4

Measurement
Solubilization

of Biosurfactant-Enhanced
and Biodegradation
of Hydrocarbons

Raina M. Miller and Yimin Zhang

1. Introduction
Biosurfactants are a chemically unique class of compounds produced by many
bacterial and fungal genera. There are several thorough reviews concerning types
of biosurfactants produced (1,2). This chapter addressesexperimental methodology concerning one potential application of biosurfactants: Their use in accelerated
remedration of hydrocarbon-contammated environments (see also Chapter 3). This
methodology includes specifics concerning the measurementof the effect of brosurfactantson both hydrocarbon solubtlization and hydrocarbon biodegradation.
Aspects of biosurfactants that may be important in their application to remediatron include the producing organism, biosurfactant chemical structure, and
environmental conditions. Generally, microorganisms within a genus produce
biosurfactants with similar structure although there can be species-level differences in the amount of biosurfactant produced. Since biosurfactants are genusspecific, it is difficult to predict the effect of a brosurfactant m the environment
because the effect that a brosurfactant has on the producing genus may be different than the effect it has on a different genus (3). In terms of chemical structure, biosurfactants can be divided mto several broad groups: glycoltprds,
lipopeptides, lipopolysaccharides, phosphohpids, and fatty acids/neutral lipids
(1,2). Generally, biosurfactant molecular weights range from approx 500-1500,
although Pseudomonas strains growing on hexadecane have been reported to
produce protein-containing surface-active substanceswith molecular weights of
up to 14,300 (2). The capacity for enhancement of solubilization and biodegradation is dependent on biosurfactant structure, although there is no information
yet available concerning a systematic study of structure and function (4).
Finally, with respect to environmental conditrons, rt has been found that both
yield and compositton of biosurfactant are affected by growth condttions,
From

Methods
Edlted

by

m Botechnology,
D Sheehan

Vol 2 Boremedratron

Humana

Press

Inc , Totowa,

Protocols
NJ

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Adder and Zhang

n-tcludmg carbon source, culture medium nutrients (e.g. nitrogen, phosphate,

iron), temperature, pH, and aeration (5,6). Therefore, it IS something of an art to
maxrmlze biosurfactant productron rn the laboratory. Such laboratory results
suggest that m sztu brosurfactant production would also be affected by environmental condrttons. Unfortunately,
there IS little informatron concernmg zn sztu
production of brosurfactants to confirm this supposrtron.
Few brosurfactants have been well-studied with respect to remedratron. The
best-studied biosurfactant is rhamnolipid
produced by Pseudomonas spp., and
some information
1s available concerning Baczllus-surfactm,
Rhodococcustrehalose lipid, and Candidu-sophorose
lipid systems. The procedures
described here can be used to test the effect of any surfactant (either brologrcal or synthetic) on solubihzation
and biodegradation
of model hydrocarbons.

2. Materials
1 Hydrocarbon-degrading bacteria can be isolated from environmental samples by
enrichment or can be obtained from the American Type Culture Collectron
(Rockvrlle, MD) For alkane degraders, degradatrve genes are chromosomal, and
thus stable, so cultures may be mamtamed on nutnent agar (Drfco, Detron, MI)
plates For polyaromatrc hydrocarbon (PAH) degraders cultures should be mamtained on mmeral salts medmm plates amended with a PAH as sole carbon and
energy source because degradattve genes may be plasmid-associated and are not
always stable. All cultures are stored at room temperature and transferred monthly
2 Biosurfactants are not yet available commerctally and therefore must be produced
m the laboratory As an example, rhamnoliprds can be produced and recovered
from many Pseudomonas aerugznosa strams. Rhamnohpid yields of up to 2 g/L can
be achteved during growth on either proteose peptone-glucose-ammomum
salts
(PPGAS) medium (7) or mineral salts medium wrth 2% glucose (8) Rhamnolrprd
isolation, purifrcatton, and rdentrfrcatron have been descrrbed m detarl (9)
Srmrlarly protocols for productron and purifrcatton of other brosurfactants have
been developed (5).
3. Model hydrocarbons can be obtained from Aldrich (Milwaukee, WI) and 14Clabeled hydrocarbons can be purchased from Sigma (St Louts, MO) An orgamc
solvent, such as hexane or chloroform, 1s used to prepare hydrocarbon and
[r4C]hydrocarbon mrxtures. The recommended amount of radroactrvrty for each
solublhzatron or biodegradation assay 1sapprox lo5 dpm (0 045 ~CI)
4 Phosphate buffer (0 lM, pH 7 0) contaming 8.67 g Na2HP04 and 5 38 g
NaH,P04 Hz0 per liter 1sused for all solubrhzatron tests.
5 Mineral salts medium 0 4% Na2HP04, 0.15% KH2P04, 0 1% NH&l, 0.02%
MgS04*7H20, 0.0005% iron ammomum citrate, and 0 001% CaCl, pH 7 2. Mineral
salts medium alone can be sterthzed by autoclavmg. Mineral salts medium containing a brosurfactant IS sterthzed by ftltratron usmg a 0 2-pm filter
6 Protem IS determined by the method of Lowry (10) Three stock solutrons are prepared and stored at room temperature, solution A* 2% Na.&YOs, solution B 1%

