Soil samples from a contaminated site at a depth of 60 cm were collected from an “oil production and exploration company” situated in the district of Chakwal, Pakistan (32.988682°N, 72.925756°E). These soil samples were ground using a mortar and pestle, air-dried, sieved via a 2 mm mesh, homogenized, and analyzed for various physiochemical parameters (Table 1) as described by Mohanty and Patra (2011). Plant root and shoot samples were also collected from contaminated soil pots for the measurement of growth parameters such as root and shoot length and fresh and dry weight of root and shoot after 60–120 days.

For the experiment, locally available perennial species of Para grass (Brachiaria mutica) plants were used as a phytoremediation entity (Dietz and Schnoor, 2001; Collins, 2007). The standard method of propagating Brachiaria mutica is by planting enrooted cuttings as they are easy to handle and economical too (Schaff et al., 2002). The cuttings of these plants were collected from the natural fields in nearby areas and soaked in clean water for 10 days to increase their survival and growth rate. As soon as an initial root emerged from soaked cuttings, they were planted in a pot according to the treatment plan. Different concentrations of solubilized Triton X-100 were used as a surfactant. All the treatments were used in triplicates. The detailed treatment plan is described as follows:

C1: soil + plant

T1: soil + crude oil + plant

T2: soil + crude oil + plant + bacteria

T3: soil + crude oil + plant + surfactant (0.01%)

T4: soil + crude oil + plant + surfactant (0.1%)

T5: soil + crude oil + plant + surfactant (1.0%)

T6: soil + crude oil + plant + surfactant (0.01%) + bacteria

T7: soil + crude oil + plant + surfactant (0.1%) + bacteria

T8: soil + crude oil + plant + surfactant (1.0%) + bacteria

The physical investigation of the soil revealed that it belonged to the taxonomic category of loamy sand. The soil was slightly acidic, with mean pH values of 6.5 and 4.5. The pH of the soil plays an important role in the adsorption of heavy metals and regulates the hydrolysis and solubility of metal carbonate, phosphate, and hydroxide (Afzal et al., 2011). Although EC has no direct effect on plant growth, it can be used to determine the number of nutrients available for plant uptake and the salinity level of soil that can inhibit microbial growth (Anwar ul Haq et al., 2018).

The total microbial count was determined by using the pure plate technique after 60 days of overtime zero according to Samuel et al. (2018) with minor modifications. From each pot, 1 g of soil was taken. Colonies that developed after incubation were counted, and then, the total bacteria count was measured and expressed as CFU (colony-forming units) per gram of each soil sample.

The simplest and most obvious agronomic parameters such as shoot and root length (cm) and shoot and root, both dry and fresh, weight (g) were measured by using a meter rod and analytical balance, respectively, at the time of harvest after the 4-month experiment.

The most applicable technique to get quality and functional group-related information about petroleum-related materials is the Fourier-transform infrared (FTIR) analysis technique. The technique uses IR (infrared) light to scan and observe the chemical properties of test samples. Samples were analyzed by FTIR spectroscopy to check the different functional groups of crude oil-contaminated soil (COCS). Samples were ground and mixed with 1 mg of contaminated soil with 200 mg of KBr (IR grade). With a hydraulic press system, the samples were transformed into a pellet. The pellet sample was analyzed by using an FTIR spectrophotometer (Thermo Fisher Scientific, United States). An absorbance spectrum was collected in the range of 400–4,000 cm⁻¹ (Aziz et al., 2015).

The parameters for plant growth and the amount of TPH are presented as mean ± SE of three replicates. The statistical significance of differences among treatments at p < 0.05 was analyzed.

