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Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: a green chemical for microbial enhanced oil recovery (MEOR) – Scientific Reports

Effect of different carbon source on biosurfactant production

Biosurfactants are amphiphilic compounds produced by microorganisms, either on the surface of microbial cells or secreted extracellularly. Their main physiological function is to enable microorganisms to grow on water-insoluble substrates, such as crude oil, by reducing the interfacial tension between the substrate and water. This allows for better absorption and metabolism of the substrate36. Biosurfactants can be produced by microorganisms using various carbon sources. The selection of a carbon source is an important step in the production process as different carbon sources can result in variations in biosurfactant structure and production mechanisms. Glucose, glycerol, molasses, and crude oil are commonly used carbon sources for biosurfactant production. Each carbon source has different properties, such as carbon chain length and solubility in water. Microbes can produce biosurfactants on both soluble and insoluble substrates37. Different bacterial genera have been reported to utilize different carbon sources for biosurfactant production. For example, Pseudomonas can produce biosurfactants better on carbon sources like n-hexane, paraffin, and glycerol, while Bacillus subtilis have been reported to preferably utilize waste sunflower oil and cassava flour waste 38,39. Furthermore, Jain et al. 22 reported that the utilization of starch as a carbon source by Klebsiella sp. RJ-03 resulted in the highest production of biosurfactants with better properties.

To evaluate the performance of biosurfactant production from different carbon sources, the interfacial tension and emulsification index of the CFS were measured. The CFS mentioned in this paper contains biosurfactant produced by P. taiwanensis and will be referred to as CFS throughout the entire paper. The findings revealed that the lowest interfacial tension and highest emulsification index were observed when crude oil was used as the carbon source. Specifically, the interfacial tension decreased from 15.6 to 14.8 mN/m, 15.6 mN/m, 14.6 mN/m, and 11.9 mN/m when glucose, glycerol, molasses, and crude oil were used as carbon sources, respectively. The emulsification index values were 24.85%, 20.4%, 26.15%, and 59.69% for glucose, glycerol, molasses, and crude oil, respectively, compared to the control with an emulsification index of 8.47%. These results were obtained when comparing the biosurfactant produced in CFS to that of the SMSS medium, which served as the control. Pseudoxanthomonas taiwanensis demonstrated its versatility in utilizing a wide range of carbon sources, including both short-chain (glucose) and long-chain (crude oil) carbon compounds with different solubilities in water. The strain exhibited the highest biosurfactant activity when crude oil was used as the carbon source, suggesting its potential as a promising candidate for MEOR applications. Crude oil was then employed as a carbon source to produce biosurfactants for further study in this research.

Growth profile, biosurfactant yield, Interfacial tension, and emulsification activity

Pseudoxanthomonas taiwanensis exhibits a logarithmic growth phase between 12 and 24 h, followed by a stationary phase up to 72 h, and then continue to dead phase until 96 h of incubation time. Figure 1a showed the production of biosurfactant by this strain continues for up to 72 h of incubation and decrease sharply until the end of incubation. This result demonstrated that the highest yield of biosurfactant at 0.6 g/L was produced at the end of stationary phase indicating that the biosurfactant belonged to secondary metabolite. Biosurfactant synthesis is carried out by bacteria to facilitate the uptake of crude oil as a carbon source. The production of biosurfactant by P. taiwanensis shows an increase under limiting growth conditions in the stationary phase. Overproduction of biosurfactant occurs when the culture reaches the stationary phase due to limitations in the carbon-to-nitrogen (C/N) ratio39. In their review on rhamnolipid production, Reis et al.40 revealed that rhamnolipid production increases under environmental stress conditions such as nutrient deficiency, even at low cell density conditions.

Figure 1
figure 1

Profile of growth curve and biosurfactant production (a) and emulsification index (EI24) and interfacial tension (IFT) (b) during the growth of P. taiwanensis. (-– Log of cell number; -□- dry weight of crude biosurfactant; -▲-EI24; –-IFT).

