Separation of Rhamnolipid Biosurfactants
Jennifer Lilly
Department of Chemical & Biomolecular Engineering
Honors Research Project
4200:497
Submitted on April 22, 2011 to
The Honors College
Approved:
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Table of Contents
Executive Summary .............................................................................................................................. 3
Introduction ........................................................................................................................................... 5
Background ........................................................................................................................................... 5
Experimental Methods ......................................................................................................................... 8
Results ................................................................................................................................................... 9
Discussion and Analysis..................................................................................................................... 16
Conclusions ......................................................................................................................................... 18
Acknowledgements ............................................................................................................................ 18
Literature Cited ................................................................................................................................... 18
Appendix A: Calibration Curves ...................................................................................................... 20
Appendix B: Raw Rhamnolipid Concentration Data ...................................................................... 23
Appendix C: Glycerol HPLC Data ................................................................................................... 25
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Executive Summary
During the last few decades, a growing interest in biological surfactants has led to an
increase in research for cost-efficient production of biosurfactants compared to traditional
synthetic surfactants. Biosurfactants offer the benefit of high biodegradability and they are
produced from cheap hydrophobic and hydrophilic substrates. There are many potential
industrial applications for these biosurfactants, but the high costs of separation and purification
have prevented their use. Some of the more commonly studied biosurfactants are the
rhamnolipids, which are produced by the bacterium called Pseudomonas aeruginosa. The
purpose of this project is to select and evaluate methods to separate rhamnolipid biosurfactants
from fermentation broth.
There are several means of separation that can be found in biosurfactant literature:
solvent extraction, adsorption, ion-exchange chromatography, centrifugation, and membrane
filtration. Solvent extraction is a practical approach to separation, but a cleaner, less costly
method is still desired. Because adsorption and ion-exchange chromatography are low capacity
separation methods, they are not practical for large-scale processing. Centrifugation and
membrane filtration are better options, but there are still some limitations due to the high
viscosity of the fermentation broth.
Centrifugation on a large scale would require too high an acceleration for separation to be
practical in industry with such a viscous broth. The problem with membrane filtration is due to
the low critical micelle concentration of rhamnolipids. The highly concentrated rhamnolipids
above the critical micelle concentration and other broth ingredients aggregate and form micelles
too large to fit through the pores of the ultrafiltration membrane, which makes for a timeconsuming separation. The first step to solving this problem was to adjust the bulk phase
properties of the broth without significantly diluting it. This was attempted by varying the
temperature and pH of the broth prior to separation.
Temperature was varied and appeared to have no effect on the broth separation. On the
other hand, pH had a considerable effect on the phase behavior of the rhamnolipid broth. Once
the broth viscosity has been reduced via acidification and the samples were centrifuged, the
rhamnolipid concentration of each phase was evaluated. The evaluation was carried out using a
colorimetric method known as the anthrone analysis (see Experimental Methods section for a
detailed procedure). Filtration was still difficult after the rhamnolipid broth viscosity had been
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adjusted using hydrochloric acid. As dilution would be necessary for the broth to pass through a
filtration membrane, it was ruled out as a separation option in comparison to centrifugation.
It can be concluded that acidification to a pH of 2.0-3.0 and centrifugation of rhamnolipid
broth results in 50% of the rhamnolipid mass being present in the bottom liquid layer and 50% of
the rhamnolipid present in the solid phase. An interesting observation during water washing the
solid phase, was the effect of pH on the rhamnolipid distribution between the solid re-dissolved
in the sodium bicarbonate solution and the water wash. With a pH of 1.80 after acidification,
significantly more rhamnolipid was found in the water wash than in the re-dissolved solid phase
(9.95 g/L in solid phase vs. 150.36 g/L in wash); whereas a pH of 2.37 after acidification resulted
in more rhamnolipid in the solid phase than water wash (44.62 g/L in solid phase vs. 79.30 g/L in
wash). Several species of rhamnolipid were present in the culture broth investigated in this
study. Though a total of seven species were identified in this broth, HPLC/MS revealed that the
only species of rhamnolipid which appeared to selectively remain in the solid phase for all
samples was R-R-C8 -C8 . All other species demonstrated no selectivity between layers.
This research has taught me the basics of biochemistry, how to design an experiment, and
how to statistically analyze data as a result from working on this project. I have also learned
how to use several new analytical tools, including HPLC, mass spectroscopy, and
spectrophotometry for colorimetric methods. In addition to the technical skills obtained with this
research, I have gained confidence as an engineer by working independently and utilizing my
creativity on a project with such a wide scope. Though a cost-efficient separation method has
yet to be determined, the phase behavior of rhamnolipids observed in this study will prove
helpful in finding a cost-effective method of separation. This research will bring biosurfactants
one step closer to being applied in industry. Once these biosurfactants become mainstream, they
will have replaced synthetic surfactants and lessened our impact on the environment.
Future work related to this project includes looking in to separation methods other than
extraction and evaluating different solvents to be used for crystallization as a method of
rhamnolipid purification. The uses of polymers or activated carbon are both potential means of
rhamnolipid separation which should be investigated. A variety of solvents, such as hexane or
ethyl acetate, should be studied as potential crystallization solvents and solubility curves for
rhamnolipids in these solvents should be produced.
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Introduction
Synthetic surfactants are used in a variety of applications, including detergents, cosmetics
and personal hygiene, food, agriculture, paper and pulp, textiles, and in the oil industry. Though
these surfactants have been used in these industries for years, they have brought about
environmental concerns due to their aquatic toxicity and slow rate of degradation. As part of the
recent green initiative, which is still gaining momentum, much research is being carried out in an
attempt to replace these petrochemical products with ones derived from renewable resources.