Removal of crude oil is due to the activities of naturally occurring soil microorganisms in non-vegetated soil, and this soil microbial activity increases if plants are present in the soil that aid the breakdown of the pollutants (Juck et al., 2000) The roots play an important role because they provide a large surface area for the degradation of crude oil and provide nutrients which are essential for the growth of microorganisms (Ma et al., 2016). Hyperaccumulator plants such as Pterisvittata are very effective and potentially useful for the removal of crude oil-contaminated soil. Soil pH is affected by a variety of factors, including mineral concentration and texture of the soil (Njoku et al., 2008). The soil which is used in the experiment was alkaline. It has been studied that crude oil contamination increases soil acidity and makes soil toxic. Reduced pH has been studied as phytoextraction, an effective technique for the removal of heavy metals that are present in contaminated soil. Naturally, the plant absorbs ammonium which decreases the pH and has been shown to lower the rhizosphere (Villegas and Fortin, 2001). During the research, the moisture content of the soil changed across a wider range due to variables such as wind, watering frequency, humidity, and plant transpiration rate.

All studied growth parameters showed significantly reduced growth under contaminated soil environments. Shoot length as a function of experimental time is presented in Figure 1). The value of shoot length after 60 days showed (62–86 cm, 43.43 g) no non-significant increase compared to that measured at the harvesting time (65.73 cm, 53.94 g). This confirmed a stunted growth behavior under crude oil-contaminated environments. Similarly, throughout the growth period of the plant, root growth was inhibited. However, plants augmented with a mixed consortium of bacteria help in growth along with different levels of chemical surfactant (Triton X-100). The maximum growth of the plant (root and shoot length and dry biomass) was observed in the pot having an application of surfactants (0.1%) augmented with the bacterial consortium (Figures 1, 2). After 60–120 days, plant dry biomass was observed and found in the form of root and shoot dry weight, and it was not increased significantly as affected in the co-contaminated soil and showed premature death. Growth rates continuously increased with time but by a lesser amount as compared to initial harvesting at 60 days (Figure 2).

The contaminated soil (crude oil) showed injurious effects on plant growth, thus leading to adverse effects on all studied growth traits, particularly the germination process. The process of germination, being sensitive, may be influenced by the presence of soil contaminants. Within the rhizosphere, heavy metals inhibit the water uptake by young seedlings (Kranner and Colville, 2011; Ko et al., 2012). Oil coating on the soil surface may reduce the diffusion of O₂ and water uptake by growing plants (Baker, 1970), and consequently, it lowers the rate of plant growth in undercontaminated soil environments (PeraltaVidea et al., 2002; Mehdi et al., 2021). Furthermore, total biomass was significantly reduced under a polluted soil medium (Monalisa and Patra, 2012). The present findings were consistent with the findings of Peralta-Videa et al. (2002), who found that the simultaneous presence of hydrocarbons significantly affects plant growth. The length of both root and shoot along with the dry biomass of the plant was hampered due to stress exerted by both types of contaminants in the growth medium. Concerning plant sensitivity to petroleum hydrocarbons, seedling growth was stunted under higher levels of total petroleum hydrocarbons (TPHs) than previously reported (Kirk et al., 2005; Aslam et al., 2021).

The concurrent existence of heavy metals along with oil-based hydrocarbon pollutants may boost toxicity in plants. The agronomic parameters are one of the indicators while investigating the overall health of a plant. Our data regarding root and shoot biomass showed that plant roots were more sensitive to the toxic effects of pollutants in the soil as compared to shoots. The direct contact between soil pollutants and the surface of roots may be the major factor in root sensitivity (Kummerová et al., 2013) overshoots. Both direct as well as indirect phytotoxicity mechanisms of heavy metals and petroleum hydrocarbons were evident in affecting the physiology of plants by changing the physical, chemical, and biological aspects of the soil (Baker, 1970; Kabata-Pendias, 2011). Maximum above-ground yield is a prerequisite for an efficient phytoextraction process; on the other hand, the development of a dense plant root network generates a productive habitat for rhizosphere-based microbes involving rhizodegradation.