The interfacial tension and emulsifying index activity of the CFS were monitored in conjunction with the bacterial growth at 12-h intervals. During the 72-h incubation period, the interfacial tension of the CFS decreased from 26.77 to 12.33 mN/m. Similarly, the emulsifying index activity of the CFS increased as the biosurfactant accumulated in the culture up to 72 h, but declined with further incubation time (Fig. 1b). These findings indicate that P. taiwanensis produces and secretes biosurfactants starting from the early stages of growth until 72 h in batch culture using the SMSS medium. Consequently, the biosurfactant produced by P. taiwanensis exhibits potential in reducing the interfacial tension between water and crude oil.

The characteristic of biosurfactant produced by P. taiwanensis

The bacteria were cultured in SMSS medium using crude oil as a carbon source to produce biosurfactant. Dilution experiments were conducted to evaluate the effectiveness of the biosurfactant in CFS to reduce interfacial tension (IFT). The IFT values of diluted CFS at different concentrations (100%, 50%, and 10%) were compared to water as a control. The decrease in interfacial tension (IFT) by the supernatant at a 10% concentration is recorded as 3.2 dyne/cm, or a reduction of about 16.49% compared to the control. Meanwhile, at a 100% concentration, the IFT decreases by 6.76 dyne/cm, which is a reduction of about 34.21%. This indicates that increasing the supernatant concentration from 10 to 100% only results in a doubling of the IFT reduction (from 16.49 to 34.21%). These results confirm that the supernatant at a 10% concentration is more effective in reducing IFT compared to a 100% concentration. This implies that the supernatant produced by P. taiwanensis is highly effective at lowering IFT even at minimal concentrations, offering benefits for its application in MEOR. Furthermore, using the biosurfactant through direct application of the CFS is more economically viable than employing crude biosurfactant. The comparison of IFT values with a chemically produced surfactant, SDS, indicates that the biosurfactant activity is comparable to SDS at a concentration of 400 ppm. SDS is commonly used in Chemical Enhanced Oil Recovery (CEOR) methods41.

Biosurfactant possesses the capability to alter the wettability of rock surfaces by adsorbing at the fluid-rock interface, resulting in a stronger affinity of the rock surface towards a particular liquid, particularly water. Wettability is recognized as a crucial factor in oil recovery42 and significantly impacts the interactions between fluids and rocks in multiphase systems. In this study, the effect of wettability was evaluated by measuring the contact angle on glass plates coated with crude oil using water and the CFS. The glass plate coated with crude oil displayed a water-wet system in contact with water (θ = 68.0°), while the biosurfactant exhibited an even stronger water-wet system (θ = 59.0°) indicating that biosurfactant produced by P. taiwanensis capable to increase the water wettability of the surface.

Biosurfactants offer potential for enhancing the mobilization of heavy oil, including the biosurfactant produced by P. taiwanensis. These specialized molecules have the ability to dissolve in heavy oil, reducing their viscosity and facilitating the mobilization of heavy oil fractions27. Experimental results demonstrated that the biosurfactant mixed with heavy oil had a spreading distance of 3.8 cm, twice that of water (1.5 cm) as a control (Fig. 2). This suggests that the biosurfactant from P. taiwanensis is likely soluble in oil, contributing to its effectiveness in mobilizing and improving the spreading of heavy oil (19° API).

Figure 2
figure 2

Evaluation of the effect of biosurfactant on a heavy oil (19◦ API) mobility. (a) Oil with water = 1.5 cm, (b) oil with SMSS medium = 2 cm, (c) Oil with CFS = 2.4 cm and (d) oil with extract biosurfactant = 3.8 cm.

