Investigating the mechanism of interfacial tension reduction through the combination of low-salinity water and bacteria

In the enhanced oil recovery (EOR) process, interfacial tension (IFT) has become a crucial factor because of its impact on the recovery of residual oil. The use of surfactants and biosurfactants can reduce IFT and enhance oil recovery by decreasing it. Asphaltene in crude oil has the structural ability to act as a surface-active material. In microbial-enhanced oil recovery (MEOR), biosurfactant production, even in small amounts, is a significant mechanism that reduces IFT. This study aimed to investigate fluid/fluid interaction by combining low biosurfactant values and low-salinity water using NaCl, MgCl2, and CaCl2 salts at concentrations of 0, 1000, and 5000 ppm, along with Geobacillus stearothermophilus. By evaluating the IFT, this study investigated different percentages of 0, 1, and 5 wt.% of varying asphaltene with aqueous bulk containing low-salinity water and its combination with bacteria. The results indicated G. Stearothermophilus led to the formation of biosurfactants, resulting in a reduction in IFT for both acidic and basic asphaltene. Moreover, the interaction between asphaltene and G. Stearothermophilus with higher asphaltene percentages showed a decrease in IFT under both acidic and basic conditions. Additionally, the study found that the interaction between acidic asphaltene and G. stearothermophilus, in the presence of CaCl2, NaCl, and MgCl2 salts, resulted in a higher formation of biosurfactants and intrinsic surfactants at the interface of the two phases, in contrast to the interaction involving basic asphaltene. These findings emphasize the dependence of the interactions between asphaltene and G. Stearothermophilus, salt, and bacteria on the specific type and concentration of asphaltene.

investigation introduces a novel approach for enhancing the efficiency of low-salinity water and MEOR, which will be further elaborated upon in subsequent sections.This study evaluated different salts at varying salinities, such as low-salinity water, combined with Geobacillus stearothermophilus (strain bio14), and various synthetic oils with different asphaltene percentages and types.Several MEOR studies have focused on reducing IFT and improving recovery using Bacillus strains [62][63][64][65] .Among these strains, the Geobacillus genus is notable for its critical features, such as being gram-positive, rod-shaped, chemo-organotrophic, and aerobic or facultatively anaerobic 13 .Moreover, Geobacillus strains are thermophilic, allowing them to tolerate high-temperature environments like oil fields [66][67][68] .Zargari et al. 69 first introduced G. Stearothermophilus (strain bio14) for MEOR purposes, demonstrating acceptable tolerance to temperature and salinity.Subsequently, Abdi et al. 13 used G. Stearothermophilus (strain bio14) along with the smart water method to reduce IFT and enhance recovery factors.

Salt and salinity
In this study, three salts, namely NaCl, MgCl 2 , and CaCl 2 , were investigated at concentrations of 1000 and 5000 ppm.These salts were evaluated in two sections: low-salinity water and a combination of low-salinity water and bacteria.NaCl, MgCl 2 •6H 2 O, and CaCl 2 •2H 2 O salts from MERCK were used to prepare the aqueous phase containing the salt.

