Study on the Synergistic Enhancement of Crude Oil Recovery by Bacillus Co-Culture Systems

Abstract

Microbial-enhanced oil recovery (MEOR) is a promising technology for oilfield development. To improve MEOR efficiency, two functional strains—Bacillus mucilaginosus ZZ-8 and Bacillus amyloliquefaciens ZZ-11—were isolated and purified. The growth characteristics, biosurfactant production, and crude oil emulsification performance of these strains were systematically evaluated through single-strain cultures and a co-culture system (ZZ-8: ZZ-11 = 1:1). The results demonstrated that the co-culture system exhibited superior growth and functional performance compared to monocultures. The cell-free supernatant significantly reduced oil–water interfacial tension, decreasing the contact angle from 53.56 ± 1.3° to 28.78 ± 0.82°, thereby enhancing crude oil detachment from rock surfaces and improving oil displacement efficiency. Gas chromatography (GC) analysis further confirmed the co-culture system’s pronounced degradation of long-chain alkanes (C17–C35). In oil sand washing experiments, the 1:1 mixed-strain fermentation broth achieved a crude oil elution rate of 84.39%, representing an 89.80% increase over uninoculated medium. This study not only validates the synergistic effect of the B. mucilaginosus–B. amyloliquefaciens co-culture system in enhancing oil recovery but also provides a theoretical foundation and innovative strategy for its practical application in MEOR technology.

1. Introduction

Following primary and secondary oil recovery stages, an increasing number of oilfields are gradually transitioning to a low-yield phase, during which conventional technologies can only extract 20% to 30% of the original oil in place (OOIP) [1]. With the continuous growth in global oil and gas demand and the declining availability of easily exploitable resources, the economic and efficient development of this “residual oil” has become a major challenge for the petroleum industry [2]. To enhance ultimate recovery, enhanced oil recovery (EOR) techniques have increasingly become a research focus, encompassing methods such as chemical flooding, gas injection, thermal recovery, and microbial-enhanced oil recovery [3]. Microbial-enhanced oil recovery (MEOR) is a type of tertiary oil recovery technology that relies on the actions of microorganisms and/or their metabolites. Due to its low cost, effectiveness, and environmental friendliness, MEOR is recognized as a promising green oil recovery technology [4,5], and its application in oilfields is becoming increasingly widespread.

The efficacy of microbial-enhanced oil recovery largely depends on the performance of functional bacteria [6]. While laboratory research and field tests have demonstrated promising results, MEOR strategies vary significantly in their application. Compared to endogenous microbial approaches, exogenous microbial-enhanced oil recovery offers distinct advantages: it allows for better prediction of bacterial activity based on reservoir characteristics, requires shorter oil production times, eliminates the need for a reservoir shut-in period, and enables comprehensive monitoring throughout the process [7]. For instance, Ren Fuping et al. [8] isolated strains from reservoir water samples in the Baolige area of the Huabei Oilfield, demonstrating their ability to reduce surface tension, emulsify, and degrade crude oil. Similarly, Marghmaleki et al. [9] reported that Alcaligenes faecalis increased oil recovery by 8.2% during shut-in conditions and by 5.2% under rapid displacement conditions. Gudina et al. [10] further highlighted the role of Bacillus subtilis strains, which enhanced oil recovery by 6% to 24%, depending on the composition of the crude oil. These findings underscore the idea that MEOR does not follow a one-size-fits-all solution. Given this variability, indoor simulations and mechanistic analyses are critical for the successful field application of MEOR technologies.

Many microbial strains isolated from oil reservoirs, including Acinetobacter junii BD, Pseudomonas aeruginosa L6-1, and Bacillus subtilis M15-10-1, have been employed as exogenous microorganisms in microbial-enhanced oil recovery research [11,12]. Direct injection of microbially produced surfactants is an effective exogenous MEOR strategy and serves as an excellent alternative to surfactant-based chemical flooding in enhanced oil recovery. Biosurfactants, which are secondary metabolites composed of glycolipids, lipopeptides, phospholipids, and polysaccharide-protein complexes [13], are regarded as the most promising microbial metabolites for MEOR. These surfactants enhance oil recovery through multiple mechanisms: (1) reducing interfacial tension (IFT) between oil and water, (2) altering the wettability of rock surfaces from lipophilic to hydrophilic, and (3) facilitating in situ emulsion formation within the reservoir [14,15]. It is known that Bacillus is an excellent strain for industrial production of lipopeptide surfactants [16]. Among them, Bacillus subtilis and Bacillus licheniformis are the most reported strains in the field of microbial oil recovery. At present, the main problem of MEOR is that there are not many singular high-quality strains with strong recovery function [17,18]. Therefore, the screening and performance optimization of oil recovery microorganisms with excellent performance are the key factors to determine the normal growth and reproduction of microorganisms and improve oil recovery.

