Crude Oil Biodegradation by a Biosurfactant-Producing Bacterial Consortium in High-Salinity Soil

Abstract

Bioremediation is a promising strategy to remove crude oil contaminants. However, limited studies explored the potential of bacterial consortia on crude oil biodegradation in high salinity soil. In this study, four halotolerant strains (Pseudoxanthomonas sp. S1-2, Bacillus sp. S2-A, Dietzia sp. CN-3, and Acinetobacter sp. HC8-3S), with strong environmental tolerance (temperature, pH, and salinity), distinctive crude oil degradation, and beneficial biosurfactant production, were combined to construct a bacterial consortium. The inoculation of the consortium successfully degraded 97.1% of total petroleum hydrocarbons in 10 days, with notable removal of alkanes, cycloalkanes, branched alkanes, and aromatic hydrocarbons. Functional optimization showed that this consortium degraded crude oil effectively in a broad range of temperature (20–37 °C), pH (6–9), and salinity (0–100 g/L). In salt-enriched crude-oil-contaminated soil microcosms, the simultaneous treatment of bioaugmentation and biostimulation achieved the highest crude oil degradation rate of 568.6 mg/kg/d, compared to treatments involving abiotic factors, natural attenuation, biostimulation, and bioaugmentation after 60 days. Real-time PCR targeting the 16S rRNA and alkB genes showed the good adaptability and stability of this consortium. The degradation property of the constructed bacterial consortium and the engineered consortium strategy may have potential use in the bioremediation of crude oil pollution in high-salinity soil.

1. Introduction

Crude oil pollution has surged drastically due to social modernization, leading to severe environmental and health impacts. Microbial remediation of petroleum pollution is known as the most environmental and thorough way to remove oil contaminants [1,2,3,4,5]. Notably, bacteria are crucial degraders among microorganisms due to their widespread presence and roles in physiology and ecology [6,7,8,9]. Large numbers of bacterial strains were isolated from various habitats to mineralize petroleum hydrocarbon compounds. For instance, Acinetobacter species are ubiquitous in the habitats of crude oil pollution, excelling in alkane degradation [1,10,11]; Bacillus strains are always tolerant of tough environmental factors (nutrient, temperature, salinity, etc.), with different degradation capabilities for short- and middle-chain alkanes and aromatic hydrocarbons [4,7,12]; and members of Dietzia have been reported to utilize middle- and long-chain alkanes, branched alkanes, and polycyclic aromatic hydrocarbons [6,13,14].

In the event of an oil spill, degradation usually occurs through abiotic processes (such as volatilization and weathering) or natural attenuation by indigenous microbes [15,16,17]. Nevertheless, the cleanup process is often slow and long. To enhance degradation, bioremediation strategies using effective bacteria to transform petroleum hydrocarbons into nontoxic inorganic compounds are often employed [4,5,7,15]. Specifically, biostimulation and bioaugmentation are two crucial strategies to promote the bioremediation of crude oil pollution [18,19,20,21]. Biostimulation involves optimizing environmental conditions—such as nutrients (mainly nitrogen and phosphorus), temperature, oxygen, and biosurfactants—to promote microbial growth. Bioaugmentation is carried out by artificial inoculation of degrading bacteria to the contaminated site. In practical applications, bioremediation often benefits from diverse bacterial populations with unique metabolic pathways. Thanks to good environmental suitability, broad substrate spectra, and abundant metabolic enzymes, constructed bacterial consortia are recognized as more efficient at petroleum hydrocarbon degradation than individual strains [8,16]. For example, a mixed consortium of three strains (Serratia proteamaculans, Alcaligenes, and Rhodococcus erythropolis) achieved a significant increase in crude oil degradation to 85.26% [8]. Further, in a soil microcosm, a mixed consortium of six hydrocarbon-utilizing strains, when combined with bioaugmentation and biostimulation (using NH₄NO₃ and Na₂HPO₄), achieved the highest hydrocarbon degradation efficiency of 93.67% over 45 days, compared to 82.56% with bioaugmentation alone and 29.28% with biostimulation alone [16].

During the process of petroleum hydrocarbon depletion, high hydrophobicity, low solubility, and weak bioavailability of hydrocarbon molecules are among the factors that restrict biodegradation by bacteria [4,5,15]. Bacterial cells access hydrocarbons through three possible mechanisms. First, bacteria generate surface activity compounds (biosurfactants), such as glycolipids, phospholipids, and lipopeptides, to emulsify hydrocarbons to reduce surface and interfacial tension. Second, bacteria enhance the hydrophobicity of their cell surfaces to directly interact with hydrocarbons [10,22,23]. Third, bacteria form biofilms by secreting extracellular polymeric substances (EPS) with high molecular weight. The EPS matrix contains amphiphilic moieties and provides a physicochemically distinct microenvironment that favors the solubilization of hydrocarbons [4,10,24]. Biosurfactants are particularly notable for their low toxicity, easy availability, and excellent biocompatibility. These properties increase the bioavailability of petroleum hydrocarbons and promote bacterial growth, making biosurfactants promising candidates for crude oil biodegradation and bioremediation [23,24,25,26].

Although bioremediation of crude oil is very promising, one of the main obstacles in recent research concerns its application under conditions with high salinity, which can inhibit the activity of most microbes and diminish their value as crude oil degraders. The slow efficiency of crude oil biodegradation can be attributed to extreme environmental conditions, which primarily damage cell membranes, accompanied by intracellular water loss and enzymatic denaturation [27,28,29]. For instance, four Pseudomonas aeruginosa strains, isolated from oil-contaminated saline soil, were combined to form a bacterial consortium. After 120 days, the inoculation of the consortium in soil dosed with 10 g/kg crude oil at 0, 150, and 300 mM NaCl led to crude oil degradation of 49.5, 47.0, and 42.3%, respectively [29]. Further, through bacterial communities in the surface and subsurface layers of saline soils contaminated with crude oil in the Yellow River Delta Natural Reserve, petroleum hydrocarbon fractions had a significant negative effect on bacterial biodiversity (Shannon, Simpson, and Chao1 indices) [30]. In high-salinity environments, therefore, it is vital to have a toolkit for petroleum cleanup, and potential bioremediation agents are necessary. The Yellow River Delta region is the youngest coastal land in China, as well as a famous petroleum production and soil salinization area. The salt content, primarily consisting of Cl⁻ and Na⁻, is reported to range from 6 to 30 g/kg in 50.88% of the soil area, with an average of 10.45 g/kg [30,31]. Located in the Yellow River Delta, Shengli Oilfield faces severe soil salinization due to its high evaporation-to-precipitation ratio. This condition makes it an excellent site for researching crude-oil-degrading bacteria [30,31,32].

