# Isolation and Characterization of Viscosity-Reducing and Biosurfactant-Producing Bacteria in Low-Permeability Reservoir

Authors: Bian Ziwei; Wu Hanning; Wei Lusha; Zhi Zena; Wu Yifei
Journal: International Journal of Energy Research
 (2023)

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## Abstract

Microbialenhanced oil recovery has gained more attention in recent years due to its low cost and eco-friendliness. However, studies on the application of this technique in the low-permeability reservoirs are few. In this study, eight strains from the Ordos Basin low-permeability reservoir that produce biosurfactant and reduce viscosity were identified. Strains could produce biosurfactants (lipopeptide and glycolipids) to emulsify the oil and had good tolerance on temperature (25°C-50°C), salinity (1 g/L-50g/L), and pH (5-10). After the actions of A-3, SC4534(2), SC4561, and JSC4535, the content of long-chain n-alkanes were decreased by 51.2%, 28.3%, 35.2%, and 28.9%, respectively. The naphthalene in aromatic hydrocarbons was also effectively degraded by the strains that were screened, and the degradation rate was higher than 84%. Additionally, all strains were able to reduce oil viscosity, which was reduced by 27-51% (in seven days). 16S rRNA gene sequencing analysis indicated that seven strains belong to the genusBacillus and one belongs to the genus Rhodospirillaceae. The results revealed that the existence of high-efficiency strains that can significantly improve the properties of crude oil in low-permeability oil fields though biodegradation and use of biosurfactants, which is of great significance for the application of MEOR in low-permeability oil fields.


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## Body

## 1. Introduction

Low-permeability reservoirs are those with permeability less than 50 mD [1]. 46% of China’s total oil and gas resources are low-permeability resources. To ensure the sustainable development of oil and gas in China, the efficient development of oil and gas resources in low permeability reservoirs is of considerable strategic significance. However, such resources often have unsatisfactory recovery rates due to their complex geological conditions [1, 2]. Microbial-enhanced oil recovery (MEOR), which uses microbial propagation and metabolic products to change the properties of oil and reservoirs and hence increase oil recovery, has gradually gained attention due to its low cost and environmental friendliness [3, 4]. Microorganisms in oil reservoirs utilise hydrocarbon as a source of carbon and energy, which affects the quality of oil [5]. The application of MEOR in China is more frequent in areas with better physical properties rather than low-permeability oilfields [6–8].Obtaining strains that produce metabolites with high efficiency and high tolerance is crucial for the application of MEOR. The previous work on the MEOR process has indicated that microbes enhance the properties of crude oil in two key ways. On the one hand, microorganisms can break down macromolecular hydrocarbons (such as long-chain hydrocarbons and aromatic hydrocarbons) into smaller molecules through degradation, which reduces the absolute viscosity of crude oil and increase fluidity [9, 10]. Dai et al. [11] obtained oil-degrading bacteria (Bacillus sp., Brevibacillus sp., and Acinetobacter sp.) that can degrade bio-refractory hydrocarbons in heavy oil by synergistic metabolism. Another study reported that a strain Ochrobactrum anthropi, screened from the sea bottom sediments in China, has the ability to degrade crude oil. The degradation rate of crude oil can reach 58.25% under the optimal growth conditions [12]. On the other hand, some bacteria, such as Bacillus sp., Pseudomonas sp., Rhodococcus sp., Yeasts (Candida sp.), and Acinetobacter sp., can produce biosurfactant (including glycolipids, lipopeptides, and phospholipids) that can reduce interfacial tension and emulsify the crude oil to increase fluidity [13–15]. Sharma et al. [16] screened a strain of Pseudomonas aeruginosa that produces rhamnolipid, which can reduce the surface tension of medium from 71.80 to 23.76 mN/m and show immense hydrocarbons emulsification capacity (50–60%). Huang et al. [17] also found that biosurfactant-producing bacteria (Serratia marcescens) can treat fracturing flowback fluids.The work presented in this paper focuses on the screening and assessment of the biosurfactant-producing and viscosity-reducing bacteria in low-permeability oilfields in the Ordos Basin. The dominating bacteria that can thrive when using crude oil as the only carbon source are identified through a series of screening experiments. The performance of the strains was evaluated by the analysis of change in crude oil composition, viscosity, and the tolerance (temperature, salinity, and pH). The strains were identified by 16S rRNA sequencing and characterized through SEM.


## 2. Materials and Methods

### 2.1. Samples Collection and Media

Crude oil and injection waters samples were obtained from Hujianshan oil field (36°49′-37°53′N, 107°35′-108°22′E), west of the Ordos Basin, China [18]. Oil samples came from different layers in different blocks. Both came from the Chang 4+5 layer in 154 Block and the Chang 6 layer in Zhuanjing Block. Both oil-bearing layers belong to the Triassic and are typical low-permeability reservoirs. The water sample was collected from Chang 4+5 reservoir. Table 1 provides the essential information on the geological and geochemical factors of the two blocks.Table 1
The geological and geochemical factors of Zhuanjing and 154 blocks in Hujianshan oilfield.
FactorsZhuanjing block154 blockLithological characteristicsSandstoneSandstoneInitial temperature∗ (°C)67.867.5Salinity (g/L)46.0137.48Average porosity (%)12.7912.54Average permeability (mD)1.23.6pH66Groundwater typeCaCl2CaCl2Depth (m)21432160∗This is the well temperature before water flooding. The current temperature of the oil well is about 40-50°C.Three culture media were used in the experiments. To enrich and separate the strains in oil and water samples, nutrient broth (NB) (g/L, beef extract 3.6; peptone 10; NaCl 5.6, addition 20 agar for solid NB) was used. The minimal salt medium (MSM) (g/L, NaNO3 1.5; (NH4)2SO4 1.5; K2HPO4 1; MgSO4 0.5; KCl 0.5; CaCl2 0.002; FeSO4 0.001) was used for biodegradation test. Crude oil as carbon source was added separately. Blood agar plates are LB supplemented with 5% (w/v) sheep blood. Prior to usage, all media were autoclaved at 120°C for 20 or 40 min (for crude oil). To prevent precipitation of MSM, reagents must be sterilized separately and maxed at a lower temperature.


### 2.2. Isolation of Strains

#### 2.2.1. Enrichment and Purification of Strains

Unless otherwise specified, all experimental procedures were carried out on the ultraclean workbench to ensure sterility. In order to enrich the dominating bacteria, five milliliters oil and water samples were added in NB media (100 mL), respectively, and cultured at 42°C with 160 rpm for four days. Then, two milliliters of culture medium were transferred to fresh medium for recultivation. Repeat the previous procedure three times. Additionally, ten milliliters oil and water samples were added to 100 mL of MSM, respectively, to screen strains that can use crude oil as the only carbon source. The enrichment method was consistent with the above. On NB agar plates, the enriched solution was plated after being diluted with sterile water (10-1-10-6). Colonies of bacteria were isolated based on color and morphology, then purified by streaking then three times on plates. The purified strain was inoculated into NB medium for enrichment, then mixed with 50% glycerol, and kept at -20°C.


#### 2.2.2. Screening of Bacteria Using Oil as the Sole Carbon Source

The strains selected in the above experiment were inoculated on MSM, incubated at 42°C with 160 rpm for seven days, and the turbidity of the medium was observed.


#### 2.2.3. Growth Monitoring (as Optical Density)

The strains obtained in Section2.2.1 were inoculated in NB medium, and the optical density (OD600) was measured using UV-vis spectrophotometry (Spectrumlab 752pro, USA) by taking 3 mL of solution at regular interval. The growth curves of the strains were obtained with time as the abscissa and OD600 as the ordinate [12].


### 2.3. Screening of Biosurfactant-Producing Bacteria

#### 2.3.1. Emulsification Rate for 24 Hours Emulsification Index

Three milliliters of n-hexane and three milliliters cell-free supernatant (8000 rpm, 10 min) were vortexed for two minutes to mix. The height of emulsified layer was measured immediately and again after 24 hours. The results were calculated asmean±standard deviation The E24 was calculated by the following formula [17]:
(1)E24=heightoftheemulsifiedlayertotalheightoftheliquidlayer×100%.


#### 2.3.2. Hemolysis Assay

The conserved strains were inoculated on NB plates and cultivated for two days to obtain moderate colonies. The colonies were selected using inoculation loop in the center and inoculated on the blood plates, and then incubated for 72 h at 42°C. After that, the clearing zone was measured and its hemolytic activity was evaluated. Hemolytic activity was assessed based on the halo diameter: (-) no halo, (+) complete hemolysis with diameter of <1 cm, (++) complete hemolysis with diameter of >1 cm but <3 cm, and (+++) hemolysis with diameter of >3 cm [19].


#### 2.3.3. Extraction of Biosurfactant

Six strains (A-3, SC4542, SC4534(2), H-1, SC4561, and SC4551) with superior hemolysis and emulsification rate had biosurfactants extracted and identified. A single strain was inoculated on 100 mL of medium and fermented for 48 h at 160 rpm and 45°C. Then, 50 mL of fermentation broth was centrifuged for 30 min at 8000 rpm and the pH of the supernatant was adjusted to 2.0 with hydrochloric acid. Finally, the supernatant was kept at 4°C overnight. Chloroform: methanol was used in a 2 : 1 ratio for liquid-liquid extraction. The mixture was magnetically stirred for 1 hour and then kept aside for 30 minutes. The lower organic phase was separated and evaporated at room temperature (about 20°C) in a ventilated area to obtain a biosurfactant [20, 21].