The critical micelle concentration (CMC) is a key parameter that indicates the concentration at which the biosurfactant forms micelles, which are important for reducing interfacial tension and enhancing oil solubility in water43. In this study, the CMC point of the biosurfactant-containing CFS produced by P. taiwanensis was determined to be 0.33 g/L. It was found that the CMC value of the biosurfactant produced by P. taiwanensis using crude oil as a carbon source in this study was lower than the CMC value reported in a previous study using heavy crude oil as the carbon source23 where the CMC was determined to be 0.73 g/L. Differences in purity, composition, and variations in growth medium components have been identified as factors that can influence the functional group compositions of the biosurfactant and consequently result in different CMC values, as mentioned by Desai and Banat36 and Satpute et al.44. The differences in chemical characteristics were further confirmed through its structural analysis (Fig. 3). The CMC value is important as it correlates with the amount of biosurfactant required for efficient oil recovery. Moreover, it should be noted that a significant increase in the biosurfactant concentration will lead to higher production costs.

Figure 3
figure 3

FTIR spectrum (A) and LCMS Chromatogram of isolated biosurfactant produced by biosurfactant-producing bacteria P. taiwanensis (B). Black : biosurfactant produced using light oil obtained from this study; Yellow: biosurfactant produced by using heavy oil (Astuti et al., 2019).

The IR spectrum analysis of the biosurfactant produced by P. taiwanensis reveals the presence of six distinct and sharp peaks (Fig. 3A). The dominant absorption peaks in the IR spectrum correspond to specific functional groups. The peaks observed in the range of 3215–3414 cm−1 indicate the stretching of O–H bonds (i), while those in the range of 2926–2956 cm−1 represent the stretching of C–H bonds in the aliphatic chain (CH2 and CH3 groups) of the hydrophobic region of the molecule (ii). The presence of the carboxyl group is indicated by absorption peaks at 2856 cm−1 (iii) and 1654 cm−1 (iv), corresponding to the O–H and C=O bonds, respectively. The hydrophilic part of the molecule is characterized by the carboxylate group (R–COOH). In the fingerprint region (1500–500 cm−1), the IR spectrum shows peaks at 1384–1402 cm−1 (v), indicating the presence of CH2 and CH3 bonds in alkyl groups, and at 1014–1114 cm−1 (vi), indicating the C–O stretch in the hydrophilic region of the glycoside groups.

Comparing these findings with the standard rhamnolipid biosurfactant reported by Eraqi et al.45, it can be observed that the IR spectrum of the biosurfactant produced by P. taiwanensis using light crude oil as a carbon source exhibits similarities, such as the O–H stretch at 3430 cm−1, the C–H stretch of CH2 and CH3 groups at 2938 cm−1, the C=O stretch of carboxyl groups at 1629 cm−1, and the C–O stretch at 1042 cm−1. Based on these results, it can be inferred that the biosurfactant produced by P. taiwanensis using crude oil as a carbon source has molecules that are structurally like the glycolipid group. Additionally, the FTIR results of the biosurfactant produced by P. taiwanensis in this study exhibit a composition of groups that is nearly identical to the biosurfactant produced by the same strain in previous studies using heavy oil as a carbon source (Fig. 3A). These results agree with the study of which revealed that variation in carbon source did not affect the chemical structure significantly but influenced properties like yield, surface tension, viscosity, emulsification properties, molecular weight, and thermo-stability22.

Furthermore, the mass spectrometric analysis of the biosurfactant (Fig. 3B) confirmed the above findings. A total of thirty molecules were detected, seven of which can be categorized as glycolipids, with peak signals observed within the m/z range of 325–669 which are equivalent to their molecular weight. In accordance with Singh et al.46, it is noted that the majority of glycolipid biosurfactants possess molecular weights within the range of 302–803 Da. Additionally, a similarity search against the PubChem NCBI database revealed that the LC–MS spectra at m/z 653 and m/z 325 closely correspond to a compound resembling rhamnolipid.

The stability of the biosurfactant produced by P. taiwanensis against pH, temperature, and salinity was assessed through the EI24 value, which represents its emulsification activity (Table 1). A polynomial equation (Eq. 5) based on a 2-factor interaction (2FI) model is employed as the simplest numerical model to explain the relationship between controllable variables and the response. This numerical model establishes a correlation between pH, temperature, and salinity, designated as X1, X2, and X3, respectively, with biosurfactant emulsification activity (EI24).