Biosurfactants, which are produced by microorganisms, have the potential to replace
synthetic surfactants if the production process could be made economically competitive to that of
synthetic surfactants. They offer the benefits of high biodegradability and they are produced
from cheap hydrophobic and hydrophilic substrates, such as hydrocarbons, vegetable oils,
carbohydrates, or even waste from the food industry (Costa et al. 2008). Rhamnolipids, which
are produced by bacteria called Pseudomonas aeruginosa, are one of the most hopeful
biosurfactants being studied. These biosurfactants solubilize or emulsify hydrocarbons and
facilitate penetration through hydrophilic cell walls for degradation by enzymes. In spite of the
biodegradability and wide range of operational conditions of rhamnolipids, the high cost of
separation of the final product have kept this biosurfactant from being used on a large scale.
While several strategies have been implemented to reduce the production cost of
rhamnolipid biosurfactants, the scope of this study focuses solely on the reduction of separation
and purification cost. The objective of this project is to evaluate and develop methods for
separation, collection, and purification of the rhamnolipid from its fermentation broth.
Parameters such as temperature and pH are varied to alter the bulk phase properties of the broth,
which facilitates the separation process. Furthermore, the solubility of rhamnolipid in several
solvents is investigated for the purpose of crystallization as a means of purification.
Background
Microbial Surfactants
Microbial surfactants are surface-active biomolecules which are produced by a variety of
microorganisms. These surfactants have gained importance in various industries due to their
unique properties, such as high biodegradability, lower toxicity, and effectiveness at extremes of
temperature, pH, and salinity (Mukherjee et al. 2006). Although the exact mechanism is not
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certain, it is also known that biosurfactants enhance cellular uptake and use of substrates with
low degrees of aqueous solubility (Mata-Sandoval et al. 1999). When associated with the cell,
microbial surfactants promote transport across the membranes and as extracellular compounds
they help to solubilize the substrate. The production of every microbial metabolite is governed
by initial raw material costs, availability of suitable and economic production and recovery
procedure, and the product yield of the producer microorganisms. There are three areas of focus
in striving to make the biosurfactant production process economically attractive (Mukherjee et
al. 2006). There is research being carried out to find cheap or waste substrates to lower the
initial raw material costs associated with this process. Another area of study is the development
of efficient bioprocesses, optimizing the culture conditions and cost-effective separation
processes for maximum biosurfactant production and recovery. The last area of research is the
development of overproducing mutant or recombinant strains for enhanced biosurfactant yield.
Rhamnolipid
Rhamnolipid is a type of microbial surfactant produced by bacteria called Pseudomonas
aeruginosa and can be characterized by its viscous, sticky yellowish-brown appearance and
fruity odor (Abdel-Mawgoud et al. 2008). This surfactant is composed of a rhamnose ring and a
fatty acid chain, as can be seen in Figure 1. Rhamnolipids are typically composed of a mixture
of several species of rhamnolipid, such as R-C10 -C10, R-C10 , R-R-C10 -C10, R-R-C10 (MataSandoval et al. 1999). The produced surfactant can be used in the crude form either as cell-free
or cell-containing culture broth of the grown bacteria. Rhamnolipid is a potential candidate for
use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry.
Monorhamnolipid (RhC10C10)
Dirhamnolipids (Rh2C10Cn), n= 8, 10, 12
Figure 1. Structures of rhamnolipid produced from P. aeruginosa (Mata-Sandoval 1999)
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Rhamnolipid Separation
The work which inspired the separation technique used in this study was US Patent
5,656,747. This patent outlined a process for quantitatively purifying glycolipids comprising of
acidification of the rhamnolipid broth to a pH less than or equal to 5.0, heating to 60-130ºC,
cooling to less than or equal to 50ºC, and centrifugation to separate the glycolipid-containing
phase (Mixich et al. 1993). The patent indicated that the rhamnolipid broth would separate into
two distinct phases upon centrifugation, yet three phases were found in this study. According to
Mixich, the rhamnolipid was all contained in the bottom phase, whereas the rhamnolipid was
split 50:50 between the bottom liquid phase and solid phase in this study. It was also stated in
the patent that this process would not work without the heating step after acidification, which
was found to be false during the temperature variation portion of this project.
Rhamnolipid Measurement and Characterization
One type of analytical method used for measuring glycolipid concentration is the
colorimetric method carried out with an assay. An anthrone assay was used for rhamnolipid
measurement in this study, which is a common method for estimating sugar moiety in
glycolipids (Sarachat et al. 2010). The anthrone reaction produces different shades of green
depending on rhamnolipid concentration and the UV/Vis spectrophotometer is used to measure
absorbance. A calibration curve using standard rhamnose concentrations was used to correlate
absorbance to rhamnose concentration. See the Experimental Methods section for details.
Similar colorimetric methods utilizing carbazole (Knutson et al. 1968) or m-hydroxydiphenyl
(Blumenkrantz et al. 1973) reactions can be used to measure rhamnolipid concentration, but were
not explored in this study.
High-performance liquid chromatography and mass spectroscopy are both common
methods for rhamnolipid moiety characterization (Chayabutra and Ju 2001). Chayabutra and Ju
determined the optimal tuning parameters and fragmentation energy for rhamnolipid
characterization by infusing the samples at a rate of 1 μL/min through a standard C18 column
with a THF/acetonitrile mobile phase. Similar operating conditions were used in this study, but a
mobile phase of ammonium acetate/acetonitrile was used instead of THF/acetonitrile. See the
Experimental Methods section for details. Fragment ion numbers used to identify species of
rhamnolipid were obtained from M. Heyd et al. (Heyd et al. 2008).