Although the joint application of crude oil degraders and biosurfactant producers has been proposed in previous research, little attention has been paid to applying this remediation strategy to evaluate the bioremediation potential in crude-oil-contaminated saline soil. This study aimed to construct a bacterial consortium using four halotolerant isolates capable of degrading crude oil and producing biosurfactants, and to evaluate the performance of the constructed bacterial consortium on the petroleum hydrocarbon degradation in flask experiments, non-saline and salt-enriched soil microcosms with five treatments (abiotic factor, natural attenuation, bioaugmentation, biostimulation, and bioaugmentation with biostimulation). The functional gene abundance was also detected to confirm the utility of this indicator in the monitoring of the bioaugmentation process.

2. Materials and Methods

2.1. Soil, Crude Oil, Medium, and Chemical

To screen and isolate crude-oil-degrading bacteria, the sites located around the oil wells in Gudao Town (37°52′48″ N; 118°48′36″ E, Figure S1) of the typical oil-producing area of Shengli Oilfield in the Yellow River Delta region, were selected for sampling. At the sampling site, crude-oil-contaminated saline soil samples were taken in triplicate at a depth of 5–20 cm. The soil samples were sealed in respective plastic bags, kept in an icebox, and sent to the laboratory within 4 h for subsequent analysis. The heavy crude oil used in the media was from Shengli Oilfield with API 20.32, viscosity 6235 mPa·s, and density 0.932 g/cm³.

Mineral salt medium (MSM) and Luria–Bertani (LB) were employed in strain isolation and enrichment, respectively. The LB composition was as follows (g/L): tryptone 10, yeast extracts 5, NaCl 40, and pH 7.0. The MSM composition was as follows (g/L): (NH₄)₂SO₄ 1, Na₂HPO₄ 0.8, KH₂PO₄ 0.2, MgSO₄·7H₂O 0.2, CaCl₂·2H₂O 0.05, FeCl₃·3H₂O 0.005, (NH₄)₆Mo₇O₂₄·4H₂O 0.001, and NaCl 40, pH 7.6. Analytical-grade chemical reagents were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China), while tetradecane (C₁₄) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and paraffin oil from Sinopharm Chemistry Reagent Co., Ltd. (Shanghai, China).

2.2. Isolation and Identification of Bacteria

Isolation of halotolerant crude-oil-degrading bacteria from crude-oil-contaminated saline soil was carried out by the enrichment culture method. The saline soil sample (10 g) was incubated in 100 mL of MSM medium supplemented with crude oil (1%, w/v) for 7 d at 30 °C, 180 rpm. Subsequently, aliquots of 10 mL were transferred to fresh MSM (NaCl 60 g/L) supplemented with crude oil (3%, w/v) for 7 d, as described above. Finally, aliquots of 10 mL were transferred to fresh MSM (NaCl 80 g/L) supplemented with crude oil (5%, w/v) for 7 d to screen dominant halotolerant crude-oil-degrading bacteria. 1 mL of individual culture was diluted tenfold serially to 10⁻⁸ and aliquots of 100 μL were spread on MSM-crude oil agar plates. The plates were cultivated in a 30 °C incubator, and morphologically different colonies were purified on fresh MSM-crude oil agar plates through multiple subcultures. Total genomic DNA was extracted from a single strain using the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) with universal primers 27F and 1492R, following the procedure outlined in [6]. The PCR procedure was conducted with the following settings: 94 °C for 5 min; 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 2 min, 30 cycles; 72 °C for 10 min, and stored at 4 °C. TransStart FastPfu DNA Polymerase (TransGen Biotech, Beijing, China) was used to amplify the products. PCR products were sequenced by Invitrogen Ltd. (Shanghai, China) and submitted to GenBank for homology analyses using the Basic Local Alignment Search Tool (BLAST) program (www.ncbi.nlm.nih.gov/BLAST/. The 16S rRNA gene sequences of S1-2 and S2-A were submitted to GenBank with accession numbers of MT043748 and MT043746, respectively. Phylogenies and evolutionary relationships of the bacterial strains were conducted based on the neighbor-joining method using MEGA 7.0 software.

2.3. Biosurfactant Production Detection of Bacteria

Four effective crude-oil-degrading bacteria were studied in this research. Specifically, strains S1-2 and S2-A were newly isolated from crude-oil-contaminated saline soil, while Dietzia sp. CN-3 and Acinetobacter sp. HC8-3S were previously isolated from crude-oil-contaminated sediments in Bohai Bay [1,6]. To evaluate their ability to produce biosurfactants using various carbon sources, each strain—S1-2, S2-A, CN-3, or HC8-3S—was inoculated into MSM with different substrates, including a straight-chain alkane (tetradecane), a liquid hydrocarbon mixture (paraffin oil), and a complex mixture (crude oil). The cultures were incubated for 7 d at 30 °C at 180 rpm. Subsequently, cell-free supernatants and cell pellets were collected by centrifugation (10,000 rpm, 10 min), and they were applied severally in the emulsification activity and cell surface hydrophobicity assays to assess biosurfactant production performance.

2.3.1. Emulsification Activity

The emulsification activity of supernatant samples was determined based on [14]. 2 mL of C₁₄ and 2 mL of cell-free supernatant were added to graduated round tubes, with maximum vortex for 2 min, and subsequently the mixtures were maintained at room temperature for 24 h. The emulsification index (E₂₄) was calculated by the following formula:

$${\mathrm{E}}_{24} (\%)={\displaystyle \frac{\mathrm{Height} \mathrm{of} \mathrm{emulsion} \mathrm{layer} \left(\mathrm{mm}\right)}{\mathrm{Total} \mathrm{height} \mathrm{of} \mathrm{liquid} \mathrm{layer} \left(\mathrm{mm}\right)}}\times 100$$

(1)

Similarly, E₂₄ was also evaluated using paraffin oil or crude oil to determine the emulsification activity of different petroleum hydrocarbons.

