Hydrocarbon degradation and bioemulsifier production by thermophilic Geobacillus pallidus strains

Bioresource Technology 102 (2011) 9155–9161

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Hydrocarbon degradation and bioemulsifier production by thermophilic Geobacillus pallidus strains Chenggang Zheng a,⇑, Jianglin He b, Yongli Wang c, Manman Wang a, Zhiyong Huang a,⇑ a

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China Chengdu Institute of Geology and Mineral Resources, Chengdu, Sichuan 610081, China c Lanzhou Institute of Geology, Chinese Academy of Sciences, Lanzhou 730000, China b

a r t i c l e

i n f o

Article history: Received 5 May 2011 Received in revised form 20 June 2011 Accepted 20 June 2011 Available online 29 June 2011 Keywords: Geobacillus pallidus Hydrocarbon Biodegradation Bioemulsifier Emulsifying activity

a b s t r a c t Geobacillus pallidus XS2 and XS3 were isolated from oil contaminated soil samples in Yumen oilfield, China, and were able to produce bioemulsifiers on different hydrocarbons. Biodegradation assays exhibited that approximately 70% of PAH (250 mg/L) or 85% of crude oil (500 mg/L) was removed by the thermophilic bacteria after 20 days. The bioemulsifiers of the two strains were isolated and obtained a productive yield of 4.24 ± 0.08 and 3.82 ± 0.11 g/L, respectively. GPC analysis revealed that the number-average molecular weights (Mn) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. Chemical composition studies exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). Emulsification assays approved the effectiveness of bioemulsifiers over a wide range of temperature, pH and salinity. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Petroleum is a complex mixture of hydrocarbons and other organic compounds, which has been recognized as substrates supporting microbial growth. A wide range of studies have dealt with biotransformation, biodegradation, and bioremediation of petroleum hydrocarbons and microbial enhanced oil recovery (MEOR), and interest in exploiting petroleum-degrading organisms for biotechnology application has become central to petroleum microbiology (Atlas and Cerniglia, 1995; Prince and Clark, 2004). Numerous studies have been implemented on the metabolic pathways, both aerobic and anaerobic, for alkane, cycloalkane, and aromatic and polycyclic aromatic hydrocarbon (PAH) biodegradation (Li et al., 2010). Biodegradation by microorganisms modifies waxy crude oils and facilitates the mobility of the heavy oil, but conditions for reservoir applications require the use of thermophiles, resistant to organic solvents, with proper metabolites and reduced oxygen requirements (Annweiler et al., 2000). Thermophilic hydrocarbon degraders, predominantly Geobacilli, are capable of degrading alkanes when the temperature is raised above 40 °C under aerobic and/or facultative anaerobic conditions. These properties of Geobacilli have been of special interest not only in MEOR, but also in the bioremediation of polluted soils mainly in naturally hot environments (Perfumo et al., 2007) or in composting as a bioremediation process (Beaudin et al., 1999).

Hydrocarbon-degrading microorganisms having co-existing capacity to produce bio-surface-active agent (biosurfactant) can effectively be used for speedy metabolic activity (Kumar et al., 2006). Biosurfactants are a diverse group of surface-active chemical compounds produced by a wide variety of microorganisms and can be divided into two categories: low-mass molecules that act to lower surface and interfacial tensions and high-molecularmass polymers, also known as bioemulsifier, that are more effective in stabilizing oil–water emulsions without remarkable surface tension reduction (Batista et al., 2006). Their superior properties, such as low toxicity and high biodegradability, are undoubtedly environmentally acceptable and make them good candidates for enhanced oil recovery and bioremediation besides various other industrial applications (Banat et al., 2000; Batista et al., 2006). Two thermophilic hydrocarbon-degrading and bioemulsifierproducing bacteria were isolated from crude oil contaminated soil samples in Yumen oilfield of China and identified as Geobacillus pallidus. Both of the two bacteria were able to grow on various hydrocarbons and produce bioemulsifiers. In the present study, we studied the hydrocarbon degradation patterns of the two bacteria as well as the chemical composition and emulsifying properties of their bioemulsifiers. 2. Methods 2.1. Isolation and identification of thermophilic bacteria