#### 2.3.4. Thin Layer Chromatography

The composition of biosurfactants was identified by thin layer chromatography (TLC). Chloroform: methanol (1 : 1 v/v) was used as developing agent (80 mL), and silica plate (MACHEREY-NAGEL, Germany) was used for the experiment. Following are the standard sample and color developing agent:(1)
To determine the presence of lipopeptides, 0.5% ninhydrin reagent was used as the indicator. If it showed purplish red, lipopeptides was present(2)
To determine the presence of glycan, 500μL of phenol and 1 mL concentrated sulfuric acid were diluted in 20 mL of ethanol as a chromogenic agent. Rhamnolipid was used as control. If it appeared brownish black, glycan was present(3)
Iodine fumes (volatilizing iodine in a sealed beaker) were used for lipid detection. If it showed yellow, lipid was presentThe sample should be fully dried and sprayed with color developing agent, and then heated in the oven for 5 min at 100°C for lipopeptide and 90°C for glycolipids to show the color.


#### 2.3.5. FT-IR Spectrometric Analysis

The biosurfactants obtained in Section2.3.4 were dried by vacuum freezing dryer (BiLon FD80A, China). The pellets were scanned on a FT-IR (Fourier Transform Infrared) spectrophotometer (Shimadzu IRAffinity-1S, Japan) over a range of 4000-0 cm-1.


### 2.4. Temperature, Salinity, and pH Sensitivity Tests

The biosurfactant-producing strains, screened in Section2.3, were inoculated at 160 rpm for three days in NB medium. The temperatures were between 25°C and 65°C, with a step of 10°C. A series of tests at 50°C was also added in response to the experimental results.To assess the salinity sensitivity of strains, the same experiments were also carried out at 42°C with different NaCl concentration (1, 3, 5, 7, 9, 10, 20, 30, and 50 g/L). The growth of strains was examined via OD600, obtained through UV-vis spectrophotometry (Spectrumlab 752pro, USA) [22].Additionally, the pH sensitivity test was also carried out at fixed temperature of 42°C and NaCl concentration of 7 g/L. The pH was adjusted with HCl (1 mol/L) and NaOH (1 mol/L). After 3 days of incubation, the growth was checked with a pH range of 4 to 10.


### 2.5. The Effect of Dominant Bacteria on Crude Oil

#### 2.5.1. Surface Tension

The biosurfactants-producing strains selected in Section were inoculated on NB medium for fermentation (50 mL) and the fermentation broth was centrifuged at 6000 rpm for five minutes after 7 days of incubation. The surface tension of the fermentation broth was measured using a surface tension meter (POWEREACH, China). The results were calculated asmean±standard deviation.


#### 2.5.2. Viscosity

Ten milliliters of sterile oil and five milliliters of strains solution were added in 100 mL of MSM and inoculated at 160 rpm, 42°C for 7 days and 14 days. After incubation, the crude oil was transferred to a centrifuge tube (three parallel samples for each strain), which was then rest at 42°C overnight to allow oil and water to completely separate. The medium without bacteria was as control. The change of crude oil viscosity before and after the action of the strains was measured by a rotational rheometer (Anton Paar MCR 302, Austria). The results were calculated asmean±standard deviation. The viscosity reduction ratio is calculated by the following formula:
(2)viscosityreductionratio=VC−VSVC×100%.VC is the viscosity of control (mP·s); VS is the viscosity of the oil after the action of the strains.


#### 2.5.3. GC-MS Analysis of the Degradation Effect of Strains

Fifty milliliter MSM with five milliliters of sterile oil and two milliliters of strains solution were inoculated at 160 rpm, 42°C for 7 days. After that, the crude oil was extracted with n-hexane after the culture broth had been centrifuged to remove bacteria and impurities [23]. The components of crude oil were determined by GC-MS (Thermo Fisher ISQ7000, USA). Helium was used as carrier gas with a flow rate of 1 mL/min. The heating program was set as follows. The initial temperature was 50°C with a hold time of 1 min. The temperature was increased to 120°C at 20°C/min and then increased to 250°C at 4°C/min. Finally, the temperature was increased to 300°C at 6°C/min with a hold time of 30 min. MS condition ionization method: EI, 70 eV; filament current, 100 A; multiplier voltage, 1200 V. Data collection method: selective ion scan (85, 123, 191, and 217). The degradation ratio of different components is calculated by the following formula:
(3)Degradationratioofdifferentcomponents=PC−PSPC×100%.PC is the sum of the peak areas of different components in the control group; PS is the sum of the peak areas of different components of strains.


### 2.6. Bacteria Identification

#### 2.6.1. Morphological Features

The strain was streaked on NB media to obtain a single colony, and the bacterial morphology was observed by Scanning Electron Microscope (SEM).


#### 2.6.2. 16S rRNA Identification of Dominant Bacteria

The 16S rRNA gene sequencing analysis was used to identify these strains. The primers 27F (5′ AGAGTTTGATCCTGGCTCAG 3′) and 1492R (5′ GGTTACCTTGTTACGACTT 3′) were used for PCR. The sequence was compared to the 16S rRNA nucleotide sequences at National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) [24].


## 2.1. Samples Collection and Media

Crude oil and injection waters samples were obtained from Hujianshan oil field (36°49′-37°53′N, 107°35′-108°22′E), west of the Ordos Basin, China [18]. Oil samples came from different layers in different blocks. Both came from the Chang 4+5 layer in 154 Block and the Chang 6 layer in Zhuanjing Block. Both oil-bearing layers belong to the Triassic and are typical low-permeability reservoirs. The water sample was collected from Chang 4+5 reservoir. Table 1 provides the essential information on the geological and geochemical factors of the two blocks.Table 1
The geological and geochemical factors of Zhuanjing and 154 blocks in Hujianshan oilfield.
FactorsZhuanjing block154 blockLithological characteristicsSandstoneSandstoneInitial temperature∗ (°C)67.867.5Salinity (g/L)46.0137.48Average porosity (%)12.7912.54Average permeability (mD)1.23.6pH66Groundwater typeCaCl2CaCl2Depth (m)21432160∗This is the well temperature before water flooding. The current temperature of the oil well is about 40-50°C.Three culture media were used in the experiments. To enrich and separate the strains in oil and water samples, nutrient broth (NB) (g/L, beef extract 3.6; peptone 10; NaCl 5.6, addition 20 agar for solid NB) was used. The minimal salt medium (MSM) (g/L, NaNO3 1.5; (NH4)2SO4 1.5; K2HPO4 1; MgSO4 0.5; KCl 0.5; CaCl2 0.002; FeSO4 0.001) was used for biodegradation test. Crude oil as carbon source was added separately. Blood agar plates are LB supplemented with 5% (w/v) sheep blood. Prior to usage, all media were autoclaved at 120°C for 20 or 40 min (for crude oil). To prevent precipitation of MSM, reagents must be sterilized separately and maxed at a lower temperature.


## 2.2. Isolation of Strains

### 2.2.1. Enrichment and Purification of Strains

Unless otherwise specified, all experimental procedures were carried out on the ultraclean workbench to ensure sterility. In order to enrich the dominating bacteria, five milliliters oil and water samples were added in NB media (100 mL), respectively, and cultured at 42°C with 160 rpm for four days. Then, two milliliters of culture medium were transferred to fresh medium for recultivation. Repeat the previous procedure three times. Additionally, ten milliliters oil and water samples were added to 100 mL of MSM, respectively, to screen strains that can use crude oil as the only carbon source. The enrichment method was consistent with the above. On NB agar plates, the enriched solution was plated after being diluted with sterile water (10-1-10-6). Colonies of bacteria were isolated based on color and morphology, then purified by streaking then three times on plates. The purified strain was inoculated into NB medium for enrichment, then mixed with 50% glycerol, and kept at -20°C.


### 2.2.2. Screening of Bacteria Using Oil as the Sole Carbon Source

The strains selected in the above experiment were inoculated on MSM, incubated at 42°C with 160 rpm for seven days, and the turbidity of the medium was observed.


### 2.2.3. Growth Monitoring (as Optical Density)

The strains obtained in Section2.2.1 were inoculated in NB medium, and the optical density (OD600) was measured using UV-vis spectrophotometry (Spectrumlab 752pro, USA) by taking 3 mL of solution at regular interval. The growth curves of the strains were obtained with time as the abscissa and OD600 as the ordinate [12].


## 2.2.1. Enrichment and Purification of Strains

Unless otherwise specified, all experimental procedures were carried out on the ultraclean workbench to ensure sterility. In order to enrich the dominating bacteria, five milliliters oil and water samples were added in NB media (100 mL), respectively, and cultured at 42°C with 160 rpm for four days. Then, two milliliters of culture medium were transferred to fresh medium for recultivation. Repeat the previous procedure three times. Additionally, ten milliliters oil and water samples were added to 100 mL of MSM, respectively, to screen strains that can use crude oil as the only carbon source. The enrichment method was consistent with the above. On NB agar plates, the enriched solution was plated after being diluted with sterile water (10-1-10-6). Colonies of bacteria were isolated based on color and morphology, then purified by streaking then three times on plates. The purified strain was inoculated into NB medium for enrichment, then mixed with 50% glycerol, and kept at -20°C.