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Experimental Methods
Rhamnolipid Separation Technique
For all trials, a variation of the same basic separation method was used, which includes
acidification, centrifugation, and decantation. The fermentation broth used remained constant
throughout the study. The Pseudomonas aeruginosa broth was prepared using glycerol and
vegetable oil as a carbon source. The rhamnolipid concentration of the fermentation broth was
around 50 g/L and remained in the same refrigerator throughout the study. Each separation trial
began with 30 minutes of centrifugation at 7,000 rpm at room temperature to pack the dead cells
at the bottom of the centrifugation tube. Next, the pH was adjusted to 2-3 using 6 N
hydrochloric acid. The tubes were centrifuged again at 7,000 rpm for 30 minutes. At this point,
the broth had separated into three distinct phases, two which were liquid and one which was
solid. The top liquid phase was always lighter in color and had better clarity than the darker
bottom liquid layer. The layers were separated into separate vials via pipette and stored in the
refrigerator for further analysis. While this separation method remained constant, temperature
was varied prior to and after acidification of samples and different pH values were targeted
during acidification. In some later trials, a water wash was used to obtain residual rhamnolipid
trapped in the solid pellet after centrifugation. All solid samples tested for rhamnolipid
concentration were weighed in an aluminum dish, dried in a 60°C oven, re-weighed, and redissolved in 15 mL of DI water to carry out the following analyses.
Rhamnolipid Measurement
An anthrone analysis was used to determine the rhamnolipid concentration of samples.
First, the sample pH was adjusted to 2-3 using 6 N hydrochloric acid if it was not already within
the appropriate pH range. The lipids were extracted using ethylacetate (20 mL/5 mL of sample)
in a rotator overnight. Depending on the dilution of the sample, the desired amount of ethyl
acetate was removed from each glass vial and transferred into a glass test tube via pipette. The
test tubes were air dried at room temperature overnight. The residue was re-dissolved in 1.7 mL
of 0.05M sodium bicarbonate solution. Then, 3.3 mL of anthrone solution (2 g/L anthrone in
concentrated sulfuric acid) were added to each test tube. The test tubes were placed in an ice
bath immediately after the anthrone solution was added to counteract the large exotherm. The
test tubes were removed from the ice bath and allowed to return to room temperature. The
mixture was then heated at 95°C for 16 minutes in a thermoblock. The absorbance of each
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sample was measured at 625nm using a spectrophotometer. The absorbance is directly
proportional to the rhamnolipid concentration and a calibration curve was developed using
standards prepared by dissolving different concentrations of rhamnose in the sodium
biocarbonate solution and preparing them using the same anthrone analysis method. Duplicate
samples were run in order to obtain a standard deviation for each study.
Rhamnolipid concentration was also measured using High Performance Liquid
Chromatography/Mass Spec. The samples were prepared as follows: 0.5 mL sample (diluted 10 500 times depending on expected concentration), 0.4 mL acetonitrile, and 0.1 mL 40mM
ammonium acetate in water. The samples were syringed through a 0.2 μm filter before being
transferred into the HPLC vials. The column used was a Supelcogel LC-18. The mobile phase
had a concentration gradient over the 40 minute run time at 0.4 mL/min. The mobile phase
consisted of 4 mM ammonium acetate in water (phase A) and acetonitrile (phase B). For the first
20 minutes of the run time, the phase A to phase B ratio was 60:40 and the ratio was increased to
95:5 for the remainder of the run. The column temperature was set at 24°C, the injection volume
was 20μL, and the autosampler temperature was set at 4°C. Blank vials with mobile phase were
run between each sample because there were issues with the rhamnolipid sticking to the column.
The UV-ABS spectra were generated at 230nm. The MS mode was set to negative, standard,
normal, and smart. The nebulizer was set at 40 psi and the dry gas was used at 8 L/min at
365°C. The target mass was set at 550 m/z with a 75% stability and 100% trap drive.
Glycerol Measurement
The glycerol concentration of the samples was also measured using HPLC. Samples
were diluted 10 times with the mobile phase, 0.1% phosphoric acid, and filtered. An injection
volume of 25μL was used and the glycerol peak occurred at around 24 minutes using the water based column. Glycerol composition was only measured for one set of samples as it did not
provide any useful information.
Results
The first study related to the separation of rhamnolipids for this project was an attempt to
duplicate the results of the glycolipid separation reported in US Patent 5,656,747 (Mixich et al.
1993). The rhamnolipid separation technique described in the Experimental Methods section
was carried out, but the sample was heated to 100°C, and then placed in an ice bath after it was
acidified. White cloudy lipids precipitated out of the dark brown broth after the hydrochloric
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acid was added dropwise. When heated, the color changed to a lighter brown and the lipids redissolved. After cooled in an ice bath, a sandy precipitate formed and the remained light brown
in color. After centrifugation, the patent described the broth separating into two phases, the
glycolipids present in the lower phase. The sample prepared in Dr. Ju’s lab, on the other hand,
resulted in three phases after centrifugation, two of which were liquid and one which was solid,
as can be seen in Figure 2. About 50% of the sample volume was composed of the top liquid
layer, 40% of the total sample volume was the bottom liquid layer, and 10% of the total sample
volume was the solid phase. Because only two phases were expected, the phase which contained
the rhamnolipid was not known. The three phases were separated and stored in the refrigerator
for rhamnolipid measurement.
