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Hydrocarbon degrading bacteria at Oil Springs, Texas.

Abstract. -- Hydrocarbon degrading bacteria capable of heterotrophic growth in air were isolated from the oily mousse (biodegraded oil) from two separate sites at Oil Springs in east Texas. This is an area of long-standing crude oil seepage into a freshwater system. Of the four species of bacteria isolated from one site, and the five from the other, only two species were common to the oil at both sites. The isolates of these common species, however, were biotypically different from each other. Samples from both sites contained strains capable of growth on crude oil as a sole source of carbon (unconditional strains) along with strains that could degrade oil if proteose peptone was available in the growth medium (conditional strains). Additionally, the groups of isolates as a whole from each sample displayed remarkable similarities in the sizes of extrachromosomal elements, adhesion to crude oil, and hydrocarbon substrate utilization. The results indicate that common selection pressures may have produced groups of bacteria of different genera with similar physiologies at both sites.


Hydrocarbon degrading bacteria are widely distributed in nature (ZoBell 1946; Atlas 1981; Leahy & Colwell 1990). They may constitute up to 100% of the viable bacterial community in areas exposed to hydrocarbons (Atlas 1981). These communities develop at least partly from the autochthonous bacteria when the area is first exposed to hydrocarbons. The numbers and proportions of the hydrocarbonoclastic bacteria in the community will increase after exposure (Colwell & Walker 1977; Atlas 1981; Floodgate 1984; Cooney 1984). Eventually, only a few genera may dominate in the community (Llanos & Kjoller 1976); or, community diversity may remain unchanged (Pinholt et al. 1979; Olsen & Sizemore 1981) or even increase (Hood et al. 1975). These communities characteristically degrade hydrocarbons at higher rates than similar bacterial communities in unexposed areas (Leahy & Colwell 1990). In general they develop as the result of the availability of hydrocarbons for bacterial growth, apparently favoring strains that can grow on hydrocarbons over those that cannot. The predominance of hydrocarbon-utilizing bacteria may become permanent in areas subject to chronic exposure.

Petroleum-polluted environments also contain a much higher number of plasmid-bearing bacterial species than similar unpolluted areas (Hada & Sizemore 1981; Leahy & Colwell 1990; Ogunseitan et al. 1987). In Pseudomonas spp., the genes responsible for hydrocarbon degradation often reside on plasmids. In these species the genes responsible for degradation of toluates, camphor, salycilate, alkanes, and naphthalene are found on, respectively, the TOL, CAM, SAL, OCT, and NAH plasmids (Chakrabarty 1976). In other known hydrocarbon-degrading bacteria such as Acinetobacter sp. HOl-N and A. calcoaceticus the genes are located chromosomally (Singer & Finnerty 1984).

Interest in the degradative activities of hydrocarbonoclastic bacteria in marine and terrestrial environments has produced many studies to date. Not as much information has been obtained, however, on similar bacteria found in freshwater systems. Furthermore, most studies have been carried out in areas following accidental spills where selection pressures exerted by the oil may be of relatively short duration. Knowledge of the bacteria present in areas subjected to long-term, chronic exposure is lacking and would be of scientific interest since bacteria in these areas likely have formed stable associations. Oil Springs, Texas is such an area of long-standing hydrocarbon exposure (Pate 1987). This report characterizes hydrocarbon degrading bacteria capable of heterotrophic growth in air from the oily mousse at two sites there.

Materials and Methods

Site description and sampling. -- The oil seepage was located in a forested area about 25 miles southeast of the Stephen F. Austin State University campus. Historical records indicate that as early as 1790 pioneers used the oil on the surface in this area for axle grease while traveling westward (Pate 1987). The first producing well in Texas also was drilled in this area in 1866.

Samples of oily mousse (biodegraded oil/water mixture) were collected from two locations: 1) a site where crude oil seeps into a small basin of freshwater and 2) the bank of a stream receiving crude oil seepage. This stream also receives the discharge from the basin and was sampled approximately 100 m upstream of that point. The two sites are separated by a distance of approximately 200 m.

Isolation and identification of bacterial species. -- Samples were returned at ambient temperature (approximately 25[degrees]C) to the lab. Approximately one gram was used to inoculate nutrient broth containing 10 gm/l crude oil as an enrichment for hydrocarbon-utilizing strains. These 24 h cultures then were streaked onto nutrient agar (Difco) to produce bacterial colonies. Preliminary sorting of isolates was carried out on the basis of colony and cellular morphologies, cellular arrangements, and Gram stain reaction. Each unique isolate then was identified by using a combination of the following tools: Bergey's Manual of Systematic Bacteriology, John G. Holt (ed.), Williams and Wilkins, Baltimore / London; 20E and Rapid NFT Identification Systems (BioMerieaux-Vitek), and Microscan Rapid Identification System (Baxter).

