Optimization of culture medium for anaerobic production of

Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Optimization of culture medium for anaerobic production
of rhamnolipid by recombinant Pseudomonas stutzeri Rhl
for microbial enhanced oil recovery
F. Zhao1,2, M. Mandlaa1,2, J. Hao3, X. Liang1,2, R. Shi1, S. Han1 and Y. Zhang1
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
110016, China.
E-mail: yzhang@iae.ac.cn
2013/2584: received 28 December 2013,
revised 23 March 2014 and accepted 7 April
2014
doi:10.1111/lam.12269
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, KH2PO4 and yeast extract were significant
factors. The proposed optimized medium contained the following: 4655 g l1
glycerol; 3 g l1 NaNO3; 525 g l1 K2HPO43H2O; 571 g l1 KH2PO4;
040 g l1 MgSO47H2O; 013 g l1 CaCl2; 10 g l1 KCl; 10 g l1 NaCl; and
269 g l1 yeast extract. Using this optimized medium, we obtained an
anaerobic yield of rhamnolipid of 312 011 g l1 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; how-
ever, 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, 231--237 © 2014 The Society for Applied Microbiology
231
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 (Sober
on-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 m1).
Glucose (decreased from 5673 to 3343 mN m1),
sucrose (decreased from 5636 to 3443 mN m1) and
molasses (decreased from 5683 to 3803 mN m1) 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). NaNO3 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, NaNO3 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, X1 (glycerol), X4 (KH2PO4) and X9 (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 321 015A 0075B 0063C
þ 0026AB 0081AC þ 0021BC
017A 011B 0097C
2
2
ð1Þ
2
Table 1 The Plackett–Burman design for screening significant variables in anaerobic production of rhamnolipid
Code
Variables (g l1)
Low level (1)
High level (+1)
Effect
Coefficient
t -Value
P -value
X1
X2
X3
X4
X5
X6
X7
X8
X9
Glycerol
NaNO3
K2HPO4.3H2O
KH2PO4
MgSO4.7H2O
CaCl2.2H2O
NaCl
KCl
Yeast extract
20
2
46
35
02
010
08
08
08
35
4
59
45
06
015
12
12
15
023141
005211
002134
019268
005173
020842
015669
008022
009211
011571
002605
001067
009634
002587
010421
007835
004011
004605
933
280
075
518
139
280
421
216
433
0011*
0107
0534
0035*
0299
0107
0052
0164
0049*
*P < 005, 5% significance level.
232
Letters in Applied Microbiology 59, 231--237 © 2014 The Society for Applied Microbiology
F. Zhao et al.
Anaerobic production medium for rhamnolipid
Table 2 Steepest ascent experiment design and response values
Trials
Glycerol
(g l1)
KH2PO4
(g l1)
Yeast
extract
(g l1)
1
2
3
4
5
6
30
35
40
45
50
55
40
45
50
55
60
65
12
16
20
24
28
32
YRHL*
(g l1)
144035
192017
218407
283182
308373
232801
*YRHL 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, KH2PO4 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 R2 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 (KH2PO4) and C (yeast extract)
were 4655, 571 and 269 g l1, respectively. The maximum predicted value of the rhamnolipid yield was
326 g l1.
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.
Table 3
ANOVA
Validation of the model
The model predicted that the maximum anaerobic yield
of rhamnolipid in the optimized medium was 326 g l1.
To validate this prediction, we performed five independent anaerobic fermentations using the optimized medium. The average rhamnolipid yield using the optimized
medium was 312 011 g l1 which was 085-fold
higher than that obtained using the initial medium
(168 g l1). 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,
437 014 g l1 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 l1) 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
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)
B (KH2PO4)
C (yeast extract)
AB
AC
BC
A2
B2
C2
Residual
Lack of fit
Pure error
Cor Total
9
1
1
1
1
1
1
1
1
1
5
3
2
14
044697
0178965
0045417
0031728
0002754
0026224
0001763
0104288
0043134
0034625
0006735
0003761
0002974
0453705
0049663
0178965
0045417
0031728
0002754
0026224
0001763
0104288
0043134
0034625
0001347
0001254
0001487
3686887
1328594
337162
2355402
204462
1946812
1308557
7742059
3202168
2570486
00005*
<00001*
00021*
00047*
02121
00069*
03044
00003*
00024*
00039*
0843246
05826
*P < 005, 5% significance level.