⇑ Corresponding authors. E-mail addresses: [email protected] (C. Zheng), [email protected] (Z. Huang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.074

Soil samples and crude oil were collected from Yumen Oilfield, China. The strains were isolated by the enrichment culture

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technique (Kumar et al., 2006). Briefly, a 5-g sample of soil was inoculated into 100 ml of basal salt medium (BSM) containing (g/L) Na2HPO4 6.0, KH2PO4 3.0, NH4Cl 4.0, yeast extract 2.5, MgSO4 0.1, trace element solution 2.5 ml. The trace element solution was prepared as described previously (Yakimov et al., 1995). Crude oil (0.050%, w/v, or PAH, 0.025% w/v) was used as carbon source and incubated at 60 °C on a rotary shaker at 200 rpm for 7 days. After 7 days, 1 ml of the culture was transferred to fresh medium containing crude oil and re-incubated for another 7 days. Then the culture was diluted and plated on BSM agar plates containing crude oil as sole carbon source. The microbial colonies obtained were further purified by streaking on Luria Bertani (LB) agar and the strains with ability to grow on a wide range of hydrocarbons over a wide range of temperature and salinity conditions were selected for further study. The strains were stored at 70 °C in BSM mixed with sterile glycerol at a final concentration of the 25% (v/v). The strains were identified by partial 16S rDNA sequencing. The 16S rDNA gene was amplified by PCR using primers corresponding to Escherichia coli positions 27F and 1387R (27f, 50 -AGA GTT TGA TYM TGG CTC AG-30 ; 1387r, 50 -GGG CGG WGT GTA CAA GGC-30 ) (Dang and Lovell, 2000). PCR amplification was performed in a total volume of 100 ll. Each PCR mixture contained 1 ll template DNA, 10 ll Ex Taq reaction buffer, 100 lM of each dNTP, 2.5 U of Ex Taq DNA polymerase and 1 ll of each primer. The amplified 16S rDNA gene was constructed to the pMD 19-T vector and transformed into Escherichia coli BL21. The plasmid DNA was isolated from positive clones identified by blue–white spot screening and the 16S rDNA gene insert was sequenced by Takara Bio Inc. in Dalian, China. The resulting sequence was compared with sequences in the GenBank database of NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) using the nucleotide–nucleotide blast (BLASTn) network service. Resulting tree was displayed with Mega 4.0 which was calculated by using the neighbor-joining method. The phenotypic characteristics of the two isolates were determined according to the ‘‘Bergey’s Manual of Determinative Bacteriology, 9th edition’’ (Holt et al., 1994).

2.2. Cell growth and hydrocarbon degradation Quantitative degradation of representative PAH (phenanthrene, PHE and fluorene, FLU) and crude oil was studied. The strains were grown in batch culture in a 250-ml flask containing 100 ml of BSM supplemented with PAH or crude oil. The experimental flasks were inoculated with 2% (v/v) inoculum prepared with LB medium and were incubated in dark on a rotary shaker at 200 rpm for 20 days. At time intervals (2 or 5 days), samples were withdrawn for the measurements of dry cell growth (biomass) and hydrocarbon degradation. The culture was centrifuged at 12,000 rpm and 4 °C for 30 min. The cells were collected and washed twice with distilled water to remove the residual carbon source. Then the cells were dried in an oven at 110 °C to constant weight and the biomass was determined. The remaining culture broth was used for residual PAH or crude oil quantification. PHE, FLU and crude oil from the culture medium were monitored spectrophotometrically according to methods described previously (Khan et al., 2001; Miyata et al., 2004; Zhao et al., 2011). A boiled treatment control flask served as controls.