## 2.2.2. Screening of Bacteria Using Oil as the Sole Carbon Source

The strains selected in the above experiment were inoculated on MSM, incubated at 42°C with 160 rpm for seven days, and the turbidity of the medium was observed.


## 2.2.3. Growth Monitoring (as Optical Density)

The strains obtained in Section2.2.1 were inoculated in NB medium, and the optical density (OD600) was measured using UV-vis spectrophotometry (Spectrumlab 752pro, USA) by taking 3 mL of solution at regular interval. The growth curves of the strains were obtained with time as the abscissa and OD600 as the ordinate [12].


## 2.3. Screening of Biosurfactant-Producing Bacteria

### 2.3.1. Emulsification Rate for 24 Hours Emulsification Index

Three milliliters of n-hexane and three milliliters cell-free supernatant (8000 rpm, 10 min) were vortexed for two minutes to mix. The height of emulsified layer was measured immediately and again after 24 hours. The results were calculated asmean±standard deviation The E24 was calculated by the following formula [17]:
(1)E24=heightoftheemulsifiedlayertotalheightoftheliquidlayer×100%.


### 2.3.2. Hemolysis Assay

The conserved strains were inoculated on NB plates and cultivated for two days to obtain moderate colonies. The colonies were selected using inoculation loop in the center and inoculated on the blood plates, and then incubated for 72 h at 42°C. After that, the clearing zone was measured and its hemolytic activity was evaluated. Hemolytic activity was assessed based on the halo diameter: (-) no halo, (+) complete hemolysis with diameter of <1 cm, (++) complete hemolysis with diameter of >1 cm but <3 cm, and (+++) hemolysis with diameter of >3 cm [19].


### 2.3.3. Extraction of Biosurfactant

Six strains (A-3, SC4542, SC4534(2), H-1, SC4561, and SC4551) with superior hemolysis and emulsification rate had biosurfactants extracted and identified. A single strain was inoculated on 100 mL of medium and fermented for 48 h at 160 rpm and 45°C. Then, 50 mL of fermentation broth was centrifuged for 30 min at 8000 rpm and the pH of the supernatant was adjusted to 2.0 with hydrochloric acid. Finally, the supernatant was kept at 4°C overnight. Chloroform: methanol was used in a 2 : 1 ratio for liquid-liquid extraction. The mixture was magnetically stirred for 1 hour and then kept aside for 30 minutes. The lower organic phase was separated and evaporated at room temperature (about 20°C) in a ventilated area to obtain a biosurfactant [20, 21].


### 2.3.4. Thin Layer Chromatography

The composition of biosurfactants was identified by thin layer chromatography (TLC). Chloroform: methanol (1 : 1 v/v) was used as developing agent (80 mL), and silica plate (MACHEREY-NAGEL, Germany) was used for the experiment. Following are the standard sample and color developing agent:(1)
To determine the presence of lipopeptides, 0.5% ninhydrin reagent was used as the indicator. If it showed purplish red, lipopeptides was present(2)
To determine the presence of glycan, 500μL of phenol and 1 mL concentrated sulfuric acid were diluted in 20 mL of ethanol as a chromogenic agent. Rhamnolipid was used as control. If it appeared brownish black, glycan was present(3)
Iodine fumes (volatilizing iodine in a sealed beaker) were used for lipid detection. If it showed yellow, lipid was presentThe sample should be fully dried and sprayed with color developing agent, and then heated in the oven for 5 min at 100°C for lipopeptide and 90°C for glycolipids to show the color.


### 2.3.5. FT-IR Spectrometric Analysis

The biosurfactants obtained in Section2.3.4 were dried by vacuum freezing dryer (BiLon FD80A, China). The pellets were scanned on a FT-IR (Fourier Transform Infrared) spectrophotometer (Shimadzu IRAffinity-1S, Japan) over a range of 4000-0 cm-1.


## 2.3.1. Emulsification Rate for 24 Hours Emulsification Index

Three milliliters of n-hexane and three milliliters cell-free supernatant (8000 rpm, 10 min) were vortexed for two minutes to mix. The height of emulsified layer was measured immediately and again after 24 hours. The results were calculated asmean±standard deviation The E24 was calculated by the following formula [17]:
(1)E24=heightoftheemulsifiedlayertotalheightoftheliquidlayer×100%.


## 2.3.2. Hemolysis Assay

The conserved strains were inoculated on NB plates and cultivated for two days to obtain moderate colonies. The colonies were selected using inoculation loop in the center and inoculated on the blood plates, and then incubated for 72 h at 42°C. After that, the clearing zone was measured and its hemolytic activity was evaluated. Hemolytic activity was assessed based on the halo diameter: (-) no halo, (+) complete hemolysis with diameter of <1 cm, (++) complete hemolysis with diameter of >1 cm but <3 cm, and (+++) hemolysis with diameter of >3 cm [19].


## 2.3.3. Extraction of Biosurfactant

Six strains (A-3, SC4542, SC4534(2), H-1, SC4561, and SC4551) with superior hemolysis and emulsification rate had biosurfactants extracted and identified. A single strain was inoculated on 100 mL of medium and fermented for 48 h at 160 rpm and 45°C. Then, 50 mL of fermentation broth was centrifuged for 30 min at 8000 rpm and the pH of the supernatant was adjusted to 2.0 with hydrochloric acid. Finally, the supernatant was kept at 4°C overnight. Chloroform: methanol was used in a 2 : 1 ratio for liquid-liquid extraction. The mixture was magnetically stirred for 1 hour and then kept aside for 30 minutes. The lower organic phase was separated and evaporated at room temperature (about 20°C) in a ventilated area to obtain a biosurfactant [20, 21].


## 2.3.4. Thin Layer Chromatography

The composition of biosurfactants was identified by thin layer chromatography (TLC). Chloroform: methanol (1 : 1 v/v) was used as developing agent (80 mL), and silica plate (MACHEREY-NAGEL, Germany) was used for the experiment. Following are the standard sample and color developing agent:(1)
To determine the presence of lipopeptides, 0.5% ninhydrin reagent was used as the indicator. If it showed purplish red, lipopeptides was present(2)
To determine the presence of glycan, 500μL of phenol and 1 mL concentrated sulfuric acid were diluted in 20 mL of ethanol as a chromogenic agent. Rhamnolipid was used as control. If it appeared brownish black, glycan was present(3)
Iodine fumes (volatilizing iodine in a sealed beaker) were used for lipid detection. If it showed yellow, lipid was presentThe sample should be fully dried and sprayed with color developing agent, and then heated in the oven for 5 min at 100°C for lipopeptide and 90°C for glycolipids to show the color.


## 2.3.5. FT-IR Spectrometric Analysis

The biosurfactants obtained in Section2.3.4 were dried by vacuum freezing dryer (BiLon FD80A, China). The pellets were scanned on a FT-IR (Fourier Transform Infrared) spectrophotometer (Shimadzu IRAffinity-1S, Japan) over a range of 4000-0 cm-1.


## 2.4. Temperature, Salinity, and pH Sensitivity Tests

The biosurfactant-producing strains, screened in Section2.3, were inoculated at 160 rpm for three days in NB medium. The temperatures were between 25°C and 65°C, with a step of 10°C. A series of tests at 50°C was also added in response to the experimental results.To assess the salinity sensitivity of strains, the same experiments were also carried out at 42°C with different NaCl concentration (1, 3, 5, 7, 9, 10, 20, 30, and 50 g/L). The growth of strains was examined via OD600, obtained through UV-vis spectrophotometry (Spectrumlab 752pro, USA) [22].Additionally, the pH sensitivity test was also carried out at fixed temperature of 42°C and NaCl concentration of 7 g/L. The pH was adjusted with HCl (1 mol/L) and NaOH (1 mol/L). After 3 days of incubation, the growth was checked with a pH range of 4 to 10.


## 2.5. The Effect of Dominant Bacteria on Crude Oil

### 2.5.1. Surface Tension

The biosurfactants-producing strains selected in Section were inoculated on NB medium for fermentation (50 mL) and the fermentation broth was centrifuged at 6000 rpm for five minutes after 7 days of incubation. The surface tension of the fermentation broth was measured using a surface tension meter (POWEREACH, China). The results were calculated asmean±standard deviation.


### 2.5.2. Viscosity

Ten milliliters of sterile oil and five milliliters of strains solution were added in 100 mL of MSM and inoculated at 160 rpm, 42°C for 7 days and 14 days. After incubation, the crude oil was transferred to a centrifuge tube (three parallel samples for each strain), which was then rest at 42°C overnight to allow oil and water to completely separate. The medium without bacteria was as control. The change of crude oil viscosity before and after the action of the strains was measured by a rotational rheometer (Anton Paar MCR 302, Austria). The results were calculated asmean±standard deviation. The viscosity reduction ratio is calculated by the following formula:
(2)viscosityreductionratio=VC−VSVC×100%.VC is the viscosity of control (mP·s); VS is the viscosity of the oil after the action of the strains.