Letters in Applied Microbiology 59, 231--237 © 2014 The Society for Applied Microbiology
233
F. Zhao et al.
Rhamnolipid (g l–1)/
Dry cell weight (g l–1)
(a)
Rhamnolipid yield (g l –1)
3·26
3·1275
2·995
2·8625
2·73
6·70
54·00
6·35
6·00
KH2PO4 (g l –1)
58·00
50·00
5·65
5·30 42·00
46·00
Rhamnolipid yield (g l–1)
3·26
2
3 4 5 6
Time (days)
7
8
9
10
3·1
concentration of 310 g l1 was obtained during the late
stationary phase.
2·94
2·78
2·62
3·40
54·00
3·10
2·80
Yeast extract (g l–1) 2·50
2·20 42·00
46·00
58·00
50·00
Glycerol (g l–1)
(c)
3·26
Rhamnolipid yield (g l–1)
0 0·5 1
3500
3000
2500
2000
1500
1000
500
0
Figure 2 Cell growth, nitrate consumption and rhamnolipid production by Ps. stutzeri Rhl grown in optimized medium: (♦) Concentration
of nitrate (mg l1) at different time points; (□) Dry cell weight (g l1)
at different time points; (▲) Concentration of rhamnolipid (g l1) at
different time points.
Glycerol (g l–1)
(b)
3·165
3·07
2·975
2·88
3·40
6·70
3·10
2·80
2·50
Yeast extract (g l–1)
6·35
6·00
2·20 5·30
5·65
KH2PO4 (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 KH2PO4 while keeping the concentration of yeast
extract constant at centre value of 28 g l1; (b) three-dimensional
plots for concentration of glycerol and yeast extract while keeping the
concentration of KH2PO4 constant at centre value of 60 g l1; (c)
three-dimensional plots for concentration of KH2PO4 and yeast extract
while keeping the concentration of glycerol constant at centre value
of 500 g l1.
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 l1 at 6 days. Rhamnolipid production was initiated at 1 days; the maximum rhamnolipid
234
4·00
3·50
3·00
2·50
2·00
1·50
1·00
0·50
0·00
Nitrate (mg l–1)
Anaerobic production medium for rhamnolipid
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 N2 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 l1, 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 312 011 g l1
rhamnolipid under anaerobic conditions and 157% EOR
Letters in Applied Microbiology 59, 231--237 © 2014 The Society for Applied Microbiology
F. Zhao et al.
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.
Anaerobic production medium for rhamnolipid
zeri Rhl is a facultative anaerobic denitrifying bacterial
strain. In this study, NaNO3 was selected as the optimal
nitrogen source.
Analytical procedures
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 min1 for 16 h.
The initial medium used for anaerobic production of
rhamnolipid contained the following: 30 g l1 glycerol;
25 g l1 NaNO3; 50 g l1 K2HPO43H2O; 40 g l1
KH2PO4; 040 g l1 MgSO47H2O; 013 g l1 CaCl2;
10 g l1 KCl; 10 g l1 NaCl; and 12 g l1 yeast extract.
The pH of the medium was adjusted to 65. This anaerobic medium was then boiled under a stream of oxygenfree N2 for 15 min before it was sterilized in an autoclave
(121°C, 20 min). Afterwards, filter-sterilized 25% (w/v)
Na2S9H2O (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
6 ml Ps. stutzeri Rhl seed culture was inoculated in a
200 ml anaerobic medium and then incubated at 42°C at
80 rev min1 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 NaNO3 is an optimal nitrogen
source for rhamnolipid production. Furthermore, Ps. stut-
The surface tension of the culture supernatant (10 000 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 (10 000 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% H2SO4)
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 (A421) was measured. The rhamnolipid concentrations were calculated from standard curves
prepared with L-rhamnose (0–50 mg l1).
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
(A220) and 275 nm (A275) 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 ml 08%
amino sulphonic acid were added. The correction value is
equal to A220 minus twice A275. Nitrate concentration was
determined from standard curves prepared with NaNO3
(008–4 mg l1).
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, NaNO3, K2HPO4
3H2O, KH2PO4, MgSO47H2O, CaCl2, 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, 231--237 © 2014 The Society for Applied Microbiology
235
Anaerobic production medium for rhamnolipid
F. Zhao et al.
ORE ð%Þ ¼
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 l1), KH2PO4 (53, 60 and 67 g l1),
yeast extract (22, 28 and 34 g l1). 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).
total volume of oil displaced
100
volume of original oil in core
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 ¼ ORE ð%Þ at the end of the second
water flooding ORE ð%Þ at the end
ð4Þ
of bacterial injection
Acknowledgements
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.
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 lm2 and volumes
of 5986, 5971 and 5958 cm3, 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 min1. 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
100 ð2Þ
volume of produced liquid
Oil recovery efficiency (ORE) was calculated using the
following equation:
236
Conflict of Interest
We declare that we have no conflict of interest.
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