Biosurfactant-Enhanced

Solubiliza tlon

61

CuS04; solution C: 2% sodmm tartrate. A workmg solution (solution D) is freshly

prepared before each use and contains 50 mL solution A, 0 5 mL solution B, and
0 5 mL solution C. The 2N Folm & Ciocalteus phenol reagent (Sigma) IS diluted
to 1N and stored at 4C A standard curve is prepared usmg bovine serum albumin
(BSA) (Sigma) in 0 1N NaOH.

3. Methods
3.1. Quantitation

of Biosurfactant

Biosurfactants can be quantified by surface and interfacial tension. This is a

generic quantitation and thus does not dlstmguish among different types of surfactants that may be present Biosurfactants can be compared m terms of the
amount they reduce surface or mterfacial tension, and the critical mlcelle concentration (cmc), which 1s the lowest surfactant concentration above which no
further decrease in surface tension or mterfaclal tension takes place

3.1.1. Measurement of Surface Tension

Surface tenslon measures the force required to move a ring immersed m a
surfactant solution upward through the surface of the liquid mto air. Surface tension in a sample is measured with a surface tensiometer, for example a Model
21 Tensiomat (Fisher, Pittsburgh, PA), which uses the du Nouy rmg method
(see Notes 1 and 2). The cmc is determined by measuring the surface tension in
a series of samples diluted m 0. lM, pH 7.0 phosphate buffer. A standard plot is
made of log (surfactant concentration) vs surface tension and 1s used to estimate
the cmc (Fig 1)
To determine the surfactant concentration in an unknown sample, dilute the
sample until the surface tension measured is above the minimum surface tension
(the surfactant concentration in solution is below the cmc). The concentration is
by the
then determined using a standard plot as shown in Fig 1 and multiplying
appropriate dilution factor (see Note 3).

3 7.2. Measurement of InterfacIal Tension

Interfacial tension measures the force required to move a ring immersed in
one liquid, in this case a hydrocarbon, upward through a 1iquld:hquid interface
mto a second liquid, in this case water. The procedure of determining
surfactant concentration in a sample by measurement of mterfaclal tension is similar
to measurement of surface tension (see Notes l-3)

3.2. Solubilization

Tests

Dissolve a mixture of hydrocarbon and [4C]hydrocarbon

m chloroform and
add it to test tubes (16 x 100 mm). After evaporation of solvent, add 2 mL of

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Miller and Zhang

80

20

(
1

cmc
I
10

Rhamnolipld

concentration

r
100

(mg/L)

Fig. 1. Standard plot of surface tension against the log concentratton of monorhamnohptd The surface tension was measured m 0. lM, pH 7 0, phosphate buffer
biosurfactant solutron in O.lM, pH 7.0, phosphate buffer The final mass of
hydrocarbon should be approx 8 pmol, and the hydrocarbon specific activity
should be 6 nCi/pmol (approx lo5 dpm). Incubate the test tubes at 23C with
gyratory shaking at 200 rpm. After 24 h, filter each solutron through a
Whatman GF/D filter, pore size 10 pm (Fisher), and add 0.2-5 mL of a general purpose scmtrllatron
cocktall,
such as Scmtrverse
BD (Fisher).
Radioactivity
in each sample is determined using a liquid scmtillatton counter
(see Notes 4-8).

