Microbial Degradation of Model Petroleum at Low Temperatures J. D. W A L K E R A N D R. R. C O L W E L L

Department of Microbiology, University of Maryland, College Park, Maryland 20742

Abstract Two areas of Chesapeake Bay, Colgate Creek in Baltimore Harbor and Eastern Bay, axe presently under study, with routine sampling of water and sediment for petroleumdegrading microorganisms (bacteria, yeasts, and fungi) by direct plating and enrichment culture. Selected physical and chemical parameters are recorded for each sampling site, and water and sediment samples are extracted for hydrocarbons. Numbers of petroleumdegrading microorganisms enumerated by direct plating were found to correlate with the concentration of benzene-extractable material and were higher for the Colgate Creek than for the Eastern Bay site. Petroleum-degrading microorganisms were isolated from water and sediment samples at environmental temperatures of 0~ 5 ~, and IO~ A salts medium supplemented with nitrate and phosphate was used to provide optimum conditions for petroleum degradation, whereas Chesapeake Bay water was used to simulate natural environmental conditions. Use of a model petroleum permitted quantitative measurement of utilization of individual hydrocarbons ranging in complexity from simple alkanes to polynuclear aromatic hydrocarbons. Higher growth yields and maximum hydrocarbon degradation was observed for microorganisms in the salts medium at 0 ~ 5 ~ and 10~ although significant quantities of hydrocarbons were utilized in some samples grown in a medium for which Chesapeake Bay water was the diluent. Bacterial hydrocarbon degradation accounted for most of the model petroleum utilization at 0 ~ and 5~ However, oscillations of bacterial populations, with significant growth of yeasts, was observed at 10~ Photomicroscopy and scanning electron microscopy revealed aggregates of bacteria, yeasts, and fungi associated with oil globules. From preliminary identification and classification of the hydrocarbon-utilizing bacteria, members of the genera Vibrio, Aeromonas, Pseudomonas, and Acinetobacter were present in the enrichment cultures. From results of this study, it is concluded that utilization of model petroleum at low temperatures is a function of the types and numbers of microorganisms present in an original inoculum taken from the natural environment.

Introduction Chesapeake Bay is t h e largest a n d m o s t i m p o r t a n t estuary w i t h r e s p e c t t o a q u a t i c f a u n a in t h e m i d - A t l a n t i c c o a s t a l region [ 7 ] . A research p r o g r a m u n d e r way in o u r l a b o r a t o r y was originally designed t o i d e n t i f y a n d e n u m e r a t e t h e

63 MICROBIAL ECOLOGY, Vol. 1, 63-95 (1974) 9 by Springer-Verlag New York Inc.

64

J.D. Walker and R.R. Colwell

petroleum-degrading microorganisms naturally occurring in Chesapeake Bay throughout the year, i.e., the annual cycle o f these microorganisms. The capability of these microorganisms to degrade petroleum, both in nature and under laboratory conditions, is also being assessed [12], and studies to date have demonstrated that microbial populations in Chesapeake Bay are capable of utilizing aliphatic and aromatic hydrocarbons [5]. Although the average water temperature o f Chesapeake Bay is about 15~ for 6 months o f the year the temperature o f the water is ~< 10~ [9]. As recently as 1969, ZoBell [7] pointed out that the contribution o f psychrophilic marine microorganisms to petroleum degradation was unknown. A few reports have since appeared which treat the problem o f petroleum degradation at low temperature [1, 11, 18]; however, the information is incomplete, at best. Since the interests of our laboratory includes the study o f petroleum degradation on a seasonal basis and because of the paucity o f reports on petroleum degradation at low temperatures, we undertook a study o f the ability o f mixed cultures o f microorganisms present in Chesapeake Bay water and sediment, when the temperature of the natural environment was between 0 ~ and 10~ to utilize model petroleum at 0 ~ 5 ~ and 10~ Materials and Methods

Sampling Water and sediment samples were collected with Niskin (General Oceanics, Miami, Florida) [8] and Ponar Samplers (Wildlife Supply Co., Saginaw, Michigan), respectively. Dilutions of samples and inoculations, following aseptic procedures, were performed on board ship as described previously [ 12]. .

