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Journal of Industrial Microbiology, 8 (1991) 237-246

Elsevier 237

SIM00369

Emulsification of hydrocarbons by subsurface bacteria


D.S. Francy 1, J.M. Thomas 1, R.L. Raymond 2 and C.H. Ward 1
1National Center for Ground Water Research and Department of Environmental Science and Engineering, Rice University, Houston,
Texas, and 2DuPont Environmental Remediation Services, Aston, Pennsylvania, U.S.A.
(Received 1 February 1991; accepted 24 June 1991)

Key words: Microbial emulsifier; Biosurfactant; Bioemulsifier

SUMMARY

Biosurfactantshave potential for use in enhancementofin situ biorestoration by increasingthe bioavailabilityof contaminants. Microorganismsisolated
from biostimulated, contaminated and uncontaminated zones at the site of an aviation fuel spill and hydrocarbon-degradingmicroorganismsisolated from
sites contaminated with unleaded gasoline were examinedfor their abilities to emulsifypetroleum hydrocarbons.Emulsifyingability was quantified by a
method involvingagitation and visual inspection. Biostimulated-zonemicrobes and hydrocarbon-degradingmicroorganismswere the best emulsifiers as
compared to contaminated and uncontaminated zone microbes. Biostimulation(nutrient and oxygenaddition) may have been the dominant factor which
selected for and encouragedgrowth of emulsifiers; exposure to hydrocarbonwas also important. Biostimulated microorganisms were better emulsifiers
Of aviation fuel (the contaminant hydrocarbon)than of heavier hydrocarbonto which they were not previouslyexposed. By measuring surface tension
changes of culture broths, 11 out of41 emulsifierstested were identified as possible biosurfaetant producers and two isolates produced large surfacetension
reductions indicating the high probability of biosurfactant production.

INTRODUCTION biosurfactants. This could be accomplished by stimulating


indigenous biosurfactant-producing microorganisms to
In situ biorestoration is a process wherein micro- increase their production or by introducing specially
organisms are stimulated to metabolize aquifer contami- adapted biosurfactant-producing bacteria into the con-
nants in place. One factor which often limits its effec- taminated subsurface. Biosurfactants are produced by a
tiveness is the limited solubility of organic contaminants. wide variety of microorganisms, and have been used for
A hydrophobic contaminant may sorb to organic fractions a variety of industrial applications, including microbial
of subsurface materials or become trapped in pore spaces enhanced oil recovery and oil spill clean-up [6,20,24]. The
of the aquifer matrix. enhancement of microbial biosurfactant production has
The use of synthetic surfactants may enhance mobili- been investigated for various applications and includes
zation of contaminant hydrocarbons. Surfactants could altering the type and/or amount of the nitrogen source
enhance in situ biorestoration by: (1) aiding in release of [9,12,15], supplying growth additives [15] or trace ele-
hydrophobic contaminants sorbed to organic matter; ments [7,11], or altering the pH of the growth medium
(2) reducing interfacial tensions thereby releasing con- [141.
taminant droplets trapped in pore spaces by capillary Little work has been conducted to assess the extent
forces; and (3) increasing the surface area of the contami- and importance ofbiosurfactant production by subsurface
nant available for microbial attack. Surfactant soil wash- microorganisms and their potential role in contaminant
ing increased recovery of gasoline in a model aquifer test degradation. Therefore, a study was conducted to charac-
sbistem [21]; however, there was significant reduction in terize biosurfactant production by heterotrophs isolated
permeability. In addition, Ziegenfuss [26] found that soil from several subsurface samples of biostimulated, con-
washing with surfactants in static microcosms resulted in taminated and uncontaminated materials collected from
decreased mineralization of benzene and naphthalene the site of an aviation fuel spill and of a group of hydro-
due to the biological oxygen demand exerted by the sur- carbon-degrading microorganisms isolated from an
factants. unleaded gasoline spill site. The first step in this process
An alternative to the use of synthetic surfactants for involved screening microbial isolates for their abilities to
the enhancement of in situ biorestoration is the use of emulsify aviation fuel or unleaded gasoline, kerosene and
diesel fuel. This was used to identify microorganisms with
Correspondence: D.S. Francy, U.S. Geological Survey, 975 W. the potential to produce biosurfactants.
Third Avenue, Columbus, OH 43212, U.S.A. However, emulsification of the hydrocarbons in a

