwa@gmail.com; rozaimah@ukm.edu.my
Abstract. Discharge of crude oil (or its products) during the extracting, refining, and transporting
into the environment have caused serious environmental distress due to their highly hydrophobic
resistance, and persistence in nature and very difficult to be remediated from the environment.
Therefore, an environmentally conscious approach to enhance the bioavailability (or solubility)
of petroleum hydrocarbon pollutants in soil involves the utilization of biosurfactants.
Biosurfactants play a crucial role in enhancing the desorption and solubilization of petroleum
hydrocarbons, facilitating their assimilation by microorganisms. This research investigated the
application of biosurfactant supplementation derived and purified from rhizobacteria of Scirpus
grossus, which are capable of producing biosurfactants and degrading hydrocarbons, in the
context of phytoremediation. The crude oil sludge used in this study was obtained from an
industrial area containing 56,600±3;900 mg/kg of total petroleum hydrocarbon (TPH). The crude
oil sludge was inoculated with biosurfactant, sodium dodecyl sulfate (SDS) as commercial
surfactant and only with the presence of S. grossus in the vegetated tanks and monitored for 90
days in a greenhouse. The results indicated that the growth of S. grossus with the addition of
biosurfactant was improved and new saplings were produced. After a 90-day exposure period,
the removal efficiency of TPH from the soil demonstrated significant increases, reaching 90.3%,
84.1%, and 73.7% when treated with biosurfactant+S. grossus, SDS+S. grossus, and S. grossus
only respectively. These percentages were notably higher compared to the non-planted control
crates (CC) where the removal efficiency was only 17.9%. These results provide evidence that
the introduction of biosurfactant through inoculation can elevate the bioavailability of organic
pollutants, consequently augmenting their microbial degradation in the soil.
Keywords: biosurfactant; bioremediation; crude oil sludge, phytoremediation, bioavailability
International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
IOP Publishing
doi:10.1088/1755-1315/1307/1/012009
2
1. Introduction
Oil refinery serves as an industrial process plant dedicated to refining crude oil, transforming it into over
2500 valuable refined products. These include liquefied petroleum gas, gasoline, heating oil, diesel fuel,
fuel oils, lubricating oils, and feedstocks essential for the petrochemical industry [1,2]. The
comprehensive range of refinery operations encompasses all aspects of handling, refining, and storing
petroleum, preparing the products for shipment to various industries before reaching consumers [3,1].
However, large amounts of unwanted leakage of petroleum oil and its products produced during the
refinery process have polluted about 80% of lands [4]. The pollution of petroleum oil to the soil promotes
extensive changes in the chemical and physical properties of soil and subsequently introduces negative
effect to human health and the environment [5,3]. According to [3], the total petroleum hydrocarbon
(TPH) content in crude oil sludge typically ranges from approximately 15% to 50% (percentage of
mass). Concurrently, the water content falls within the range of 30% to 85%, and the solid content ranges
from 5% to 46%. The complexity of crude oil sludge composition has led to environmental distress due
to their high hydrophobicity (poor biodegradation efficiency), resistance, and persistence in nature and
very difficult to remediate from the environment [6].
Thus, an effective and eco-remediation method is required and must be implemented successfully to
detoxify and remove the polluted areas. Countless of environmental remediation approaches are being
studied and implemented which involve the use of different techniques or a combination of parallel or
consecutive treatment [6]. Even so, bioremediation through combination of biosurfactant and
phytoremediation have been prioritized for alternative treatment because they cost less and are more
eco-friendly than thermal or chemical treatment [7]. Biosurfactants are metabolic by products produced
extracellularly or as part of the cell membrane by microorganism (bacteria, yeast, fungi) [8]. Many
recent studies have demonstrated that biosurfactants produced by the hydrocarbon degradable bacteria
have more strength to remediate such pollution and can apply for diverse application in oil industry
including microbial enhanced oil recovery, and clean-up of oil containers and storage tanks [9]. In
addition, the chemical and physical structure of biosurfactant produced by bacteria have such as less
toxicity, biodegradability, selectivity, higher specificity, and high surface activity has boosted their
application [10]. Phytoremediation, or phytotechnology, is a process that employs plants to detoxify
either organic pollutants (such as petroleum hydrocarbons) or inorganic contaminants (such as heavy
metals) from water and soils [11,5]. This approach has gained widespread and successful application,
particularly in developed countries such as Europe, the USA, and Japan. It is utilized for the treatment
of both organic and inorganic wastes, including liquid forms like wastewater and solid forms such as
sludge or contaminated soil [11].
