Degradation of aliphatic and aromatic hydrocarbon mixture by a Rhodococcus sp.

As an important energy, the widespread use of petroleum products has brought a lot of benefits, yet accidental spills have been an inevitable part of the exploration, exploitation, and transportation activities of petroleum. An infamous example is the three-month-long release of approximately 4.9 million barrels of light crude oil into the Gulf of Mexico due to the Deepwater Horizon oil rig explosion (Camilli et al. 2010). The composition of petroleum is complicated, which includes aliphatics, aromatics, and other recalcitrant compounds such as resin and asphaltene, some of which are reported to be toxic, recalcitrant, and carcinogenic (Varjani 2017). Compared with physical and chemical methods such as combustion, photolysis, landfill, and ultrasonic decomposition, biodegradation is expected to be an economic and environment-friendly alternative for the removal of petroleum pollutants (Durairaj et al. 2023; Ma et al. 2025).

Research studies on the biodegradation of petroleum hydrocarbons during the past decades have led to the isolation and identification of microorganisms with an outstanding ability to degrade petroleum pollutants including bacteria, fungi, and even algae. Among them, bacteria are considered as primary and the most active degraders of spilled oil in an environment (Brooijmans et al. 2009). At least 100 genera of bacteria are capable of petroleum degradation (Li 2018). Strains, such as Pseudomonas spp., Rhodococcus spp., and Alcanivorax spp. have been shown to rapidly degrade long-chain alkanes in diesel, while the degradation rates of more complex aromatic hydrocarbons are slower. Particularly, Rhodococcus strains dominate contaminated ecosystems and can efficiently degrade crude oil and a large variety of chemicals, including alkanes and aromatics (Martina et al. 2020). Key factors influencing the microbial degradation efficiency of petroleum pollutants include temperature, oxygen availability, pH, and nutrient levels. Optimizing microbial consortia and applying genetic engineering techniques can further enhance degradation rates.

Hitherto, most studies have focused on biodegradation of the individual petroleum hydrocarbons (Margesin et al. 2013). While these studies have provided indispensable knowledge on underlying mechanisms of hydrocarbon biodegradation, there are a few reports on biodegradation of the hydrocarbon mixtures in recent years. The existing studies have mainly focused on the biodegradation of polyaromatic hydrocarbons (PAHs). For example, Zhang et al. (2013) reported biodegradation of the mixtures of naphthalene, phenanthrene, fluorene, anthracene, fluoranthene, and pyrene by two phenanthrene-degrading bacteria, Micrococcus sp. PHE3 and Sphingobium sp. PHE9. There is a lack of reports on the degradation of the mixtures of aliphatic and aromatic hydrocarbons although aliphatic and aromatic hydrocarbons generally co-exist in petroleum. In order to better remove the petroleum hydrocarbons in oil-contaminated sites, it is essential to understand the influence of aliphatics and aromatics on each other breakdown by a bacterial degrader.

Previously, an aerobic dioxin-degrading bacterium, Rhodococcus sp. strain p52 was isolated from petroleum-contaminated soil by Peng et al. (2013). The purpose of the present study is to assess the biodegradation capacity of strain p52 towards petroleum pollutants. We focused on the degradation of a mixture of aliphatic hydrocarbons and aromatic hydrocarbons by Rhodococcus sp. strain p52. Anthracene and phenanthrene were selected as model aromatic contaminants due to their poor solubility, making them less bioavailable for microbial biodegradation and hence enlisted in the USEPA priority pollutant list. Hexadecane and tetradecane were major components of various crude oil and diesel fuels and were opted as model aliphatic hydrocarbons. Furthermore, the removal of the diesel fuel by strain p52 was investigated.

Phenanthrene, anthracene, n-hexadecane, n-tetradecane, and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA) and were of the highest purity available. Methyl tertiary butyl ether (MTBE) and carbon tetrachloride were purchased from Sinopharm (Beijing, China) and were high-performance liquid chromatography grade. Automotive diesel fuel (No.0) with a minimum cetane number of 49 was purchased from Sinopec (Jinan, China). Rhodococcus sp. strain p52 used in the study was previously isolated from petroleum-contaminated soil in our lab (Peng et al. 2013).