Media The media used in this study for isolation of heterotrophic and petroleum-utilizing aerobic microorganisms have been described elsewhere [12]. The liquid culture medium which was employed in this study was prepared with either sterilized Chesapeake Bay water or a salts solution (10.0 g NaCl, 0.5 g MgSO4, and 1.0 g NH4NO 3 per liter of distilled water and the appropriate ratio of KH2PO4:K2HPO4, vide infra) as indicated, Sterile solutions of 10% KH2PO 4 and K2HPO 4 were added to the sterile salts solution at ambient temperature to prevent precipitation. In order to culture fungi and yeasts, the pH of the medium was adjusted to 5.5 with a ratio of 9.4 ml KH2PO4:0.6 ml K2HPO 4 per liter of medium, along with the addition of streptomycin (50 ug/ml). In the case of the medium used to culture bacteria, the pH of the medium was adjusted to 7.0 by using a ratio of 3.0 ml KH2PO4:7.0 ml K2HPO4 per liter, coupled with the addition of fungizone (10 ~g/ml). For mixed cultures of bacteria, yeasts, and fungi, the pH of the medium was adjusted to 7.0 and all antibiotics were omitted. In each instance, the medium was overlaid with sterile model petroleum (1%, v/v) prior to inoculation. Microbial growth in liquid media was determined by plating appropriate aliquots or dilutions of the sample onto plate count agar (pH 7.0) prepared with estuaxine salts (10.0 g

Microbial Degradation of Petroleum

65

NaC1, 2.3 g MgC12, and 0.3 g KCI per liter distilled water) and an estuarine yeast and fungi medium (pH 5.5) composed of 10.0 g dextrose, 1.0 g yeast extract, 4.0 g proteose peptone, 20.0 g Difco purified agar (Difco Laboratories, Detroit, Michigan), and 10.0 ml 85% lactic acid per liter of estuarine salts.

Culture Systems To measure microbial growth on model petroleum, 250-ml flasks (samples were run in duplicate) containing 100 ml salts medium or sterile Chesapeake Bay water were inoculated with sediment or water immediately after sample collection while on board ship. After the return to the laboratory, the inoculated flasks were placed in a New Brunswick G-26 Psychrotherm (New Brunswick Scientific, New Brunswick, New Jersey) and reciprocally shaken at sixty 1.25 in. strokes per minute at 0~ 5~ or 10~ Growth was monitored by plating every 7 days. An uninoculated control, used to monitor weathering of the model petroleum, was examined periodically for contamination.

Chemical Analysis Water and sediment samples were extracted with benzene as described previously [12]. At the end of stationary phase, the culture in the flasks not employed for the estimation of microbial growth was used to determine the extent, if any, of microbial utilization of the model petroleum. One hundred microliters n-octane was added to each flask prior to extraction, followed by extraction with 50 ml n-hexane (twice)for quantitative extraction (99%+) of the model petroleum. The extract was dried over 5 g anhydrous NaSO 4 and filtered through Whatman No. 1 filter paper. The paper, along with the NaSO4, was rinsed with two 25-ml volumes of n-hexane. The extract was concentrated to about 8 ml by flask rotary evaporation at 35~ and, subsequently, to 1.5 ml by evaporation under nitrogen. Samples were stored under nitrogen in culture tubes fitted with Teflon-sealed caps. Analyses were accomplished within 48 hr using gas-liquid chromatography (GLC).