For different concentrations of Triton X-100, the fractions of oil decreased with the low concentration of Triton X-100 (Figure 3). The fractions of degraded oil ranged from 6 to 40 percent. The maximum removal of oil fractions from the soil after 30 days of treatment was as follows: 26% at 0.01% of Triton X-100, < 27% at 0.1% of Triton X-100, and > 25.4% at 1% of Triton X-100. The maximum removal of oil fractions from the soil after 60 days of treatment was as follows: 32% at 0.01% of Triton X-100, < 33% at 0.1% of Triton X-100, and > 32.4% at 1% of Triton X-100. The removal of oil fractions from the soil after 90 days of treatment was as follows: 34% at 0.01% of Triton X-100, < 36.2% at 0.1% of Triton X-100, and < 37% at 1% of Triton X-100. The removal of oil fractions from the soil after 120 days of treatment was observed as follows: 38.98% at 0.01% of Triton X-100, < 40.33% at 0.1% of Triton X-100, and > 38% at 1% of Triton X-100. However, the increase in the concentration of Triton X-100 from 0.01 to 1% led to the increased removal of total petroleum hydrocarbons at moderate concentration. This is indicative of the inhibiting effect of Triton X-100 above 0.1% on the growth of plants. In the current study, the population of microorganisms was increased in the samples with both crude oil and Triton X-100. This suggests that Triton X-100 could be used as a carbon source and was not harmful to the microbes. Some reports have shown that both nonionic and anionic surfactants increase the dissolution of hydrocarbons by forming micelles. The surfactants begin to combine into micelles at critical micelle concentration (CMC), and the interior of the micelles provides a hydrophobic environment to dissolve soluble compounds, such as hydrocarbons (Roy et al., 1994). The higher reduction of TPH was observed with a higher concentration of Triton X-100 due to the nonionic character of surfactants that increases the solubility of hydrocarbons.

For microbial consortium treatment, a variety of methods are used, including seed coverings, soil injections, rhizosphere injections, and leaf injections. However, the effects of such injection methods on the effectiveness of phytoremediation have not been evaluated. In this study, the production of most biomass of plants, the reduction of toxicity of hydrocarbon degradation, and the bacterial colony were identified as soil based compared with other methods (Mahmood et al., 2021; Afzal et al., 2012). However, other previous studies have reported that seed coverage with a microbial consortium works better compared to other vaccines (Müller and Berg, 2008; Müller et al., 2009). In addition, inoculation is a well-established method and is often used to detect seed germination quickly and consistently. Seed extraction has been used successfully in the phytoremediation of organic pollutants such as hydrocarbons (Afzal et al., 2011). If you add a beneficial microbial consortium to contaminated soil, the inoculated bacteria must be able to adapt quickly to the new environment and survive. In addition, the survival and persistence of the inoculated beneficial bacterial consortium are essential for the growth and health of plants in an effective repair system where plants and microorganisms interact with each other (Afzal et al., 2011; Ahmad et al., 2012).

The colony-forming unit (CFU) g⁻¹ of crude oil-contaminated soil in the presence of surfactant as amendment ranged between 6.7 × 10⁷ at the end of 2 months and 2.2 × 10⁴ at the end of the fourth month of applying 0.01% of surfactant Triton X-100. The crude oil-polluted soil amended with 0.1% of surfactant Triton X-100 ranged from 5.8 × 10⁷ at the end of 2 months and 6.4 × 10⁷ at the end of the fourth month of treatment, while that of crude oil-polluted soil amended with 1.0% of surfactant Triton X-100 ranged from 6.2 × 10⁷ at the end of 2 months and 8.2 × 10⁷ at the end of the fourth month of treatment, respectively (Table 2). By applying Triton X-100 combined with crude oil, the total colony count showed an increased bacterial population. On the contrary, under control conditions (without oil contaminants), the bacterial population was slightly lower than that in other treatment samples. The samples with Triton X-100 showed non-significant differences regarding colony count. During the 120 days of the growth period, the control sample did not show an increasing trend.

The application of biosurfactants has been published in improving the bioavailability of hydrocarbons within the soil and, as a result, increased microbial-based pollutant degradation (Marchant and Banat, 2012). This may be due to the ability of rhamnolipids to enhance both types of interactions (hydrophilic and hydrophobic) that lead to blocking the formation of H-bonds, lowering surface tension, and ultimately improving mobilization of oil contents, thus producing oil micelles in water (Marchant and Banat, 2012). Degradation of crude oil in soil enhances nutrient supplementation in contaminated soils. Such a type of microbial degradation was performed by various catabolic pathways. In the case of aerobic oxidation of hydrocarbons, the hydroxylase enzyme performs the terminal or sub-terminal O₂ integration, while under anaerobic metabolism, nitrate or sulfate ions act as electron donors, and the addition of the C group promotes alkane activation (Callaghan et al., 2009).