Figure 2. Three distinct phases formed after heating, acidification, and centrifugation
After conducting the anthrone analysis procedure outlined in the Experimental Methods
section, it was determined that a majority of the rhamnolipid was present in the lower liquid
phase, which was termed the “bottom phase” in this study. A rhamnose material balance using
the results from the rhamnolipid measurement was carried out in Table 1. A calibration curve
for this analysis is provided in Figure 5 in Appendix A and the raw rhamnolipid concentration
data is provided in Table 7 in Appendix B. It can be noted that a majority (89.43%) of the total
rhamnolipid was found in the bottom liquid layer. Also, 33.1% of the rhamnolipid expected
(calculated using the concentration of the initial broth) is unaccounted for in the summation of
the three layers analyzed, meaning significant error is associated with this number.
Table 1. Rhamnose material balance for replication of patent procedure
Top Liquid Layer
Bottom Liquid Layer
Solid Pellet
Sum
Concentration (g/L)
0.439±2.14
29.0±1.55
1.50±0.04
31.0±3.73
Mass (g)
0.004±0.161
0.290±0.161
0.030±0.161
0.325±0.161
RhL Distribution
1.35±33.1%
89.4±33.1%
9.22±33.1%
100.±33.1%
*The 33.1% error is due to not re-dissolving all the RhL in the pellet, as pH was not readjusted.
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Given the results from the replicated patent procedure, temperature was chosen as the
first variable to observe its effect on the rhamnolipid separation. The patent procedure was
carried out again heating to 100°C before acidification instead of after, refrigerating for one
week after acidification, heating only to 50°C after acidification, and without any heating or
cooling. The results for these four variations on the patent procedure are presented in Tables 2 -5
and summarized in Figure 3.
Table 2. Rhamnose material balance for patent procedure heating to 100°C before
acidification instead of after
Top Liquid Layer
Bottom Liquid Layer
Solid Pellet
Sum
Concentration (g/L)
4.55±0.604
36.1±4.60
2.26±0.162
42.9±5.37
Mass (g)
0.023±4.47E-3
0.180±4.47E-3
0.023±4.47E-3
0.226±4.47E-3
RhL Distribution
10.1±1.86%
79.9±1.86%
10.0±1.86%
100.±1.86%
*The 1.86% error is due to not re-dissolving all the RhL in the pellet, as pH was not readjusted.
The patent said that the procedure does not work if the sample is heated before acidifying
instead of heating after. The results observed proved otherwise, as three phases were formed and
the bottom liquid layer still contained the largest percentage of total rhamnolipid. The only
difference observed for this trial was that the cells were packed more tightly after the initial
centrifugation, which is mostly likely due to the lower viscosity of the sample.
Table 3. Rhamnose material balance for patent procedure refrigerating for one week after
acidification
Top Liquid Layer
Bottom Liquid Layer
Solid Pellet
Sum
Concentration (g/L)
0.322±0.0928
32.8±1.66
3.34±0.187
36.4±1.94
Mass (g)
0.002±0.0314
0.164±0.0314
0.033±0.0314
0.199±0.0314
RhL Distribution
0.81±13.1%
82.4±13.1%
16.8±13.1%
100.±13.1%
*The 13.1% error is due to not re-dissolving all the RhL in the pellet, as pH was not readjusted.
For this refrigeration trial, the bottom liquid phase was more viscous than the previous
runs and was almost gel-like. The pellet also occupied a larger volume after centrifugation. The
upper liquid layer was thin like previous runs, but was a deeper orange color. The white cloudy
precipitate in the bottom liquid layer looked the same as previously observed.
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Table 4. Rhamnose material balance for patent procedure heating only to 50°C after
acidification
Top Liquid Layer
Bottom Liquid Layer
Solid Pellet
Sum
Concentration (g/L)
14.0±1.53
37.9±3.75
1.89±0.154
53.8±5.43
Mass (g)
0.070±0.048
0.189±0.048
0.019±0.048
0.278±0.048
RhL Distribution
25.1±20.0%
68.1±20.0%
6.80±20.0%
100.±20.0%
*The 20.0% error is due to not re-dissolving all the RhL in the pellet, as pH was not readjusted.
It can be noted that when only heating to 50°C, the solids did not completely dissolve as
they did when the patent procedure was run at 100°C. Also, the solids formed a more compact
layer after being cooled to room temperature for this trial. The solids took up about half the
volume as previously seen.
Table 5. Rhamnose material balance for patent procedure without heating or cooling
Top Liquid Layer
Bottom Liquid Layer
Solid Pellet
Sum
Concentration (g/L)
0.675±0.103
34.6±3.33
4.53±0.203
39.8±3.64
Mass (g)
0.004±0.009
0.190±0.009
0.045±0.009
0.239±0.009
RhL Distribution
1.55±3.71%
79.5±3.71%
19.0±3.71%
100.±3.71%
*The 3.71% error is due to not re-dissolving all the RhL in the pellet, as pH was not readjusted.
Figure 3. Summary of Temperature Effect on Rhamnolipid Distribution
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The results from this study indicate that temperature has little effect on the rhamnolipid
distribution. For all cases, the bottom liquid layer contained about the same percentage of the
total amount of rhamnolipid for each sample. It can be noted that the lower temperature samples
appeared to have less rhamnolipid in the top liquid layer and more rhamnolipid in the solid
phase. For this reason, separation without heating or cooling was used for further studies.