The equation is as follows:

$$begin{aligned} {text{E24 }} = & { 22}.{7 }{-} , 0.{137}X_{1} + { 12}.{97}X_{2} + { 1}.{55}X_{3} {-} , 0.000{19}X_{1} X_{1} {-}{ 1}.{377}X_{2} X_{2} {-} , 0.{3}0{5}X_{3} X_{3} & + 0.0{text{259X}}_{{1}} {text{X}}_{{2}} {-} , 0.0{text{132X}}_{{1}} {text{X}}_{{3}} + , 0.{text{511X}}_{{2}} {text{X}}_{{3}} end{aligned}$$


This regression model was analyzed using ANOVA to assess its significance and fitness. The statistical analysis reveals that the model is highly significant, confirmed by the Fisher test value (Fmodel = 25.57), which is greater than the tabulated value (F0.05, 9, 35 = 2.16). Thus, with a 95% confidence level in the Fisher F-test, the regression model explains a significant portion of the variation in the response values. The regression model exhibits high accuracy, with R2 and adj-R2 values of 86.80% and 83.41%, respectively. The significance of each coefficient was determined using the Student’s t-test. All parameters, except X3, were found to be significant since their p-values are less than 0.05.

A two-dimensional contour plot (Fig. 4) was generated to visualize the effect of pH and temperature on the biosurfactant stability, while salinity was held at a fixed level. The results indicated that extreme pH values (< 3 or > 9) led to lower EI24 values, suggesting a decrease in biosurfactant stability. However, temperature and salinity did not significantly affect the biosurfactant stability. It is known that excessively low or high pH can cause the biosurfactant to precipitate and distort its structure, resulting in the loss of its ability to reduce surface tension47,48.

Figure 4
figure 4

Contour plot of stability test of the biosurfactant produced by P. taiwanensis againts environmental factors (pH, temperature and salinity) after 24 h. Hold values: Temperatur 80 °C; pH 7; Salinity 6%.

The thermostability of the biosurfactant was also evaluated by subjecting the supernatant to high temperatures up to 120 °C. It was found that the biosurfactant performance only slightly decreased, indicating its thermostable nature. Additionally, the biosurfactant demonstrated stable emulsification activity in a wide pH range of 4–10. Furthermore, the addition of NaCl at concentrations ranging from 2 to 10% did not significantly affect the emulsification activity, which remained stable with an EI24 value of more than 40%. These findings align with previous studies that reported the stability of biosurfactants at high temperatures and under varying pH and salinity conditions16,35,49,50. Based on the Box Behnken model equation, the predicted optimum values for each variable were determined to be pH 6.4, temperature 40 °C, and 7% salinity, resulting in an EI24 value of 65.21%.

The biosurfactant produced by P. taiwanensis demonstrates significant stability and efficacy under optimized conditions (pH 6.4, temperature 40 °C, and 7% salinity), making it a promising candidate for MEOR. Its optimal stability in a neutral pH range, high temperature tolerance up to 120 °C, and resistance to salinity levels between 2 and 10% underscore its versatility for use in diverse reservoir environments. This biosurfactant’s ability to maintain high emulsification activity (EI24 value of 65.21% at optimal conditions) is crucial for enhancing oil recovery by effectively reducing interfacial tension and improving oil mobilization. The adaptability to various pH levels, temperatures, and salinities minimizes the need for stringent environmental control, potentially reducing operational costs and the environmental footprint associated with MEOR operations. Its broad range of stability, combined with the environmental benefits of being biodegradable, positions the P. taiwanensis biosurfactant as an environmentally friendly and efficient alternative to synthetic surfactants for improving oil recovery in a variety of reservoir conditions.

Rhamnolipids are often produced by Pseudomonas aeruginosa and are well-known for their capacity to lower water surface tension. They are also recognized for their strong emulsifying properties and stability at a variety of pH levels, salt concentrations, and temperatures51,52,53,54. In contrast, despite their structural similarities to rhamnolipids, P. taiwanensis glycolipids are characterized by their higher efficiency at lower concentrations in lowering interfacial tension using CFS, which is critical in MEOR. This efficiency could make them more cost-effective than typical rhamnolipids. They are also more advantageous for MEOR applications due to their capacity to mobilize heavy oils and to increase the wettability of oil-coated surfaces to water.

