Optimization of culture medium for anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl for microbial enhanced oil recovery

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Optimization of culture medium for anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl for microbial enhanced oil recovery F. Zhao 1,2, M. Mandlaa 1,2, J. Hao 3, X. Liang 1,2, R. Shi 1,S.Han 1 and Y. Zhang 1 1 Key Laboratory of Institute of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China 2 University of Chinese Academy of Sciences, Beijing, China 3 The Second Oil Production Factory, Daqing Oilfield Company Limited, Daqing, China Significance and Impact of the Study: The ex situ application of rhamnolipid for microbial enhanced oil recovery (MEOR) is costly and complex in terms of rhamnolipid production, purification and transportation. Compared with ex situ applications, the in situ production of rhamnolipid in anaerobic oil reservoir is more advantageous for MEOR. This study is the first to report the anaerobic production optimization of rhamnolipid. Results showed that the optimized medium enhanced not only the anaerobic production of rhamnolipid but also crude oil recovery. Keywords anaerobic, glycerol, microbial enhanced oil recovery, Pseudomonas stutzeri, response surface methodology, rhamnolipid. Correspondence Ying Zhang, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang , China. yzhang@iae.ac.cn 2013/2584: received 28 December 2013, revised 23 March 2014 and accepted 7 April 2014 doi: /lam Abstract Response surface methodology was employed to enhance the anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl. Glycerol is a promising carbon source used to anaerobically produce rhamnolipid. In a Plackett Burman design, glycerol, KH 2 PO 4 and yeast extract were significant factors. The proposed optimized medium contained the following: 4655 g l 1 glycerol; 3 g l 1 NaNO 3 ; 525 g l 1 K 2 HPO 4 3H 2 O; 571 g l 1 KH 2 PO 4 ; 040 g l 1 MgSO 4 7H 2 O; 013 g l 1 CaCl 2 ;10 gl 1 KCl; 10 gl 1 NaCl; and 269 g l 1 yeast extract. Using this optimized medium, we obtained an anaerobic yield of rhamnolipid of g l 1 with a 085-fold increase. Core flooding test results also revealed that Ps. stutzeri Rhl grown in an optimized medium enhanced the oil recovery efficiency by 157%, which was 66% higher than in the initial medium. Results suggested that the optimized medium is a promising nutrient source that could effectively mobilize oil by enhancing the in situ production of rhamnolipid. Introduction Rhamnolipid is a biodegradable and ecologically safe biosurfactant with low toxicity, high surface activity and low critical micelle concentration compared with chemical surfactants (Desai and Banat 1997; Arutchelvi and Doble 2010). Thus, rhamnolipid can be potentially applied in microbial enhanced oil recovery (MEOR) process (Sen 2008), such as emulsifying crude oil, improving reservoir rock wettability, reducing crude oil viscosity and removing wax. In MEOR, rhamnolipid is generally produced using a bioreactor and then injected into an oil reservoir; however, this process of rhamnolipid production, purification and transportation is costly and complex (Kosaric 1992). Furthermore, aerobic fermentation is limited by oxygen supply rate in the bioreactor and poses risks of severe foaming (Chayabutra et al. 2001). Compared with the previous method, the in situ production of rhamnolipid in oil reservoirs is advantageous for MEOR because of several factors, such as low costs, simple implementation and wide scope (Youssef et al. 2007). Despite these advantages, in situ applications require the production of large quantities of rhamnolipid in anaerobic oil reservoirs (Albino and Nambi 2010). As such, methods that can be used to improve the capability of anaerobic rhamnolipid Letters in Applied Microbiology 59, The Society for Applied Microbiology 231