2.3.2. Cell Surface Hydrophobicity

Cell surface hydrophobicity (CSH) was tested through bacterial adherence to different carbon sources according to [33]. The cell pellets obtained from different cultures were washed twice with PUM buffer (K₂HPO₄ 22.2 g, KH₂PO₄ 7.26 g, urea 1.8 g, MgSO₄ 0.2 g, and distilled H₂O 1 L, pH 7.2) and resuspended in the same buffer. The OD₆₀₀ was regulated to 0.6 to be as A1. Subsequently, 0.2 mL of C₁₄, paraffin oil, or crude oil was added to 1.2 mL of the corresponding cell resuspension in glass tubes at maximum vortex for 2 min. The mixtures were left at room temperature for 1 h to achieve phase separation. Subsequently, the aqueous phase was carefully displaced, and its OD₆₀₀ was measured and recorded as A2. CSH was calculated by the following equation:

$$\mathrm{Bacterial} \mathrm{adherence} (\%)=(1 -{\displaystyle \frac{\mathrm{A}2}{\mathrm{A}1}})\times 100$$

(2)

2.4. Bacterial Consortium Construction and Tolerance to Environmental Stressors

To enhance crude oil biodegradation and bioremediation in a high-salt environment, four halotolerant strains (S1-2, S2-A, CN-3, and HC8-3S) with diverse petroleum hydrocarbon degradation features and biosurfactant-producing performance were selected to construct a mixed bacterial consortium. Their tolerances to various environmental stressors (temperature, pH, and salinity) were primarily assessed herein. The temperatures were set up as follows: 4, 10, 20, 30, 37, and 42 °C. The pH levels (5, 6, 7, 8, 9, and 10) and salinities (0, 20, 40, 60, 80, and 100 g/L NaCl) were also surveyed. Each strain in LB medium under the above-mentioned conditions was cultivated for 3 d, and OD₆₀₀ was tested regularly.

The four single strains were incubated severally in LB medium, and their cell pellets were harvested, washed, and re-suspended in MSM. Through mixing the same volumes of four inoculations (OD₆₀₀ = 1.0), we successfully constructed a functional bacterial consortium. Specifically, the initial bacteria number was 5.66 × 10⁹ CFU/mL by the plate counting method. We also monitored the growth curves of this consortium under different temperatures, pH levels, and salinities to evaluate their environmental adaptability.

2.5. Crude Oil Degradation Detection

To examine the ability of these four strains (S1-2, S2-A, CN-3, and HC8-3S) to degrade thick crude oil, we investigated each strain’s performance and that of the constructed consortium. A set of flasks containing MSM + crude oil (1%, w/v) were prepared and autoclaved at 121 °C for 20 min before use. Each strain and the consortium were inoculated individually at the logarithmic phase. Subsequently, their own cell pellets were harvested, washed, and reinoculated to MSM + crude oil (1%, w/v) at 30 °C, 180 rpm for 10 d, severally. The controls contained crude oil without bacterial cells.

Crude oil was extracted by n-hexane to obtain total petroleum hydrocarbons (TPHs), including alkanes (n-alkane, cycloalkane, and branched alkane) and aromatic hydrocarbons [6]. The TPH components were analyzed qualitatively and quantitatively by gas chromatography–mass spectrometry (GC-MS, 7890, Agilent, Santa Clara, CA, USA). The capillary column was the HP-5 model (30 m × 0.25 mm × 0.25 μm). High-purity helium was the carrier gas, with a rate of 1 mL/min. The inlet temperature was 300 °C. The column temperature procedure was set up at 50 °C for 3 min, increased to 300 °C at 8 °C/min and held for 10 min. Mass spectrometer conditions were performed as follows: ion source temperature 250 °C, electron energy 70 eV, and scanning mass range 50–550 mu. Individual hydrocarbon compounds were identified by matching the retention time of standard substances (C₇–C₄₀, 1000 mg/L; PAH, 2000 mg/L) and mass spectra in the reference library. All experimental data were collected in triplicate, with standard deviations represented as error bars.

2.6. Impact of Growth Conditions on Crude Oil Biodegradation

Crude oil biodegradation is often hindered by diverse environmental stressors, especially temperature, pH, and salinity. Optimizing cultural conditions is essential for effective bioremediation. For temperature optimization, the consortium was inoculated into MSM + crude oil (1%, w/v) and cultivated at 20, 30, 37, and 42 °C. For pH optimization, the pH values of the initial media were adjusted to 5, 6, 7, 8, 9, and 10 while maintaining the optimal temperature. Similarly, salinity levels were set at 0, 20, 40, 60, 80, and 100 g/L NaCl, with incubation at optimal temperature and pH conditions. Crude oil was extracted and analyzed every 2 d.

2.7. Crude-Oil-Contaminated Soil Microcosms

To evaluate the bioremediation potential of the bacterial consortium, soil microcosm experiments were conducted. The soil sample was collected from non-contaminated, non-saline farmland in Dongying (37°45′27″ N; 118°32′19″ E). The top layer (0–15 cm) of the soil was collected and sieved through a 2 mm mesh. The soil characteristics were as follows: pH 7.62, moisture content 12.60%, organic carbon 1.78%, and organic nitrogen 0.13%. The soil was then mixed with crude oil (50 g/kg) and different amounts of NaCl (0 or 20 g/kg). Crude oil contamination and salinity levels were set according to previous reports from contaminated sites of the Yellow River Delta region [30,32].