Oils
To investigate the types of asphaltenes, asphaltene was extracted from two crude oil samples obtained from the Bangestan oil field in southern Iran, each with distinct specifications (refer to Table 1, Figs. 1, and 2).These extracted asphaltenes, designated as oils A and B, were subsequently used in the production of synthetic oils.Based on the analysis of the total acid number (TAN) and total base number (TBN) of the crude oil, asphaltene A was classified as acidic, whereas asphaltene B was classified as basic.For this study, five samples of synthetic oil, as indicated in Table 2, were utilized.
To investigate the different components of oil in line with the objectives of this study, five samples of synthetic oil with varying compositions were prepared and evaluated, as outlined in Table 2. To ensure comparability  C-H , CH3 Figure 1.Fourier-transform infrared spectroscopy (FTIR) of crude oils 13 .
among the different asphaltene samples, their percentages in the oil were determined to be equal.The determination of asphaltene percentages in the synthetic oils (1% and 5% by weight) was based on the following considerations: 1. To assess the effects of incorporating asphaltene into the oil, a sample of synthetic oil without asphaltene was included.Additionally, oils containing 1 wt.% of asphaltene (a percentage close to zero) were used to demonstrate the effect of asphaltene presence.Furthermore, two different asphaltene samples were employed to examine the influence of asphaltene type, specifically acidity and alkalinity.2. To highlight differences in asphaltene percentages, the percentages were deliberately selected to be distinct from one another, enabling a clear differentiation.3. The asphaltene percentages in the synthetic oils were intentionally reduced compared with those in the crude oils from which the asphaltenes were extracted.This choice allows for the evaluation of the study results on a realistic and field scale, aligning with the characteristics of the selected crude oils.Consequently, percentages below 6.4 wt.% (which is less than the asphaltene percentage of crude oil B, as indicated by the SARA analysis in Table 1) were considered.4. The asphaltene percentages in the oil were determined to maintain the colloidal instability index (CII) (Eq. 1) within a stable range, with values below 0.7 70 .For 1 wt.% and 5 wt.% asphaltene oils, the calculated CII values were 0.34 and 0.39, respectively, indicating the stability of asphaltene in the oil.

Bacteria characteristics
The bacteria used in this study were G. Stearothermophilus bio14 strains were isolated from reservoir fluid samples obtained from hydrocarbon reservoirs in southwestern Iran.The samples were collected from various well-sampling lines.The evaluation of the isolated bacterium has been conducted in previous studies, including those by Zargari et al. 69 , Sarafzadeh et al. 71,72 , and Abdi et al. 13 .These bacteria exhibit moderate halotolerance, ranging between 5 and 15%, indicating their moderate halophilic nature.G. Stearothermophilus (strain bio14) (1) CII = Asphaltenes + Saturates Aromatics + Resins  www.nature.com/scientificreports/ is well-known for its ability to produce biosurfactants 73 , which are resistant to elevated temperatures and salt levels 74 .These properties make the bacteria a compelling choice for this study.The injection of their biosurfactant offers significant advantages in carbonates and sandstones with moderate temperatures, including emulsification, reduction in IFT, alteration of wettability, and enhancement of pore-scale displacement 73 .

Methodology
As shown in Fig. 3, the stages of the research can be summarized into six steps.

Preparation of synthetic oils
The first stage involved the extraction of asphaltene from crude oils using the IP-143 protocol method.Initially, a mixture of crude oil and heptane was prepared at a ratio of 1:40.The solution was stirred with a magnetic stirrer for 12 h to precipitate asphaltene.Following the 12-h stirring period, the solution was placed in the dark for an additional 12 h.This stirring and resting process was repeated three times.Subsequently, the crude oil and normal heptane solution were filtered using a vacuum filtration setup and Whatman 42 paper.The remaining solid particles on the Whatman paper were filtered and then transferred to the Soxhlet system for washing.The Soxhlet washing process consisted of two steps, starting with normal heptane, followed by toluene.The purpose of the normal heptane wash was to remove solid particles separated from the asphaltene.Note that both stages of Soxhlet washing were continued until the fluid within the Soxhlet extraction chamber became completely transparent.After the toluene was rinsed, the solution from the Soxhlet boiling flask was transferred to a glass container for toluene evaporation.The remaining solid particles after toluene evaporation constituted asphaltene, which would be used in subsequent stages.
In the second stage, a combination of n-heptane, toluene, and asphaltene was employed to prepare the synthetic oil samples based on Table 2. First, the desired percentage of asphaltene was added to toluene and thoroughly mixed for 30 min using a magnetic stirrer operating at a rotational speed ranging from 200 to 300 rpm.Subsequently, n-heptane was added to the resulting mixture, and the combined solution was stirred with a magnetic stirrer at a speed of 200-300 rpm for 15 min to ensure uniformity.