In this study, Bacillus mucilaginosus and Bacillus amyloliquefaciens were isolated through screening, and the potential applications of these two bacteria, as well as their co-culture system, in enhanced oil recovery were investigated. First, the growth, biosurfactant production capabilities, and emulsifying properties of both single and co-culture systems were assessed. The cell density and biosurfactant production of these systems were quantitatively analyzed. Building on this, the variations in crude oil components before and after microbial treatment were compared and analyzed using gas chromatography. Finally, a microbial oil washing experiment was conducted to evaluate the mechanisms and potential of the co-culture system in improving oil recovery.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals, reagents, and hydrocarbons used were of analytical grade (AR) and purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). The crude oil samples had the following basic physicochemical properties: relative density (0.8583), viscosity (19.38 mPa·s), wax content (23.56%), and combined asphaltene and resin content (13.0%).

2.2. Strains, Culture Conditions, and Preparation of Inoculums

The two bacterial strains utilized in this study were isolated from petroleum-contaminated soil samples. Subsequently, molecular identification was performed through 16S rDNA sequencing, PCR amplification, and sequencing techniques, which unambiguously characterized their respective genus and species affiliations.

In this study, two different nutrient media were used: LB medium (g/L) containing yeast extract 5.0, tryptone 10.0, NaCl 5.0, pH adjusted to 7.0–7.5 with NaOH and HCl; the crude oil inorganic salt medium (g/L) contained crude oil 2.0, K₂HPO₄ 1.0, KH₂PO₄ 1.0, MgSO₄ 0.5, NH₄Cl, CaCl₂ 0.02, and FeCl₃ trace. The pH was also adjusted to 7.0–7.5 with NaOH and HCl.

Before each experiment, glycerol-preserved strains of Bacillus mucilaginosus ZZ-8 and Bacillus amyloliquefaciens ZZ-11 were inoculated into LB medium at 1% (v/v) and incubated at 37 °C with shaking at 140 rpm for 24 h. The bacterial suspension was then centrifuged at 6000× g for 5 min. After discarding the supernatant, the pellet was resuspended in phosphate-buffered saline (PBS, pH 7.3) to an optical density (OD) of 1.0 to prepare the inoculum for subsequent experiments [19].

2.3. Biosurfactant Activity Detection

The surface activity changes-surface tension (SFT) and interfacial tension (IFT) of cell-free samples were regularly measured by the “hanging drop method”, using the Drop Shape Analyzing system-DSA 100 (KRÜSS, Hamburg, Germany). IFT measurements were compared with crude oil samples. All measurements were conducted in triplicate under ambient conditions (25 ± 2.0 °C, 1 atm), and mean values are presented [20].

2.4. Water Separation Measurement

The emulsifying performance of biosurfactants against crude oil was evaluated by measuring water separation rate [21]. Specifically, 5 mL of cell-free fermentation broth and 5 mL of crude oil were introduced into a 25 mL stoppered graduated cylinder. The system was pre-incubated at 45 °C for 20 min to equilibrate the temperature. After tight sealing, the cylinder was vigorously shaken for 5 min to facilitate emulsion formation. Subsequently, the cylinder was allowed to stand statically in the same incubator at 45 °C. The timing was initiated immediately upon standing, and the volume of separated water was recorded at predetermined time intervals. The water separation rate was calculated accordingly. All experiments were performed in triplicate. The equation of E_d is

$${\mathrm{E}}_{\mathrm{d}}={\displaystyle \frac{{\mathrm{V}}_{\mathrm{W}}}{\mathrm{V}}}$$

(1)

where

E_d is the water separation ratio (%);

V_w is the volume of separated water (mL);

V is the total volume of the aqueous phase (mL).

2.5. Oil Spreading Method

The oil displacement method demonstrates the presence of biosurfactants by dispersing an oil film. This procedure follows the protocol described by Jaysree et al. [22]: A 9 cm-diameter culture dish is filled with distilled water, and 100 μL of crude oil is added to the water surface to form an oil film. Then, 20 μL of the supernatant is gently added to the center of the oil layer. If biosurfactants are present in the supernatant, a clear zone (halo) will form due to oil displacement. The diameter of the halo is positively correlated with the biosurfactant’s activity.