(30 m  0.2 mm  0.2 lm). Split injections were conducted using nitrogen as carrier gas. The column temperature increased from 50 °C to 310 °C at a rate of 8 °C per minute. An interface temperature of 310 °C and an ion source temperature of 320 °C were utilized. Individual components present in the n-alkane fraction were determined by matching the retention time with authentic standards. 2.4. Isolation and characterization of bioemulsifier After the incubation, the culture was centrifuged at 8000 rpm for 30 min at 4 °C. The residual hydrocarbon was carefully removed by extraction and the supernatant obtained was precipitated with three volumes of cold methanol for the isolation of bioemulsifier. The precipitated bioemulsifier was re-dissolved in distilled water (1.0%, w/v), dialyzed against distilled water for 24 h, lyophilized and then weighed (Joshi et al., 2008). Surface tension of the bioemulsifier was measured by ring Du Nuoy method using a K100 tensionmeter (Kruss, Germany) at room temperature (Lin et al., 1994). The number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersity index (PDI) of the bioemulsifier were measured by gel permeation chromatography (GPC) using pullulan standards as described previously (Cao et al., 2010). Total protein content of the bioemulsifier was determined according to the method described by Lowry et al. (1977). Total carbohydrate content of the bioemulsifier was determined according to the phenol–sulfuric acid method (Dastgheib et al., 2008). Lipid was analyzed by diethyl ether extraction (Kuyukina et al., 2001).

2.5. Emulsification assays The emulsification assays were performed as described previously (Cooper and Goldenberg, 1987). Emulsification index (E24) was employed to quantify the emulsifying activity and the values of E24 were determined in the following procedures. A total of 5 ml culture supernatant or bioemulsifier solution was poured into a test tube containing 5 ml of n-hexadecane. The mixture was vortexed for 5 min and then allowed to settle at room temperature for 24 h. Emulsification index (E24) was calculated as the ratio of volume of emulsified zone to total liquid volume. The hydrocarbon compounds used in the emulsification assays also included the following: n-hexane, n-octane, n-hexadecane, liquid paraffin, toluene, xylene and crude oil. Stability study was conducted to investigate the effect of several environmental parameters on emulsifying activity of the bioemulsifier. For the thermal stability study, the culture supernatant was incubated in a water bath for 1 h from 30 to 100 °C and allowed to cool to the room temperature. For studying pH stability, pH of the culture supernatant was adjusted to different values (2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0) using 1.0 N NaOH or 1.0 N HCl. Then the emulsification index was measured. To determine the effect of salinity (NaCl) on the emulsifying activity, different concentrations of NaCl were added (1–30%, w/v) to culture supernatant and mixed until complete dissolution was achieved. The emulsification activity of each treatment was assessed as described above, with n-hexadecane used as the substrate.

2.6. Statistical analyses 2.3. Analysis of n-alkane fraction in crude oil by gas chromatography The n-alkane fraction of crude oil after biodegradation was analyzed by gas chromatography (GC) using a FID detector (HP 6890), equipped with a PONA quartz capillary column

Analyses were performed in triplicate samples and the mean values with standard error were presented. The data were subjected to one-way analysis of variance (ANOVA) and Duncan’s multiple range tests using Microsoft Excel software.