According to [12], biosurfactant alone are competent of elevating the biodegradation process but the
significant increase in the rate of biodegradation was detected when compared to the treatments when
biosurfactants and phytoremediation were combined. This is because certain bacterial strains, isolated
from the rhizospheres of plants, possess the ability to metabolize petroleum-derived compounds. These
bacteria can produce biosurfactants, making them valuable for the decontamination of polluted
environments. However, only a few studies have addressed the combination treatment of biosurfactant,
and phytoremediation of soils contaminated with real waste of petroleum hydrocarbons, and they usually
test on artificially contaminated soils as single treatment. Therefore, the aim of this current study was to
assess the effectiveness of purified biosurfactant in conjunction with Scirpus grossus plants for the
degradation of total petroleum hydrocarbon (TPH) in real crude oil sludge waste.
2.0 Materials & Methods
2.1. Chemicals and Crude Oil Sludge
All chemicals employed in this study, including Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB)
for bacterial growth, were procured from Fisher Scientific (M) Sdn. Bhd. The real crude oil sludge was
International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
IOP Publishing
doi:10.1088/1755-1315/1307/1/012009
3
obtained from a Malaysian-based oil refining industry. The crude oil sludge underwent homogenization
and was securely sealed in a clean container before the commencement of the study.
2.2 Plant Preparation
S. grossus was taken from its natural habitat (wetlands) at Tasik Chini, Pahang. The growth of microbial
population near the roots of S. grossus is increasing due to the large, extensive, and widely branched
root system [13]. All plants were propagated in a greenhouse located in Universiti Kebangsaan Malaysia
where the plant growth was closely monitored.
2.3 Preparation of Biosurfactant, Supernatant and Commercial Surfactant
In this present study, a biosurfactant producing microorganisms, known as Bacillus sp. strain SB1,
Bacillus sp. strain SB3 and Lysinibacillus sp. strain SB6 were selected to perform the experiments based
on its capability to extract biosurfactant and shows great performance for hydrocarbon degradation
process. These strains were isolated from roots of S. grossus contaminated by petroleum hydrocarbons
as conducted in a previous study [9] and stored in our laboratory. The bacteria producing biosurfactant
was mixed with carbon and nitrogen source in 100 ml minimal salt medium (MSM). The incubation was
carried out in a shaker for 7 days at 150 rpm. Then, the extraction process of purified biosurfactant was
adapted from [9]. The commercial surfactant, sodium dodecyl sulfate was prepared accordingly. The
concentration of purified biosurfactant and commercial surfactant were amended to the crude oil sludge
was 1.5 L in each treatment tank of 30 kg crude oil sludge.
2.4 Experimental Phytoremediation Design and Sampling
Experimental design as shown in Table 1 consists of 5 treatments in 3 replicates (R1, R2, R3). The five
treatments were: (1) no crude oil sludge, only S. grossus + garden soil (Plant Control, (PC)) ;(2) crude
oil sludge only without S. grossus (Control Contaminant, (CC)); (3) crude oil sludge + S. grossus (SC);
(4) crude oil sludge + S. grossus + biosurfactant (CSB); (5) crude oil sludge + S. grossus + commercial
surfactant (CSC). The treatment study was conducted in polyethylene tanks, each with a dimension of
60 cm × 40 cm × 30 and about 30 kg of crude oil sludge was placed in each treatment tank. Water supply
to the soil was provided around 50% amount during the experimental period. The treatment was
conducted about 90-day exposure. The soil from each tank was sampled for TPH analysis from CC, SC,
SCB and SCC on day 0, day 60 and day 90 and stored at 4°C for TPH analysis. The growth of S. grossus
was monitored physically (healthy or died) during the exposure period.
2.5 Analysis of TPH
The TPH analysis involved combining 10 g of the soil sample with 2 g of anhydrous sodium sulfate.