2.6. Contact Angle Measurement

The contact angle was measured to assess biosurfactant activity, following the method described by Al-Sulaimani et al. (2012) [23]. A DSA100 system was utilized to compare the contact angles between a blank control (uninoculated medium) and fermentation broth samples containing biosurfactants. All measurements were conducted at room temperature (25 ± 2.0 °C) and atmospheric pressure (1 atm). Each experimental group was tested in triplicate, and the results were averaged to ensure statistical reliability.

2.7. Gas Chromatographic Analysis of Saturated Hydrocarbons

The residual oil components after degradation by single bacterial strains and microbial consortia were determined using an Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). The analysis was performed under the following conditions: helium carrier gas at a flow rate of 2 mL/min; an initial column temperature of 60 °C held for 1 min, followed by temperature programming at 8 °C/min to 290 °C, before ramping at 30 °C/min up to 320 °C and held for 7 min. A petroleum hydrocarbon mixed standard (1000 μg/mL) was used as an internal standard for calibration of the GC measurements [24].

2.8. Evaluation of the Oil Washing Efficiency

The oil washing efficiency of the oil sands by nanoemulsion was tested based on the weight-loss method [25]. Crude oil and quartz sand (Henan Minghai Environmental Protection Technology Co., Ltd., Zhengzhou, China) were homogenously mixed at a mass ratio of 1:5, followed by aging in an oven at 60 °C for 7 days. A predetermined quantity of oil-sand mixture was then immersed in 30 mL of fermentation broth and maintained under static conditions for 24 h. The oil washing rate was calculated by [26]:

$$\mathsf{\eta } = {\displaystyle \frac{m_1-m_2}{m_3}}$$

where $\mathsf{\eta }$ is the oil washing rate, m₁ is the mass of oil sands before oil washing, m₂ is the mass of oil sands after oil washing, and m₃ is the mass of oil sands after aging process.

3. Results and Discussion

3.1. Strain Identification

Based on 16S rRNA gene sequence analysis, strain ZZ-8 showed >98% homology with Bacillus mucilaginosus. Combined with Gram staining (Figure 1A) and microscopic examination, the strain was identified as Bacillus mucilaginosus. When cultured on LB solid medium, the colonies appeared colorless, transparent, glossy, smooth, and rounded, with a sticky texture and neat edges (Figure 1C). Gram staining confirmed it as Gram-negative. Strain ZZ-11 exhibited high homology with Bacillus amyloliquefaciens. Gram staining (Figure 1B) and microscopic analysis identified it as Bacillus amyloliquefaciens. On LB solid medium, the colonies were filthy white, round or irregular, with a rough, sticky surface (Figure 1D). Gram staining confirmed it as Gram-positive.

3.2. Biosurfactant Production

The growth properties and functional expression ability of microorganisms are key factors affecting their oil recovery performance. In this study, the growth characteristics, surfactant production and emulsification ability of microorganisms can be indirectly assessed by systematically quantifying their crude oil recovery performance. Spectrophotometry was used to measure the absorbance of bacterial liquid (OD600) at 600 nm to characterize the growth of microorganisms (Figure 2A); surface tension measurements and oil drainage circle experiments were used to characterize the biosurfactant production (Figure 2B,C). Oil–water interfacial tension is one of the most important indexes for measuring the efficiency of the surfactant in oil washing. In general, the smaller the oil–water interfacial tension is, the easier the formation crude oil can be driven out. The active role of microbial-generated biosurfactant at the oil–water interface was evaluated by measuring the interfacial tension. After 3 d of incubation, single strains ZZ-8 and ZZ-11 showed similar performance in terms of growth performance, surface tension, and interfacial tension. Both strains were able to significantly reduce the surface tension of the fermentation broth from the initial 58.496 mN/m to 21.977 mN/m (ZZ-8) and 22.069 mN/m (ZZ-11), with a reduction of 62.4% and 62.3%, respectively, suggesting that they have strong surfactant synthesis ability. Compared with the monoculture, the co-culture system with 1:1 compounding ratio showed optimal performance, having the highest cell density (OD 600: 2.23), strongest biosurfactant production (surface tension = 18.46 mN/m, oil drainage circle diameter = 45.00 mm), and best regulation of oil–water interfacial performance (interfacial tension = 0.68). These results indicate that co-culture can synergistically promote bacterial growth, enhance biosurfactant synthesis, and improve emulsification performance. The results were consistent with those reported in the literature [27,28], confirming that both biosurfactant production and crude oil emulsification capacity were positively correlated with bacterial growth.