Gas-Liquid Chromatography The chromatograms were obtained on a Shimadzu Model GC-4BMPF gas chromatograph (American Instrument Co., Silver Spring, Maryland) equipped with a single flame ionization detector. Two types of glass columns were used: a 3 mm • 1.5 m column packed with 3% OV-17 on 80/100 mesh Ohimalite and 3 mm x 1.5 m column packed with 3% OV-1 on 80/100 mesh Shimalite. Nitrogen, the carrier gas, was run through the columns at a flow rate of 40 ml/min. Temperature was programmed from 60 ~ to 300~ at 5~ The hydrocarbons were identified by retention time. Standards of each hydrocarbon were also employed. Individual components were quantitated by calculating the peak area using a Hewlett-Packard Model 3373B Integrator. The percent of individual hydrocarbon remainhag after weathering or microbial degradation was calculated by comparing each component peak with its corresponding peak in the control sample and calculating the difference in peak areas. Column efficiency and detector responses were measured by running a known hydrocarbon m!xture, No. 19251, obtained from Applied Science (State College, Pennsylvania) containing C14-C20 saturated and unsaturated hydrocarbons. A relative error of less than 2.5% for all components was obtained.

Physical and Chemical Parameters In order to determine whether there was a correlation of the total viable, aerobic, and heterotrophic microbial populations present in each of the samples examined with specific

66

J.D. Walker and R.R. Colwell

environmental conditions, selected physical and chemical parameters were measured at the time of sampling. Measurements were taken as described elsewhere [ 12 ].

Chemicals All solvents were of reagent grade spectro-quality for GLC analysis. Normal, cyclic, and aromatic hydrocarbons, 99+% purity, were obtained from Chemical Samples Company (Columbus, Ohio). Pure samples of 1,2-benzanthracene, perylene, and pyrene were provided by the Sun Oil Company (Marcus Hook, Pennsylvania) through the courtesy of R. L. Raymond.

Scanning Electron Microscopy Specimens were aseptically transferred from actively growing cultures to sterile plastic coverslips and stained with 2% osmium tetroxide vapors for 3 hr. Then specimens were frozen in liquid nitrogen and transferred to an Edwards tissue dryer at -80~ After specimens had dried, they were warmed to ambient temperature. Then the plastic coverslips were cut and attached to stubs with double stick tape. The specimens were then coated with 500 A of gold palladium and viewed under the scanning electron microscope.

Composition of Model Petroleum The model petroleum was designed to permit measurement of degradation of a combination of simple and complex hydrocarbons. The composition of the model petroleum used in this study is given in Table 1. Although the model petroleum is very simple in composition, compared with natural petroleum, it provides a mixture that can be conveniently and accurately analyzed, both qualitatively and quantitatively, by GLC, i.e., each component can be readily quantitated (Fig. 1). Furthermore, the composition of the model petroleum can be altered, depending on the nature of the hydrocarbons under examination.

Results

Sampling Stations and Experimental Protocol Sediment and water from Colgate Creek in Baltimore Harbor contained more benzene-extractable compounds than did the Eastern Bay samples (Table 2). The benzene.extractable compounds from the Eastern Bay samples are probably not petroleum in nature, whereas those of Colgate Creek were identified as paraffins, alkylbenzenes, alicyclic, and aromatic hydrocarbons typical of petroleum [13]. In addition, the numbers of petroleum-utilizing microorganisms were significantly greater for the Colgate Creek samples than those from Eastern Bay (Table 2).

Effect of Temperature on Microbial Growth and Model Petroleum Degradation Colgate Creek and Eastern Bay samples inoculated into model petroleum and incubated at 0~ reached stationary phase at 42 - 49 days (Figs. 3 and 4). Parallel cultures reached stationary phase between 28 - 35 days at 5~ (Figs. 5

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Microbial Degradation of Petroleum

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Table 1 Composition o f the Model Petroleum Used in This Study Hydrocarbon

Percent

Peak number a

7.80 7.80 7.80 7.80 7.80 7.80 7.80 7.80 7.80 7.80 7.80

4 6 7 8 10 11 12 14 15 16 17

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85.80 Branched alkanes

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3.90

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2

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1,2-Benzanthracene Perylene Pyrene

0.39 0.39 0.39

19 20 21

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and 6). The initial stationary phase for cultures incubated at 10~ was reached at approximately 28 days for cultures in salts medium and approximately 7 - 21 days for cultures in Bay water (Figs. 7 and 8). However, the latter resulted in lower growth yields at the first stationary phase (Fig. 8). Oscillations o f each population occurred after the first stationary phase (Figs. 7 and 8). In general, higher growth yields in salts medium were obtained for those cultures incubated