At this point, the rhamnolipid concentration in the separate phases was known, but the
splits of types of rhamnolipid and other broth components, such as glycerol and salts, were still a
mystery. There was a clear difference in color and viscosity between the two liquid layers, so it
was decided that HPLC/MS would be used to determine if any other components were
selectively splitting between phases. The MS results revealed that rhamnolipids R-R-C10 -C8, RR-C10 -C8 , R-R-C10 -C10, R-R-C12 -C10, R-R-C12 -C12, and R-R-C10 -C12 were present in all the
sample layers. The only type of rhamnolipid which appeared to selectively remain in the solid
phase for all samples was R-R-C8 -C8 , which is demonstrated by the sample MS spectra in Figure
4. Other rhamnolipid peaks were the same for the top and bottom, demonstrating no selectivity.
Fragment ion numbers used to identify species of rhamnolipid were obtained from M. Heyd et al.
(Heyd et al. 2008). The quantitative rhamnolipid HPLC results did not match the rhamnolipid
concentrations obtained using the anthrone analysis exactly; however, the same trends were
observe. The only useful piece of information which can be obtained from this data is that a
majority of the rhamnolipid from each group of samples is found in the bottom liquid phase,
supporting previous findings.
Intens.
x107
Broth
3
2
1
0
Bottom
Layer
Top
Layer
5
10
15
20
25
30
Time [min]
Figure 4. MS Spectra Demonstrating Absence of R-R-C 8-C8 in Top and Bottom Layers
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Another interesting note from the rhamnolipid HPLC/MS study was that the top and
bottom liquid phases settled significantly while sitting in the refrigerator for a week after they
were prepared. It was noticed that sub-phases within each phase had formed after the week, so
samples were taken from the sub-phases to observe the rhamnolipid split. The bottom of the
bottom phase sample looked identical to the bottom of the top phase samples, indicating that the
longer the samples are left to settle while refrigerated, the better rhamnolipid split is achieved.
The top of the top phase samples were almost completely free of rhamnolipid, so settling time at
low temperature needs to be optimized. The rhamnolipid HPLC/MS results did reveal that some
compounds were selectively separating, but it was too difficult to identify these compounds
without having an idea of what all was in the broth. A protein analysis of the broth, top and
bottom phases, and post-ethyl acetate extraction top and bottom phases is recommended to try to
identify these selective compounds.
The HPLC/MS was also used to examine glycerol composition of each phase. Glycerol
composition remained constant throughout all phases, which indicates that glycerol has no
selective separation amongst liquid phases during the acidification and centrifugation process.
Table 14 in Appendix C summarizes the glycerol HPLC results. It can be noted that the heated
top phase had a significantly lower glycerol concentration than the non-heated top layer (samples
5-7). Also, the heated bottom layer has around the same glycerol concentration as the nonheated bottom phase (samples 4 and 8). The calibration curve for this data can be found in
Appendix A.
There was a significant amount of rhamnolipid unaccounted for in the material balances
and the HPLC results were inconclusive as to what compounds were splitting selectively.
Therefore, the anthrone analysis was carried out on the solid samples, but dissolved in sodium
bicarbonate instead of DI water after washing them with DI water. It was thought that at a low
pH, the rhamnolipid may have been trapped in the solid pellet was not soluble in water and that
using sodium bicarbonate would give a more accurate rhamnolipid concentration for the solid
phase. Also, larger amounts of ethyl acetate were used in the ethyl acetate extraction to see if
some of the early peaks seen in the MS results could be removed.
Table 6 describes the rhamnolipid distribution of duplicate samples which were prepared
following the patent procedure without heating or cooling. The DI water used to wash the solid
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pellet was analyzed for rhamnolipid concentration. The calibration curves for these samples can
be found in Appendix A and the raw data is located in Appendix B.
Table 6. Rhamnose material balance for samples in which the solid phase was
washed with DI water
Top
Bottom
Solid
Wash
Total
Conc. (g/L)
0.319
15.9
1.32
37.6
55.2
Trial 1
Mass (g)
0.003±0.004
0.127±0.004
0.026±0.004
0.094±0.004
0.251±0.004
RhL Distribution
1.27±1.59%
50.8±1.59%
10.5±1.59%
37.4±1.59%
100.±1.59%
Conc. (g/L)
2.55±0.444
4.91±1.04
9.09±8.17*
38.3±16.7*
54.8
Trial 2
Mass (g)
0.015±0.013
0.020±0.013
0.109±0.013
0.096±0.013
0.240±0.013
RhL Distribution
6.39±5.23%
8.19±5.23%
45.5±5.23%
39.9±5.23%
100.±5.23%
*Replicate samples were not run for Trial 1 & the large error associated with the replicate solid and wash
samples in Trial 2 is believed to be due to pH differences as discussed later.
These results revealed that the solid phase did contain more rhamnolipid than original
results had indicated. Not all of the rhamnolipid trapped in the solid phase was recovered
without the water wash and dissolution in sodium bicarbonate. About half of the total mass of
rhamnolipid can be found in the solid phase after centrifugation and half can be found in the
bottom liquid layer. These results are more like those reported in Patent 5,656,747 (Mixich et al.
1993). The solid phase was still found to contain a significant portion of the initial mass of
rhamnolipid, but the rest was in the bottom liquid layer unlike the results reported in the patent.
It can be noted that pH varied slightly between replicate samples. With a pH of 1.80 after
acidification, significantly more rhamnolipid was found in the water wash than in the redissolved solid phase (9.95 g/L in solid phase vs. 150.36 g/L in wash); whereas a pH of 2.37
after acidification resulted in more rhamnolipid in the solid phase than water wash (44.62 g/L in
solid phase vs. 79.30 g/L in wash).