Media and growth conditions. -- The components of each growth medium are listed in Table I. All incubations were carried out at 25[degrees]C with vigorous shaking.

Extrachromosomal DNA isolation and agarose electrophoresis. -- An isolation procedure designed to recover both large and small plasmids was used. Bacteria were grown overnight on Luria agar at 25[degrees]C. Approximately one square centimeter of cells was scraped from the plate and resuspended in 0.3 ml of lysis buffer containing 50 mM Tris-HCl and 3% (w:v) sodium dodecyl sulfate adjusted to pH 12.6 with 10 M NaOH. Cells were incubated at 60[degrees]C for 30 minutes. The resulting lysate was extracted with an equal volume of Tris-buffered phenol: chloroform (1:1, pH 7.5). The aqueous phase was recovered and electrophoresed through 1% (w:v) agarose gels in TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA). DNA was stained with ethidium bromide and visualized with 254 nm wavelength UV light.

Hydrocarbon utilization. -- Individual isolates were tested for the ability to utilize crude oil as a sole source of carbon and energy for growth by inoculating 100 ml of OSS broth (Table 1) with 0.1 ml of an 18-24 hour-old culture growing in PPSS broth (Table 1). OSS broth cultures were incubated seven days after which the remaining hydrocarbon was measured by infrared spectrophotometry according to Standard Methods for the Examination of Water and Wastewater (American Public Health Association 1990). In some experiments identical tests were conducted in PPSS broth. The ability to utilize diesel fuel, mineral oil, naphthalene, and toluene as sole sources of carbon and energy for growth was determined after incubation for seven days in DSS, MOSS, NSS and TSS broths (Table 1), respectively. Growth was considered positive if the cultures became turbid and dense populations of cells were detected microscopically.

Microbial Adhesion to Hydrocarbon (MATH) Assays. -- MATH assays were carried out by a modification of the method of Rosenberg et al. (1980). Cultures that were 18-24 hours old were washed once and resuspended in SS (Table 1). The density of the washed cultures was adjusted with SS to approximately 0.1 absorbance units at 600 nm wavelength. Two-tenths of a ml of crude oil was added to a 1.0 ml portion of the resulting suspensions which were then vigorously mixed for 120 seconds after which the hydrocarbon phase was allowed to separate from the aqueous phase for 15 minutes. The absorbance of the aqueous phase was measured. The percentage decrease in absorbance is a direct measurement of the percentage of cells removed from the suspension by adhesion to the crude oil.


The four species of bacteria isolated from the basin and the five species from the stream site samples are listed in Table 2. All of these, with the exception of Erwinia, are common in freshwater habitats.

It is probable that other species were present in the samples and were not culturable by the enrichment and isolation procedure. The organisms that were isolated, therefore, probably do not constitute the entire bacterial community present in the oil.

A typical electrophoretic gel is shown in Figure 1. All of the strains tested, except Pseudomonas aeruginosa CRFY1, contained the smaller of the two size classes of extrachromosomal elements present. Additionally, Enterobacter cloacae OS2A and CRFY2, Serratia marcescens CR2B, and Erwinia americana CRFW all possessed the larger size class of element. No other size classes of elements were detected.

Each isolate was tested for its ability to utilize crude oil as a sole source of carbon and energy for growth in OSS broth (Table 2). Only P. aeruginosa OSB1 and Alcaligenes faecalis OS2B from the basin site and E. americana CRFW, P. aeruginosa CRFY1, and Flavobacterium odoratum CRLY from the stream site utilized the oil. The other species were unable to grow or degrade the oil in this medium. The inability of the species to grow in OSS apparently was due to inability to utilize the oil as a carbon source and not to a missing growth factor since all could grow in SS supplemented with 1% (w:v) glucose.

When similar experiments were conducted in OPPSS broth, which contains proteose peptone as an additional carbon source, degradation patterns changed for most of the isolates (Table 2). Degradation of the oil by the two P. aeruginosa isolates was reduced in this broth. This likely was due to repression of enzyme activity by constituents of the proteose peptone, a phenomenon which has been observed in this species during paraffin oxidation (van Eyk & Bartles 1968). Degradation by all of the other isolates increased markedly, except for F. odoratum CRLY and Bacillus cereus OS3 which remained unchanged. It is unknown whether the oil was utilized for growth or was degraded by cooxidation. All of the isolates grew vigorously in OPPSS and so failure to degrade the oil was not due to growth inhibition by components of the oil.