Cultivation of bacteria
Brain Heart Infusion (BHI) broth medium was used to cultivate single colonies of G. Stearothermophilus bio14 strains.The medium composition included 6 g/L BHI, 14.5 g/L gelatin, 3 g/L dextrose, 2.5 g/L Na 2 HPO 4 , 5 g/L KCl, and 6 g/L peptic digest of animal tissue.The medium was agitated at 150 rpm and 37 °C for 48 h until the OD600 nm value reached 2. Although the ideal condition for biosurfactant production is a medium like MSSO, Bacillus can still produce biosurfactants in the BHI medium, as reported previously 75 .Moreover, numerous studies have employed BHI as an effective medium for biosurfactant production by different bacteria types [76][77][78][79] .In previous investigations, BHI was proven to be effective in cultivating G. Stearothermophilus under harsh conditions 13,69,71,72 .According to Zargari et al. 69 , the isolation process was carried out.Initially, the cultured bacterial solution was evaluated for IFT.However, because of the turbidity of the bacterial solution, analyzing the IFT and capturing the image of the oil droplet within the bacterial bulk was not feasible.Therefore, the bacterial solution was mixed with deionized water at a one-to-one ratio to enable IFT evaluation.Deionized water was used to avoid the presence of polar components such as salt and to ensure reliable results.Additionally, in line with the study's objective of combining bacteria with low-salinity water, an aqueous fluid compatible with low-salinity water was employed.Ultimately, the diluted bacterial solution reached an OD600 nm value of 0.8, which was used for IFT assessment.We conducted a limited-scale experiment using the previously mentioned biosurfactant to investigate its impact on reducing IFT and to understand the underlying mechanism.

Performing IFT tests
In the fourth step, the three salts (NaCl, MgCl 2 , and CaCl 2 ) were prepared in two different environments: lowsalinity water and a combination of low-salinity water with bacteria at salinity levels of 1000 and 5000 ppm.To prepare the aqueous solutions, salt was added to either deionized water or a bacterial solution.A magnetic stirrer operating at a maximum speed of 200-300 rpm was used to dissolve the salt and achieve a homogeneous solution within a time frame of up to 5 min.The IFT of the aqueous phase/oil was assessed using the Krüss DSA100 (Germany) device under ambient conditions, following the schematic shown in Fig. 4.This device comprises a drip injection pump, an aquarium base and holder, a glass aquarium, a light source, and a camera connected to a computer.The pendant drop method, as illustrated in Fig. 4, was employed to calculate the IFT.A microsyringe filled with oil, connected to an injection pump, was used to inject a specific volume of oil at a controlled flow rate determined by the computer settings.This injection process resulted in the formation of an oil drop at the tip of the needle within the aquarium, which was filled with aqueous phases.Using the device's light source, the camera captured an image of the oil drop formed at the needle's tip.Subsequently, the drop picture was analyzed using the Fiji-win 64 ImageJ software (version 1.53e), and the IFT was computed using Eq. ( 2).The IFT between the two phases in Eq. ( 2). is governed by the equilibrium established among gravity, capillary forces, and the shape factor (H).The shape factor (H) is calculated using the parameter S = d e /d s and the Young-Laplace equation, where d e represents the equatorial droplet and d s represents the diameter of the droplet at a distance de from the top of the droplet 80,81 .The densities of the assessed fluids employed for calculating the IFT were determined using the standard test method ASTM D1217-15 under ambient conditions.The gravitational acceleration (g) was also computed using the International Gravity Formula (IGF) 82,83 .The reported results in the current study were derived from the mean of two independent repetitions to demonstrate the reproducibility of the results.
�ρ : The disparity in density between the oil and the aqueous phase.g: Gravitational acceleration.d e : Equatorial diameter of the droplet.H: shape factor.

Statistical analysis
The presented IFT results represent the mean value obtained from a minimum of two replicates.A summary of the statistical analysis conducted for all repetitions is provided in Table 3.Throughout the discussion section, the results are presented as the mean of multiple replicates, with error bars indicating the standard error (SE).Statistical analysis of all IFT data was performed using SigmaPlot software (version 14).Various statistical methods, including the Student-Newman-Keuls method, Tukey test, Dunn's method, and Dunnett's method for analysis of variance (ANOVA) on ranks, were applied to analyze the significance level, and all tests yielded a significance level of P < 0.05.