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A 2.0

3.1. Isolation and identification of thermophilic bacteria

250 1.5 200 150

1.0

100 0.5 50 0

0.0 0

5

15

20

25

300 250

1.5 200 1.0

150 100

0.5 50 0.0

Remaining fluorene, mg/L

B 2.0

0 0

5

3.2. Degradation pattern of PAHs

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15

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25

Cultivation time, d

C 2.0

600 500

1.5 400 300

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Remaining crudeoil, mg/L

Dry biomass, g/L

Cell growth and PAHs degradation were verified by determining the biomass and a decrease in PAH concentration (Fig. 2). The boiled treatment control flasks served as controls and no significant decrease in PAH concentration was observed in the control flasks after 20 days. Both of the two strains exhibited the similar degradation kinetics of PAH (PHE and FLU). During the cultivation, rapid cell growth and a sharp decrease of PAH were monitored in the initial 6 days. After 20 days, approximately 76.58 ± 0.63% of initial amount of PHE was degraded by Geobacillus pallidus XS2 while the degradation was around 71.20 ± 3.1% for Geobacillus pallidus XS3 (Fig. 2A). The removal efficiency of FLU (20 d) for the two thermophilic bacteria were 68.52 ± 1.85% and 71.48 ± 4.07%, respectively (Fig. 2B). The experimental conditions for PAH degradation varied in different literature that PAH concentration ranging from 200 to 500 mg/L was employed for assays (Churchill et al., 1999; Sarma et al., 2004). The PAH degradation in the present study was comparable to that performed by B. subtilis BUM and P. aeruginosa P-CG3 (250 mg/L, approximately 70% degradation rate in 30 days) with the supplement of rhamnolipids (Zhao et al., 2011).

10

Cultivation time, d

Dry biomass, g/L

Yumen Oilfield is the earliest oilfield in China and has been exploited for more than 100 years. In order to isolate the hydrocarbon-degrading bacteria, soil samples were collected from Yumen Oilfield, China. The soil samples were obtained in the sites which were heavily contaminated with hydrocarbons for decades. A total of 12 strains were isolated with the ability to degrade crude oil. The strains XS2 and XS3 were selected for further study for their ability to grow on different hydrocarbons over a wide range of temperature and produce extracellular surface active compound(s). Taxonomic identification of the strains XS2 and XS3, was performed by amplification and sequencing of the 16S rDNA sequences. The strains XS2 and XS3 were found to be 100% homology with each other and the 16S rDNA sequence of the strain XS2 was deposited in the GenBank database under accession number HQ637544. The 16S rDNA sequence analysis revealed that the strains XS2 and XS3 had 100% homology with Geobacillus pallidus strains FN562406, EU935594 and AB543491 (Fig. 1). The phenotypic characteristics were assayed and both of two strains were gram positive, spore forming, with aerobic/anaerobic activities, and with rod cells. These features confirmed strains XS2 and XS3 as Geobacillus pallidus.

300

Remaining phenanthrene, mg/L

3. Results and discussion

Dry biomass, g/L

Fig. 1. Phylogenetic neighbor joining tree obtained with the 16S rDNA sequences of strains XS2 and XS3.

0

0.0 0

5

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Cultivatin time, d Fig. 2. Cell growth and hydrocarbon degradation of strains XS2 and XS3 grown on BSM.

However, no exogenous biosurfactants were needed for enhancement of PAH degradation by Geobacillus pallidus XS2 and XS3.

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3.3. Degradation pattern of crude oil After cultivation on crude oil (500 mg/L) for 20 days, the oil remaining in the BSM was extracted using n-hexane. The

degradation rates of hydrocarbon by Geobacillus pallidus XS2 and XS3 were 87.32 ± 0.74% and 86.40 ± 2.21%, respectively (Fig. 3C). The biodegradation rates of crude oil (a complex mixture of aliphatic and aromatic hydrocarbons) were approximate 10–15%

Fig. 3. GC-FID analysis of crude oil (Yumen) after biodegradation by Geobacillus pallidus XS2 and XS3.

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higher than those of PAH, which indicated the PAHs were more refractory to degradation. It had been reported that the supplementation with limiting nutrients and/or addition of biosurfactants could contribute to hydrocarbon degradation resulting in a maximum of 90% removal of n-hexadecane by thermophilic Geobacilli within 40 days (Perfumo et al., 2007). A detailed GC-FID study was therefore conducted to investigate the variation of aliphatic component after biodegradation by the two thermophilic strains. It was observed in the GC chromatograms in Fig. 3 that Geobacillus pallidus XS2 reduced n-alkanes in the range n-C8 to nC18 when compared to the control whereas an increase of n-alkanes ranging from n-C20 to n-C38 was monitored for Geobacillus pallidus XS3. To investigate the preferences in n-alkanes degradation, the area of each of the normal n-alkanes was normalized to the area of the n-C19 (Fig. 4). The normalized distributions in bar charts resulted in ratio of n-C21/n-C22+ from 1.74 in the control to 1.51 and 0.66 for strains XS2 and XS3, respectively. The n-alkane variation indicated that the strain XS3 consumed more short-chain n-alkanes than the long-chain ones, compared with XS2, though the two strains exhibited quite similar biodegradation rates of crude oil. A wide range of n-alkanes from n-C5 to n-C38 could be utilized as the growth substrates for thermophilic Geobacilli as described previously (Marchant et al., 2006). However, there was not even a clear regulation between the use of short-chain and longerchain n-alkanes (Marchant and Banat, 2010).