The experiments in OSS and OPPSS broths provide a basis for separating each isolate into one of three categories: unconditional degrader, conditional degrader, or non-degrader. The unconditional degraders can utilize oil as a sole source of carbon and energy. Isolates in this category are P. aeruginosa OSB1 and A. faecalis OS2B from the basin site and E. americana CRFW, P. aeruginosa CRFY1, and F. odoratum CRLY from the stream site. The conditional degraders can degrade oil when an additional carbon source is present, in this case proteose peptone. The isolates in this category are E. cloacae OS2A from the basin site and S. marcescens CR2B and E. cloacae CRFY2 from the stream site. The only non-degrader in either medium was B. cereus OS3 from the basin site.

Several attempts were made to induce growth of the conditional degraders and the non-degraders by culturing them in OSS broth in various combinations with the unconditional degraders (data not shown). All attempts failed; therefore, these species apparently cannot utilize the metabolites produced by the unconditional degraders during growth on oil. All species, however, could grow together in OPPSS broth and so growth inhibition of one species by another was not a factor in these results.

No correlation was found between utilization of crude oil by an isolate and the ability to adhere to crude oil (Table 2). Among the unconditional degraders, only A. faecalis OS2B and F. odoratum CRLY adhered. Neither of the P. aeruginosa isolates adhered, a finding which is consistent with previous reports on this organism (Rosenberg, 1991). However, during growth of these organisms the medium became foamy and the oil appeared to be emulsified. A bioemulsifier may therefore have been produced, allowing these strains to utilize the oil without adhering to it. The other unconditional degrader, E. americana CRFW, did not adhere. Of the conditional degrader strains, only S. marcescens CR2B was observed to adhere to crude oil; this finding is consistent as well with previous experiments with this species (Rosenberg 1991). The non-degrader strain (B. cereus OS3) also was non-adherent as has been observed previously (Rosenberg 1991).

The unconditional degrader strains were tested for the ability to utilize various components of crude oil. Mineral oil was chosen to represent a mixture of n-aliphatic compounds; diesel fuel was chosen to represent a mixture of straight and branched chain hydrocarbons; and naphthalene and toluene represented specific aromatics. Results are given in Table 3. There was remarkable homogeneity among the strains. All utilized diesel fuel; all but F. odoratum CRLY utilized mineral oil; and none was able to utilize naphthalene or toluene under the experimental conditions. The conditional degraders and the non-degraders were not tested.


The oily mouse from the basin and stream sites contained different groups of bacteria. Although both samples contained a Pseudomonas aeruginosa and an Enterobacter cloacae strain, the strains from each were biotypically different from each other. No other cultured species were common to both samples. These assemblages of bacteria, therefore, appear to have formed independently.

The unconditional degrader strains represent species which are known to utilize crude oil for growth (Bartha & Atlas 1977) except for Erwinia, which to our knowledge has not been reported to do so. This isolate is unusual, therefore, and its presence in the oily mousse is unexpected since this genus is associated with plants in nature.

Notable similarities were found in the physiological categories of bacteria in the samples from both sites. Both contained unconditional and conditional degrader strains. Furthermore, among the unconditional degraders, there was almost complete homogeneity with respect to the substrates (diesel, mineral oil, naphthalene, or toluene) that can be utilized for growth. The unconditional degrader strains may be important hydrocarbon oxidizing strains at the sampled sites, while perhaps the conditional degrader strains also consume or cooxidize a portion while utilizing an additional carbon source for growth. The laboratory experiments do not indicate that the unconditional degrader strains can supply carbon for growth of the conditional strains in the form of hydrocarbon oxidation products.

The presence of extrachromosomal elements in the isolates from both sites is consistent with previous observations in other systems (Hada & Sizemore 1981; Leahy & Colwell 1990; Ogunseitan et al. 1987). In those systems, extrachromosomal DNA genes may have contributed to the ability of the isolates to degrade oil. It is unknown at this point whether there is a similar function for the DNA in the Oil Springs isolates.

The presence of adherent strains in the samples is readily understood as cells of these organisms likely posses hydrophobic cell surfaces or fimbriae, or produce adhesins (Rosenberg 1991), which would maintain physical contact between the cell and the crude oil substratum. For example, the hydrophobic pigment, prodigiosin, of Serratia marcescens has been shown to increase adhesion of the cells to hydrocarbons (Rosenberg 1984) as well as has the serraphobin outer surface protein of this organism (Bar-Ness & Rosenberg 1989). Serratia marcescens CR2B produces the red pigment prodigiosin characteristic of the species and probably adheres at least for that reason.

The presence of hydrocarbon non-adherent strains may be explained by their adherence to organic particles within the oily mousse or co-adhesion to the adherent strains. Pseudomonas aeruginosa for example is well-known to produce exopolysachharides which allow it to form biofilms on a variety of surfaces. Cells of other species can become trapped in these biofilms as well (Wilderer & Characklis 1989).