The water precipitation rate is a crucial index for evaluating the stability of oil-in-water emulsions formed by biosurfactants interacting with crude oil. This rate is negatively correlated with emulsion stability; specifically, a lower water precipitation rate indicates more stable emulsion and stronger emulsification performance of the system. Similarly, Jin et al. (2024) assessed the emulsification performance of in situ emulsified activated polymer with associative binding (ISEPAM) by monitoring the water precipitation rate [29]. As illustrated in Figure 3, the water precipitation rates of all experimental groups exhibited an increasing trend over time. Notably, the ZZ-11, ZZ-8, and 1:1 co-culture systems stabilized during the later stages of the reaction, with final water precipitation rates of 47%, 44%, and 16%, respectively. These results suggest that the 1:1 co-culture system demonstrates superior emulsion stability performance.

In order to further investigate the emulsification degradation of crude oil via the ZZ-11 and ZZ-8 co-culture systems, the emulsification degradation of crude oil under the 1:1 co-culture condition was systematically analyzed. As shown in Figure 4, in the initial state (0 days), the crude oil floated uniformly on the surface of the medium, showing a typical sheet-like aggregation state, and the interface between the oil and water phases was clearly recognizable. After 3 days of incubation, compared with the sterile blank control group, the 1:1 co-culture system showed significant microbial value-added characteristics, and the medium appeared obviously turbid, while the crude oil was emulsified and dispersed to form tiny oil droplets with uniform particle size, and some areas showed the mixed-phase state of oil and water. On the 7th day of incubation, the emulsification effect continued to increase, and the size of oil droplets was further reduced, while the lower layer of the bacterial liquid showed a characteristic brown color due to the accumulation of crude oil degradation products. The above experimental results confirmed that ZZ-11 and ZZ-8 compounded in the ratio of 1:1 could not only grow synergistically with crude oil as the only carbon source but also significantly promote the physical dispersion and biodegradation of crude oil through bioemulsification.

3.3. Contact Angle Measurement

Any change in the “oil–water–rock” interface in reservoirs leads to changes in surface wettability, which is an important mechanism for enhanced recovery [30,31]. The change in wettability is related to interfacial tension, and biosurfactants can change hydrophilic surfaces to lipophilic surfaces, and vice versa [32]. It has been shown that when the contact angle is in the range of 20.8–43°, the system exhibits a strongly water-wet state. The stronger the water-wetness of the reservoir rock, the more favorable it is for the crude oil to be stripped from the rock surface, thus promoting the crude oil to be driven out from the pore cracks and ultimately achieving the effect of improving the crude oil recovery. Therefore, this study evaluated the effect of the biosurfactant (cell-free supernatant) produced under the 1:1 compounding system on the wettability of the hydrophobic surface of coverslips. The results of contact angle measurements (Figure 5A,B) showed that the contact angle was significantly reduced to 28.78 ± 0.82° after treatment with cell-free supernatant compared to the initial contact angle of uninoculated medium (53.56 ± 1.3°). This result is in line with the trend reported in the literature: a study by Joshi et al. [33] showed that the contact angles of uninoculated medium and cell-free supernatant were 55.67 ± 1.6° and 19.54 ± 0.7°, respectively; Al-Sulaimani et al. [30] found that the biosurfactant produced by Bacillus subtilis W19 (0.25% w/v) resulted in a decrease in distilled water’s contact angle from 70.6 ± 0.3° to 25.32 ± 0.06°; and Al-Wahaibi et al. [34] also reported that the biosurfactant produced by Bacillus subtilis B30 in glucose or molasses medium reduced the contact angle of hydrophobic surfaces from 58.7 ± 0.85° to 28.4 ± 1.03° and 27.2 ± 0.72°, respectively. This study demonstrated that the biosurfactant could significantly enhance the hydrophilicity of the hydrophobic surface, a property that has potential application for enhanced crude oil recovery.