### 2.5.3. GC-MS Analysis of the Degradation Effect of Strains

Fifty milliliter MSM with five milliliters of sterile oil and two milliliters of strains solution were inoculated at 160 rpm, 42°C for 7 days. After that, the crude oil was extracted with n-hexane after the culture broth had been centrifuged to remove bacteria and impurities [23]. The components of crude oil were determined by GC-MS (Thermo Fisher ISQ7000, USA). Helium was used as carrier gas with a flow rate of 1 mL/min. The heating program was set as follows. The initial temperature was 50°C with a hold time of 1 min. The temperature was increased to 120°C at 20°C/min and then increased to 250°C at 4°C/min. Finally, the temperature was increased to 300°C at 6°C/min with a hold time of 30 min. MS condition ionization method: EI, 70 eV; filament current, 100 A; multiplier voltage, 1200 V. Data collection method: selective ion scan (85, 123, 191, and 217). The degradation ratio of different components is calculated by the following formula:
(3)Degradationratioofdifferentcomponents=PC−PSPC×100%.PC is the sum of the peak areas of different components in the control group; PS is the sum of the peak areas of different components of strains.


## 2.5.1. Surface Tension

The biosurfactants-producing strains selected in Section were inoculated on NB medium for fermentation (50 mL) and the fermentation broth was centrifuged at 6000 rpm for five minutes after 7 days of incubation. The surface tension of the fermentation broth was measured using a surface tension meter (POWEREACH, China). The results were calculated asmean±standard deviation.


## 2.5.2. Viscosity

Ten milliliters of sterile oil and five milliliters of strains solution were added in 100 mL of MSM and inoculated at 160 rpm, 42°C for 7 days and 14 days. After incubation, the crude oil was transferred to a centrifuge tube (three parallel samples for each strain), which was then rest at 42°C overnight to allow oil and water to completely separate. The medium without bacteria was as control. The change of crude oil viscosity before and after the action of the strains was measured by a rotational rheometer (Anton Paar MCR 302, Austria). The results were calculated asmean±standard deviation. The viscosity reduction ratio is calculated by the following formula:
(2)viscosityreductionratio=VC−VSVC×100%.VC is the viscosity of control (mP·s); VS is the viscosity of the oil after the action of the strains.


## 2.5.3. GC-MS Analysis of the Degradation Effect of Strains

Fifty milliliter MSM with five milliliters of sterile oil and two milliliters of strains solution were inoculated at 160 rpm, 42°C for 7 days. After that, the crude oil was extracted with n-hexane after the culture broth had been centrifuged to remove bacteria and impurities [23]. The components of crude oil were determined by GC-MS (Thermo Fisher ISQ7000, USA). Helium was used as carrier gas with a flow rate of 1 mL/min. The heating program was set as follows. The initial temperature was 50°C with a hold time of 1 min. The temperature was increased to 120°C at 20°C/min and then increased to 250°C at 4°C/min. Finally, the temperature was increased to 300°C at 6°C/min with a hold time of 30 min. MS condition ionization method: EI, 70 eV; filament current, 100 A; multiplier voltage, 1200 V. Data collection method: selective ion scan (85, 123, 191, and 217). The degradation ratio of different components is calculated by the following formula:
(3)Degradationratioofdifferentcomponents=PC−PSPC×100%.PC is the sum of the peak areas of different components in the control group; PS is the sum of the peak areas of different components of strains.


## 2.6. Bacteria Identification

### 2.6.1. Morphological Features

The strain was streaked on NB media to obtain a single colony, and the bacterial morphology was observed by Scanning Electron Microscope (SEM).


### 2.6.2. 16S rRNA Identification of Dominant Bacteria

The 16S rRNA gene sequencing analysis was used to identify these strains. The primers 27F (5′ AGAGTTTGATCCTGGCTCAG 3′) and 1492R (5′ GGTTACCTTGTTACGACTT 3′) were used for PCR. The sequence was compared to the 16S rRNA nucleotide sequences at National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) [24].


## 2.6.1. Morphological Features

The strain was streaked on NB media to obtain a single colony, and the bacterial morphology was observed by Scanning Electron Microscope (SEM).


## 2.6.2. 16S rRNA Identification of Dominant Bacteria

The 16S rRNA gene sequencing analysis was used to identify these strains. The primers 27F (5′ AGAGTTTGATCCTGGCTCAG 3′) and 1492R (5′ GGTTACCTTGTTACGACTT 3′) were used for PCR. The sequence was compared to the 16S rRNA nucleotide sequences at National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) [24].


## 3. Results

### 3.1. Screening and Isolation of Bacteria

#### 3.1.1. Bacteria Using Oil as Sole Carbon Source

70 strains were obtained from two kinds of samples after months of screening and purification. Detailed information is shown in Table2. All strains were then monitored for growth. Fast-growing strains can shorten the cultivation time and reduce the time cost. During this process, it was found that about 20 strains that were screened and preserved for the first time could not be rejuvenated successfully; hence, they were not used in the subsequent test. The growth curves are shown in S2, and 27 strains with fast growth rates are shown in Table 3.Table 2
Information of selected single strains.
LayerCulture mediumNameQuantityChang 6Oil sample, NB mediumLetter+number16Chang4+5Water sample, NB mediumSC45+ dilution gradient+number24Oil sample, NB mediumOC45+ dilution gradient+number17Water sample, MSMJSC45+ dilution gradient+number13Oil sample, MSMJOC45+ dilution gradient+numberTable 3
Strains with fast growth rates.
SourcesStrainsWater samples (Chang4+5)SC4532, SC4534(2), SC4535(1), SC4536, SC4537, SC4541, SC4542, SC4543, SC4544, SC4561, SC4562, JSC4501, JSC4521, JSC4532, and JSC4535Oil samples (Chang4+5)OC411, OC4512, OC4524, OC4526, OC4534, and OC4537Oil samples (Chang 6)A-3, D-1, H-1, H-2, I-1, and I-2When the bacteria can survive on crude oil as the only source of carbon, they require fewer additional nutrients and are more tolerable. This property is crucial for reducing costs and improving the efficacy of microbial flooding in the field. The fast-growing strains were inoculated into MSM and a total of 14 strains that could turn the medium turbid quickly (Table4).Table 4
The results of hemolysis and E24.
StrainsHemolysisE24 (%)StrainsHemolysisE24 (%)SC4532—27.0±0.04JSC4535—50±0SC4534(2)+++57.1±0.03OC4511—20.3±0.03SC4535(1)—46.9±0.04OC4524+19.3±0.03SC4542+++54.8±0.11OC4541—17.6±0.04SC4551++51.7±0.03A-3++23.2±0.15SC4561++60±0.02H-1+68±0.03JSC4501+27.0±0.1I-1—24.8±0.06


#### 3.1.2. Biosurfactant-Producing Bacteria

Erythrocyte-lysing capabilities of biosurfactant-producing strains can cause clearing zones around strains colonies [25]. According to hemolysis and E24, eight of the 14 strains screened from MSM have the ability to produce biosurfactants (Table 4). SC4561, SC4534(2), SC4551, and SC4542 showed great biosurfactant-producing ability, shown in Figure 1.Figure 1
Hemolytic circles formed by SC4542, SC4534(2), SC4551, and SC4561.


### 3.2. Characterization of Biosurfactants

#### 3.2.1. TLC

The experimental results of thin layer chromatography are shown in Figure2. Five strains A-3, SC4542, SC4534(2), H-1, and SC4551 exhibited clear purple red, demonstrating the presence of lipopeptide (Figure 2(a)). Two strains SC4542 and SC4534(2) produced very light brown spots in Figure 2(b), while the other strains produced obvious brown-black spots at the front of the developing agent, but the color was also lighter than standard rhamnolipids, indicating that little glycan were present. The lipid reaction (Figure 2(c)) showed that all strains had obvious yellow bands except. In general, the 6 strains may produce lipopeptides and lipid biosurfactants.Figure 2
The results of thin layer chromatography ((a) lipopeptide; (b) glycan; (c) lipid).
(a)(b)(c)


#### 3.2.2. FT-IR

Figure3 displays the FT-IR results, which were of two different types. First, all the tested have an obvious absorption peak at 1637.56 cm-1, which is the evidence of the existence of amide group (H2N-C=O), indicating that all the strains produced lipid [26]. In Figure 3(a), absorption peaks for the O-H stringing vibration at 3400-3200 cm-1 and the absorption peaks at 1193-1226 and 1082-1078 cm-1 were characteristic of pyranose [27], indicating that SC4534 (2), SC4542, and SC461 strains produce glycolipid. However, in Figures 3(b), C-H stretching vibration peak at 2850-2950 cm-1, without obvious O-H absorption peak, and absorption peaks at 1458.18 cm-1 are caused by C-H on the aliphatic peptide chain [20, 21], indicating that A-3, H-1, and SC4551 may produce lipopeptide. Therefore, the biosurfactant produced by SC4534(2), SC4542, and SC4561 was mainly glycolipid, and A-3, H-1, and SC4551 produced lipopeptide, which was basically consistent with the TLC results.Figure 3
FT-IR spectrum of biosurfactant produced by 6 strains ((a) SC4534(2), SC4542, and SC4561; (b) A-3, H-1, and SC4551).
(a)(b)