Biodegradation of petroleum hydrocarbons or diesel fuel was carried out using washed cells of Rhodococcus sp. strain p52. Strain p52 was pre-cultured in 1,000-mL Erlenmeyer flasks containing a 300 mL Luria-Bertani (LB) medium. For each test, cells were harvested at the logarithmic phase by centrifugation at 6,000 rpm for 6 min at 4 °C, then washed twice with a potassium phosphate buffer (0.068 M), and finally resuspended in a 50 mL phosphate buffer to reach a final OD₆₀₀ of 0.8, which was corresponding to 0.3218 g/L cell dry weight. Anthracene (2 g L⁻¹) and phenanthrene (50 g L⁻¹) were dissolved in DMSO as a stock solution. For the biodegradation of individual substrates, anthracene and phenanthrene were respectively added to the phosphate buffer to a final concentration of about 48 mg L⁻¹, and hexadecane and tetradecane to a final concentration of about 450 mg L⁻¹. For biodegradation of the mixtures of the aliphatic and aromatic hydrocarbons, pairs of compounds were added to the phosphate buffer and subjected to tests, respectively, including anthracene with hexadecane, phenanthrene with hexadecane and phenanthrene with tetradecane. The final concentration of each substrate was the same as that for the biodegradation of individual substrates. Additionally, while investigating the effect of different amounts of hexadecane on the biodegradation of phenanthrene, the concentration of hexadecane was adjusted to the desired final concentration according to each test. Automotive No.0 diesel fuel was added to the phosphate buffer to a final concentration of about 4.4 g L⁻¹. All reaction systems (50 mL) were incubated in 500 mL Erlenmeyer flasks in the dark at 30 °C on a reciprocal shaker at 180 rpm (Yang et al. 2014). The phosphate buffer with tested compounds or diesel fuel but without the bacterium suspension served as control. All experiments were conducted in triplicate. The results were expressed as mean ± standard error of the triplicate samples at each sampling interval. The specific activity for the degradation of different hydrocarbons was calculated according to Li et al. (2004).

The hydrocarbons (triplicate samples of each test) were extracted with 50 mL of MTBE at each analysis interval and the extracts were dried by anhydrous Na₂SO₄. The concentration of the hydrocarbons was analyzed by Agilent 7890B gas chromatography with a flame ionization detector (GC-FID) accordingly, as described by Wu et al. (2010) with minor modifications: the GC-FID was equipped with an HP-5MS fused silica capillary column (30 m × 0.25 mm ID × 0.25 μm thickness, Agilent Technologies, USA) with the injector and detector temperature of 280 °C, and 300 °C, respectively. The oven temperature programme was set as follows: initially held at 120 °C for 2 min, then raised from 120 to 300 °C at a rate of 15 °C min⁻¹, and finally held at 300 °C for 10 min.

Before the extraction, 10 mL of the HCl solution (3 mol L⁻¹) was added to the reaction system in each flask for demulsification. The samples were extracted three times with 50 mL carbon tetrachloride. Then the three extracts were combined and dried by anhydrous Na₂SO₄. Diesel fuel was analyzed by an infrared oil-measuring instrument (Oil 460, Beijing ChinaInvent, China). The measurements were carried out in the range 4,000–2,500 cm⁻¹ which include the absorption of aromatic and aliphatic hydrocarbons. The sample extract was diluted 20 times for measurement.