Before the introduction of the mixture of bacteria, the number of indigenous bacteria in the crude oil refinery was determined to be 6.7 × 107CFU/g dry soil. A combination of the Triton x-100 and the bacterial consortium was also made to test the feasibility of using this integrated method in soil preparation. Table 3 shows the efficiency of crude oil during the refining process. In the control group (i.e., unprocessed soil), only 6% of crude oil is degraded which may be due to short-chain bonds and oxidation in the air. The efficacy of T2 group degradation (i.e., soil modified by mixed bacterial consortium inoculation) (29%) was significantly higher than that of the T1 group during all remedial tests, showing that the mixed bacterial consortium was able to adapt quickly to the new contaminated environment of oil and has shown a high degree of degradation of crude oil. The conclusion can also be confirmed by further short-term bioremediation tests. In Figure 3, the number of bacteria increased from 6.7 × 107 to 8.2 × 107 CFU/ g dry soil during the 120-day bioremediation. Mixed bacteria can grow well and, in turn, facilitate the deterioration of crude oil depending on their strength. In the case of the T6 group (i.e., soil modified with a combination of bacterial consortium inoculation and Triton x-100), the degradation efficacy was further improved compared to that of T1 and T2 and up to 40% after 4 days of treatment. Alarcón et al. (2008) found that crude oil depletion was significantly higher (39%) after 180 days of treatment, which was significantly lower than our experimental results. The introduction of the Triton x-100 was used to improve the efficiency of the destruction of crude oil by indigenous microbes (Figure 4). The reason could be that the Triton x-100 could be used as a carbon source by native bacteria and promotes the growth of native bacteria, which was consistent with the results obtained. During the first 60 days of repair, the efficiency of the deterioration increased rapidly. From the beginning, after incubation, there was a high concentration of carbon dioxide in the contaminated soil, and the microorganism was unable to reduce long-chain chains or alkene directly into the petroleum hydrocarbon priority (Chang et al., 2009). From the 60th day to the 120th day of repair, the efficiency of the deterioration tends to be flat. Possible causes may be as follows: 1) crude oil that was easily destroyed is already completely consumed by a mixture of bacteria (Zahed et al., 2010); 2) toxic medium metabolites can be produced and collected (Zhu and Aitken 2010); and 3) with the use of a carbon source, the activity of the bacterial consortium has decreased significantly (Yang et al., 2010).

Under control treatment, the value of log CFU/g of contaminated soil showed lower values than the pots amended with surfactant and ranged between 2.2 × 10⁴ at the end of the second month and 2.8 × 10⁴ at the end of the fourth month of application, respectively. The growth of indigenous oil-degrading microbiota may be activated by surfactant application (Abioye et al., 2010). Similar results were reported when the organic amendment was applied to contaminated soils (Al-Kindi and Abed, 2016). The population of indigenous oil-degrading microbiota rapidly increases in response to increased hydrocarbon availability(Song and Bartha., 1990). The key reason behind the increased log CFU for bacteria under surfactant as an organic amendment for the decontamination of crude oil-based contaminated soil is the availability of more hydrocarbon as a basic nutrient for microbial activities. The addition of surfactant as an amendment improved petroleum hydrocarbon biodegradation by enhancing their availability and solubility to natural soil microbiota.