2 Anaerobic production medium for rhamnolipid F. Zhao et al. production should be developed. For example, a culture medium should be optimized to enhance rhamnolipid production. Studies have focused on the medium optimization of aerobic production of rhamnolipid (Abalos et al. 2002; Chen et al. 2007; Wu et al. 2008), but few studies have reported on the medium optimization of anaerobic production of rhamnolipid. Response surface methodology (RSM) is a collection of different statistical techniques, including designing experiments, building models and evaluating the effects of factors to trigger desirable responses (Li et al. 2002). In this study, RSM was used to optimize the medium composition in which the engineered bacterial strain Ps. stutzeri Rhl s was grown to produce rhamnolipid anaerobically. Core flooding tests were also conducted to evaluate the enhanced oil recovery efficiency (ORE) of Ps. stutzeri Rhl in the optimized medium and in the initial medium. Results and discussion Selection of carbon source and nitrogen source Rhamnolipid is a series of congeners containing one or two rhamnoses attached to different lengths of b-hydroxy fatty acid chains (Soberon-Chavez et al. 2005). Using different carbon sources, micro-organisms may produce different structures and proportions of rhamnolipid congeners with different surface activities. Among the four examined carbon sources, glycerol exhibited an evident effect on surface activity and reached minimum surface tension (decreased from 6340 to 3063 mn m 1 ). Glucose (decreased from 5673 to 3343 mn m 1 ), sucrose (decreased from 5636 to 3443 mn m 1 ) and molasses (decreased from 5683 to 3803 mn m 1 ) also affected surface activity. Moreover, glycerol is soluble in water and can be easily absorbed and metabolized by micro-organisms (Hauser and Karnovsky 1957). Anaerobes grow slower and exhibit lower cell density than aerobes. Therefore, glycerol is a promising carbon source for anaerobic production of rhamnolipid. As a major by-product of biodiesel, glycerol is also a promising and inexpensive carbon source for the production of biosurfactants (Morita et al. 2007; Silva et al. 2010). Anaerobes perform anaerobic respiration and consume oxidants other than oxygen, such as nitrate, sulphate and carbonate. Among these oxidants, nitrate is the most commonly used by facultative anaerobes (Chayabutra et al. 2001). NaNO 3 is also an optimal nitrogen source for rhamnolipid production (Wu et al. 2008). Moreover, Ps. stutzeri Rhl is a facultative anaerobic denitrifying bacterial strain. In this study, NaNO 3 was selected as the nitrogen source to produce rhamnolipid anaerobically. Evaluation of significant variables A 12-run Plackett Burman (PB) design was used to identify and evaluate the most significant variables. The twolevel values of nine variables and the analysis of variance (ANOVA) are shown in Table 1. According to the analysis of P values, X 1 (glycerol), X 4 (KH 2 PO 4 ) and X 9 (yeast extract) were the significant variables for anaerobic production of rhamnolipid (P < 005; Table 1). The contrast coefficients of the significant variables were positive, indicating that these variables positively affected rhamnolipid yield. Optimization of significant variables The steepest ascent experimental results are shown in Table 2. The maximum yield was near the fifth step. The concentrations of significant variables in the fifth step were used as the central point in a Box Behnken design. The following quadratic regression equation was obtained by multiple regression analysis: Y(RHL) ¼ A 0075B 0063C þ 0026AB 0081AC þ 0021BC ð1þ 017A 2 011B C 2 Table 1 The Plackett Burman design for screening significant variables in anaerobic production of rhamnolipid Code Variables Low level ( 1) High level (+1) Effect Coefficient t -Value P -value X 1 Glycerol * X 2 NaNO X 3 K 2 HPO 4.3H 2 O X 4 KH 2 PO * X 5 MgSO 4.7H 2 O X 6 CaCl 2.2H 2 O X 7 NaCl X 8 KCl X 9 Yeast extract * *P < 005, 5% significance level. 232 Letters in Applied Microbiology 59, The Society for Applied Microbiology