3.4. Gas Chromatographic Analysis Results of Crude Oil

Microbial degradation of crude oil and biosurfactant generation are the core mechanisms in the microbial oil drive process. The interaction of microbial activity and its metabolites with crude oil and rock cores jointly promotes the enhancement of oil recovery [35,36]. In order to gain a deeper understanding of the degradation characteristics of petroleum hydrocarbons by single bacteria and their composite flora, the petroleum hydrocarbon fractions before and after degradation were analyzed by GC. With reference to previous studies [37], the carbon atom number ranges of short-chain alkanes (C8–C17), long-chain alkanes (C18–C30) and heavy long-chain alkanes (C31–C36) were clearly defined. Through Figure 5A, it was found that the carbon number in crude oil was mainly distributed from C11 to C28, while the vast majority of hydrocarbon fractions in crude oil were degraded after 5 d of microbial degradation. Specifically, the ZZ-8 strain showed specific degradation ability for long-chain alkanes (C22–C33), and both its relative content and absolute concentration showed obvious decreasing trends (Figure 5B). In contrast, strain ZZ-11 showed efficient degradation properties for C13–C15 short-chained alkanes and C17–C23 long-chained alkanes (Figure 6C), a functional trait with similarities to those reported in the literature for Alcanivorax borkumensis SK2 and Rhodococcus sp. P2–29 strains [38]. Notably, the 1:1 composite colony showed significant degradation of long-chain alkanes (C17–C35), specifically a significant decrease in the proportion of C20–C24 hydrocarbons (especially C20) (Figure 6). These results confirmed that the composite bacterial colony constructed in this study was able to grow and metabolize crude oil as the sole carbon source and demonstrated efficient degradation of long-chain alkanes.

3.5. Oil Sand Washing Experiment

As a promising oil repellent, oil washing ability is often used as an important index to evaluate the performance of enhanced oil recovery (EOR) [39]. For this reason, the present study evaluated the elution effect of a single strain and its 1:1 compounded fermentation solution on crude oil in oil-bearing sands by an oil washing experimental system. The experimental results showed (

Table 1. Oil washing efficiency of different strains of fermentation broth.

Fermentation Solution	Oil Washing Efficiency/%
ZZ-8	40.83
ZZ-11	34.32
Culture medium	8.60
1:1	84.39

) that ZZ-8 and ZZ-11 fermentation broths could elute 40.83% and 34.32% of crude oil from oily sands, respectively, whereas only 8.60% of crude oil could be eluted from the medium under the same conditions, which shows that the strain fermentation broths significantly contributed to the elution efficiency of crude oil. It was further found that the mixed strain fermentation broth with 1:1 compounding exhibited even better oil washing performance, with a crude oil elution rate as high as 84.39% (Figure 7). Experimental observations showed (Figure 8) that after treatment with the mixed strains, most of the crude oil adhering to the sand was eluted, the sand revealed its original color, and the solution was turbid; after eluting the crude oil with the culture medium, the sand still adhered to the black crude oil, and the water phase was more clarified. Compared with the culture medium, the elution rate of the mixed strain increased by 89.80%, which confirms that the biosurfactant produced by its metabolism has excellent oil washing ability and has potential application in tertiary oil recovery. In addition, it has been reported that Bacillus cereus LY-1 can efficiently degrade high-carbon-number saturated alkanes (e.g., the degradation rate of C24 reached 76.43%) and significantly change the component distribution of oil sands: the resin content was reduced from 60.41% to 55.03%, and at the same time, we observed an increase in the ratio of saturated hydrocarbons to aromatic hydrocarbons, which can reduce the complexity of petroleum hydrocarbons and enhance mobility [40]. This finding further supports the important role of microbial metabolites in enhancing crude oil recovery.

4. Conclusions

In this study, the co-culture experiments of Bacillus mucilaginosus ZZ-8 and Bacillus amyloliquefaciens ZZ-11 revealed that, compared with the single-strain culture system, the microbial growth activity, biosurfactant production, and emulsifying properties were significantly improved. During the co-culture process, the contact angle of the uninoculated medium was 53.56 ± 1.3°, whereas the cell-free supernatant of the co-culture system was significantly reduced to 28.78 ± 0.82°. This change indicates that the co-culture system produced substances with potent surface activity, which significantly enhanced the wetting ability of the system. In terms of crude oil elution rate, the fermentation broth of the 1:1 co-culture system performed well, with a high crude oil elution rate of 84.39%, which was significantly higher than that of the single strains (40.83% for ZZ-8 and 34.32% for ZZ-11) and the medium control (8.60%). This fully indicates that the biosurfactant produced by this co-culture system has excellent oil washing performance and broad application prospects in the field of tertiary oil recovery. This study confirms the great potential of the co-culture system in enhancing microbial oil recovery performance; however, the mutualistic mechanism of the oil recovery functional bacteria in the co-culture process still needs to be further studied and elucidated.