Crude-oil-contaminated soil microcosms were set up in sterile glass bottles. Each bottle was filled with 200 g of crude-oil-contaminated (50 g/kg) and salinized (0 or 20 g/kg NaCl) soil. Soil samples were sterilized at 121 °C for 20 min, and moisture was maintained at approximately 20%. Five treatments were conducted: (1) abiotic control (AC), sterile crude-oil-contaminated soil, to detect crude oil loss from abiotic factors; (2) natural attenuation (NA), non-sterile crude-oil-contaminated soil, to measure crude oil loss due to indigenous microbiota and abiotic factors; (3) biostimulation (BS), with additional nutrients added into the non-sterile crude-oil-contaminated soil, to detect crude oil removal by indigenous microbiota in the assistance and stimulation of nutrient amendments; (4) bioaugmentation (BA), in which the constructed bacterial consortium was cultivated into the sterile crude-oil-contaminated soil, to investigate the impacts of the inoculated consortium on petroleum removal; (5) bioaugmentation + biostimulation (BA + BS), a combination of the consortium and nutrients amendments in the sterile crude-oil-contaminated soil, to study the combined effect on crude oil degradation. Additionally, high-salinity (20 g/kg NaCl) crude-oil-contaminated soil microcosms were prepared as S-AC, S-NA, S-BS, S-BA, and S-BS + BA to evaluate remediation in a high-salt environment.

Biostimulation involved adding nutrient amendments consisting of sodium nitrate (82 g/L) and potassium dihydrogen phosphate (6 g/L) [34]. Bioaugmentation used a 10 mL inoculum of the consortium, with bacterial 16S rRNA gene copy numbers at 6.53 × 10⁹ g⁻¹. During the experiment, soil samples were periodically stirred to ensure uniform distribution of petroleum hydrocarbons. The microcosms were cultivated at 30 °C for 60 d, with 10 g of soil samples collected after 0, 15, 30, 45, and 60 d. Subsequently, carbon tetrachloride and magnesium silicate were used to extract the residual TPHs in the soil samples according to previous study [35]. TPH concentrations were measured with an infrared spectrometric oil detector (OIL 460, Beijing ChinaInvent Instrument Tech. Co., Ltd., Beijing, China), and variations in petroleum hydrocarbon compounds were analyzed by GC–MS, as described above.

2.8. Degradation Kinetics of TPHs

The degradation kinetics of TPHs in crude oil-contaminated soil microcosms were conducted herein. Particularly, the degradation kinetics analyses assumed that the majority of environmental factors (microorganism, nutrient, oxygen, etc.) were unlimited and maintained constant [36,37]. In this study, based on the first-order kinetic model [38], the TPH degradation was predicted by the following formula:

$$\ln {\mathrm{C}}_{\mathrm{t}}={\ln \mathrm{C}}_0-\mathrm{kt}$$

(3)

In detail, t represents time (d), C₀ represents the initial concentration of TPH in soil, C_t represents the TPH concentration in soil at any time (t), and k represents the degradation rate constant (d⁻¹).

Further, t_1/2 is the half-life period (d), indicating the time to degrade 50% of the initial TPHs in soil. We calculated t_1/2 based on the following formula [38]:

$${\mathrm{t}}_{1/2}=\ln 2/\mathrm{k}$$

(4)

2.9. Quantification of Degradation-Related Genes

Real-time quantification PCR (RT-qPCR) was performed to assess microbial growth and the abundance of genes related to petroleum hydrocarbon degradation in different treatments. The bacterial populations were assessed through the analysis of 16S rRNA gene abundance. Meanwhile, the alkB gene was selected for its strong correlation between abundance and alkane degradation efficiency [39]. The primer pairs for RT-qPCR are listed in Table S1 [40,41,42]. RT-qPCR was conducted using the ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The 20 μL reaction mixture contained 10 μL of 2×TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China), 1 μL of DNA template, 0.4 μL of passive reference dye, and 0.5 μM of each primer. Each sample was measured in triplicate. The RT-qPCR program was as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Product specificity was detected by melting curve from 60 °C to 95 °C. Standard curves for RT-qPCR assays were established through serial tenfold dilutions [43].

2.10. Statistical Analysis

Data are represented as mean ± standard deviation (SD) in triplicates. Statistical analyses were performed by IBM SPSS statistics 22. Before proceeding to apply ANOVA for comparison of groups, data were tested for assumption of normality and homogeneity of variance by the Shapiro–Wilks test and Levene’s test, respectively. The post hoc test of Tukey was applied to differentiate the means. One-way ANOVA was employed to compare the significant differences in emulsification activity, cell surface hydrophobicity, and degradation efficacy of petroleum hydrocarbons under diverse treatments. Values of p < 0.05 were considered significant differences.

3. Results

3.1. Isolation and Identification of Halotolerant Crude Oil-Degrading Bacteria

Through acclimatization in MSM media with high salt and crude oil concentrations, strains S1-2 and S2-A were newly isolated from crude-oil-contaminated saline soil at Shengli Oilfield for further analysis. Strain S1-2, a Gram-negative bacterium, formed yellow, smooth, viscous, slightly opaque colonies with entire margins (0.5–1 mm) on LB agar plates. S2-A, a Gram-positive, rod-shaped, spore-forming bacterium, produced gray-white, flat, dry colonies (1.5–2 mm) on LB agar plates. Molecular identifications of these four strains were performed (see Figure 1) through phylogenetic tree and evolutionary relationship, demonstrating that S1-2 showed 99.86% identity with Pseudoxanthomonas mexicana AMX 26B, and S2-A revealed 98.67% identity with Bacillus endozanthoxylicus 1404. Based on phenotypic characteristics and phylogenetic analysis, the two newly isolated strains of S1-2 and S2-A were identified as Pseudoxanthomonas and Bacillus, respectively.

3.2. Biosurfactant Production with Different Carbon Sources

Carbon sources provide bacteria with carbon and energy for growth and influence the types and amounts of metabolic products. Based on the phenomena of petroleum hydrocarbons dispersed and emulsified into small droplets in the S1-2, S2-A, CN-3, or HC8-3S inoculations, investigations of their abilities to produce biosurfactants were carried out through emulsification activity and cell surface hydrophobicity assays.