Discussion and Analysis
As seen throughout this study, it can be concluded that acidification of rhamnolipid broth
to a pH of 2.0-3.0 and centrifugation at 7,000 rpm for 30 minutes will cause the solution to
separate into 3 distinct layers. The top liquid layer typically accounted for 50% of the sample
volume, the bottom liquid layer typically accounted for 40% of the sample volume, and the solid
phase accounted for 10% of the volume of the sample. The layers varied in color and
consistency. The top layer was always a lighter color and appeared to be less viscous than the
thick, dark bottom liquid layer. Upon settling in the refrigerator, a milky white layer formed on
Lilly 16
top of the liquid, a yellow tinted oil-like layer was apparent at the interface of the two liquid
phases, and the bottom liquid layer was still dark brown. This phase behavior needs to be further
investigated.
As temperature was varied both before and after acidification, there seemed to be no
effect on the rhamnolipid distribution amongst layers. The patent procedure was carried out
heating to 100°C before acidification instead of after, refrigerating for one week after
acidification, heating only to 50°C after acidification, and without any heating or cooling. The
anthrone analysis initially revealed that, on average, around 80% of the initial mass of
rhamnolipid in the sample ended up in the bottom liquid layer, leaving around 12% in the solid
phase and 8% in the top liquid layer. While temperature appeared to have no effect on
rhamnolipid splits, it was noted that refrigeration sped up settling time of samples.
Due to the large amount of error encountered in the rhamnose material balances of this
study, it was originally postulated that acidification to a pH of as low as 2.0 was hydrolyzing the
rhamnolipid, separating the rhamnose sugar from the fatty acid. This theory was plausible
because the company which obtained the patent off which this procedure was based separates the
sugar from the lipid in downstream processing anyway, so this would not be a major concern for
their purposes. In order to test this hydrolysis theory, the aqueous layers left over from the ethyl
acetate extractions were tested for rhamnose concentration using the anthrone analysis. The
anthrone analysis results indicated that no rhamnose was present in the aqueous layers, so the
theory was disproved and the discrepancies observed in the rhamnose material balances were
attributed to experimental error.
After further investigation, it was realized that the solid pellets were re-dissolved in water
at a low pH before being analyzed. It was then postulated that the pH was too low for the
rhamnolipid to be completely soluble in the water, resulting in an inaccurate rhamnolipid
measurement for the solid phases. For this reason, the solids were dissolved in sodium
bicarbonate solution after being washed with DI water to see if the rhamnolipid distribution
results changed. Summing the mass of rhamnolipid found in the solid phase re-dissolved in
sodium bicarbonate solution and the mass of rhamnolipid found in the water wash, the solid
phase after centrifugation now accounted for 50% of the rhamnolipid mass distribution. It can be
concluded that the patent procedure without heating and cooling results in 50% of the
rhamnolipid present in the bottom liquid layer and 50% of the rhamnolipid present in the solid
Lilly 17
phase. Perhaps some broth components are forming complexes with the rhamnolipid in the
bottom liquid layer, which would explain why all of the rhamnolipid is not contained in one
phase as suggested by US Patent 5,656,747. This would also explain the large number of
extraneous peaks which could not be identified in the HPLC results.
An interesting observation during the water wash study, was the effect of pH on the
rhamnolipid distribution between the solid re-dissolved in the sodium bicarbonate solution and
the water wash. With a pH of 1.80 after acidification, significantly more rhamnolipid was found
in the water wash than in the re-dissolved solid phase (9.95 g/L in solid phase vs. 150.36 g/L in
wash); whereas a pH of 2.37 after acidification resulted in more rhamnolipid in the solid phase
than water wash (44.62 g/L in solid phase vs. 79.30 g/L in wash). It is recommended that pH is
varied between 1.0 and 5.0 while carrying out the patent procedure without heating and cooling
as an experiment to find the effect of pH on rhamnolipid distribution amongst phases.
HPLC/MS results provided some interesting information about selective separation of the
broth components. The only type of rhamnolipid which appeared to selectively remain in the
solid phase for all samples was R-R-C8 -C8. Other rhamnolipid peaks were the same for the top
and bottom, demonstrating no selectivity. Glycerol did not demonstrate much selectivity
amongst phases, but it can be noted that heated samples had a lower glycerol concentration in the
top layer than those which were not heated. There was definitely selectivity amongst phases for
unknown compounds, presumably salts, because the top liquid phase MS spectra were all
missing peaks observed in the bottom liquid phase and solid phase spectra. These spectra should
be further investigated if some of the salts present in the broth can be identified.
Different ratios of ethyl acetate to sample were studied later in the project to observe the
effect on rhamnolipid solubility. The results from this study were inconclusive, so they are not
presented in this research project. The typical ethyl acetate to sample ratio for rhamnolipid
extraction is 4:1, but 5:1, 10:1, 20:1, and 30:1 were tested in an attempt to produce a solubility
curve for rhamnolipid in ethyl acetate. The results did not match what they should have been
theoretically, so it is recommended that this study is repeated. After developing an accurate
solubility curve for rhamnolipid in ethyl acetate, curves should be produced for other solvents,
such as hexane, which may be useful for post-separation rhamnolipid crystallization.
Lilly 18
Conclusions
There are many potential industrial applications for rhamnolipid biosurfactants, but the
high costs of separation and purification have prevented their use. The purpose of this project
was to select and evaluate methods to separate rhamnolipid biosurfactants from fermentation
broth. Upon acidification and centrifugation, the rhamnolipid fermentation broth separates into
three distinct layers. It can be concluded that acidification to a pH of 2.0-3.0 and centrifugation
of rhamnolipid broth results in 50% of the rhamnolipid mass being present in the bottom liquid
layer and 50% of the rhamnolipid present in the solid phase, regardless of the temperature at
which the separation is performed. All species of rhamnolipid present in the broth demonstrated
no selectivity amongst phases with the exception of R-R-C8 -C8 , which was present only in the
solid phase. Because patents indicate that all rhamnolipid should be contained in one phase
using this process, it can be deduced that some broth component is forming a complex with the
rhamnolipid in the bottom liquid layer. Further investigation needs to be conducted to better
describe this phase behavior. Moreover, rhamnolipid solubility in various solvents should be
determined to prepare for post-separation rhamnolipid crystallization.