3 F. Zhao et al. Anaerobic production medium for rhamnolipid Table 2 Steepest ascent experiment design and response values Trials Glycerol KH 2 PO 4 Yeast extract Y RHL * *Y RHL represented for average rhamnolipid anaerobic yield of triplicate experiments. where Y(RHL) is the predicted rhamnolipid yield, and A, B and C are the coded values of glycerol, KH 2 PO 4 and yeast extract, respectively. ANOVA (Table 3) was conducted to evaluate the statistical significance of Eqn (1). The P values of the model (00005) and the lack of fit (05826) suggested that the obtained experimental data exhibited a good fit with the model. The determination coefficient R 2 was 09851, indicating that the model could explain 9851% of the variability in the rhamnolipid yield. In Eqn (1), the optimal values of A (glycerol), B (KH 2 PO 4 ) and C (yeast extract) were 4655, 571 and 269 g l 1, respectively. The maximum predicted value of the rhamnolipid yield was 326 g l 1. The response surface plot and the corresponding contour plot were used to demonstrate the interactions between two significant variables by keeping the other variables at central point level for anaerobic production of rhamnolipid (Fig. 1). Xu et al. (2008) reported that elliptical contours will be formed when there is a great interaction between the examined significant variables. Validation of the model The model predicted that the maximum anaerobic yield of rhamnolipid in the optimized medium was 326 g l 1. To validate this prediction, we performed five independent anaerobic fermentations using the optimized medium. The average rhamnolipid yield using the optimized medium was g l 1 which was 085-fold higher than that obtained using the initial medium (168 g l 1 ). This excellent correlation between predicted and measured values suggested that the model was accurate and reliable. No rhamnolipid was produced in the control culture without inoculating strain. However, g l 1 of rhamnolipid was produced using the optimized medium under aerobic conditions. Ps. stutzeri Rhl is a facultative anaerobic denitrifying bacterial strain; as such, this strain grows faster (025 days of lag and 05 days of log phases) and exhibits higher cell density (maximum biomass concentration of 564 g l 1 ) under aerobic conditions than anaerobic conditions. Therefore, Ps. stutzeri Rhl has a higher rhamnolipid yield under aerobic conditions in the optimized medium. Studies have shown that RSM designs are used to optimize media for aerobic production of rhamnolipid (Abalos et al. 2002; Chen et al. 2007; Wu et al. 2008). However, studies have yet to be conducted to optimize anaerobic production of rhamnolipid. Time course of rhamnolipid production and nitrate consumption in the optimized medium The change in rhamnolipid production, nitrate consumption and biomass of Ps. stutzeri Rhl grown in anaerobic bottles containing the optimized medium are shown in Fig. 2. Ps. stutzeri Rhl performs anaerobic respiration Table 3 ANOVA for quadratic regression model of RSM Factors Degree of freedom (DF) Sum of square (SS) Mean square (MS) F-value P-value Model * A (glycerol) <00001* B (KH 2 PO 4 ) * C (yeast extract) * AB AC * BC A * B * C * Residual Lack of fit Pure error Cor Total *P < 005, 5% significance level. Letters in Applied Microbiology 59, The Society for Applied Microbiology 233

4 Anaerobic production medium for rhamnolipid F. Zhao et al. (a) Rhamnolipid yield (g l 1 ) (b) Rhamnolipid yield (g l 1 ) (c) Rhamnolipid yield (g l 1 ) KH 2 PO 4 (g l 1 ) Glycerol (g l 1 ) Yeast extract (g l 1 ) Glycerol (g l ) Yeast extract (g l 1 ) KH 2 PO 4 (g l 1 ) Figure 1 Response surface contour plots of rhamnolipids yield for two independent variables. (a) Three-dimensional plots for concentration of glycerol and KH 2 PO 4 while keeping the concentration of yeast extract constant at centre value of 28 gl 1 ; (b) three-dimensional plots for concentration of glycerol and yeast extract while keeping the concentration of KH 2 PO 4 constant at centre value of 60 gl 1 ; (c) three-dimensional plots for concentration of KH 2 PO 4 and yeast extract while keeping the concentration of glycerol constant at centre value of 500 gl 1. using nitrate as an electron acceptor. In