As shown in Figure 2a, the maximum emulsification index (E₂₄) of 73.03 ± 2.76% was achieved in S2-A with paraffin oil as the carbon source, followed by 70.48 ± 2.06% with crude oil in S1-2. In contrast, E₂₄ was the minimum (46.98 ± 4.33%) in HC8-3S with tetradecane as the carbon source, which was slightly lower than when using paraffin oil. Particularly, S2-A showed satisfying emulsification ability (E₂₄ > 65%) with all the substrates tested, including tetradecane, paraffin oil, and crude oil, which were significantly higher than those of CN-3 and HC8-3S (p < 0.05).

Furthermore, cell surface hydrophobicity (CSH) was performed to monitor hydrocarbon adhesion to the bacterial cell surface (Figure 2b). The variation ranges of CSH, varying from 31.12% to 79.52%, were quite different from those of E₂₄. HC8-3S displayed the highest CSH (79.52 ± 5.1%) with crude oil, followed by 71.61 ± 4.38% with paraffin oil, indicating a high cell affinity to the complex hydrocarbons—significantly higher than those of the other three strains for the same substrates (p < 0.05). In contrast, S2-A exhibited a minimum hydrophobicity toward all tested substrates, nearly significantly lower than that of S1-2, CN-3, and HC8-3S (p < 0.05).

3.3. Construction of a Halotolerant Bacterial Consortium

Four bacterial strains with different crude oil degradation features and biosurfactant producing capacity were applied to construct a halotolerant bacterial consortium for enhancing degradation efficiency and environmental tolerances in crude oil pollution. The environmental stressor tolerances (temperature, pH, and salinity) of these four strains were tested: S1-2, S2-A, CN-3, and HC8-3S (temperature: 4–42 °C; pH: 5–10; and salinity: 0–100 g/L). Due to the NaCl requirements, these four strains were classified as moderately halophilic and tolerant of a NaCl concentration of 5–20% [27]. Growth curves under different temperature, pH, and salinity conditions show that four strains were able to grow harmoniously in this consortium in broad ranges of temperature (4–42 °C), pH (5–10), and salinity (0–100 g/L).

3.4. Crude Oil Degradation by Single Strains and the Constructed Bacterial Consortium

To investigate crude oil degradation potential, each strain (S1-2, S2-A, CN-3, or HC8-3S), as well as the constructed bacterial consortium, were inoculated respectively into flasks with crude oil (1%, w/v) as the sole carbon source for 10 d. As one of the most complex mixtures in nature, crude oil is mainly composed of TPHs identifiable by GC–MS, as well as great amounts of unknown chemicals. Herein, TPHs were classified as different carbon number hydrocarbons (C₇–C₃₅) or different hydrocarbon components (alkane, cycloalkane, branched alkane, and aromatic hydrocarbon).

Table 1. Main compounds identified in crude oil by GC–MS.

Retention Time (s)	Compounds	Formula	Retention Time (s)	Compounds	Formula
239.82	Toluene	C7H8	1351.00	Phytane	C20H42
251.95	1,3-dimethylcyclohexane	C8H16	1419.85	Nonadecane	C19H40
356.53	Ethylbenzene	C8H10	1451.83	1-methylphenanthrene	C15H12
366.99	p-Xylene	C8H10	1493.19	Eicosane	C20H42
541.12	Decane	C10H22	1563.27	Heneicosane	C21H44
667.04	Undecane	C11H24	1630.37	Docosane	C22H46
772.60	Naphthalene	C10H8	1694.68	Tricosane	C23H48
783.42	Dodecane	C12H26	1756.41	Tetracosane	C24H50
891.44	Tridecane	C13H28	1815.79	Pentacosane	C25H52
969.77	2,6,10-trimethyldodecane	C15H32	1872.49	Hexacosane	C26H54
992.43	Tetradecane	C14H30	1928.17	Heptacosane	C27H56
1025.22	1,6-dimethylnaphthalene	C12H12	1981.49	Octacosane	C28H58
1087.38	Pentadecane	C15H32	2033.11	Nonacosane	C29H60
1177.12	Hexadecane	C16H34	2084.01	Triacontane	C30H62
1219.37	2,6,10-trimethylpentadecane	C18H38	2140.42	Hentriacontane	C31H64
1262.20	Heptadecane	C17H36	2205.1	Dotriacontane	C32H66
1267.30	Pristane	C19H40	2280.72	Tritriacontane	C33H68
1315.42	Undecylcyclohexane	C17H34	2370.29	Tetratriacontane	C34H70
1343.01	Octadecane	C18H38	2477.33	Pentatriacontane	C35H72

summarizes the crude oil compounds, retention time, and chemical formulae. In detail, alkanes ranged from n-decane (C₁₀) to n-pentatriacontane (C₃₅); cycloalkanes contained 1,3-dimethylcyclohexane and undecylcyclohexane; 2,6,10-trimethyldodecane, 2,6,10-trimethylpentadecane, pristane, and phytane were the dominant branched alkanes; and toluene, ethylbenzene, p-xylene, naphthalene, 1,6-dimethylnaphthalene, and 1-methylphenanthrene were the main aromatic hydrocarbons.

Despite previous research indicating that bacterial consortia generally degrade crude oil more efficiently than individual strains, few studies have assessed the degradation of specific crude oil compounds. After 10 d cultivation, the consortium obtained a TPH degradation ratio of 97.1% (Figure S2), significantly higher (p < 0.05) than that of the single strains (91.4% for S1-2, 85.8% for S2-A, 89.5% for CN-3, and 87.2% for HC8-3S), with a natural depletion control at only 5.9%. The degradation efficiencies of different carbon number hydrocarbons (C₇–C₃₅) and components (alkanes, cycloalkanes, branched alkanes, and aromatic hydrocarbons) are displayed in Figure 3a,b. As shown in Figure 3a, four single strains had distinctive degradation characteristics, especially for short-chain alkanes (C₇–C₁₀) and long-chain alkanes (C₂₁–C₃₀ and C₃₁–C₃₅). Notably, the consortium exhibited superior degradation rates for most components compared to individual strains, particularly for long-chain alkanes (C₃₁–C₃₅), where it achieved 88.7% degradation efficiency—22% higher than S2-A and 12.2% higher than S1-2. For the different crude oil components in Figure 3b, S1-2 utilized a broad range of hydrocarbons (alkanes, cycloalkanes, branched alkanes, and aromatic hydrocarbons) with degradation efficiencies above 86.5%; CN-3 excelled in degrading alkanes, cycloalkanes, and branched alkanes well; while S2-A was proficient in alkanes and cycloalkanes; and HC8-3S depleted alkanes and aromatic hydrocarbons well. Although all the individual strains degraded alkanes efficiently (> 90%), the consortium achieved the highest degradation rate of 98.3%. Overall, the consortium depleted four components of alkanes, cycloalkanes, branched alkanes, and aromatic hydrocarbons with high efficiency (> 91%), achieving significantly better enhancements (p < 0.05) than the individual strains and highlighting the effective synergy of four crude-oil-degrading strains.