3.3. Biodegradation

Tests

Hydrocarbon biodegradation
can be determined by measurement of hydrocarbon mmerahzatton or by measurement of protein mcrease as an mdrcation of
cell growth (see Notes S-10)

3.3.1 Mmerallza t/on

For mineralization
experiments, a mixture of hydrocarbon and [4C]hydrocarbon dissolved in chloroform IS used to coat the bottom of sterrle modrfred

125mL microfernbach flasks (Wheaton, Milvllle,

NJ). These flasks have

screw tops that have been modrfted by the addmon of two Luer-lock needles
(II) Thts allows the flask to be attached to a vacuum pump and a strrpping
chain to allow flushing and trapping of 14C02 and t4C-volatile compounds
(Fig. 2). Evaporate the solvent and add 20 mL of filter-sterilized
mineral salts

Biosutfactant-Enhanced

Solubillzation

63

Open to air
\
To vacuum pump

Fig. 2. Flushing apparatus to collect 14C02 and C-volattle compounds The stripping chain conststs of six 20-mL scmtillation vials that can be removed and placed m
a scmtillatton counter for assay of radtoacttvlty Vials 1 and 4 remam empty as backflow traps Vials 2 and 3 contam a general purpose scmttllatton cocktail to trap 14Cvolatile compounds and vials 5 and 6 contain a phenethylamme-based cocktall, such
as 0x0~01 (National Dtagnosttcs, Manville, NJ) to trap 14C02 Shown on the left 1sthe
mtcrofernbach flask. The flask cap has been modified by the addmon of two Luer-lock
needles and replacement of the plastic septum wtth a Teflon septum After flushing, a
plugged 1-mL syringe 1splaced into the Luer-lock to keep the flask airtight

medium contammg blosurfactant to each flask. The final mass of hydrocarbon

added should be approx 80 pmol, specific activity 0.6 nCi/pmol (approx lo5
dpm). Each flask is then inoculated and capped For solution studies, a 2.5%
moculum is recommended
If the inoculum is a biosurfactant-producing
cul-

ture, the cells should be washed prior to inoculation to remove any associated
biosurfactant. The flasks are incubated with gyratory shakmg (200 rpm) at
23C and are flushed perrodically to collect 14C02 and C-volatile organic
compounds (see Note 11).
3.3.2, Protein Measurement
Add hydrocarbon (40 pmol) in chloroform to a series of sterile 50-mL test
tubes. After the solvent 1sevaporated, add 10 mL of mineral salts medium containing biosurfactant to each test tube Inoculate and incubate the tubes as
described above. Periodically, take a 0.5 mL sample from each test tube and
determine the protein content. Briefly heat the sample for 10 mm with 0.05 mL
of 1N NaOH. Then mix 0.4 mL of the sample with 2 mL solution D and mcubate at room temperature for 10 mm. Add Folm and Ciocalteus phenol reagent
(0.2 mL) and incubate samples at room temperature for 30 min. Determine the
optical density (OD) for each sample spectrophotometrlcally using a wave-

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Miller and Zhang

length of 650 nm (see Note 12). The protein content of each sample is calculated
using a BSA standard curve.