### 3.3. Growth in Different Temperatures, Salinity, and pH

The reservoir is an extreme environment (high temperature, anaerobic conditions, high salinity, high pressure, etc.), which is not conducive to the survival of microorganisms. The dominant bacteria that can be applied to MEOR must be able to adapt to this harsh environment.Figure4 displays the results of the sensitivity test on JSC4535 and JSC4501 strains. Both strains were screened by MSM. Temperature sensitivity tests show that all strains can grow fast between 25 and 50°C, with 35°C being the optimal temperature for the strain was. The growth decreased rapidly at 50°C. The optimal growth salinity of all strains was 10 g/L and JSC4535 can also survive under 50 g/L. All strains thrived when pH was over 7 shows that the screened bacteria have strong alkaline tolerance.Figure 4
Growth curve of strains from water sample (Chang4+5 layer, MSM).Figure5 shows the result of 4 strains screening from water sample in Chang 4+5 layer. The optimal growth temperature of SC4551 was 35°C-45°C. The ideal salinity was 10 g/L. The fastest growth rate was got at pH=5. The growth of strain SC4561 decreased with temperature, and it grew well at temperatures below 45°C. When the salinity was 50 g/L, the strain hardly survived, and the optimal salinity was 3 g/L. The strain has best optical density under pH=9. SC4534(2) had the best growth temperature at 45°C and 10 g/L of salinity. The concentration of the bacterial solution was high at pH upon 5. The results of SC4542 strain were special, the OD600 of the bacterial solution is relatively small in all test. The ideal temperature was 35°C and the best pH was 8. When the salinity is 10 g/L, the bacterium survived best.Figure 5
Growth curve of strains from water sample (Chang4+5 layer).The optimum growth temperature for H-1 was 35°C, and at 50°C, the OD600 value fell sharply. The optimal salinity was 3 g/L. A-3 have the best growth under 35°C and pH=7. The best salinity is 7 g/L (Figure 6).Figure 6
Growth curve of strains from oil sample (Chang 6 layer).There are some anomalies in the sensitivity test. Some strains, as SC4551, SC4534(2), and JSC4501, would exhibit higher OD at high temperatures than low temperatures. The OD value of the SC4542 strain drops sharply at the salinity of 30 g/L, which may due to that the strain concentrate on the surface of the medium to form a film rather than dispersing in the medium (Figure7(a)). The OD600 of H-1 altered dramatically at pH=6, and the curve fluctuated significantly. This is primarily because the state of the strain was completely different under the two pH conditions (Figure 7(b)).Figure 7
The state of the strain in the medium ((a) film on the medium surface; (b) the different states of H-1 atpH=6 and pH=7).
(a)(b)


### 3.4. Biodegradation of Crude Oil

#### 3.4.1. Changes of Surface Tension

The results of surface tension measurement of fermentation broth showed that the highest reduction in surface tension was from 58.37 mN/m to 40.51 mN/m (Table5). Four strains, A-3, H-1, SC4534(2), and SC4561, exhibited more obvious effect in reducing surface tension, while other two strains, JSC4501 and JSC4535, had poor effects on the surface tension of crude oil or fermentation broth and even caused the surface tension to increase.Table 5
Changes in surface tension of fermentation broth.
StrainsSurface tension mN/mReduction rate %Control58.37±1.41N.D.SC4534(2)50.56±0.2813.38A-340.51±0.3630.6SC455157.83±0.350.93H-143.76±1.5625.03SC454252.14±0.0510.67SC456152.31±4.0010.38JSC453557.86±0.860.87JSC450170.49±0.30-20.76


#### 3.4.2. Viscosity Reduction Ratio

Figure8 depicts the effects of the strains on the viscosity of crude oil. All strains but SC4542 had viscosity decreased by 27-51% after 7 days of incubation (Figure 8(a)) and had viscosity decreased by 27-47% after 14 days of incubation (expect for SC4534(2)) (Figure 8(b)). Overall, the viscosity reduction rate did not increase with increasing incubation time; on the contrary, some strains, including SC4534(2), SC4551, and SC4561, had the drop in the rate of viscosity reduction. Strains SC4542 and JSC4535 had a longer period of action, the viscosity reduction of 7 days incubation (5.71% and 38.66%, respectively) is less that of 14 days (32.59% and 47.2%, respectively).Figure 8
Viscosity and viscosity reduction ratio after bacterial action ((a) 7 days; (b) 14 days).
(a)(b)


#### 3.4.3. GC-MS Analysis

Through the analysis of the crude oil components by GC-MS before and after the action of the strain, long-chain n-alkanes were more effectively degraded by A-3, SC4534(2), SC4561, SC4551, and JSC4535 (Figure9(a) and 9(b)), and A-3 had the best effects. The degradation rates of n-alkanes by these strains were 51.2%, 28.3%, 35.2%, 5.45%, and 28.9%, respectively. At the same time, they also had a slight degradation effect on cholestane (m/z=217) (Figure 9(b)). Strains JSC4501, SC4542, and H-1 only had a little degradation effect on short-chain alkanes (Figure 10(a)) but they can degrade tricyclic terpenes (m/z=191) (Figure 10(b)) and SC4542 has the best effect.Figure 9
Degradation by SC4561, A-3, SC4534(2), SC4551, and JSC4545 of n-alkanes ((a) degradation of n-alkanes; (b) degradation of cholestane).
(a)(b)Figure 10
Degradation by JSC4501, H-1, and SC4542 ((a) degradation of n-alkanes; (b) degradation of terpenes).
(a)(b)Seven strains had a good degradation effect on aromatic hydrocarbons, methylnaphthalene, and ethyl (Figure11). The JSC4501, JSC4535, SC4542, SC4551, SC4561, A-3, and SC4534(2) all had degradation rate of 90.8%, 90.6%, 87.2%, 85.9%, 85.7%, 85.3%, and 84.7%, respectively. In addition, the strain SC4561, SC4534(2), A-3, and JSC4501 also slightly degraded methyl chrysene, though less than that of naphthalene.Figure 11
Degradation of part of naphthalene.


### 3.5. Identification of Dominant Bacteria

The morphological characteristics and gene identification of different dominant bacteria are shown in Table6. The species of SC4542 were identified through ANI (Average Nucleotide Identity) since it did not identify though 16S rRNA. Six of seven strains were Bacillus, and one belonged to Alphaproteobacteria (JSC4535). SEM showed the morphological characteristics of eight strains (Figure 12). All strains were rod-shaped but had different morphologies. JSC4535, SC4542, and SC4561 were elongated with aspect ratios ranging from 5 : 1 to 9 : 1. While H-1, SC4534(2) were stubby, with an aspect ratio of almost 2 : 1.Table 6
Results of 16S rRNA gene sequences.
StrainsGenBank numberSimilarity (%)TaxonomyJSC4501MW559321.199%Bacillus cereus K1M30SC4534(2)MF576500.199%Bacillus sp. (in: Bacteria) strain DSC4561MW559317.199%Bacillus cereus strain T3M32SC4551MN784674.199%Bacillus sp. (in: Bacteria) strain BRIA-3HM055598.198%Bacillus licheniformis strain GD2aJSC4535NR_028632.199%Tistrella mobilis strain JCM 21370H-1AJ438305.299%Brevibacillus sp. R-12868SC4542CP026673.199%Bacillus licheniformis strain 14ADL4Figure 12
Strain morphology by SEM.


## 3.1. Screening and Isolation of Bacteria

### 3.1.1. Bacteria Using Oil as Sole Carbon Source

70 strains were obtained from two kinds of samples after months of screening and purification. Detailed information is shown in Table2. All strains were then monitored for growth. Fast-growing strains can shorten the cultivation time and reduce the time cost. During this process, it was found that about 20 strains that were screened and preserved for the first time could not be rejuvenated successfully; hence, they were not used in the subsequent test. The growth curves are shown in S2, and 27 strains with fast growth rates are shown in Table 3.Table 2
Information of selected single strains.
LayerCulture mediumNameQuantityChang 6Oil sample, NB mediumLetter+number16Chang4+5Water sample, NB mediumSC45+ dilution gradient+number24Oil sample, NB mediumOC45+ dilution gradient+number17Water sample, MSMJSC45+ dilution gradient+number13Oil sample, MSMJOC45+ dilution gradient+numberTable 3
Strains with fast growth rates.
SourcesStrainsWater samples (Chang4+5)SC4532, SC4534(2), SC4535(1), SC4536, SC4537, SC4541, SC4542, SC4543, SC4544, SC4561, SC4562, JSC4501, JSC4521, JSC4532, and JSC4535Oil samples (Chang4+5)OC411, OC4512, OC4524, OC4526, OC4534, and OC4537Oil samples (Chang 6)A-3, D-1, H-1, H-2, I-1, and I-2When the bacteria can survive on crude oil as the only source of carbon, they require fewer additional nutrients and are more tolerable. This property is crucial for reducing costs and improving the efficacy of microbial flooding in the field. The fast-growing strains were inoculated into MSM and a total of 14 strains that could turn the medium turbid quickly (Table4).Table 4
The results of hemolysis and E24.
StrainsHemolysisE24 (%)StrainsHemolysisE24 (%)SC4532—27.0±0.04JSC4535—50±0SC4534(2)+++57.1±0.03OC4511—20.3±0.03SC4535(1)—46.9±0.04OC4524+19.3±0.03SC4542+++54.8±0.11OC4541—17.6±0.04SC4551++51.7±0.03A-3++23.2±0.15SC4561++60±0.02H-1+68±0.03JSC4501+27.0±0.1I-1—24.8±0.06


### 3.1.2. Biosurfactant-Producing Bacteria

Erythrocyte-lysing capabilities of biosurfactant-producing strains can cause clearing zones around strains colonies [25]. According to hemolysis and E24, eight of the 14 strains screened from MSM have the ability to produce biosurfactants (Table 4). SC4561, SC4534(2), SC4551, and SC4542 showed great biosurfactant-producing ability, shown in Figure 1.Figure 1
Hemolytic circles formed by SC4542, SC4534(2), SC4551, and SC4561.