The cometabolic enhancement of aromatic hydrocarbon degradation that diverts resources from aliphatic degradation (Varjani & Upasani 2017); metabolic regulation that prioritizes the breakdown of more complex aromatic compounds by inducing specific enzyme expressions (Prince & Walters 2016); substrate competition, where aromatics outcompete aliphatics for microbial surface adsorption sites, reducing aliphatic bioavailability (McGenity et al. 2012); and enzyme activity shifts, as aromatics promote oxygenase expression, which is less effective for aliphatic hydrocarbon degradation (Johnsen et al. 2005). These factors, in turn, reduce the degradation efficiency of aliphatic hydrocarbons. Some studies reported that the CYP gene, alkB may relate to hydrocarbon degradation (Liang et al. 2015; Chen et al. 2017), and alkB-1 and alkB-2 were transcribed in the presence of tetradecane, dotriacontane, naphthalene, and phenanthrene in Rhodococcus sp. strain p52 (Yang et al. 2014). Likewise, during the simultaneous degradation of phenol and hexadecane by Acinetobacter strains, phenol remarkably inhibited hexadecane degradation while hexadecane had positive influences on phenol degradation (Sun et al. 2012). Thus, further works about the degradation mechanism need to focus on the hydrophilic or hydrophobic characteristic of the cell surface and confirm the genes involved in hydrocarbon degradation.

In addition, the degradation rate of aliphatic hydrocarbons is significantly higher than that of aromatic hydrocarbons, consistent with Varjani & Upasani (2017), who reported greater biodegradability of linear alkanes compared wtihpolyaromatics. Furthermore, tetradecane's degradation rate exceeds that of hexadecane by 5.99 mg h⁻¹ g⁻¹ dry cells, while phenanthrene degrades 2.01 mg h⁻¹ g⁻¹ dry cells faster than anthracene. The superior degradation of hexadecane by Rhodococcus sp. strain p52 is due to its higher hydrophobicity and logK_o/w, promoting better microbial adhesion and bioavailability. Additionally, longer-chain alkanes provide more energy upon oxidation, boosting microbial activity. Previous studies highlight microbial surface hydrophobicity and carbon chain length as key factors in alkane degradation (Eibes et al. 2010; Margesin et al. 2013). The faster degradation of phenanthrene relative to anthracene is attributed to phenanthrene's non-linear ‘bent’ structure, enhancing bioavailability and interaction with microbial enzymes, while anthracene's linear structure and higher molecular stability make it more resistant to degradation. Phenanthrene's smaller molecular surface area and uneven electron density further increase its susceptibility to oxidative attack, accelerating degradation (Mueller et al. 1989; Cerniglia 1992; Kanaly & Harayama 2000; Johnsen et al. 2005; Zhang et al. 2013).

To further learn the effect of aliphatic hydrocarbons, the degradation of phenanthrene was carried out with different amounts of hexadecane (Figure 3(b)). After seven days, the removal of phenanthrene increased from 66.6% to 70.6% and 78.8% with the increase of hexadecane from 50 to 200 and 500 mg L⁻¹, respectively. The results indicated that the degradation of phenanthrene was enhanced with the increase of hexadecane concentration. As suggested by Eibes et al. (2010), the dispersion of NAPL could lead to an increase in contact area between microorganisms and substrate and thus facilitate the transport of the pollutant. With the increased concentration of NAPL, the emulsifying effect would be aggravated, which may offer microorganisms better contact with microscopic droplets or adhesion to microscopic droplets, hence favouring the uptake of pseudo-solubilized substrate or substrate dissolved in NAPL (Yap et al. 2010).

Rhodococcus sp. strain p52 exhibited strong degradation capabilities for both aliphatic hydrocarbons (hexadecane and tetradecane) and aromatic hydrocarbons (phenanthrene and anthracene). Importantly, the degradation of aromatic hydrocarbons was enhanced in the presence of aliphatic hydrocarbons, likely due to increased bioavailability and improved cell surface hydrophobicity, while the presence of aromatics reduced aliphatic degradation, likely due to the cometabolic effect, metabolic regulation, substrate competition, and enzyme activity shift. These findings suggest a complex interaction between different hydrocarbon types during microbial degradation, with implications for the optimization of bioremediation strategies.

The study also demonstrated that Rhodococcus sp. strain p52 has the potential for the bioremediation of petroleum-contaminated environments, particularly when dealing with mixed hydrocarbon pollutants such as diesel. The strain was able to remove 63.7% of diesel within 96 h, showing its practical application in treating oil spills and industrial effluents.

Future research should focus on the underlying genetic and enzymatic mechanisms responsible for these interactions to further enhance the efficiency of hydrocarbon biodegradation. Additionally, scaling up this process for field applications will be crucial in developing effective bioremediation technologies.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.