4. Notes
1 Some medium components dtssolved m solution, e g , glucose, do not interfere
with the du Nouy rmg method. Other medium components, such as peptone, or solvents, such as methanol, can reduce surface tension m the absence of a surfactant
Therefore, care must be taken to use the proper surface tension controls Also, some
floating materials, such as froth or 011,can affect measurement of surface tension
In this case, a separation may be required to remove the floating materials
Alternatively, btosurfactants may be quantified by methods other than surface tension For example, rhamnohpids may be determined by measurement of L-rhamnose by the orcmol method (12) Another alternative is to quantify rhamnohpid by
high performance liquid chromatography (HPLC). We have developed the following HPLC protocol using a Waters LCM-1 system operating with a UV detector at
214 nm The column used is a Nova-Pak C18, 3 9 x 150 mm (Milhpore, Bedford,
MA) Elution 1s tsocratrc and the mobile phase used is acetomtnle-water (40 60) at
a flow rate of 1 mL/mm. These methods may be adapted to quantitatton of other
biosurfactants
2 Surface and mterfacial tension measurements are dependent on temperature
Therefore, all samples should be equilibrated at room temperature before measurement
3 In some cases, the identity of a btosurfactant IS unknown and a simple screemng of
a crude surfactant solution is desired. The concentration of btosurfactant can, therefore, not be measured. Such crude surfactant solutions may be compared on the
basis of surface tension
4. Filtration of biosurfactant-hydrocarbon
solutions m solubthzatton expenments may
cause hydrocarbon sorption to the filter This can lead to underestimation of the
apparent solubihty measured Of the various commercially-available
filters tested,
it has been found that glass fiber filters have the least hydrocarbon sorption
5. Special care must be taken m solubiltzatton studies mvolvmg a volatile hydrocarbon. In this case, an antight container with a Teflon-lined cap must be used to prevent loss of the hydrocarbon during mcubation
6 The solubihzation of hydrocarbons by surfactants IS markedly affected by envnonmental conditions, such as the buffer system, ionic strength and pH For example,
monorhamnohpid (4 mM) increased the apparent solubrhty of hexadecane to 40
mg/L m distilled water Under identical condmons except that 0 5M phosphate
buffer was used instead of distilled water, the apparent solubihty of hexadecane
was 360 mg/L It is therefore recommended that consistent, predetermined, expertmental condmons be maintained throughout and between experiments
7 Commerctally
available [ 14C]hydrocarbons contam impurtties that range from
l-2% of the total radioactivrty Given the low water solubihties of these hydrocarbons, these impurities can lead to measurement of solubihty values that are much
higher than values reported m the literature To avotd this problem, the [14C]hydro-

Biosutfactant-Enhanced

8.

10.

11

12.

Solubilization

65

carbon may be purified by thin layer chromatography or by HPLC prior to use

Alternatively, use of radiolabeled hydrocarbons can be avoided by measurement of
hydrocarbon with alternate techmques, such as HPLC or gas chromatography
These methods are also useful m determination of relative hydrocarbon solubihttes
m multtcomponent hydrocarbon solutions
This chapter addresses measurement of surfactants m batch solution systems.
However, behavior of surfactants in soil systems needs to be investigated In this
case, the effect of brosurfactant sorptron by so11must be considered because sorptron can srgnifrcantly reduce the effective amount of brosurfactant available m solution (413). Thus, much higher biosurfactant concentrations may be required m
these experiments
One common way to measure biodegradatton 1s to measure substrate dtsappearante. Because hydrocarbons are not miscible with water, the alternate methods of
14C02 and protein measurement are recommended If substrate disappearance is
measured, a separate flask or test tube should be sacrificed at each time-pomt and
extracted with organic solvent (14).
Surfactants do not confer degradatrve abihty; rather, they aid m increasing mass
transfer of hydrocarbons mto the aqueous phase and they may possibly aid m dehvery of hydrocarbons to cells Thts is much easrer to demonstrate for hydrocarbons
with very low aqeous solublhty. However, for hydrocarbons with relatively htgh
water solubrlity, such as naphthalene (32 mg/L) or phenanthrene (1 6 mg/L), it may
be useful to limit the hydrocarbon surface area available when determmmg the
Impact of surfactants on solubilization and partmularly on biodegradation m solution. By limiting hydrocarbon surface area, the mass transfer of the hydrocarbon
mto solution IS slowed. Hydrocarbon surface area can be limited by carefully
applying the hydrocarbon-solvent solutron to a small area of the flask rather than
coating the entire bottom of the flask
Mmerahzation can alternatively be measured by placing NaOH traps inside screw
top flasks to trap 14C02 The NaOH traps are periodically replaced and assayed for
radroacttvity. This minimizes effort required to set up the mmeralization experiment. However, this does not allow separation of 14C02 and C-volatiles This separation can be important, especially when studying volatrle hydrocarbons that may
be trapped m the NaOH solutton.
Some mtcrobtal metabohtes, e g., Pseudomonas pigments, are released into the
growth media and interfere with protein determination at 650 nm. To remove mterfering pigments prior to protein analysis, cells in the sample are centrifuged,
washed, and resuspended m distilled water The sample IS then subjected to the normal protein analysis.