## 3.1.1. Bacteria Using Oil as Sole Carbon Source

70 strains were obtained from two kinds of samples after months of screening and purification. Detailed information is shown in Table2. All strains were then monitored for growth. Fast-growing strains can shorten the cultivation time and reduce the time cost. During this process, it was found that about 20 strains that were screened and preserved for the first time could not be rejuvenated successfully; hence, they were not used in the subsequent test. The growth curves are shown in S2, and 27 strains with fast growth rates are shown in Table 3.Table 2
Information of selected single strains.
LayerCulture mediumNameQuantityChang 6Oil sample, NB mediumLetter+number16Chang4+5Water sample, NB mediumSC45+ dilution gradient+number24Oil sample, NB mediumOC45+ dilution gradient+number17Water sample, MSMJSC45+ dilution gradient+number13Oil sample, MSMJOC45+ dilution gradient+numberTable 3
Strains with fast growth rates.
SourcesStrainsWater samples (Chang4+5)SC4532, SC4534(2), SC4535(1), SC4536, SC4537, SC4541, SC4542, SC4543, SC4544, SC4561, SC4562, JSC4501, JSC4521, JSC4532, and JSC4535Oil samples (Chang4+5)OC411, OC4512, OC4524, OC4526, OC4534, and OC4537Oil samples (Chang 6)A-3, D-1, H-1, H-2, I-1, and I-2When the bacteria can survive on crude oil as the only source of carbon, they require fewer additional nutrients and are more tolerable. This property is crucial for reducing costs and improving the efficacy of microbial flooding in the field. The fast-growing strains were inoculated into MSM and a total of 14 strains that could turn the medium turbid quickly (Table4).Table 4
The results of hemolysis and E24.
StrainsHemolysisE24 (%)StrainsHemolysisE24 (%)SC4532—27.0±0.04JSC4535—50±0SC4534(2)+++57.1±0.03OC4511—20.3±0.03SC4535(1)—46.9±0.04OC4524+19.3±0.03SC4542+++54.8±0.11OC4541—17.6±0.04SC4551++51.7±0.03A-3++23.2±0.15SC4561++60±0.02H-1+68±0.03JSC4501+27.0±0.1I-1—24.8±0.06


## 3.1.2. Biosurfactant-Producing Bacteria

Erythrocyte-lysing capabilities of biosurfactant-producing strains can cause clearing zones around strains colonies [25]. According to hemolysis and E24, eight of the 14 strains screened from MSM have the ability to produce biosurfactants (Table 4). SC4561, SC4534(2), SC4551, and SC4542 showed great biosurfactant-producing ability, shown in Figure 1.Figure 1
Hemolytic circles formed by SC4542, SC4534(2), SC4551, and SC4561.


## 3.2. Characterization of Biosurfactants

### 3.2.1. TLC

The experimental results of thin layer chromatography are shown in Figure2. Five strains A-3, SC4542, SC4534(2), H-1, and SC4551 exhibited clear purple red, demonstrating the presence of lipopeptide (Figure 2(a)). Two strains SC4542 and SC4534(2) produced very light brown spots in Figure 2(b), while the other strains produced obvious brown-black spots at the front of the developing agent, but the color was also lighter than standard rhamnolipids, indicating that little glycan were present. The lipid reaction (Figure 2(c)) showed that all strains had obvious yellow bands except. In general, the 6 strains may produce lipopeptides and lipid biosurfactants.Figure 2
The results of thin layer chromatography ((a) lipopeptide; (b) glycan; (c) lipid).
(a)(b)(c)


### 3.2.2. FT-IR

Figure3 displays the FT-IR results, which were of two different types. First, all the tested have an obvious absorption peak at 1637.56 cm-1, which is the evidence of the existence of amide group (H2N-C=O), indicating that all the strains produced lipid [26]. In Figure 3(a), absorption peaks for the O-H stringing vibration at 3400-3200 cm-1 and the absorption peaks at 1193-1226 and 1082-1078 cm-1 were characteristic of pyranose [27], indicating that SC4534 (2), SC4542, and SC461 strains produce glycolipid. However, in Figures 3(b), C-H stretching vibration peak at 2850-2950 cm-1, without obvious O-H absorption peak, and absorption peaks at 1458.18 cm-1 are caused by C-H on the aliphatic peptide chain [20, 21], indicating that A-3, H-1, and SC4551 may produce lipopeptide. Therefore, the biosurfactant produced by SC4534(2), SC4542, and SC4561 was mainly glycolipid, and A-3, H-1, and SC4551 produced lipopeptide, which was basically consistent with the TLC results.Figure 3
FT-IR spectrum of biosurfactant produced by 6 strains ((a) SC4534(2), SC4542, and SC4561; (b) A-3, H-1, and SC4551).
(a)(b)


## 3.2.1. TLC

The experimental results of thin layer chromatography are shown in Figure2. Five strains A-3, SC4542, SC4534(2), H-1, and SC4551 exhibited clear purple red, demonstrating the presence of lipopeptide (Figure 2(a)). Two strains SC4542 and SC4534(2) produced very light brown spots in Figure 2(b), while the other strains produced obvious brown-black spots at the front of the developing agent, but the color was also lighter than standard rhamnolipids, indicating that little glycan were present. The lipid reaction (Figure 2(c)) showed that all strains had obvious yellow bands except. In general, the 6 strains may produce lipopeptides and lipid biosurfactants.Figure 2
The results of thin layer chromatography ((a) lipopeptide; (b) glycan; (c) lipid).
(a)(b)(c)


## 3.2.2. FT-IR

Figure3 displays the FT-IR results, which were of two different types. First, all the tested have an obvious absorption peak at 1637.56 cm-1, which is the evidence of the existence of amide group (H2N-C=O), indicating that all the strains produced lipid [26]. In Figure 3(a), absorption peaks for the O-H stringing vibration at 3400-3200 cm-1 and the absorption peaks at 1193-1226 and 1082-1078 cm-1 were characteristic of pyranose [27], indicating that SC4534 (2), SC4542, and SC461 strains produce glycolipid. However, in Figures 3(b), C-H stretching vibration peak at 2850-2950 cm-1, without obvious O-H absorption peak, and absorption peaks at 1458.18 cm-1 are caused by C-H on the aliphatic peptide chain [20, 21], indicating that A-3, H-1, and SC4551 may produce lipopeptide. Therefore, the biosurfactant produced by SC4534(2), SC4542, and SC4561 was mainly glycolipid, and A-3, H-1, and SC4551 produced lipopeptide, which was basically consistent with the TLC results.Figure 3
FT-IR spectrum of biosurfactant produced by 6 strains ((a) SC4534(2), SC4542, and SC4561; (b) A-3, H-1, and SC4551).
(a)(b)


## 3.3. Growth in Different Temperatures, Salinity, and pH

The reservoir is an extreme environment (high temperature, anaerobic conditions, high salinity, high pressure, etc.), which is not conducive to the survival of microorganisms. The dominant bacteria that can be applied to MEOR must be able to adapt to this harsh environment.Figure4 displays the results of the sensitivity test on JSC4535 and JSC4501 strains. Both strains were screened by MSM. Temperature sensitivity tests show that all strains can grow fast between 25 and 50°C, with 35°C being the optimal temperature for the strain was. The growth decreased rapidly at 50°C. The optimal growth salinity of all strains was 10 g/L and JSC4535 can also survive under 50 g/L. All strains thrived when pH was over 7 shows that the screened bacteria have strong alkaline tolerance.Figure 4
Growth curve of strains from water sample (Chang4+5 layer, MSM).Figure5 shows the result of 4 strains screening from water sample in Chang 4+5 layer. The optimal growth temperature of SC4551 was 35°C-45°C. The ideal salinity was 10 g/L. The fastest growth rate was got at pH=5. The growth of strain SC4561 decreased with temperature, and it grew well at temperatures below 45°C. When the salinity was 50 g/L, the strain hardly survived, and the optimal salinity was 3 g/L. The strain has best optical density under pH=9. SC4534(2) had the best growth temperature at 45°C and 10 g/L of salinity. The concentration of the bacterial solution was high at pH upon 5. The results of SC4542 strain were special, the OD600 of the bacterial solution is relatively small in all test. The ideal temperature was 35°C and the best pH was 8. When the salinity is 10 g/L, the bacterium survived best.Figure 5
Growth curve of strains from water sample (Chang4+5 layer).The optimum growth temperature for H-1 was 35°C, and at 50°C, the OD600 value fell sharply. The optimal salinity was 3 g/L. A-3 have the best growth under 35°C and pH=7. The best salinity is 7 g/L (Figure 6).Figure 6
Growth curve of strains from oil sample (Chang 6 layer).There are some anomalies in the sensitivity test. Some strains, as SC4551, SC4534(2), and JSC4501, would exhibit higher OD at high temperatures than low temperatures. The OD value of the SC4542 strain drops sharply at the salinity of 30 g/L, which may due to that the strain concentrate on the surface of the medium to form a film rather than dispersing in the medium (Figure7(a)). The OD600 of H-1 altered dramatically at pH=6, and the curve fluctuated significantly. This is primarily because the state of the strain was completely different under the two pH conditions (Figure 7(b)).Figure 7
The state of the strain in the medium ((a) film on the medium surface; (b) the different states of H-1 atpH=6 and pH=7).
(a)(b)