68

J.D. Walker and R.R. Colwetl

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Fig. 1. Gas chromatographic tracing of hydrocarbons comprising the model petroleum separated on an OV-17 column programmed to run from 60 ~ to 300~ at 5~ at IO~ rather than at 0 ~ or 5~C. However, for some microbial populations, growth at 5 ~ and 10~ were approximately equal, e.g., Colgate Creek sediment and Eastern Bay sediment (Figs. 3, 5, and 7). In the Chesapeake Bay water medium, the growth yield, in general, increased with temperature, in the range 0~ - IO~ (Figs. 4, 6, and 8). Degradation of hydrocarbons of the model petroleum was greater at O~ except in the case of the Colgate Creek sediment samples cultured in Chesapeake Bay water medium which produced about the same degradation at all three temperatures (see Tables 3 - 5). Degradation of hydrocarbons was greater at IO~ than at 5~ for microorganisms of Colgate Creek sediment and water cultured in salts medium and for Eastern Bay sediment cultured in Bay water; otherwise, patterns of hydrocarbon degradation for these two temperatures were similar (Tables 4 and 5).

Effect of Inoculum Source and Different Media on Microbial Growth and Model Petroleum Degradation Bacterial utilization of model petroleum at 0 ~ and 5~ was greater using a medium supplemented with phosphate and nitrate (Tables 3 and 4). In most cases, this medium also resulted in the greatest utilization, at 10~ of the model petroleum hydrocarbons (Table 5). However, selected hydrocarbons were degraded to the same extent by cultures grown in the salts medium or Bay water, viz., hydrocarbon degradation by microorganisms from Colgate Creek water and Eastern Bay sediment. Higher cell yields were obtained from cultures supplemented with nitrate and phosphate and incubated at 0 ~ 5 ~ and 10~ with the highest cell yield

Microbial Degradation of Petroleum

71

obtained for the Colgate Creek water sample (Figs. 3, 5, and 7). Chesapeake Bay water inoculated with Colgate Creek and Eastern Bay water samples provided highest yields at 0~ as well at 10 ~ and 5~ (Figs. 4 and 6). Eastern Bay water samples produced similar yields at 0 ~ and 5~ when cultured in oil-salts medium and Chesapeake Bay water (Figs. 3 - 6). Cultures inoculated with Eastern Bay water samples and incubated at 10~ failed to grow because of the low indigenous populations (Table 2). Comparable microbial growth was noted in both the presence and absence of antibiotics at 0 ~ and 5~ Also, at 35 days, an unidentified yeast was observed in the Eastern Bay samples cultured in Bay water at 0 ~ and 5~ There was a lack of significant growth of fungi and yeast in the model petroleum at 0 ~ and 5~ Similar studies carried out at IO~ revealed considerable growth of yeasts (Figs. 7 - 10). These data reflect the seasonal variations in the indigenous populations at the time of sampling (Table 2). The presence of higher populations of yeast in April than in February (Table 2) resulted in growth of these microorganisms in model petroleum

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72

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(Figs. 7 and 8) and permitted evaluation of the effects of antibiotics on growth (Figs. 9 and 10) and hydrocarbon degradation (Table 5) by these populations. Growth and hydrocarbon utilization by Cladosporium resinae in the presence and absence of 0.1 /ag/ml fungizone was found to be similar (Fig. 9). Although low concentrations (0.I /ag/ml) of fungizone inhibited growth of the yeasts on the model petroleum, concentrations of 10.0 /ag/ml were necessary to inhibit growth of C. resinae (Fig. 9). The greatest amount of growth and of model petroleum utilization was observed for C. resinae cultured in the presence of 1.0/ag/ml fungizone (Fig. 9 and Table 5). Streptomycin was found to be ineffective in inhibiting growth and hydrocarbon degradation in the case of bacteria present in Colgate Creek sediment (Fig. I0, Table 5). Higher concentrations of streptomycin decreased the initial rate of growth and the time required to reach stationary phase (Fig. 10). However, concentrations of streptomycin used (5.0 or 50.0/ag/ml) permitted increased growth of yeasts (Fig. 10) as well as greater hydrocarbon degradation (Table 5). Cladosporium resinae was also uneffected by streptomycin and could be easily counted on plates designed to be selective for yeasts and fungi (Fig. I 1).