Fig. 2, nitrate was constantly consumed during cell growth and rhamnolipid production. An almost parallel relationship was observed among cell growth, rhamnolipid production and nitrate utilization. Using the optimized medium in the anaerobic bottle, we obtained the maximum biomass concentration of 352 g l 1 at 6 days. Rhamnolipid production was initiated at 1 days; the maximum rhamnolipid Rhamnolipid (g l 1 )/ Dry cell weight (g l 1 ) Time (days) Figure 2 Cell growth, nitrate consumption and rhamnolipid production by Ps. stutzeri Rhl grown in optimized medium: ( ) Concentration of nitrate (mg l 1 ) at different time points; ( ) Dry cell weight at different time points; ( ) Concentration of rhamnolipid at different time points. concentration of 310 g l 1 was obtained during the late stationary phase. Enhanced oil recovery evaluated by core flooding tests Core flooding experiments were performed using Daqing Oilfield-injected water and crude oil at similar temperatures in an oil reservoir to evaluate the enhanced oil recovery efficiency (ORE) of the optimized medium. The first water flooding resulted in 575, 606 and 612% of the oil recovered in core A (with the optimized medium), core B (with the initial medium) and core C (contrast), respectively, because of the volumetric sweep efficiency. At the end of the second water flooding, the OREs of cores A, B and C were 732, 697 and 616%, respectively. The engineered bacterial strain Ps. stutzeri Rhl could produce rhamnolipid and N 2 using the medium in cores A and B, which are effective in mobilizing oil in the cores. The enhanced oil recovery (EOR) of Ps. stutzeri Rhl grown in the optimized medium was 157%, which was 66% higher than that in the initial medium. This difference in results may be attributed to the optimized medium enhanced rhamnolipid production in the core. The ex situ application of biosurfactants for MEOR entails high costs of complex bioprocessing techniques and transportation. By comparison, the in situ production of biosurfactants is more advantageous for future MEOR applications. Youssef et al. (2007) demonstrated the in situ production of lipopeptide biosurfactants in oil reservoirs by injecting two Bacillus strains and their nutrients. The average lipopeptide concentration in produced fluids is c. 90 mg l 1, which is approximately nine times of the minimum concentration required to mobilize the entrapped oil from sandstone cores. Using the optimized medium in this study, we found that the recombinant Ps. stutzeri Rhl strain could produce g l 1 rhamnolipid under anaerobic conditions and 157% EOR Nitrate (mg l 1 ) 234 Letters in Applied Microbiology 59, The Society for Applied Microbiology

5 F. Zhao et al. Anaerobic production medium for rhamnolipid in the core flooding model. Therefore, the optimized medium could be a promising nutrient that could effectively mobilize oil in oil reservoirs by enhancing the in situ production of rhamnolipid. Materials and methods Micro-organism and cultivation Few wild-type strains can produce rhamnolipid under anaerobic conditions (Albino and Nambi 2010). In this study, an anaerobic rhamnolipid-producing recombinant strain P. stutzeri Rhl (F. Zhao, R. Shi, Y. Zhang, unpublished data) was used. This strain was constructed by cloning the rhamnosyltransferase gene rhlabri from P. aeruginosa SQ6 (GenBank Accession Number: KF850544) into a facultative anaerobic denitrifying bacterial strain Ps. stutzeri DQ1 (GenBank Accession Number: KF850545), which was isolated from Daqing Oilfield-produced water. A seed culture was incubated at 42 C at 200 rev min 1 for 16 h. The initial medium used for anaerobic production of rhamnolipid contained the following: 30 g l 1 glycerol; 25 gl 1 NaNO 3 ; 50 gl 1 K 2 HPO 4 3H 2 O; 40 gl 1 KH 2 PO 4 ; 040 g l 1 MgSO 4 7H 2 O; 013 g l 1 CaCl 2 ; 10 gl 1 KCl; 10 gl 1 NaCl; and 12 gl 1 yeast extract. The ph of the medium was adjusted to 65. This anaerobic medium was then boiled under a stream of oxygenfree N 2 for 15 min before it was sterilized in an autoclave (121 C, 20 