## 3.4. Biodegradation of Crude Oil

### 3.4.1. Changes of Surface Tension

The results of surface tension measurement of fermentation broth showed that the highest reduction in surface tension was from 58.37 mN/m to 40.51 mN/m (Table5). Four strains, A-3, H-1, SC4534(2), and SC4561, exhibited more obvious effect in reducing surface tension, while other two strains, JSC4501 and JSC4535, had poor effects on the surface tension of crude oil or fermentation broth and even caused the surface tension to increase.Table 5
Changes in surface tension of fermentation broth.
StrainsSurface tension mN/mReduction rate %Control58.37±1.41N.D.SC4534(2)50.56±0.2813.38A-340.51±0.3630.6SC455157.83±0.350.93H-143.76±1.5625.03SC454252.14±0.0510.67SC456152.31±4.0010.38JSC453557.86±0.860.87JSC450170.49±0.30-20.76


### 3.4.2. Viscosity Reduction Ratio

Figure8 depicts the effects of the strains on the viscosity of crude oil. All strains but SC4542 had viscosity decreased by 27-51% after 7 days of incubation (Figure 8(a)) and had viscosity decreased by 27-47% after 14 days of incubation (expect for SC4534(2)) (Figure 8(b)). Overall, the viscosity reduction rate did not increase with increasing incubation time; on the contrary, some strains, including SC4534(2), SC4551, and SC4561, had the drop in the rate of viscosity reduction. Strains SC4542 and JSC4535 had a longer period of action, the viscosity reduction of 7 days incubation (5.71% and 38.66%, respectively) is less that of 14 days (32.59% and 47.2%, respectively).Figure 8
Viscosity and viscosity reduction ratio after bacterial action ((a) 7 days; (b) 14 days).
(a)(b)


### 3.4.3. GC-MS Analysis

Through the analysis of the crude oil components by GC-MS before and after the action of the strain, long-chain n-alkanes were more effectively degraded by A-3, SC4534(2), SC4561, SC4551, and JSC4535 (Figure9(a) and 9(b)), and A-3 had the best effects. The degradation rates of n-alkanes by these strains were 51.2%, 28.3%, 35.2%, 5.45%, and 28.9%, respectively. At the same time, they also had a slight degradation effect on cholestane (m/z=217) (Figure 9(b)). Strains JSC4501, SC4542, and H-1 only had a little degradation effect on short-chain alkanes (Figure 10(a)) but they can degrade tricyclic terpenes (m/z=191) (Figure 10(b)) and SC4542 has the best effect.Figure 9
Degradation by SC4561, A-3, SC4534(2), SC4551, and JSC4545 of n-alkanes ((a) degradation of n-alkanes; (b) degradation of cholestane).
(a)(b)Figure 10
Degradation by JSC4501, H-1, and SC4542 ((a) degradation of n-alkanes; (b) degradation of terpenes).
(a)(b)Seven strains had a good degradation effect on aromatic hydrocarbons, methylnaphthalene, and ethyl (Figure11). The JSC4501, JSC4535, SC4542, SC4551, SC4561, A-3, and SC4534(2) all had degradation rate of 90.8%, 90.6%, 87.2%, 85.9%, 85.7%, 85.3%, and 84.7%, respectively. In addition, the strain SC4561, SC4534(2), A-3, and JSC4501 also slightly degraded methyl chrysene, though less than that of naphthalene.Figure 11
Degradation of part of naphthalene.


## 3.4.1. Changes of Surface Tension

The results of surface tension measurement of fermentation broth showed that the highest reduction in surface tension was from 58.37 mN/m to 40.51 mN/m (Table5). Four strains, A-3, H-1, SC4534(2), and SC4561, exhibited more obvious effect in reducing surface tension, while other two strains, JSC4501 and JSC4535, had poor effects on the surface tension of crude oil or fermentation broth and even caused the surface tension to increase.Table 5
Changes in surface tension of fermentation broth.
StrainsSurface tension mN/mReduction rate %Control58.37±1.41N.D.SC4534(2)50.56±0.2813.38A-340.51±0.3630.6SC455157.83±0.350.93H-143.76±1.5625.03SC454252.14±0.0510.67SC456152.31±4.0010.38JSC453557.86±0.860.87JSC450170.49±0.30-20.76


## 3.4.2. Viscosity Reduction Ratio

Figure8 depicts the effects of the strains on the viscosity of crude oil. All strains but SC4542 had viscosity decreased by 27-51% after 7 days of incubation (Figure 8(a)) and had viscosity decreased by 27-47% after 14 days of incubation (expect for SC4534(2)) (Figure 8(b)). Overall, the viscosity reduction rate did not increase with increasing incubation time; on the contrary, some strains, including SC4534(2), SC4551, and SC4561, had the drop in the rate of viscosity reduction. Strains SC4542 and JSC4535 had a longer period of action, the viscosity reduction of 7 days incubation (5.71% and 38.66%, respectively) is less that of 14 days (32.59% and 47.2%, respectively).Figure 8
Viscosity and viscosity reduction ratio after bacterial action ((a) 7 days; (b) 14 days).
(a)(b)


## 3.4.3. GC-MS Analysis

Through the analysis of the crude oil components by GC-MS before and after the action of the strain, long-chain n-alkanes were more effectively degraded by A-3, SC4534(2), SC4561, SC4551, and JSC4535 (Figure9(a) and 9(b)), and A-3 had the best effects. The degradation rates of n-alkanes by these strains were 51.2%, 28.3%, 35.2%, 5.45%, and 28.9%, respectively. At the same time, they also had a slight degradation effect on cholestane (m/z=217) (Figure 9(b)). Strains JSC4501, SC4542, and H-1 only had a little degradation effect on short-chain alkanes (Figure 10(a)) but they can degrade tricyclic terpenes (m/z=191) (Figure 10(b)) and SC4542 has the best effect.Figure 9
Degradation by SC4561, A-3, SC4534(2), SC4551, and JSC4545 of n-alkanes ((a) degradation of n-alkanes; (b) degradation of cholestane).
(a)(b)Figure 10
Degradation by JSC4501, H-1, and SC4542 ((a) degradation of n-alkanes; (b) degradation of terpenes).
(a)(b)Seven strains had a good degradation effect on aromatic hydrocarbons, methylnaphthalene, and ethyl (Figure11). The JSC4501, JSC4535, SC4542, SC4551, SC4561, A-3, and SC4534(2) all had degradation rate of 90.8%, 90.6%, 87.2%, 85.9%, 85.7%, 85.3%, and 84.7%, respectively. In addition, the strain SC4561, SC4534(2), A-3, and JSC4501 also slightly degraded methyl chrysene, though less than that of naphthalene.Figure 11
Degradation of part of naphthalene.


## 3.5. Identification of Dominant Bacteria

The morphological characteristics and gene identification of different dominant bacteria are shown in Table6. The species of SC4542 were identified through ANI (Average Nucleotide Identity) since it did not identify though 16S rRNA. Six of seven strains were Bacillus, and one belonged to Alphaproteobacteria (JSC4535). SEM showed the morphological characteristics of eight strains (Figure 12). All strains were rod-shaped but had different morphologies. JSC4535, SC4542, and SC4561 were elongated with aspect ratios ranging from 5 : 1 to 9 : 1. While H-1, SC4534(2) were stubby, with an aspect ratio of almost 2 : 1.Table 6
Results of 16S rRNA gene sequences.
StrainsGenBank numberSimilarity (%)TaxonomyJSC4501MW559321.199%Bacillus cereus K1M30SC4534(2)MF576500.199%Bacillus sp. (in: Bacteria) strain DSC4561MW559317.199%Bacillus cereus strain T3M32SC4551MN784674.199%Bacillus sp. (in: Bacteria) strain BRIA-3HM055598.198%Bacillus licheniformis strain GD2aJSC4535NR_028632.199%Tistrella mobilis strain JCM 21370H-1AJ438305.299%Brevibacillus sp. R-12868SC4542CP026673.199%Bacillus licheniformis strain 14ADL4Figure 12
Strain morphology by SEM.