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Microbial Degradation of Petroleum

73

Effect of Source of Inoculum on Petroleum-Microorganism Interactions Bacteria from Eastern Bay sediment were observed inside hydrocarbon globules (Fig. 12), whereas yeasts and bacteria from Colgate Creek sediment were observed on the surface of the hydrocarbon globules (Fig. 13). The Pullularia sp. from Eastern Bay sediment revealed lipid globules similar to those observed by Walker and Cooney [ 17] for Cladosporium resinae cultured on hydrocarbons. Those Pullularia sp. and bacteria from Eastern Bay sediment were also examined by scanning electron microscopy (Fig. 14). Effect of Hydrocarbon Structure on Microbial Degradation of Model Petroleum Most of the components of the model petroleum were degraded to approximately the same degree at 0~ (Table 3). However, cyclohexane, cumene, naphthalene, and polynuclear aromatic hydrocarbons (PAH) were utilized more extensively by microorganisms present in Colgate Creek water and sediment samples than by microorganisms present in Eastern Bay water and sediment samples cultured in salts medium with model petroleum added (Table 3). Petroleum utilization for Colgate Creek and Eastern Bay water samples cultured

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74

J.D. Walker and R.R. ColweU

Table 3 Bacterial Utilization o f Model Petroleum at O~ a

Colgate Creek sediment Hydrocarbon Cyclohexane n-Decane Cumene n-Undecane n-Dodecane rr-Tridecane Naphthalene n-Tetradecane n-Pent adecane n-Hexadecane Pristane n-Heptadecane n-Octadecane n-Nonadecane n-Eicosane Phenanthrene

1,2-Benzanthracene Perylene Pyrene

Control 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

Salts 0.83 11.59 0.70 13.97 13.94 13.78 0.51 13.72 13.76 14.12 12.58 14.52 14.74 15.00 14.94 12.12 11.86 11.00 12.40

Water 50.03 79.96 51.01 66.05 62.72 61.37 52.17 61.04 61.31 61.83 59.58 63.31 63.38 64.27 64.36 60.97 60.10 58.09 60.61

a"Salts" and "water" refer to the diluent used in preparation of the hydrocarbon medium. "Salts" refers to the mixture of salts in distilled water (see Materials and Methods). "Water" indicates that the natural water of the area sampled was used as the diluent for the medium in which degradation of the hydrocarbons was tested.

Microbial Degradation of Petroleum

75

Percent b hydrocarbons remaining after microbial degradation Eastern Bay water

Colgate Creek water

Eastern Bay sediment

Salts

Water

Salts

Water

Salts

Water

9.09 29.03 9.00 24.07 24.56 22.87 8.88 22.98 23.08 23.32 23,45 23,41 23,61 23.83 23.85 22,52 22.45 22.51 23.38

28.03 23.63 27.20 25.13 24.84 24.74 26.13 24.97 25.57 26.60 25.29 27.84 29.07 30.00 30.28 25.44 25.43 24.23 26.60

31.42 38.21 30.73 37.03 35.10 34.39 29.92 34.29 34.36 34.71 34.77 34.86 35.16 35.56 35.56 34.39 34,62 34.43 34.45

3.72 22.96 3.1,3 21.22 20.61 19.63 2.17 19.53 19.73 20.06 19.33 20.63 20.79 2 I. 13 21.21 17.71 17.38 17.01 16.69

12.22 25.35 7.42 23.46 23.59 22.80 10.06 23.23 24.18 25.90 24.34 28.36 29.22 30.33 30.76 11.56 11.40 11.16 10.70

0.42 5.63 0.34 6.01 5.98 5.99 0.26 6.05 6.15 6.33 5.96 6.55 6.70 6.85 6.89 5.81 5.65 4.96 5.62

bCalculated as percent of individual peak areas obtained from an OV-17 tracing, 0 0 0 . programmed to run from 60 to 300 C at 5 C/mm.