Effect of bacteria on IFT reduction
Based on the results presented in Fig. 5 for five samples of synthetic oil with varying properties, the coexistence of bacteria and asphaltene with different percentages and structures demonstrates their potential to reduce IFT.The existence of bacteria results in the production of biosurfactants, which contribute to IFT reduction.Asphaltene can also function as a surfactant in the system.However, when bacteria and asphaltene coexist in the same system, the nature of the interaction between these two entities may vary depending on the asphaltene's structure and properties.Acidic asphaltene (A) and basic asphaltene (B) exhibit distinct characteristics.
The presence of bacteria leads to a greater reduction in IFT when basic asphaltene is present, as shown in Fig. 5 (SYOil1%wtAsB).This behavior is similar to the interaction described by Abdi et al. 61 , where the interaction between asphaltene and ion hydration shells was discussed.According to Abdi et al. 61 , the interaction between acidic asphaltene and anions, as well as between basic asphaltene and cations, primarily governs IFT behavior in oil/low-salinity water systems.Bacteria have a complex structure with polar regions that are both positively and negatively charged, resembling the interaction between asphaltene various ions in brine.The further reduction in IFT observed for basic asphaltene in Fig. 5 suggests that the positively charged regions of bacteria have a dominant effect.This reduction is attributed to the interaction between the positively charged regions of bacteria and basic asphaltene, leading to the formation of intrinsic surfactants and increased production of biosurfactants at the oil-aqueous phase interface.

Effect of salinity and salt type
The study examined the impact of salinity and salt type on IFT under both bacterial and non-bacterial conditions, with the oils specified in Table 2.The findings of this investigation are presented in Figs. 6, 7, 8 and 9. Abdi et al. 61 proposed a mechanism explaining IFT in low-salinity water based on the interaction between the polar components of oil and ion hydration shells in water .Their findings indicate that IFT in oil/low -salinity water systems is influenced by factors such as ion hydration energy, ion concentration, and the type of polar components (acidic or basic) in the oil.This mechanism suggests that the interaction between oil and low-salinity water can result in different behaviors, as shown in Figs. 6, 7, 8 and 9.
Figure 6 demonstrates the ability of asphaltene in oil to reduce IFT.In the presence of low-salinity water, asphaltene acts as an intrinsic surfactant.When salts dissolve in water, the charged ions interact with the polar head of asphaltene, causing asphaltene to migrate toward the interface between the two phases.This behavior of asphaltene creates an intrinsic surfactant, which is attributed to the structure of the non-polar chains and polar components of the asphaltene.The presence of different ions in water can lead to varying interactions with asphaltene, affecting the results shown in Fig. 6 for SYOil5%wtAsA at 0, 1000, and 5000 ppm salinity of NaCl, CaCl 2 , and MgCl 2 salts.The predominance of acidic components in SYOil5%wtAsA leads to stronger interactions with anions in low-salinity water.This interaction is evident in Fig. 6 at a salinity of 1000 ppm, where increased Cl -ions result in reduced IFT.As the salinity of low-salinity water increases, with a simultaneous increase in both anions and cations, the repulsion and attraction forces also increase.This behavior causes simultaneous repulsion and attraction between the polar components of oil and the hydration shell of the ions, which differ at 5000 ppm salinity.
In the presence of bacteria within the system (Fig. 6), more complex interactions occur.The intricate structure of bacteria can result in diverse interactions with asphaltene.For SYOil5%wtAsA, the presence of bacteria demonstrated a greater reduction in IFT than low-salinity water.The observed discrepancy could be attributed to the generation of biosurfactants within the system by bacteria.IFT reduction is achieved through the combination of intrinsic surfactants formed by asphaltene and biosurfactants produced by bacteria.This favorable behavior of bacteria and asphaltene in reducing IFT relative to low-salinity water was observed across CaCl 2 , NaCl, and MgCl 2 salts at salinities of 0, 1000, and 5000 ppm.The presence of polar components within bacteria combined with low salinity water facilitates interactions among bacteria, oil, low salinity water ions, and bacteria.As mentioned in the previous section, the positive charge within bacteria has a more dominant effect, enhancing their interaction with negatively charged.Acidic asphaltene also exhibits a stronger interaction with anions.This tendency for interaction is intensified by increasing the number of chloride ions.In Fig. 6, the number of chloride ions follows the sequence MgCl 2 > CaCl 2 > NaCl for NaCl, CaCl 2 , and MgCl 2 salts, respectively.A higher number of Cl -ions leads to an increased interaction with bacteria and asphaltene.Consequently, as the Cl -ion count increases, more biosurfactants and intrinsic surfactants are produced, resulting in a lower IFT.