References
1 Rosenberg, E. (1986) Mtcrobtal surfactants. CRC Crlt Rev Bzotechnol 3, 109-132
2. Lang, S and Wagner, F (1987) Structure and properties of btosurfactants, m
Biosu$actants and Bzotechnology (Kosaric, N , ed ) Surfactant Sctence Series vol
25, Marcel Dekker, New York, pp. 21-45.

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Miller and Zhang

3 Ito, S and Inuoe, S (1982) Sophorohpids from Torulopsis

bombzcola
possible
relation to alkane uptake. Appl. Envzron Microbial.
43, 1278-1283
4 Mtller, R M (1995) Surfactant-enhancedbtoavatlabihty of slightly solubleorganic
compounds,m Btoremedlatton-Science
& Appkatlon
(Sktpper, H. and Turco, R.,
eds ) So11ScienceSociety of America spectalpubhcatton, Madison, WI, pp 33-54
5. Syldatk, C and Wagner, F (1987) Productton of blosurfactants, m Blosurfactants
and Biotechnology
(Kosartc, N , ed ) Surfactant Science Series Vol. 25, Marcel
Dekker, New York, pp 89-120
6. Hommel, R F. and Ratledge,C (1993) Brosynthettc mechanismsof low molecular
wetght surfactants and then precursor molecules, m Bzosurfactants,
Production,
Propertzes,
Applicatzons
(Kosaric, N , ed ), Surfactant Science Series Vol 48,
Marcel Dekker, New York, pp 3-63
7. Zhang, Y. and Miller, R. M (1992) Enhancedoctadecanedispersionand btodegradation by a Pseudomonas
rhamnoliptd surfactant (blosurfactant) Appl Envzron
Mlcroblol.

58, 3276-3282.

8. Guerra-Santos, L , Kappeli, 0 , and Aechter, A (1984) Pseudomonas aerugmosa

btosurfactant production m continuousculture with glucoseascarbon source Appl.
Environ

Mxroblol

48, 301-305

9 Syldatk, C , Lang, S., and Wagner, F. (1984) Chemical and physical characterrzanon of four inter-facial-active rhamnohprds from Pseudomonas
spec. DSM 2874
grown on n-alkanes.Z Natu$orsch.
40,5 l-60.
10. Lowry, 0. H , Rosebrough,N. J., Farr, A L., andRandall, R. J (195 1) Protein measurementwith Folm phenol reagent J Bzol Chem 193,265-275
11 Marmucct, A C. and Bartha, R (1979) Apparatus for momtormg the minerahzanon of volatile 14C-labeledcompounds Appl Envwon. Microbial.
38, 1020-1022.
12 Chandrasekaran,E V and BeMiller, J N (1980) Constrtuent analysts of glucosammoglycans,m Methods m Carbohydrate
Chemtstry (Whistler, R. L , ed ),
Academic, New York, pp. 89-96
13. Herman, D C , Arttola, .I. F , and Mtller, R. M (1995) Removal of cadmium, lead,
and zinc from sot1by a rhamnohprd brosurfactant Envzron Sci TechnoE 79,

2280-2285.
14 Jam, D K , H. Lee, and Trevors, J T (1992) Effect of addition of Pseudomonas
aeruginosa
UG2 mocula or blosurfactants on btodegradation of selectedhydrocarbons in soil. J Ind Mlcroblol
10,87-93