min). Afterwards, filter-sterilized 25% (w/v) Na 2 S9H 2 O (final concentration of 002% (w/v)) was added to remove residual oxygen. Resazurin (final concentration, 00001% (w/v)) was also added to verify whether or not the reduced medium was obtained (Javaheri et al. 1985). The oxidation reduction potential of the culture system could reach 60 mv. The optimization experiments of the medium were conducted in serum bottles (250 ml) sealed with butyl rubber stoppers. A 6ml Ps. stutzeri Rhl seed culture was inoculated in a 200 ml anaerobic medium and then incubated at 42 C at 80 rev min 1 for 8 days. Selection of optimal carbon and nitrogen sources Surface activity is an important parameter for the application of rhamnolipid, particularly in MEOR. Several carbon sources (such as glucose, sucrose, glycerol and molasses) were evaluated to screen the optimal carbon source for high surface activity of rhamnolipid product by using the one-variable-at-a-time strategy. The culture conditions were the same as described above. Wu et al. (2008) reported that NaNO 3 is an optimal nitrogen source for rhamnolipid production. Furthermore, Ps. stutzeri Rhl is a facultative anaerobic denitrifying bacterial strain. In this study, NaNO 3 was selected as the optimal nitrogen source. Analytical procedures The surface tension of the culture supernatant ( g, 10 min) was determined using a BZY-1 automatic surface tension meter (Shanghai Equitable Instruments Factory, Shanghai, China). The amount of rhamnolipid in the culture was quantified by the colorimetric determination of sugars moiety with orcinol (Candrasekaran and Bemiller 1980; Wang et al. 2007). The rhamnolipid culture broth was initially centrifuged ( g, 10 min) to separate the cells from the supernatant. Approximately 05 ml of supernatant was extracted thrice using 1 ml of ether. The upper organic phase was collected and evaporated to dryness; afterwards, 05 ml of distilled water was added. Approximately 45 ml of a solution containing 019% orcinol (in 53% H 2 SO 4 ) was then added to 05 ml of each sample with suitable dilution. After heating for 30 min at 80 C, the samples were cooled at room temperature for 15 min, and the samples absorbance at 421 nm (A 421 ) was measured. The rhamnolipid concentrations were calculated from standard curves prepared with L-rhamnose (0 50 mg l 1 ). Nitrate concentration in the culture was determined using a two-wave length approach (Edwards et al. 2000). In this method, the absorbances of the sample at 220 nm (A 220 ) and 275 nm (A 275 ) were determined to remove organic matter interference. The culture supernatant was diluted 1000 times; approximately 50 ml of the diluted sample was added to a 50 ml colorimetric tube with a stopper. Afterwards, 01 ml of 37% HCl and 01 ml08% amino sulphonic acid were added. The correction value is equal to A 220 minus twice A 275. Nitrate concentration was determined from standard curves prepared with NaNO 3 (008 4 mgl 1 ). Plackett Burman design The Plackett Burman (PB) design was used to screen and evaluate the medium components that significantly influenced anaerobic production of rhamnolipid. In this study, nine variables (including glycerol, NaNO 3, K 2 HPO 4 3H 2 O, KH 2 PO 4, MgSO 4 7H 2 O, CaCl 2, KCl, NaCl and yeast extract) were selected to investigate. Each variable exhibited two levels: 1 for a low level and +1 for a high level (Table 1). The PB experimental design was developed using MINITAB 16.0 (Minitab Inc., State College, PA) with 12 runs. Based on regression analysis, the variables with a significance level of 95% (P < 005) were considered as significant factors. Letters in Applied Microbiology 59, The Society for Applied Microbiology 235