Type of Oil
A decrease in the percentage of asphaltene A leads to different interactions with bacteria and ions in lowsalinity water.The results of Fig. 7, compared with those of Fig. 6, indicate that reducing the percentage of acidic asphaltene, along with the presence of bacteria and salt at low salinity, results in an increase in IFT.When the bulk phase is low -salinity water, the results obtained for SYOil1%wtAsA demonstrate that the behavior of NaCl, MgCl 2 , and CaCl 2 salts is similar.At low concentrations (1000 ppm), the behavior does not depend on the salt type.The low percentage of asphaltene A reduces its likelihood of being present at the interface.For 1000 ppm, www.nature.com/scientificreports/ the results show the lowest IFT, which is attributed to the increased interaction of asphaltene A with system components, leading to the formation of intrinsic surfactants.Increasing the salinity from 1000 to 5000 ppm revealed that the low percentage of asphaltene in the oil diminishes the potential for intrinsic surfactant formation and has minimal impact on the changes in IFT above 1000 ppm (Fig. 7).In the absence of salt, bacteria lead to a reduction in IFT for SYOil1%wtAsA, which is attributed to the formation of biosurfactants.However, the behavior of the system becomes more complex when salt is added.The IFT behavior, as depicted in Fig. 7, is similar to that of SYOil5%wtAsA in the presence of bacteria and a salinity of 1000 ppm salts, depending on the number of Cl ions.However, at a salinity of 5000 ppm, the repulsion between the cation and asphaltene A causes a different IFT behavior compared with that observed in SYOil5%wtAsA.Figure 8 illustrates the effect of salinity and salt type on SYOil5%wtAsB.Analysis of the TAN and TBN values presented in Table 1 reveals that the predominant polar compounds of oil B are basic.Most asphaltene in crude oil B can be considered basic.According to the mechanism proposed by Abdi et al. 61 , the interaction between B oils (SYOil5%wtAsB and SYOil1%wtAsB) and cations can have a more significant impact on IFT behavior.The IFT behavior, as shown in Fig. 8, depends on the hydration energy of the cations and the concentration of the anions.Cations promote asphaltene adsorption at the interface, whereas anions cause repulsion.The balance between attraction and repulsion is influenced by the hydration energy and the quantity of ions.At 1000 ppm www.nature.com/scientificreports/salinity, the presence of ions enhances their interaction with asphaltene, resulting in reduced IFT compared with that at 0 salinity.As salinity increases from 1000 to 5000 ppm, the interactions become more complex.The order of cation hydration energy is Mg 2+ > Ca 2+ > Na + , while the order of chlorine ion concentration is MgCl 2 > CaCl 2 > NaCl.Higher cation hydration energy leads to greater asphaltene adsorption, whereas a higher concentration of anions causes increased asphaltene repulsion at the interface.Consequently, NaCl exhibits the least interaction with asphaltene at 5000 ppm because of its lower cation hydration energy, hindering the formation of intrinsic surfactants.Despite Mg 2+ having a higher hydration energy than Ca 2+ , the higher concentration of Cl -ions in MgCl 2 results in an elevated IFT, surpassing that of CaCl 2 .
The presence of bacteria in the aqueous phase and their affinity for basic asphaltene result in the formation of biosurfactants, which reduce IFT in the absence of salt.Bacteria interact with cations and anions, leading to their absorption and repulsion at the interface between phases.The presence of cations with higher hydration energy in the bulk of the bacteria enhances their interaction.Therefore, as shown in Fig. 8, when Mg 2+ and Ca 2+ cations are present, the interaction between the biosurfactant and the hydration shell of these cations leads to an increase in IFT.However, Na + cations, due to their lower hydration energy, this repulsion is not sufficient to overcome the interaction between the anion, bacteria, and basic asphaltene, resulting in the formation of intrinsic surfactants and biosurfactants.Changes in IFT for salts in the presence of bacteria are influenced by the number of ions and the interactions between ions, bacteria, and asphaltene, as well as the interaction between asphaltene and bacteria.
The findings depicted in Fig. 9, regarding the bulk of low salinity water, demonstrate the correlation between the outcomes and the number of Cl − ions.The interaction between basic asphaltene and cations/anions results in the adsorption and desorption of asphaltene at the interface of the two phases, respectively.The impact of anions on the interaction with asphaltene intensifies with increasing salt concentrations.The trend of changes in IFT with increasing salt salinity shows an increasing trend for MgCl 2 , a decreasing trend for NaCl, and a dual behavior (decreasing-increasing) for CaCl 2 .The sequence of Cl -ions is MgCl 2 > CaCl 2 > NaCl, which influences their interaction with asphaltene and the behavior of IFT.A higher number of anions leads to greater removal of asphaltene from the interface.Therefore, the increase in IFT with increasing salinity for MgCl 2 can be attributed to the higher number of Cl -ions compared to CaCl 2 and NaCl.While NaCl has the lowest chlorine ion content among the three salts, which leads to less removal of the inherent surfactant in the two-phase interface compared with CaCl 2 and MgCl 2 .In the case of CaCl 2 , its Cl -ion content is higher than that of NaCl but lower than that of MgCl 2 , leading to a dual behavior in response to increasing salinity.
The interaction in their presence of bacteria is also influenced by the number of ions and their hydration energy.The order of cations is Na + > Mg 2+ > Ca 2+ , indicating that in the presence of a lower number of cations, a greater amount of biosurfactant can be formed at a salinity of 1000 ppm.As the salinity increases, it leads to a variety of interactions because it increases the number of Cl -ions compared to cations, resulting in the behavior of IFT not exhibiting a specific trend.