## 4. Discussion

### 4.1. The Nature of the Dominant Bacteria and Their Adaptations

Eight effective strains that can change the properties of crude oil have been obtained from the produced water and crude oil of Hujianshan oilfield, a low-permeability oilfield in the Ordos Basin, through separation and screening. According to the16S rRNA analysis, seven strains belong toBacillus. Oil wells frequently include the dominant bacterium Bacillus, which has been identified in many studies on the community structure of oil wells [28–30]. Bacillus has strong environmental adaptability due to its endospores. It has been employed in oil reservoir development, soil, and marine pollution remediation [11, 31]. The biosurfactants produced by the SC4534(2), SC4561, and SC4542 were lipopeptide and A-3, H-1, and SC4551 produced glycolipid. Biosurfactants are more friendly to the environment and less toxic compared with chemical synthesized surfactants [13, 17]. They are amphiphilic molecules and soluble in both nonpolar and polar compounds [26]. Therefore, they can spontaneously assemble at the oil-water interface to reduce surface/interfacial tension and emulsify crude oil [32]. Among them, lipopeptide including surfactin are considered to be more promising due to great surface activity [33, 34]. Bacillus sp. has been reported in numerous studies to be the main strain of lipopeptide producing bacteria [35–37].A special phenomenon in the study is that 20 strains of the 70 strains obtained at the initial stage could not be revived. This might be the result of strains having limited cultivability and surviving in extreme environments (oil well environments are extreme with unique biomes) [15, 38]. Culture-dependent approaches might not suitable for these reservoir species.The temperature and salinity sensitivity tests show that strains have a wide range of optimal growth temperatures and salinity. They can multiply quickly in the range of 25°C-50°C, and had the best growth rate at 35°C-40°C. Additionally, they also exhibited good action in culture medium with salinity under 40 g/L. Some strains can survive at salinity 50 g/L (JSC4535, SC4542, and A-3). The strains obtained in this study are more broadly adapted than those reported in Zhao et al. [39] in another oilfield in the Ordos Basin. The temperature of the oil well often changes with the exploitation method and it controls the growth of microorganisms and may inhibit the enzyme expression and activity within the optimum range [22, 40]. The temperature of the sampling well after water flooding ranges between 40 and 50°C. The salinity of the reservoir is 37-46 g/L, and the salinity will also decrease significantly after water flooding. The results indicate that these biosurfactant-producing strains have good adaptability to well conditions. The pH tolerance test showed that the eight strains screened did not produce organic acids, but could survive in strong alkaline environments. The alkali-tolerant strains are better adapted to the environment since the study area is heavily mineralized and the water drive will raise the pH of the well [41].Noteworthy phenomena were shown in sensitivity test. OD600 value changed nonlinearly, dropping suddenly (such as SC4542 and H-1). In addition, OD at 55°C was higher than 50°C in JSC4501, SC4551, and SC4534(2). We have two speculations. First, the optional density (OD600) was used to measure the concentration of the bacterial solution because that this method is suitable for measuring a large number of cultures, and is more accurate for homogeneous bacterial solutions [42, 43] However, these screening strains are aerobic bacteria which can easily live in contact with the air on the surface of the culture medium and whose solutes are not homogeneous (Figure 7), thus the OD600 value is low. Second, although 1 mL bacterial solution were added to the fresh medium at the beginning of the experiment, the concentration of 1 mL solution (OD600) was not completely consistent from transfer to transfer. When the strain multiplies a lot, the effect of the transferred solution is less, but the above phenomenon occurs at higher temperatures and salinity, where the strain multiplies less and slowly, and the influence of the 1 mL transferred solution is reflected. Furthermore, the relationship between salinity and the amount of dissolved oxygen is generally negative [44]. High salinity will inhibit growth of aerobic bacteria. Attention should be paid to the supply of oxygen in MEOR applications.


### 4.2. Effects of Dominant Strains on Crude Oil

In the biodegradation test, crude oil viscosity is reduced by 27-51% after 7 days of incubation, but prolonged incubation time may make the strain compete for nutrients and produce toxins that have a counter impact on viscosity (for example, SC4534(2), SC4551, and SC4561). The change of∑C21−/∑C21+ can reveal the priority of the strain for the utilization of n-alkanes [45]. The ∑C21−/∑C21+ of A-3, SC4534(2), SC4561, JSC4535, and SC4551 rise from 1.29 before action to 1.77, 2.04, 1.61, 1.52, and 1.48, respectively, while the value of JSC4501, SC4542, and H-1 decrease to 1.18, 1.03, and 1.17, respectively. The results indicate that the strains A-3, SC4534(2), SC4561, JSC4535, and SC4551 may grow on long-chain alkanes (C>22) preferentially, making increased content of short-chain alkanes (Figure 6), but the content of long-chain alkanes increased after the action of JSC4501, SC4542, and H-1. It is possible that the content increases due to the degradation of short-chain hydrocarbons and the action on cycloalkanes [46].


## 4.1. The Nature of the Dominant Bacteria and Their Adaptations

Eight effective strains that can change the properties of crude oil have been obtained from the produced water and crude oil of Hujianshan oilfield, a low-permeability oilfield in the Ordos Basin, through separation and screening. According to the16S rRNA analysis, seven strains belong toBacillus. Oil wells frequently include the dominant bacterium Bacillus, which has been identified in many studies on the community structure of oil wells [28–30]. Bacillus has strong environmental adaptability due to its endospores. It has been employed in oil reservoir development, soil, and marine pollution remediation [11, 31]. The biosurfactants produced by the SC4534(2), SC4561, and SC4542 were lipopeptide and A-3, H-1, and SC4551 produced glycolipid. Biosurfactants are more friendly to the environment and less toxic compared with chemical synthesized surfactants [13, 17]. They are amphiphilic molecules and soluble in both nonpolar and polar compounds [26]. Therefore, they can spontaneously assemble at the oil-water interface to reduce surface/interfacial tension and emulsify crude oil [32]. Among them, lipopeptide including surfactin are considered to be more promising due to great surface activity [33, 34]. Bacillus sp. has been reported in numerous studies to be the main strain of lipopeptide producing bacteria [35–37].A special phenomenon in the study is that 20 strains of the 70 strains obtained at the initial stage could not be revived. This might be the result of strains having limited cultivability and surviving in extreme environments (oil well environments are extreme with unique biomes) [15, 38]. Culture-dependent approaches might not suitable for these reservoir species.The temperature and salinity sensitivity tests show that strains have a wide range of optimal growth temperatures and salinity. They can multiply quickly in the range of 25°C-50°C, and had the best growth rate at 35°C-40°C. Additionally, they also exhibited good action in culture medium with salinity under 40 g/L. Some strains can survive at salinity 50 g/L (JSC4535, SC4542, and A-3). The strains obtained in this study are more broadly adapted than those reported in Zhao et al. [39] in another oilfield in the Ordos Basin. The temperature of the oil well often changes with the exploitation method and it controls the growth of microorganisms and may inhibit the enzyme expression and activity within the optimum range [22, 40]. The temperature of the sampling well after water flooding ranges between 40 and 50°C. The salinity of the reservoir is 37-46 g/L, and the salinity will also decrease significantly after water flooding. The results indicate that these biosurfactant-producing strains have good adaptability to well conditions. The pH tolerance test showed that the eight strains screened did not produce organic acids, but could survive in strong alkaline environments. The alkali-tolerant strains are better adapted to the environment since the study area is heavily mineralized and the water drive will raise the pH of the well [41].Noteworthy phenomena were shown in sensitivity test. OD600 value changed nonlinearly, dropping suddenly (such as SC4542 and H-1). In addition, OD at 55°C was higher than 50°C in JSC4501, SC4551, and SC4534(2). We have two speculations. First, the optional density (OD600) was used to measure the concentration of the bacterial solution because that this method is suitable for measuring a large number of cultures, and is more accurate for homogeneous bacterial solutions [42, 43] However, these screening strains are aerobic bacteria which can easily live in contact with the air on the surface of the culture medium and whose solutes are not homogeneous (Figure 7), thus the OD600 value is low. Second, although 1 mL bacterial solution were added to the fresh medium at the beginning of the experiment, the concentration of 1 mL solution (OD600) was not completely consistent from transfer to transfer. When the strain multiplies a lot, the effect of the transferred solution is less, but the above phenomenon occurs at higher temperatures and salinity, where the strain multiplies less and slowly, and the influence of the 1 mL transferred solution is reflected. Furthermore, the relationship between salinity and the amount of dissolved oxygen is generally negative [44]. High salinity will inhibit growth of aerobic bacteria. Attention should be paid to the supply of oxygen in MEOR applications.


## 4.2. Effects of Dominant Strains on Crude Oil

In the biodegradation test, crude oil viscosity is reduced by 27-51% after 7 days of incubation, but prolonged incubation time may make the strain compete for nutrients and produce toxins that have a counter impact on viscosity (for example, SC4534(2), SC4551, and SC4561). The change of∑C21−/∑C21+ can reveal the priority of the strain for the utilization of n-alkanes [45]. The ∑C21−/∑C21+ of A-3, SC4534(2), SC4561, JSC4535, and SC4551 rise from 1.29 before action to 1.77, 2.04, 1.61, 1.52, and 1.48, respectively, while the value of JSC4501, SC4542, and H-1 decrease to 1.18, 1.03, and 1.17, respectively. The results indicate that the strains A-3, SC4534(2), SC4561, JSC4535, and SC4551 may grow on long-chain alkanes (C>22) preferentially, making increased content of short-chain alkanes (Figure 6), but the content of long-chain alkanes increased after the action of JSC4501, SC4542, and H-1. It is possible that the content increases due to the degradation of short-chain hydrocarbons and the action on cycloalkanes [46].


## 5. Conclusion and Future Work

In the present work, sixBacillus and one Tistrella mobilis were isolated from low-permeability oil fields. They had a great growth rate on crude oil as the sole carbon source and exhibited high stability over a wide range of temperature, salinity, and pH than other strains. By biodegrading the heavier components of the crude oil (long-chain n-alkanes and aromatics) and emulsifying the crude oil through biosurfactants, these eight strains could improve flowability and reduce oil viscosity. The results in the study demonstrated the presence of high-efficiency biosurfactant-producing strains and viscosity-reducing bacteria in low-permeability oilfields. Under the combined effect of emulsification and viscosity reduction, the condition and properties of crude oil were improved, but the culture time of the strains should not be too long and the attention should be paid to the supply of oxygen, which is important for the application of MEOR in low permeability oil fields. Moreover, since the strains screened have different effects on different components in crude oil, the research on the compound use of strains will also be very important.



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*Source: 3223516-2023-02-06.xml*