6 Anaerobic production medium for rhamnolipid F. Zhao et al. Response surface methodology Steepest ascent experiments (Table 2) were performed to determine the suitable operating conditions of the significant variables, which are important to effectively simulate the real situation using RSM. A Box Behnken design was used to determine the optimal concentration of the screened variables for enhancing anaerobic production of rhamnolipid. The Box Behnken experimental design (three factors and three levels, including three replicates at the centre point) comprised 15 trials. The three levels of the three variables are described as follows: glycerol (42, 50 and 58 g l 1 ), KH 2 PO 4 (53, 60 and 67 gl 1 ), yeast extract (22, 28 and 34 gl 1 ). The response values (Y) in each trial were the average of triplicates. Experimental design and data analysis were conducted using the software package DESIGN-EXPERT (Version 8.0.5; Stat-Ease Inc., Minneapolis, MN). Core flooding tests The core flooding test was performed according to previously described methods (Xia et al. 2012; Sun et al. 2013) with some modifications. The test was performed at 42 C, which simulated the oil reservoir zone temperature at the Daqing Oilfield. Although cores A, B and C measured 302 mm in length, these cores exhibited pore volumes (PV) of 1124, 1071 and 1054 ml, absolute permeabilities of 0376, 0372 and 0368 lm 2 and volumes of 5986, 5971 and 5958 cm 3, respectively. The cores were saturated with Daqing Oilfield-injected water after vacuum pumping. Each core was saturated with crude oil. The cores were aged at 42 C for 24 h and flooded with Daqing Oilfield-injected water until the water cut in the effluent of cores was higher than 98%. After the first water flooding, 03 PV of culture solution A (Ps. stutzeri Rhl seed culture mixed with the optimized medium (1:20, v/v)) was injected into core A; 03 PV of culture solution B (Ps. stutzeri Rhl seed culture mixed with the initial medium (1:20, v/v)) was injected into core B. The 03 PV of Daqing Oilfield-injected water was injected into core C as a contrast. All of the cores were then incubated at 42 C for 27 days. The cores were flooded again with the same injected water. The flow rate of flooding was set at 02 ml min 1. The amounts of displaced liquid (ml) and water cut (%) in the effluent were measured. Water cut was calculated using the following equation: water cut ð%þ ¼ volume of water volume of produced liquid 100 ð2þ Oil recovery efficiency (ORE) was calculated using the following equation: ORE ð%þ ¼ total volume of oil displaced volume of original oil in core 100 ð3þ where the volume of the original oil in place (ml) is the volume of brine displaced by oil saturation. Therefore, enhanced oil recovery (EOR) was calculated using the following equation: EOR ð%þ ¼ORE ð%þ at the end of the second water flooding ORE ð%þ at the end of bacterial injection Acknowledgements ð4þ This work was financially supported by the National High Technology Research and Development Program of China (No. 2013AA064402) and the Program of China Daqing Oilfield Company Limited. Conflict of Interest We declare that we have no conflict of interest. Reference Abalos, A., Maximo, F., Manresa, M. and Bastida, J. (2002) Utilization of response surface methodology to optimize the culture media for the production of rhamnolipids by Pseudomonas aeruginosa AT10. J Chem Technol Biotechnol 77, Albino, J.D. and Nambi, I.M. (2010) Partial characterization of biosurfactants produced under anaerobic conditions by Pseudomonas sp ANBIOSURF-1. Adv Mater Res 93, Arutchelvi, J. and Doble, M. (2010) Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum contaminated soil. Lett Appl Microbiol 51, Candrasekaran, E.V. and Bemiller, J.N. (1980) Constituent analyses of glycosaminoglycans. In Methods in Carbohydrate Chemistry ed. Whistler, R.L. pp New York: Academic Press. Chayabutra, C., Wu, J. and Ju, L.K. (2001) Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effects of limiting nutrients and carbon substrates. Biotechnol Bioeng 72, Chen, S.Y., Lu, W.B., Wei, Y.H., Chen, W.M. and Chang, J.S. (2007) Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2. Biotechnol Prog 23, Desai, J.D. and Banat, I.M. (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61, Letters in Applied Microbiology 59, The Society for Applied Microbiology

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