Effect of asphaltene percentage
To investigate asphaltene's effect on the IFT of low -salinity water and bacteria, weight percentages of 0, 1, and 5 of asphaltene were examined (Tables 4 and 5).The interaction between the ion hydration shell in low-salinity water and asphaltene leads to an IFT reduction as the concentration of asphaltene in oil increases.This behavior was observed for both acidic and basic asphaltene samples (Fig. 10-b).The results presented in Table 4 demonstrate that, under identical conditions of salt type, salinity, and asphaltene percentage, the IFT consistently shows lower values for acidic asphaltene than for basic asphaltene.This behavior can be attributed to the higher number of Cl -anions, which exceed the number of cations in CaCl 2 , NaCl, and MgCl 2 salts.In addition, acidic asphaltene has a greater tendency to interact with the hydration shell of anions.Therefore, according to Table 4, acidic asphaltene consistently displays a lower IFT than basic asphaltene under the same conditions.
The results presented in Fig. 5 and the aforementioned findings indicate that bacteria exhibit a higher interaction tendency with anions and negative charges.In addition, asphaltene A, which is primarily acidic, exhibits a stronger interaction with the hydration shell of the anions.Consequently, the elevated concentration of Cl -ions in each of the CaCl 2 , NaCl, and MgCl 2 salts, compared with the number of cations, increases the likelihood of their interaction with asphaltene A and bacteria.This inclination of anion interaction with bacteria and asphaltene A leads to the formation of biosurfactants and intrinsic surfactants, which is primarily facilitated by the www.nature.com/scientificreports/higher number of Cl -ions in the case of acidic asphaltene.According to Table 5, the increase in the percentage of asphaltene A suggests that the likelihood of interaction with anions will likely increase, promoting the formation of intrinsic surfactants and resulting in a reduction in IFT with the increase in the percentage of asphaltene A (except one case).A comparison of the results between low-salinity water and bacterial systems for NaCl, CaCl 2 , and MgCl 2 reveals that a lower percentage of asphaltene corresponds to a lower IFT in low-salinity water.The observed phenomenon can be attributed to the existence of bacteria, the formation of biosurfactants initially dominates, leading to a decrease in IFT (in the absence of asphaltene).However, upon the addition of up to 1% asphaltene to the oil, the interaction between salt, asphaltene, and bacteria, as well as the interaction between asphaltene and bacteria, results in a diminished inclination to generate intrinsic surfactants.In the presence of bacteria and a lower percentage of asphaltene A, this decrease in intrinsic surfactant formation leads to higher IFT values compared with low-salinity water (Fig. 10-a).Conversely, an increase in the percentage of asphaltene A in the system decreases IFT in the bacterial + salt bulk system compared with low-salinity water.This decrease in IFT is attributed to the increased tendency to form more intrinsic surfactants at the two-phase interface due to the presence of a higher amount of asphaltene.Asphaltene B exhibits a distinct behavior compared with asphaltene A in its interaction with ions and bacteria.The interaction of the hydration shell of cations and anions with basic asphaltene B leads to the absorption and removal of asphaltene at the interface, respectively.However, the bacteria G. stearothermophilus has an opposite interaction with anions and cations compared to asphaltene B. Furthermore, the interaction between asphaltene B (basic) and the bacteria is more pronounced.This contrasting pattern of interactions results in a lower formation of intrinsic surfactants and biosurfactants at the two-phase interface when the percentage of asphaltene B is low, such as 1 wt.% (Fig. 10-a).In this case, the interaction between Cl − ions, which are more abundant than cations, and asphaltene B, as well as the interaction between cations and bacteria, hinders the formation of intrinsic surfactants and biosurfactants.However, as the percentage of asphaltene B increases beyond 1 wt.%, the likelihood of interaction between asphaltene and the bacteria itself increases, leading to an increased formation of intrinsic surfactants and biosurfactants at the interface.As indicated in Tables 4 and 5, the IFT values for various salts and salinities can be influenced by factors such as ion type, salinity, and asphaltene percentage.These observations apply to both low-salinity water and the combination of bacteria and low-salinity water.The findings demonstrate that the interaction between asphaltene and salt is contingent on the specific percentage of asphaltene, the type of salt, and the salinity level.