Acknowledgements
The guidance from Dr. Lu-Kwang Ju, Chemical and Biomolecular Engineering
Department Head, is greatly appreciated. Furthermore, I would like to thank the graduate
students in the Bioprocess Engineering Laboratory, specifically Maysam Sodagari and Nicholas
Callow, who were very helpful in completing the experimental design and analysis involved with
this project.
Literature Cited
(1) Abdel-Mawgoud, Ahmad Mohammad, et al. “Characterization of Rhamnolipid Produced by
Pseudomonas aeruginosa Isolate Bs20.” Appl. Biochem. Biotechnol. 13 May 2008.
(2) Blumenkrantz, Nelly and Gustav Asboe-Hansen. “New Method for Quantitative
Determination of Uronic Acids.” Analytical Biochemistry 54: 484-489, 1973.
(3) Costa, Siddhartha, et al. “Wettability of Aqueous Rhamnolipids Solutions Produced by
Pseudomonas aeruginosa LBI.” J. Surfact. Deterg. 12: 125-130, 2009.
Lilly 19
(4) Chayabutra, Chawala and Lu-Kwang Ju. “Polyhydroxyalkanoic Acids and Rhamnolipids Are
Synthesized Sequentially in Hexadecane Fermentation by Pseudomonas aeruginosa
ATCC 10145.” Biotechnol. Prog. 17: 419-423, 2001.
(5) Heyd, M., et al. “Development and trends of biosurfactant analysis and purification using
rhamnolipids as an example.” Anal. Bioanal. Chem. 391: 1579-1590, 2008.
(6) Knutson, Clarence A. and Allene Jeanes. “Determination of the Composition of Uronic Acid
Mixtures.” Analytical Biochemistry 24: 482-490, 1968.
(7) Mata-Sandoval, Juan C., et al. ”High-performance liquid chromatography method for the
characterization of rhamnolipid mixtures produced by Pseudomonas aeruginosa UG2 on
corn oil.” Journal of Chromatography A 864: 211-220, 1999.
(8) Mixich, Johann, et al. “Process for the quantitative purification of glycolipids.” US Patent
5,656,747. 12 Aug. 1997.
(9) Mukherjee, Soumen, et al. “Towards commercial production of microbial surfactants.”
TRENDS in Biotechnology 24(11): 509-515, 2006.
(10) Sarachat, Thitima, et al. “Purification and concentration of a rhamnolipid biosurfactant
produced by Pseudomonas aeruginosa SP4 using foam fractionation.” Bioresource
Technology 101: 324-330, 2010.
Lilly 20
Appendix A: Calibration Curves
Calibration Curve for Replicated Patent Procedure
Rhamnose
Concentration (g/L)
0.06
y = 0.0387x
R2 = 0.9437
0.05
0.04
0.03
y = 0.0468x - 0.0061
R2 = 0.9955
0.02
0.01
0
0
0.5 Absorbance
1
1.5
Figure 5. Anthrone analysis calibration curve for replicated patent procedure
*Trend lines were fit with and without the y-intercept set to zero. The model with the y-intercept
set equal to zero was used for all results in this report unless stated otherwise.
Calibration Curve for Patent Procedure Heating to 100C
Before Acidification Insted of After and for Refrigerating One
Week After Acidifcation
Rhamnose Concentration
(g/L)
0.06
y = 0.0409x - 0.0011
R2 = 0.9953
0.05
0.04
y = 0.0396x
R2 = 0.9934
0.03
0.02
0.01
0
0
0.5
1
1.5
Absorbance
Figure 6. Anthrone analysis calibration curve for patent procedure heating to 100°C before
acidification instead of after and patent procedure refrigerating for one week after
acidification
*Trend lines were fit with and without the y-intercept set to zero. The model with the y-intercept
set equal to zero was used for all results in this report unless stated otherwise.
Lilly 21
Rhamnose Concentration
(g/L)
Calibration Curve for Patent Procedure Heating to 50C After
Acidification and for Patent Procedure without Heating or Cooling
0.06
y = 0.046x - 0.0036
R2 = 0.9956
0.05
0.04
y = 0.0413x
R2 = 0.9749
0.03
0.02
0.01
0
0
0.5
1
1.5
Absorbance
Figure 7. Anthrone analysis calibration curve for patent procedure heating to 50°C after
acidification and patent procedure without heating or cooling
*Trend lines were fit with and without the y-intercept set to zero. The model with the y-intercept
set equal to zero was used for all results in this report unless stated otherwise.