Table 1 Characterization of the bioemulsifiers synthesized by strains XS2 and XS3.

Bioemulsifier yield, g/L Surface tension, mN/m E24, %a Mn, Da Mw, Da PDI Carbohydrate, %-mass Lipid, %-mass Protein, %-mass a

Bioemulsifier XS3

4.24 ± 0.08 42.5 ± 0.1 61 ± 0.5 271,785 362,013 1.104 68.59 22.66 8.75

3.82 ± 0.11 45.7 ± 0.1 55 ± 0.5 526,369 540,468 1.027 41.1 47.6 11.3

Against n-hexadecane.

the E24 values of 61 ± 0.5% and 55 ± 0.5%, respectively. As shown in Table 1, the number-average molecular weights (Mn) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. The result again confirmed the high molecular characteristic of the bioemulsifiers (Batista et al., 2006). Further studies on chemical composition of the bioemulsifiers were conducted and exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). The hydrophobic portion (lipid) always attached to the polysaccharide backbone (hydrophilic portion) and provided the amphiphilic structure common to surface-active agents (Lukondeh et al., 2003). The bioemulsifiers with similar composition were broadly reported previously (Luna-Velasco et al., 2007; Singh et al., 2011). Emulsification index was employed to investigate the emulsifying properties of the bioemulsifiers. Most microbial surface active compounds are substrate-specific and emulsify different hydrocarbons at different rates (Luna-Velasco et al., 2007). The bioemulsifier specificity (solution 0.4%, w/v) was tested with different hydrophobic substrates (Table 2). n-Hexane, toluene and xylene were the most well-emulsified substrates, while crude oil was the poorest for both of the two bioemulsifiers. The value of E24 was found to decrease with the increase of carbon numbers for the aliphatic hydrocarbons and liquid paraffin, a complex mixture of long chain n-alkanes, exhibited a restrictive emulsifying activity. An important property of bioemulsifiers is their effectiveness over a wide range of temperature, pH and salinity (Banat et al., 2000). Fig. 5 presents the effect of temperature, pH and salinity on emulsifying activity of bioemulsifiers in stability study. The temperatures of 30–60 °C did not affect on emulsifying activity. A slight decrease was detected with the treatment higher than 70 °C. Previous studies have proved that the high molecular bioemulsifiers were thermostable (Nitschke and Pastore, 2006). The bioemulsifi-

3.4. Characterization and emulsifying properties of the bioemulsifiers Geobacillus pallidus XS2 and XS3 were grown on BSM supplemented with different hydrocarbons. After the cultivation, the supernatants were harvested and employed for surface tension and emulsification index measurements. Neither of the strain could reduce the surface tension to the value below 40 mN/m when grew on different hydrocarbons (Data not shown). However, significant emulsifying activity was monitored that the E24 achieved the value more than 55% for both of the two strains. It was reported that low-molecular weight biosurfactants are able to reduce the surface tension below 40 mN/m (Mulligan, 2005) while the high molecular bioemulsifiers can form and stabilize emulsions without remarkable surface tension reduction (Batista et al., 2006). Therefore, it can be inferred that the bio-surface active compounds synthesized by the two thermophilic strains belonged to bioemulsifiers. Hence, the bioemulsifiers of bacteria were harvested from the BSM culture as whitish powders, denominated as bioemulsifier XS2 and XS3, and achieved productive yields of 4.24 ± 0.08 and 3.82 ± 0.11 g/L, respectively. As shown in Table 1, bioemulsifier XS2 and XS3 could reduce the surface tension from 70.5 ± 0.1 mN/m to 42.5 ± 0.1 and 45.7 ± 0.1 mN/m, and exhibited