Effect of asphaltene type
As shown in Tables 4 and 5, both acidic and basic asphaltene exhibit similar behaviors in low-salinity water.Bacterial activity can be described as the production of biosurfactants that reduce IFT in the absence of asphaltene.However, in the presence of basic asphaltene, the biosurfactant interacts inversely with the intrinsic surfactant  generated by the asphaltene.When comparing the IFT values, it is observed that an increasing percentage of basic asphaltene results in a smaller reduction in IFT for the bulk of bacteria compared with low-salinity water.The nature of the polar components found in oil, including their acidity and alkalinity, significantly influences their interaction with the aqueous phase.Asphaltene A exhibited a tendency to interact with negative charges because of the prevalence of its acidic components, whereas asphaltene B tended to interact with positive charges because of the predominance of basic components.The simultaneous presence of bacteria and salt in the aqueous phase alters the interaction between asphaltene and ions in the aqueous phase.As discussed earlier, there is a stronger interaction between bacteria and basic asphaltene and a higher tendency for bacteria to interact with negative charges.Consequently, anions in the aqueous phase, with their higher abundance compared with cations in CaCl 2 , NaCl, and MgCl 2 salts, result in a greater interaction with acidic asphaltene and bacteria.The simultaneous interaction of anions with asphaltene A and bacteria leads to the formation of intrinsic surfactants and biosurfactants, which explains the decreasing trend in IFT observed for asphaltene A as the percentage of asphaltene increases.The similarity in the behavior of IFT with an increasing percentage of asphaltene in low -salinity water and bacterial systems, as indicated in Tables 4 and 5, suggests that the interaction between anions, acidic asphaltene, and bacteria follows a similar trend.
The interaction between ions in the aqueous phase and basic asphaltene differs from that of acidic asphaltene.Cations in the aqueous bulk tend to attract asphaltene B to the two-phase interface, whereas anions repel it from the interface.Conversely, the interaction of bacteria with anions and cations is contrary to their interaction with asphaltene B. However, the interaction at the two-phase interface between bacteria and asphaltene B promotes the absorption of both biosurfactants and intrinsic surfactants.The contrasting interactions between anions and cations with bacteria and asphaltene B, as well as the interaction between asphaltene B and bacteria, result in an IFT behavior that does not exhibit a clear trend concerning increasing the percentage of asphaltene B.
The evaluation of many factors, including salinity, type of salt, TAN and TBN of the oil, percentage of asphaltene, and the presence or absence of bacteria (as shown in Tables 4 and 5), revealed that all these parameters have an impact on IFT.Consequently, a multiple linear regression model was developed to analyze the influence of these parameters on IFT.Equation (3) represents the multiple linear models, and a summary of the statistical analysis for the independent parameters is provided in Table 6.The regression model successfully passed both the normality test, conducted using the Shapiro-Wilk method, and the constant variance test, performed using the Spearman Rank Correlation method.These tests confirmed that the data is normally distributed around the regression line, indicating the suitability of the regression model for data evaluation.Furthermore, the P and F values from the ANOVA of the multiple linear regression model for IFT, as presented in Table 7, further support the appropriateness of the regression model.Additionally, the statistical evaluation of the R, R 2 , and R 2 adj parameters of the model yielded values of 0.922, 0.850, and 0.833, respectively.These values indicate that the regression model provides a good description of the relationship between the independent and dependent variables.Moreover, a summary of the statistical analysis for the independent parameters of the regression model, as outlined in Table 6, highlights the significant influence of the interaction between bacteria and asphaltene, as well as the type of polar components of asphaltene, on IFT.