Glyc Conc vs Peak Height
6
y = 0.0001x
R2 = 1
Glyc Conc. (g/L)
5
4
3
2
1
0
0
10000
20000
30000
40000
50000
Peak Height
Figure 8. Calibration curve for glycerol HPLC data
Lilly 22
Calibration Curve for Patent Procedure Without Heating or
Cooling and Water Washing Solid (Trial 1)
Rhamnose
Concentration (g/L)
0.06
y = 0.0332x
0.05
2
R = 0.9989
0.04
0.03
y = 0.033x + 0.0002
R2 = 0.999
0.02
0.01
0
0
0.5
1
1.5
2
Absorbance
Figure 9. Anthrone analysis calibration curve for patent procedure without heating or
cooling and solid phase water wash (Trial 1)
Calibration Curve for Patent Procedure Without Heating or
Cooling and Water Washing Solid (Trial 2)
Rhamnose
Concentration (g/L)
0.06
y = 0.028x
R² = 0.998
0.05
0.04
0.03
y = 0.028x - 0.000
R² = 0.999
0.02
0.01
0
0.000
0.500
1.000
1.500
2.000
Absorbance
Figure 10. Anthrone analysis calibration curve for patent procedure without heating or
cooling and solid phase water wash (Trial 2)
Lilly 23
Appendix B: Raw Rhamnolipid Concentration Data
Table 7. Raw RhL concentration data for replicated patent procedure
Sample
Top
Bottom
Solid
Final
ABS1
-0.01
0.36
0.02
0.302
ABS2
0.04
0.37
0.02
0.325
ABS3
-0.02
0.40
0.02
0.305
Con1
-0.39
27.71
1.47
23.375
Con2
2.86
28.64
1.55
25.155
Con3
-1.16
30.73
1.47
23.607
Con
0.44
29.03
1.50
24.265
Error
2.14
1.55
0.04
1.259
*Absorbance was measured three times for each sample to obtain standard error.
Table 8. Raw RhL concentration data for patent procedure heating to 100°C before
acidification instead of after
Sample
Top
Bottom
Solid
ABS1
0.283
0.407
0.131
ABS2
0.327
0.440
0.147
ABS3
0.251
0.520
0.150
Con1
4.483
32.234
2.075
Con2
5.180
34.848
2.328
Con3
3.976
41.184
2.376
Con
4.546
36.089
2.260
Error
0.604
4.602
0.162
*Absorbance was measured three times for each sample to obtain standard error.
Table 9. Raw RhL concentration data for patent procedure refrigerating for one week after
acidification
Sample
Top
Bottom
Solid
ABS1
0.016
0.393
0.214
ABS2
0.018
0.435
0.198
ABS3
0.027
0.413
0.221
Con1
0.253
31.126
3.390
Con2
0.285
34.452
3.136
Con3
0.428
32.710
3.501
Con
0.322
32.762
3.342
Error
0.093
1.664
0.187
*Absorbance was measured three times for each sample to obtain standard error.
Table 10. Raw RhL concentration data for patent procedure heating to 50°C after
acidification
Sample
Top
Bottom
Solid
ABS1
1.479
0.436
0.246
ABS2
1.793
0.511
0.232
ABS3
1.806
0.429
0.209
Con1
12.217
36.014
2.032
Con2
14.810
42.209
1.916
Con3
14.918
35.435
1.726
Con
13.981
37.886
1.892
Error
1.529
3.755
0.154
*Absorbance was measured three times for each sample to obtain standard error.
Table 11. Raw RhL concentration data for patent procedure without heating or cooling
Sample
Top
Bottom
Solid
ABS1
0.082
0.390
0.536
ABS2
0.069
0.447
0.577
ABS3
0.094
0.615
0.533
Con1
0.677
32.214
4.427
Con2
0.570
36.922
4.766
Con3
0.776
50.799
4.403
Con
0.675
39.978
4.532
Error
0.103
9.662
0.203
*Absorbance was measured three times for each sample to obtain standard error.
Lilly 24
Table 12. Raw RhL concentration data for patent procedure without heating or cooling
and washing the solid phase with DI water
Sample
Broth
Top Liquid Layer
Bottom Liquid Layer
Solid Phase
Water Wash
ABS
0.186
0.024
0.24
0.099
0.566
Con
12.350
0.319
15.936
1.315
37.582
Table 13. Raw RhL concentration data for patent procedure without heating or cooling
and washing the solid phase with DI water replicated
Sample
Broth
Top Liquid Layer
Bottom Liquid Layer
Solid Phase
Water Wash
ABS1
0.312
0.2
0.403
0.296
0.895
ABS2
0.441
0.256
0.298
1.328
0.472
Con1
17.472
2.24
5.642
3.3152
50.12
Con2
24.696
2.867
4.172
14.874
26.432
Con
21.084
2.554
4.907
9.094
38.276
Error
5.108
0.443
1.039
8.173
16.750
*The sample pH for ABS1 was 1.80 and the sample pH for ABS2 was 2.37.
Lilly 25
Appendix C: HPLC Data
Table 14. Glycerol HPLC Results
Sample
#
-5std
1std
5std
1
2
5
6
7
8
Sample
Name
0.5 g/L
1 g/L
5 g/L
Top 2/18
10X
Bot 2/18
10X
Top 3/24
10X
Post-Acid
3/24 10X
Top 1/21
10X
Bot 1/21
10X
Description
Standard
Standard
Standard
Top layer after pH adj, heat to
100°C, & centrifugation
Bottom layer after pH adj,
heat to 100°C, &
centrifugation
Top layer after pH adj &
centrifugation
After pH adjustment, before
centrifugation
Top layer after pH adj &
centrifugation
Bottom layer after pH adj &
centrifugation
Ht of Peak
(Sample 1)
3994
7858
39504
Ht of Peak
(Sample 2)
4376
8512
43770
Avg Ht
of Peak
4185
8185
41637
Glyc Conc.
(g/L)
0.5
1
5
27762
29519
28640.5
34.4
25882
27544
26713
32.1
32227
36926
34576.5
41.5
32623
36871
34747
41.8
32902
36721
34811.5
41.8
27358
28379
27868.5
33.5
*Ht of Peak (1) and Ht of Peak (2) are the same peak measured for replicate samples.