The most applicable technique to get quality and functional group-related information on petroleum-related materials is the Fourier-transform infrared (FTIR) analysis technique. The technique uses IR (infrared) light to scan and observe the chemical properties of test samples. Samples were analyzed by FTIR spectroscopy to check the different functional groups of crude oil-contaminated soil (COCS). Results regarding the surface chemistry of contaminated oil particles showed similarities amongst surfactants and spectral areas in COCS, although distinct differences also exist in absorption bands, thus pointing out the kinds of functional groups involving bonding interaction. A comprehensive investigation of band shifting and their association with peak area as well as height along with elemental data observed that the curve resolving spectra of COCS showed wide bands at about 3,730–3,001 cm⁻¹ (Sung et al., 2013). Such types of bands are linked with O–H stretching vibrations (Table 4) and are chiefly recognized as belonging to the O–H functional group corresponding to low concentrations of alcoholic, phenolic, and carboxylic acid groups of compounds within COCS. Data suggest that asymmetric stretching of both methyl and methylene functional groups is caused by two larger wavenumber bands in the range of 2,947–2,397 cm⁻¹. In the studied samples, a band that appeared in the range of 1785–1,575 cm⁻¹ was linked with the C=O diaryl ketone stretching phenomenon. All other remaining functional groups that are accountable for IR absorption are as follows: 1,507–1,387 (cis = C–H bending), 1,307–1,097 (–C–O stretch) (Gaskin and Bentham, 2010), 1,067–886 (–C–O stretch), 886–706 cm⁻¹ (–C–Cl stretch), 656–503(–C–Br stretch), and 504–440 (–O– stretch).

Figure 4 presents the FTIR spectra of soil samples with and without any amendments with their functional groups. The results present vital information about the examined soils concerning their surface functional groups as well as traits of chemical bonding. These results were used to classify the functional groups within oil-based contaminants in control soil without any amendments and along with eight different amendments. Without any amendment, it was recognized that the results of FTIR spectra showed numerous functional groups in the contaminated soil sample.

The FTIR spectra result showed that (O–H) functional groups exist within 2,997–3,726 cm⁻¹ wavenumber, (C–H) functional groups from 2,880 to 2,997 cm⁻¹, (Si–H)-based groups from 2397 to 2880cm⁻¹, (C=C) functional groups from 1,344 to 1,504 cm⁻¹, (C–O)-based groups from 1,067 to 1,105 cm⁻¹, (–O–C) functional groups from 838 to 1,067 cm⁻¹, and (Si-O-Si) functional groups from 465 to 594 cm⁻¹. Hydrocarbons group alkanes were confirmed by the presence of O–H and C–H single-bounded bonds. After remediating the contaminated soil with the integrated application of bacterial and surfactant, the result specifies a variety of functional groups in a different soil sample. With the highest spectra ranging from 2,999 to 3,743 cm⁻¹, wavenumber indicated the (O–H) functional group, (C–H) functional groups from 2,880 to 2,947 cm⁻¹, (Si–H)-based compounds from 2,366 to 2,879 cm⁻¹, (C=C)-based groups between 1,343 and 1,560 cm⁻¹, (C–O)-based groups ranging from 1,067 to 1,338 cm⁻¹, (–O–C) groups from 824 to 1,070 cm⁻¹, and (Si-O-Si) functional groups ranging from 434 to 656 cm⁻¹.

These findings confirm that some specific functional groups are accountable for the existence of petroleum hydrocarbons within tested soil samples (Aziz et al., 2015). Functional groups, particularly alcohols, methyl, phenols, aliphatic hydrocarbons, and amides, are responsible for the occurrence of petroleum-based hydrocarbons. Moreover, similar characteristic peaks showed a lower strength under higher oil content concentrations in soil samples. Overall, our findings based on FTIR results may suggest that plant species may help to degrade oil-based soil contamination after 120 days of experimentation. Such alterations strongly suggest that crude oil-based soil contamination was effectively remediated by the process of phytoremediation. Figure 4 shows fluctuations in the FTIR spectrum of COCS and COCS + surfactants at different concentrations. Wavenumbers range from 400 to 4,000 (right to left), whereas absorbance is given on the left side. It can be observed from the aforementioned graph that from 2,000 to 3,000, the asymmetric stretching of methylene is very low and the highest peak of absorbance is shown at 1,000 wavenumber, where CO stretches about 1,031.

It has been proven that surfactants used in the remediation of petroleum hydrocarbon-contaminated soil have great application potential. The effects of surfactant (Triton X-100) on the leaching of petroleum hydrocarbons from soil were investigated through various experiments, and petroleum hydrocarbon components were analyzed by FTIR. The effects of surfactants on the degradation of petroleum hydrocarbons were analyzed by the changes in microbial growth. The techniques developed through this study could be applied to field areas contaminated with crude oil.