Enhanced Removal of Hydrocarbons from Crude Oil Sludge through Phytoremediation with Biosurfactant-producing Rhizobacteria

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.


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.

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 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.

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.

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.

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.

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.with, TPH0 = total petroleum hydrocarbon on sampling day 0, and TPHSD = total petroleum hydrocarbon on each sampling day.

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].

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 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.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 soilcontaminated 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].

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].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.

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.
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 (%) = TPH 0 −TPH SD TPH 0  100 Eq. 2

Figure 1
Figure 1 Concentration and percentage removal of TPH after 90 days exposure study 60 Day 90 Day 0 Day 60 Day 90 Day 0 Day 60 Day 90 Day 0 Day 60 Day 90

Table 1
Schematic diagram for treatment process.

Table 2
Physical Observation of S. grossus to crude oil sludge contaminants