3.5. Effects of Temperature, pH, and Salinity on Crude Oil Degradation

Different environmental stressors (temperature, pH, and salinity) play significant roles in bacterial activity and physiological performance in their habitats. As a result, the optimal cultural conditions for this constructed bacterial consortium to degrade crude oil were investigated. The influence of temperature on TPH removal is displayed in Figure 4a. The optimal temperature was 30 °C, with a TPH residual ratio of 5.1%. This was followed by temperatures of 37 °C (12.2%), 20 °C (15.6%), and 42 °C (33.7%). As shown in Figure 4b, residual TPHs varied from 3.2% to 34.1% in pH ranges of 5−10, with optimal TPH removal at pH 8. Notably, even at a strong alkaline pH of 10, TPH degradation remained at 76.3%, indicating minimal restriction in degradability. As Figure 4c shows, compared to temperature and pH, salinity had apparently less effect on crude oil utilization, showing the superiority of halotolerant bacteria. Crude oil degradation efficiencies were consistently above 85%, even under a high salinity of 100 g/L, displaying wonderful salinity tolerances. The optimal salinity was 40 g/L, slightly better than 20 g/L. In a word, functional optimization demonstrated that this consortium depleted crude oil effectively in a broad range of temperatures (20–37 °C), pH (6–9), and salinities (0–100 g/L).

3.6. Crude Oil Degradation in Non-Saline and Salt-Enriched Soil Microcosms

The bioremediation potential of the constructed bacterial consortium to deplete crude oil was investigated in both non-saline and salt-enriched soil microcosms with diverse treatments: abiotic control, natural attenuation, biostimulation, bioaugmentation, and biostimulation + bioaugmentation. In non-saline soil microcosms, the crude oil depletion efficiencies of AC, NA, BS, BA, and BS + BA treatments were significantly different (p < 0.05) after 30, 45, and 60 d (Figure 5a). Specifically, the abiotic control (AC) resulted in a 15.14% depletion of crude oil after 60 d, with only a marginal increase upon extended incubation. The natural attenuation (NA) treatment achieved a 22.77% depletion, reducing the TPH concentration to 38,624 mg/kg, likely due to the activity of indigenous microbiota. Regardless of sample points, the crude oil degradation efficiencies in the BS microcosm were all significantly higher (p < 0.05) than those of AC and NA, revealing that nutrient amendments improved the growth and activity of indigenous bacteria. The bioaugmentation (BA) treatment, which involved adding the constructed bacterial consortium, enhanced crude oil removal to 60.30%, with most of the degradation occurring within the first 45 d. Particularly, the biostimulation + bioaugmentation (BS + BA) treatment achieved the highest TPH degradation of 70.19% with a high degradation rate of 585.1 mg/kg/d, demonstrating the effective synergy between bioaugmentation and biostimulation.

In salt-enriched soil microcosms, crude oil depletion was generally lower across treatments compared to their non-saline counterparts. The crude oil removal of S-NA (16.92%) or S-BS (32.32%) was significantly (p < 0.05) lower than that of NA (22.77%) or BS (37.65%) after 60 d, demonstrating that salinity inhibited the effectiveness of indigenous microbiota and nutrient amendments. On the contrary, there was no significant difference in crude oil depletion between S-BA (58.31%) and BA (60.30%) throughout the test periods, implying that the halotolerant consortium maintained its bioremediation potential in saline environments. In particular, the S-BS + BA treatment achieved the highest TPH depletion of 68.23% with a high degradation rate of 568.6 mg/kg/d, indicating the synergistic effects of bioaugmentation and biostimulation on crude oil removal and bacteria growth in high salt environments.

3.7. TPH Degradation Kinetics in Soil Microcosms

Taking advantage of a first-order kinetic mode, the TPH degradation for non-saline and salt-enriched soil microcosms under five treatments of abiotic control, natural attenuation, biostimulation, bioaugmentation, and biostimulation + bioaugmentation were predicted and analyzed. The kinetic equation, degradation rate constant (k), correlation coefficient (R²), and half-life time (t_1/2) are displayed in

Table 2. Kinetic equation, degradation rate constants (k), correlation coefficients (R²), and half-life (t_1/2) of TPH degradation in soil microcosms under different treatments.

Treatment	k (d−1)	t1/2 (d)	R2	Treatment	k (d−1)	t1/2 (d)	R2
AC	0.0026	266.60	0.9033	S-AC	0.0027	256.72	0.9437
NA	0.0041	169.06	0.8756	S-NA	0.0029	239.02	0.9413
BS	0.0078	88.87	0.9343	S-BS	0.0065	106.64	0.9507
BA	0.0169	41.01	0.9477	S-BA	0.0157	44.15	0.9727
BS + BA	0.0219	31.65	0.9694	S-BS + BA	0.0208	33.32	0.9704

. The correlation coefficients (0.8756–0.9727) were achieved through linear regression analysis, demonstrating that the first-order kinetic model properly characterized the TPH degradation.