0169-4146/91/$ 03.50 9 1991 Society for Industrial Microbiology


238

screening procedure can be caused by other mechanisms Media and chemicals


in addition to the release ofextracellular biosurfactants or The mineral salts solution used in this study was a
bioemulsifiers by intact cells. The hydrophobicity of an modification of a mineral salts solution developed by
organism is due to the presence of lipophilic and hydro- Knetting and Zajic [15]. It contained per liter deionized
philic cell wall and cell membrane components, some of water: KH2PO4, 2.0 g; K2HPO4, 5.0 g; (NH4)2804, 3.0 g;
which are surface-active. Therefore, the cell itself can NaC1, 0.1 g; FeSO 4 97H20, 0.01 g; MgSO 4 97H20, 0.2 g;
demonstrate significant emulsifying activity and act as a CaC12' 2H20, 0.01 g; and MnSO4" H20 , 0.002 g.
biosurfactant agent [25]. Secondly, the solvent action of Four hydrocarbons were used in this study. Aviation
the hydrocarbon on lipophilic cell surface components fuel, similar to the fuel spilled at Traverse City, Michigan,
may cause the cell to lose its structural integrity [16] was blended by Exxon Corporation. It contained 70~o
releasing surface-active compounds into the medium. 2,2-dimethyl butane, 20 ~o 2-methylbutane (IC5) and 10
Therefore, microbes with emulsification ability were toluene. Kerosene was obtained from Fisher Scientific,
further examined to identify the biosurfactant producers Houston, Texas. Number 2 Diesel Fuel Oil was obtained
by: (1)determining the isolates which produced cell-free from Exxon Company, U.S.A., Baytown, Texas. Unlead-
supernatant fluids with emulsifying abilities, thereby ed gasoline was purchased at a local service station. The
eliminating the isolates exhibiting emulsifying ability due hydrocarbons were filter-sterilized using filter assemblies
solely to the presence of whole microbial cells; and (2) (Gelman, Ann Arbor, MI) with 0.2-#m pore size poly-
measuring surface tension changes produced by cells propylene membranes.
grown on glucose with and without hydrocarbon amend-
ments. Surface tension reduction by actively growing cul- Microbial cultures
tures was used as an indirect indicator of biosurfactant Microorganisms from Traverse City, Michigan, were
production. Hydrocarbon was added because Duvnjak isolated by adding 1 g of subsurface material to 9.5 ml of
et al. [9] showed that a glucose-grown Corynebacterium 0.1 ~o sodium pyrophosphate in screw-cap bottles. The
produced a cell-bound surfactant which was subsequently mixture was placed on a reciprocal shaker for 1 h to
released after the addition of hexadecane. Using this produce a well-dispersed suspension. The suspension was
protocol, 13 isolates were identified as possible bio- diluted serially in phosphate-buffered saline (1.24g
surfactant producers. Na2HPO4, 0.180 g NaH2PO4" H20 and 8.5 g NaC1 per
liter deionized water) and plated in triplicate on one-half
MATERIALS AND METHODS strength Nutrient Broth (BBL, Cockeysville, MD) with
1.5 ~o Bacto-Agar (Difco, Detroit, MI) to isolate hetero-
Sampling sites and procedure trophs. The plates were incubated at room temperature
Subsurface materials were collected by a procedure (approx. 22 ~C). After the number of colonies stabilized,
developed by Dunlap et al. [8] and modified by Wilson viable cell counts in the three subsurface samples were
et al. [23] which prevented contamination by surface determined. Three successive streak plate isolations were
microbes. Three subsurface samples were collected from performed on isolates selected for emulsification tests to
a U.S. Coast Guard station in Traverse City, Michigan, ensure that pure cultures were obtained.
at the site of an aviation fuel spill. The core materials were Nineteen hydrocarbon-degrading microorganisms
collected from uncontaminated, contaminated and bio- were obtained from H.F. Ridgway of the Orange County
stimulated zones. The biostimulated sample was collected Water District, Fountain Valley, California. These
from the site of a field demonstration of patented microorganisms were isolated on a mineral salts agar
proceSs(es ) [17,18] for in situ bioremediation of aquifers incubated in the presence of gasoline vapors. They were
contaminated with petroleum hydrocarbons. At the time obtained from two subsurface sites contaminated with
of sample collection, the indigenous subsurface microflora unleaded gasoline, at Seal Beach and San Diego,
in the biostimulated zone was being stimulated to degrade California.
the contaminants by the addition of a nutrient solution Isolates from Traverse City and hydrocarbon-degrad-
composed of nitrogen, phosphorus, and oxygen in the ing microorganisms were maintained on one-half strength
form of hydrogen peroxide [22]. The contaminated Nutrient Agar slants and R2A agar slants (Difco, Detroit,
sample was obtained from the zone of contamination MI) respectively, and transferred to agar plates of the
outside the biostimulated test plot. The uncontaminated same compositions 7 days prior to use in emulsification
sample was taken from a nearby location of similar depth tests.
and moisture content. All samples were taken at depths
of approx. 194 to 201 inches and were composed of mix-
tures of sand and gravel.