Figure 5 .
Figure 5.Effect of bacteria on the IFT reduction for different oils (different percentages and types of asphaltene).

Figure 10 .
Figure 10.Schematic of changes in IFT with increasing percentage of asphaltenes.

Table 1 .
Specifications of crude oils.

Table 2 .
Characteristics of synthetic oil.

Table 3 .
Statistical analysis of all IFT data obtained in this study.Parameter Size Mean Standard deviation (SD) Standard error (SE) Max Min Median IFT (mN/m) 148 25.920 5.704 0.469 36.48013.220 26.505 Vol.:(0123456789) Scientific Reports | (2024) 14:11408 | https://doi.org/10.1038/s41598-024-62255-0www.nature.com/scientificreports/ Effect of salinity and salt type on the IFT of low-salinity water and the combination of low-salinity water with biosurfactant for SYOil5%wtAsA.Effect of salinity and salt type on the IFT of low-salinity water and the combination of low-salinity water with biosurfactant for SYOil1%wtAsA.
Vol.:(0123456789) Scientific Reports | (2024) 14:11408 | https://doi.org/10.1038/s41598-024-62255-0 Effect of salinity and salt type on the IFT of low-salinity water and the combination of low-salinity water with biosurfactant for SYOil5%wtAsB.Effect of salinity and salt type on the IFT of low-salinity water and the combination of low-salinity water with biosurfactant for SYOil1%wtAsB.

Table 6 .
Summary of the statistical analysis of independent parameters for multiple linear regression of IFT.

Table 7 .
ANOVA of the multiple linear regression model for IFT.
Degrees of Freedom (DF) The sum of Squares (SS) Mean Square (MS) F P