The physiological similarities between the bacterial populations from the two samples may be a result of selection pressures exerted by the availability of the same crude oil at both sites. Crude oil from this location is high in C-20 or larger compounds with cyclic hydrocarbons present in lesser amounts (Pike 1977). The preference of the unconditional degrader strains for diesel and mineral oil likely reflects this content of the crude oil. Non-utilization of naphthalene and toluene by the isolates might be expected since cyclic hydrocarbons are not present in high amounts in this oil. This along with the hydraulic flow that moves bacteria and oil downstream away from the seep may inhibit strains capable of utilizing the cyclic portion from colonizing the oil at the seepage sites. Common selection pressures, therefore, appear to have produced the two different groups of hydrocarbonoclastic bacteria with similar physiologies at both sites, an expected outcome in stable, similar microenvironments. The unconditional strains do not appear to provide nutrients for the conditional strains and so the interaction between them in the oily mousse, if any, is not clear. Further investigations will reveal whether some other interaction exists and whether additional similarities can be found at the two seepages among the sediment and free aquatic bacteria not cultured in this study.
Table 1. Composition of growth media.

Medium Composition (gm/liter)

1. SS (a) (N[H.sub.4])[.sub.2]S[O.sub.4],(2); [K.sub.2]HP[O.sub.4],
 (14); K[H.sub.2]P[O.sub.4], (6); MgS[O.sub.4], (0.2)
2. OSS Crude Oil, (2); in SS
3. PPSS Proteose peptone (Difco), (10); in SS
4. OPPSS Crude Oil, (2); proteose peptone, (10); in SS
5. DSS Diesel fuel, (2); in SS
6. MOSS Mineral oil, (2); in SS
7. NSS Naphthalene, (2); in SS
8. TSS Toluene, (2); in SS
9. Luria agar Tryptone, (10); yeast extract, (5); NaCl, (5); agar, (15)

(a) Spizizen, J. 1958. Transformation of biochemically deficient strains
of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. USA.

Table 2. Degradation of crude oil and microbial adhesion to hydrocarbon
(MATH) assays of bacterial isolates.

Bacterial isolate Amount of crude oil MATH assay (b)
 degraded (a) (ppm) percentage decrease
 in each medium in optical density

Basin site
1. P. aeruginosa (c) OSB1 1500-2000 1-500 0
2. E. cloacae (c) OS2A 0 1500-2000 0
3. A. faecalis OS2B 1500-2000 1500-2000 75
4. B. cereus OS3 0 0 0

Stream site
1. S. marcescens CR2B 0 1000-1500 52
2. E. americana CRFW 1-500 1500-2000 0
3. P. aeruginosa (c) CRFY1 1000-1500 1-500 0
4. E. cloacae (c) CRFY2 0 1500-2000 0
5. F. odoratum CRLY 1000-1500 1000-1500 35

(a) Starting concentration of crude oil was 2000 ppm. Values reported
are in ranges consistently observed in several trials.
(b) Measurements as described in Materials and Methods. Averages of
three trials.
(c) The two P. aeruginosa and the two E. cloacae isolates are different

Table 3. Growth of unconditional degrader strains on diesel fuel,
mineral oil, naphthalene, and toluene as sole sources of carbon and

Isolate Growth in each medium *

Basin site
1. P. aeruginosa OSB1 + + - -
2. A. faecalis OS2B + + - -

Stream site
1. E. americana CRFW + + - -
2. P. aeruginosa CRFY1 + + - -
3. F. odoratum CRLY + - - -

* growth, (+); no growth, (-). Growth was measured as described in
Materials and Methods. See Table 1 for composition of media.


This work was supported in part by special item appropriation from the State of Texas. The authors gratefully acknowledge the assistance of the following students: R. Elton Sloan, Thomas Vyles, Aaron Schultz, Garlene Kayanan, and Kalyn Sowell.

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Pate, C. S. 1987. A subsurface study of the queen city formation in Nacogdoches and Angelina counties. Unpublished thesis, Stephen F. Austin State University, Nacogdoches, 160 pp.

Pike, R. W. 1977. A study of the biodegradation of an east Texas shallow well crude oil and a deep well crude oil using an autochthounous bacterium. Unpublished thesis, Stephen F. Austin State University, Nacogdoches, 46 pp.

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Thomas G. Benoit and Robert J. Wiggers

Department of Biology, Stephen F. Austin State University, Nacogdoches, Texas 75962
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Author:Benoit, Thomas G.; Wiggers Robert J.
Publication:The Texas Journal of Science
Geographic Code:1U7TX
Date:May 1, 1995
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