Specifically, the lowest k (0.0026 d⁻¹) and longest t_1/2 (266.60 d) were obtained in AC, indicating that natural crude oil evaporation was a slow process. The TPH degradation was accelerated with a slightly higher k (0.0041 d⁻¹) and shorter t_1/2 (169.06 d). Natural attenuation (NA) slightly accelerated TPH degradation with a higher k (0.0041 d⁻¹) and a shorter t_1/2 (169.06 d). The bioaugmentation (BA) treatment, involving the addition of the constructed bacterial consortium, significantly increased k to 0.0169 d⁻¹. Notably, the biostimulation + bioaugmentation (BS + BA) treatment achieved the highest k (0.0219 d⁻¹) and the shortest t_1/2 (169.06 d). Natural attenuation (NA) slightly accelerated TPH degradation with a higher t_1/2 (31.65 d), demonstrating the substantial synergistic effects of combining bioaugmentation and biostimulation.

In salt-enriched soil microcosms, k ranged from 0.0027 to 0.0208 across the five treatments. Salinity impeded the degradation process in S-NA and S-BS but had minimal effect on S-BA and S-BS + BA. Adding NaCl decreased k from 0.0041 (NA) to 0.0029 (S-NA), a reduction of 29.27%, whereas the decrease from BA (0.0169) to S-BA (0.0157) was only 7.10%. The bacterial consortium and nutrient amendments in S-BS + BA further mitigated the impact of salinity, reducing k by only 5.02% from BS + BA.

3.8. Abundance of Functional Genes

Real-time quantitative PCR estimated bacterial abundance by targeting total 16S rRNA and alkB gene copies. Different distribution patterns of gene abundance were observed in these treatments. In non-saline soil microcosms, due to the inoculation of a constructed bacterial consortium, the 16S rRNA gene copy of BA or BS + BA was apparently higher than in NA or BS (Figure 6a) during the entire incubation period. The largest 16S rRNA gene copy was 7.02 × 10⁹ g⁻¹ in the BS + BA treatment (15 d). In salt-enriched soil microcosms, although the 16S rRNA gene copies decreased in most treatments compared with those in non-saline soil microcosms at the same sample point, S-BA (5.15 × 10⁹ g⁻¹) had significantly higher gene copies than BA (4.78 × 10⁹ g⁻¹) on 45 d.

The initial alkB copy number in NA was 2.1 × 10⁸ g⁻¹ (Figure 6b), reflecting the presence of indigenous petroleum hydrocarbon-degrading bacteria, and it declined gradually over 60 d. In the BA and BS + BA treatments, alkB copy numbers initially decreased but then increased in line with crude oil removal after 30 d. The ratio of alkB/16S rRNA, indicating the proportion of alkane-degrading bacteria, increased significantly in the BS + BA treatment, reaching 6.29 times higher than in the NA treatment after 45 d, aligning with its superior crude oil degradation performance. In salt-enriched soil microcosms, alkB copy numbers apparently decreased in S-NA and S-BS. However, the alkB/16S rRNA ratio for S-BA (0.78) or S-BS + BA (0.83) was 5.20 times or 5.53 times higher than the S-NA ratio (0.15) on 45 d, likely due to the enhanced performance of the halotolerant bacterial consortium and the synergistic effects of bioaugmentation combined with nutrient biostimulation.

4. Discussion

Bioremediation by functional bacterial strains has been considered one of the most promising approaches to treat crude oil pollution, as well as being more effective, versatile, economical, and environmentally friendly compared with physical and chemical remediation [1,4,44,45]. In this study, two halotolerant crude oil-degrading strains of Pseudoxanthomonas sp. S1-2 and Bacillus sp. S2-A were newly isolated and identified from crude-oil-contaminated saline soil at Shengli Oilfield in the Yellow River Delta region. Further, supplemented with Dietzia sp. CN-3 and Acinetobacter sp. HC8-3S, these four halotolerant bacterial strains, known for their effective crude oil degradation and biosurfactant production, were applied to construct a mixed bacterial consortium. Experiments in liquid media, non-saline, and salt-enriched soil microcosms were conducted to evaluate the consortium’s efficacy and potential. The effects of abiotic factor, natural attenuation, biostimulation, bioaugmentation, and bioaugmentation + biostimulation treatments on crude oil depletion were also analyzed, providing a valuable theoretical basis and bacterial candidates for the bioremediation of crude oil pollution in high-salinity soil.

From the perspective of strain resources, genera of Bacillus and Acinetobacter are predominant crude-oil-degrading bacteria and widely reported in many studies [1,11,12], and Dietzia has also attracted great attention in the last ten years [14,24,35]. However, relevant reports of Pseudoxanthomonas in crude oil degradation are rare. For instance, Pseudoxanthomonas sp. RN402 was able to degrade diesel oil slightly (35%) and the removal was enhanced to 80% after immobilization on polyethylene plastic pellets [46]. A comparative study of genomic analysis investigated the genomic inventories of Pseudoxanthomonas involved in the metabolic pathways of benzene, toluene, ethylbenzene, and o-, m-, and p-xylene [47]. Pseudomonas, Pseudoxanthomonas, and Pseudoallescheria had the highest relative abundances in petroleum pollution soil through 16S rRNA sequences [48], but no pure culture was obtained. In this study, due to its noticeable crude-oil-degrading performance, Pseudoxanthomonas sp. S1-2 was newly isolated from crude-oil-contaminated saline soil and applied in crude oil biodegradation and bioremediation.

Generally, bacteria employ three key strategies to exploit petroleum hydrocarbons: secreting biosurfactants, altering cell surface hydrophobicity, and forming biofilms. These strategies can appear separately or simultaneously in different strains [35,49]. Herein, the biosurfactant production ability of S1-2, S2-A, CN-3, and HC8-3S with different carbon sources were assessed through emulsification activity and cell surface hydrophobicity assays. Notably, S2-A displayed high emulsification ability (> 65%) and low CSH (< 44%) with tetradecane, paraffin oil, and crude oil, suggesting that emulsification activity played a more significant role than cell surface hydrophobicity in this case. On the contrary, cell surface hydrophobicity might be more important than emulsification activity in HC8-3S. For S1-2, both E₂₄ and CSH indicators were considerably high (> 61%) in crude oil utilization, predicting that crude oil degradation relied on the two strategies. Noticeably, bacterial biosurfactant types were closely related to the carbon source [22,24,25,50]. For example, Dietzia sp. strain DQ12-45-1b produced glycolipids, phospholipids, and lipopeptides when degrading C₁₆, C₂₄, and C₃₆, respectively [24]. The types, yields, and molecular characteristics of biosurfactants produced by S1-2, S2-A, CN-3, and HC8-3S with various petroleum hydrocarbons warrant further investigation.