This mixture was subjected to extraction using 50 ml of dichloromethane in an Ultrasonic solvent
extraction, following the modified USEPA 3550C method [14]. After extraction, the resulting extracts
underwent rotary evaporation. The TPH sample obtained was then submitted for GC-FID analysis, and
the TPH concentration in the crude oil sludge was determined using the provided equation.
TPH Concentration =
( )
() Eq.1
The percentage of TPH degradation on Day 90 was calculated by dividing the difference between the
current TPH value and the initial TPH value by the initial TPH value, as shown in the following equation:
TPH Removal (%) =
100 Eq. 2
International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
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doi:10.1088/1755-1315/1307/1/012009
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with, TPH0 = total petroleum hydrocarbon on sampling day 0, and TPHSD = total petroleum hydrocarbon
on each sampling day.
Table 1 Schematic diagram for treatment process.
Parameter
Treatment Replicate
R1
R2
R3
Plant Control (PC)
Control Contaminant (CC)
S. grossus + crude oil sludge (SC)
S. grossus + crude oil sludge + biosurfactant
(SCB)
S. grossus + crude oil sludge+ commercial
Surfactant (SCC)
X=S. grossus
2.6 Statistical data analysis
Statistical analysis of the results was conducted using Statistical Package for the Social Sciences (SPSS)
Version 21.0. The experiments were performed in triplicate to account for experimental errors in each
parameter. Mean values marked with different letters indicate significant differences, determined
through an analysis of variance (ANOVA). The significance level for comparing differences was set at
p<0.05.
3. Materials & Methods
3.1 Characterization of crude oil sludge
Several parameters such as pH, nutrients such as nitrite, nitrate and phosphorus, ammonia nitrogen and
initial TPH was previously measured [13].
3.2 Plant Growth Survival
The condition of S. grossus was observed physically throughout 90 days of exposure period as tabulated
in Table 1. Over the 90-day treatment period, noticeable differences in the appearance of plants were
observed in each treatment tank with varied conditions, as compared to those in the control group. As
presented in Table 2, the condition of S. grossus in treatment control (PC) were stay healthy from day 0
until day 90. Difference situation occurs in the treatment tank of SC, SCB and SCC when all plants
portrayed sign of toxicity and distress such as yellowing of leaves/stem and the growth performance
became slower compared with the plants in PC tank. According to [5], petroleum oil and its constituents
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
X X X
International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
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doi:10.1088/1755-1315/1307/1/012009
5
can decrease the availability of oxygen, water, and nutrients in soil, and as a result declining the seed
germination rate and affecting the plant growth as obviously shown by S. grossus in all treatments tanks
except from PC tank.
Table 2 Physical Observation of S. grossus to crude oil sludge contaminants
Parameter
Physical Observation
Day 0
Day 60
Day 90
Plant Control
(PC)
S. grossus +
crude oil
sludge + (SC)
S. grossus +
crude oil
sludge +
Biosurfactant
(SCB)
S. grossus +
crude oil
sludge+
Commercial
Surfactant
(SCC)
However, as depicted in Table 2 (yellow circle), the presence of few new saplings in SCB tank at
day 60 and increasing number of saplings were observed at the end of exposure period. Meanwhile, both
treatments tanks of SC and SCC have non new healthy sapling at the end of exposure period.
Phytoremediation, a highly effective bioremediation method, has been widely employed for the
remediation of various potentially organic contaminants, such as bauxite [15], gasoline [16], and
hydrocarbons [13]. In this current study, the growth of S. grossus as phytoremediation agent was
improved with the inoculation of biosurfactant due the increasing bioavailability of organic pollutants
in the form of total petroleum hydrocarbon (TPH). As indicated by [7], the interactions between plants
and microorganisms have been extensively researched for the purpose of hydrocarbons treatment.
Microorganisms that produce biosurfactants play a crucial role in enhancing the solubility of soil-
contaminated oil, thereby increasing its bioavailability to plants.
Improvement in the plant-microorganisms interactions promotes plant biomass and tolerance to
petroleum hydrocarbon contaminants which supports the findings of this study when the growth
performance of S. grossus is improved in the presence of biosurfactant. Interactions between plants and
microorganisms result in elevated population densities of microbes and increased metabolic activity in
the rhizosphere, particularly under challenging conditions such as soil pollution [17,7].