1.5 1.2 0.9 0.6 0.3

n-Alkanes Blank

XS2

XS3

Fig. 4. Relative distribution of n-alkanes: normalized concentration relative to n-C19.

n-C38

n-C36

n-C34

n-C32

n-C30

n-C28

n-C26

n-C24

n-C22

n-C20

n-C18

n-C16

n-C14

n-C12

n-C10

0 n-C8

Normalised to nC19 area

Bioemulsifier XS2

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Table 2 Emulsification index of bioemulsifier with different substrates. Emulsification index, %

n-Hexane n-Octane n-Hexadecane Liquid paraffin Toluene Xylene Crude oil

Bioemulsifier XS2

Bioemulsifier XS3

74 ± 1 72 ± 0.5 61 ± 0.5 50 ± 0 77 ± 1 76 ± 2 21 ± 2

71 ± 1 66 ± 0.5 55 ± 0.5 49 ± 1 74 ± 0 74 ± 0 21 ± 1

Emulsification Index %

A 70 60 50 40 30 20 10

emulsifiers by Geobacillus pallidus XS2 and XS3 showed a better halotolerance, remaining active under the salinity up to 20%. Many species from the genus Geobacillus, as recently described, have come from oil-rich environments and their capability has been well documented. However, the hydrocarbon degrading activity by Geobacillus pallidus had not been reported until 2006 (Mohamed et al., 2006) and this study has characterized hydrocarbon degradation patterns of thermophilic strains XS2 and XS3. Previous review reported no biosurfactant-producing species of Geobacillus and none of the strains showed any evidence of surface active components (Marchant and Banat, 2010). To our knowledge, this is the first report of the bioemulsifier production by Geobacillus pallidus. The production of surface active compounds by bacteria is often viewed as aiding hydrocarbon degradation and enhancing the solubility of hydrophobic substrates, which can be used broadly in biotechnology applications. Based on the findings in the present study, including the hydrocarbon-degrading and bioemulsifier-producing properties, the thermophilic bacteria exhibited a promising potential for applications within extreme environmental conditions, such as the bioremediation and MEOR (Banat et al., 2000; Batista et al., 2006). 4. Conclusion

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Emulsification Index %

B 70 60 50 40 30 20 10

Acknowledgements

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This work was supported by Project KZCX2-YW-Q05-05, Program of Knowledge Innovation, Chinese Academy of Sciences.

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C 70 Emulsification Index %

Two thermophilic bacteria were isolated from crude oil contaminated soil samples in Yumen oilfield of China and identified as Geobacillus pallidus. Both of the bacteria were able to grow on various hydrocarbons and produce bioemulsifiers. Significant hydrocarbon degradation was monitored for Geobacillus pallidus XS2 and XS3 grown on PAHs or crude oil. The high-molecular bioemulsifiers were isolated and identified as a complex of carbohydrates, lipids and proteins. The emulsifying properties of the bioemulsifiers approved the effectiveness over a wide range of temperature, pH and salinity, which were considered to be ideal candidates for industrial applications.

References

60 50 40 30 20 10 0 1

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Bioemulsifier XS2 Bioemulsifier XS3 Fig. 5. Effect of temperature, pH and salinity on emulsification activity.

ers exhibited the highest activity at pH 8–12 and the activity was significantly inhibited under the acid conditions. On the contrary, the bioemulsifiers produced by fungi were stable under acidic condition (Luna-Velasco et al., 2007). Previous literature reported that the emulsifying activity was significantly inhibited at a NaCl concentration greater than 5% (Ilori et al., 2005), whereas the bio-

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