Application of biosurfactant in enhancing oil recovery

The sand-pack column (SPC) is a laboratory-scale testing design that aims to mimic the actual conditions of oil reservoirs in the oil field for studying enhanced oil recovery (EOR) mechanisms6. The porosity within the column and the original oil in place (OOIP) showed similar values for all the samples used. This indicates a similar sand structure and permeability zones. The sand-pack column system exhibited porosity ranging from 25 to 29% and OOIP of approximately 45–50 mL. Figure 5 illustrates the amount of oil recovery that occurs during the primary and secondary recovery processes in the column. The oil recovery amount decreases after the brine flooding process of approximately 1.5–2 PV. The residual oil saturation (Sor) in the column was calculated after primary recovery using brine flooding, resulting in values of 42–49%. Subsequently, secondary recovery was conducted using either chemical or bacterial flooding using CFS, biosurfactant extract, P. taiwanensis bacterial cells, SDS, and brine water as a control. Additional oil recovery percentages obtained were 9.16%, 36.04%, 10.56%, 15.27%, and 1.33% respectively for CFS, biosurfactant extract, P. taiwanensis bacterial cells, SDS, and brine water (Table 2). The use of biosurfactant extract in the secondary recovery phase yielded the highest additional oil recovery (AOR) compared to CSF, bacterial cells and SDS. This indicates that the biosurfactant produced by P. taiwanensis is capable to enhance the recovery of residual oil within the column.

Figure 5
figure 5

Primary and secondary oil recovery of sand-pack column experiments. SDS concentration: 400 ppm.

Table 2 Oil recovery process by sand pack column and core flooding experiments.

The use of SPC as a laboratory-scale simulation tool for EOR has been reported to increase the recovery of residual oil from 6.7 to 86%6,55,56. However, the oil recovery process within the SPC does not accurately represent the AOR values at the field scale. The SPC is composed of sand that is nearly uniform in type and size, thus it does not fully represent the actual reservoir conditions. Advanced EOR simulation experiments, such as core flooding, can provide a more accurate estimation of the potential oil mobilization by microorganisms or their metabolites56,57,58.

Therefore core flooding experiment was conducted in this study to assess the effectiveness of the biosurfactant produced by P. taiwanensis in MEOR. Figure 6 presents the cumulative oil recovery achieved through primary and secondary recovery. Initially, the oil saturation (Soi) in the core was approximately 63–66%, and after injecting 2.5 pore volumes (PV) of brine, the residual oil saturation (Sor) reduced to about 50–54%. Upon initiating brine injection, there was a significant increase in oil production, and stabilization occurred after approximately 0.75 PV. Subsequently, an additional oil recovery was observed after injecting 2.5 PV of the biosurfactant solution, resulting in an AOR of 12.92% over the residual oil in the core, while AOR for control just only 2.09% (Table 2). The findings demonstrate that the biosurfactant produced by P. taiwanensis can facilitate the mobilization of crude oil trapped in rock pores. Biosurfactants reduce the interfacial tensions of reservoir fluids, altering the wettability of the rock, thus promoting the release of oil and enhancing oil recovery. These findings highlight the potential of P. taiwanensis biosurfactants in MEOR applications. Accurately simulating reservoir conditions, controlling the variability in biosurfactant performance across various oil types and reservoir rocks, and scaling up from laboratory conditions to field applications are potential problems in such investigations. To tackle these obstacles, meticulous experimental planning is frequently required. This includes employing control experiments, modifying experimental settings to more closely mimic field conditions, and carrying out several trials to guarantee the repeatability and reliability of findings. Thus, further testing at the pilot field application is required to fully understand the biosurfactant’s potential and limitation in practical MEOR applications.

Figure 6
figure 6

Primary and secondary oil recovery using biosurfactant flooding produced by P. taiwanensis on core flooding experiment.