International Conference on Environmental and Earth Sciences 2023
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doi:10.1088/1755-1315/1307/1/012009
6
3.3 Degradation of TPH
After a 90-day treatment period, the percentage removal of TPH and concentration in treatment tanks:
CC, SC, SBC, and SCC are illustrated in Figure 1. The results revealed that the SCB treatment tank
exhibited the highest TPH removal percentage (92.7%), resulting in a TPH concentration of
approximately 5021 mg/kg after the 90-day exposure period. In comparison, the TPH removal
percentages for SC and SCC treatment tanks were 73.7% and 84.1%, respectively. These figures were
notably higher than that of the non-planted treatment tank (CC), which only showed a minor reduction
(17.9%) in TPH concentration.
According to [1], one of the importance limiting factor on hydrocarbon petroleum remediation is the
low bioavailability of these compound. The inoculation of biosurfactant into the crude oil sludge
increased the uptake of TPH by the S. grossus which might be due to the low solubility of these
contaminants. The purified biosurfactant secreted by the rhizobacteria from the previous study and
released into the hydrophobic medium increase the bioavailability of the hydrocarbons. In additions, the
desorption of hydrocarbon was promoted when the attraction forces have been reduced hence the
removal of TPH is significantly higher than two others treatment tank without the presence of
biosurfactant. In other words, the degradation of hydrocarbons in the presence of biosurfactant was
elevated due to modification of physicochemical properties of contaminants [18].
Figure 1 Concentration and percentage removal of TPH after 90 days exposure study
According to a study by [19], the combination of plants with hydrocarbon-degrading bacteria has
shown promise for the effective remediation of hydrocarbon-contaminated soils. Another investigation
by [20] demonstrated that the removal of total petroleum hydrocarbons (TPH) was significantly higher
(approximately 58%) when phytoremediation was supplemented with biosurfactants compared to
sunflower cultivation without biosurfactant supplementation. Additionally, the application of
biosurfactants in the phytoremediation of gasoline-contaminated soil, as reported by [16], resulted in the
removal of up to 93.5% of TPH from the soil. Consequently, the findings from the current study
underscore the potential of S. grossus in conjunction with biosurfactant supplementation for enhancing
TPH removal. In addition, this study also emphasizes the crucial role of plant-microbe interactions in
the success of bioremediation efforts targeting petroleum contamination.
0
10
20
30
40
50
60
70
80
90
100
0
10000
20000
30000
40000
50000
60000
70000
80000
Day 0 Day 60 Day 90 Day 0 Day 60 Day 90 Day 0 Day 60 Day 90 Day 0 Day 60 Day 90
CC SC SCB SCC
Percentage Removal TPH (%)
Concenntration TPH (mg/kg)
Treatment
Concentration TPH % Removal TPH
A
A
A
A
B
B
A
B
B
A
B
B
International Conference on Environmental and Earth Sciences 2023
IOP Conf. Series: Earth and Environmental Science 1307 (2024) 012009
IOP Publishing
doi:10.1088/1755-1315/1307/1/012009
7
4.0. Conclusions
Phytoremediation studies with the inoculation of biosurfactant in hydrocarbon-contaminated soils
showed that the growth of S. grossus was improved by the appearance of new sapling throughout the
treatments period. Moreover, this biosurfactant was able to reduce the concentration of TPH from initial
TPH concentration, 62,311 mg/kg reduced to 5021 mg/kg of TPH by 93% removal of TPH at the end
of 90 days. The biosurfactant, with its ability to emulsify and disperse water-insoluble compounds such
as hydrocarbons, likely contributed to enhanced bioremediation efficiency. This integrated approach,
combining phytoremediation with the inoculation of biosurfactants, holds considerable promise for the
remediation of hydrocarbon-contaminated soils, offering an effective solution without adverse
environmental impacts.
Acknowledgements
The authors would like to extend their gratitude to the Ministry of Higher Education, Malaysia, the
Faculty of Engineering, and Built Environment (FKAB) and Universiti Kebangsaan Malaysia for
funding this research (GUP-2022-022).
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