Thanks to their metabolic diversity and reliability for field applications, it is generally more practical to use microbial consortia to deplete crude oil than single strains [45]. Although different crude oil components vary in their susceptibility to bacterial attack [3,7,35], the overall pattern displayed by this consortium was: alkane > branched alkane > cycloalkane > aromatic hydrocarbon. Usually, low-molecular-weight alkanes are more readily assimilated and degraded by bacteria, while high-molecular-weight hydrocarbons, due to their low solubility, often require biosurfactants to facilitate uptake and assimilation [15,51]. The bacterial consortium, consisting of four biosurfactant-producing strains, is likely to address these challenges effectively.

Among environmental factors, temperature, pH, and salinity play significant roles in crude oil degradation by influencing the physiology and diversity of the microorganisms, as well as the physicochemical properties (viscosity, volatility, etc.) of the pollutants [45,52,53]. Oil-spill accidents often occur in high-salinity environments, such as salt lakes, oil reservoirs, salt cavern oil storage, and so on. Salinity not only influences the growth and diversity of microorganisms but also has direct effects on biodegradation and functional enzymes [45,52,54]. The crude oil degradation efficiencies of the constructed bacterial consortium were all higher than 85%, even under a high salinity of 100 g/L, implying that this consortium of four salt-tolerate strains could address the deficiencies and take effect in crude oil mineralization. Notably, the constructed bacterial consortium had super robustness and viability in wider ranges of temperature (20–37 °C), pH (6–9), and salinity (0–100 g/L) than many single strains or consortia [1,12,52].

The successful bioremediation of crude oil contaminants relies not only on the metabolic capabilities of introduced microorganisms but also on the survival abilities of inoculants in the environments [16,55]. Bioavailability for microbial reactions could be decreased for contaminants’ sorption to soil particles, wider diffusion in soil macropores, or in nonaqueous-phase liquid form [16,29,45]. Thus, the bioavailability of crude oil contaminants is a vital factor in interfering with the bioremediation process in soil microcosms. Nutrient amendments, especially to the phosphate and nitrate salts, are important for cell growth and division, as well as biostimulation electron donors, to alter contaminant bioavailability.

Although the ecological safety of exogenous bioaugmentation is still debatable, many laboratory and field researches have proved that bioaugmentation contributes to enhancing petroleum utilization through microbial metabolic capacities and the reliability needed for field applications [21,53]. Bioaugmentation of our consortium in the non-saline soil microcosms degraded 60.30% of TPHs after 60 d, which was significantly higher (p < 0.05) than with the AC, NA, and BS treatments. Meanwhile, the 16S rRNA and alkB gene numbers had the same variation trend, displaying the good adaptability and biodegradation of this consortium. Remarkably, thanks to the simultaneous effects of biostimulation and bioaugmentation, the highest crude oil depletion (70.19%), with a degradation rate of 585.1 mg/kg/d, was found in the BS + BA microcosm, which was obviously higher than that found in plenty of researches [17,29,39,56]. For instance, a hydrocarbon-utilizing bacterial consortium comprised of three strains (Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Ochrobactrum sp.) degraded 82.13% of crude oil at a degradation rate of 439.9 mg/kg/d in oily sludge microcosms, significantly better than natural attenuation and abiotic factors [17]. However, the consortium of Rhodococcus erythropolis CD 130 and CD 167 had a lower TPH depletion (29.72%) than CD 167 (38.40%), suggesting that these two strains compete intensely or have antagonistic action in petroleum utilization [56].

Soil salinization in crude oil fields can reduce microbial respiration and, consequently, lower bioremediation rates [28,29,57]. For example, a bacterial consortium was formed using four biosurfactant-producing Pseudomonas aeruginosa strains. After 120 days of inoculation in soils with 0, 150, and 300 mM NaCl, the consortium achieved crude oil degradation rates of 49.5%, 47.0%, and 42.3%, respectively [29]. Herein, in the salt-enriched soil microcosms (NaCl 20 g/kg), the crude oil depletion of S-BA (58.31%) was satisfying, thanks to the bioaugmentation of the halotolerant consortium, with high k (0.0157 d⁻¹), bacterial numbers (4.13 × 10⁹ g⁻¹) and alkB gene copies (3.22 × 10⁹ g⁻¹). The highest TPH depletion occurred in the S-BS + BA microcosm, which exhibited a high degradation rate of 568.6 mg/kg/d after 60 d. This highlights the synergistic effects of bioaugmentation and biostimulation on crude oil removal and bacterial growth in high-salinity environments, which is better than many studies have found [17,29,56,57].

Despite numerous crude-oil-degrading strains and communities being isolated and identified from various habitats, their viability, sustainability, and competitiveness must be continuously improved to address the challenges to crude oil degradation. There is still limited knowledge regarding the molecular mechanisms (e.g., genes and enzymes) and pathways involved in crude oil degradation under high salinity. Thus, further research into the physiology, biochemistry, metabolic mechanisms, and remediation technologies related to crude oil bioremediation is essential for future advancements.

5. Conclusions

This study investigated the efficacy of a bacterial consortium constructed from four halotolerant petroleum degraders and biosurfactant producers in the bioremediation of crude oil pollution. The consortium effectively promoted the removal of C₇–C₃₅, including alkanes, cycloalkanes, branched alkanes, and aromatic hydrocarbons, with abilities to withstand broad temperatures (20–37 °C), pH levers (6–9), and salinities (0–100 g/L). In both non-saline and salt-enriched crude oil-contaminated soil microcosms, the most effective strategy for bioremediation of oil-contaminated soils is a combination of biostimulation and bioaugmentation using the halotolerant consortium of biosurfactant-producing microorganisms. The outcomes of this study contribute to establishing feasible bioremediation strategies for crude oil contaminants in high salinity.