76

J.D. Walker and R.R. Colwell

Table 4 Bacterial Utilization o f Model Petroleum at 5~ a

Colgate Creek sediment Hydrocarbon Cyclohexane n-Decane Cumene n-Undecane n-Dodecane n-Tridecane Naphthalene n-Tetradecane n-Pentadecane n-Hexadecane Pristane n-Heptadecane n-Octadecane n-Nonadecane n-Eicosane Phenanthrene 1,2-Benzanthracene Perylene

Pyrene aSee footnote in Table 3. b See footnote in Table 3.

Control 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

Salts 5.42 51.44 2.99 59.86 61.62 61.64 2.40 61.48 61.41 61.93 61.66 61.91 60.00 61.37 61.94 55.40 38.73 52.32 56.17

Water 7.27 61.31 5.61 62.18 62.64 62.49 4.08 62.23 61.99 62.06 62.39 61.38 61.88 63.59 64.60 60.99 59.23 60.70 59.76

Microbial Degradation of Petroleum

77

Percent b hydrocarbons remaining after microbial degradation Colgate Creek water

Eastern Bay sediment

Salts

Water

Salts

Water

Salts

Eastern Bay water Water

3.77 41.55 2.77 47.59 52.81 47.52 1.77 47.31 47.27 47.59 47.68 47.33 48.52 48.57 50.13 40.73 42.35 40.82 42.96

14.30 57.93 11.11 55.29 63.51 55.10 9.95 55.06 55.00 55.35 54.77 55.30 56.37 56.26 56.98 52.86 52.05 51.54 52.28

2.72 35.65 1.96 34.87 34.96 34.46 1.34 34.27 34.26 34.61 34.65 34.51 35.62 35.80 36.45 31.76 33.27 31.81 32.20

5.61 65.16 3.03 59.80 58.09 56.49 2.35 55.71 55.31 55.47 55.46 54.96 55.95 55.66 56.33 53.07 52.35 52.13 51.75

6.63 50.78 4.76 52.00 50.67 49.15 3.42 48.67 48.28 48.86 45.61 48.98 51.01 50.62 51.36 38.80 34.30 31.85 34.01

59.99 63.76 56.12 67.12 64.69 62.26 55.17 61.28 60.31 60.72 54.77 61.00 62.32 61.79 62.36 53.09 46.77 44.97 46.21

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in Bay water at 0~ was the same for both samples. Degradation of individual components was similar at 0 ~ and at 5~ However, at 5~ only cyclohexane, cumene, and naphthalene, and not PAH, were degraded to a significant degree by the Colgate Creek water and sediment samples inoculated into the salts medium. The Eastern Bay sediment samples demonstrated greatest utilization of the model petroleum. As observed at 0 ~ and 5~ significant utilization of alicyclic, aromatic, and polynuclear aromatic hydrocarbons was observed for microorganisms of Colgate Creek sediment and water cultured in salts medium with model petroleum at 10~ (Table 5). At IO~ degradation of normal and branched paraffins in oil-salts medium was greater for microorganisms of Eastern Bay sediment.

Effect of lnoculum, Temperature, and Antibiotics on Type of Petroleum-Degrading Bacteria Tables 6 through 8 provide a tentative identification of the isolates from the 0 ~ 5~ and 10~ experiments. The extensive methods of polyphasic taxonomy [4] are being applied to the isolates and will be the subject of a separate communication. All the isolates were Gram-negative rods. At 0~ only representatives of the genera Pseudomonas and Vibrio were found to be present IOa

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Microbial Degradation of Petroleum

81

in cultures inoculated with Colgate Creek samples. However, at 5 ~ and 10~ members of the genera Acinetobacter and Aeromonas were also isolated (Tables 6 - 8). Similar results were observed in classifying cultures inoculated with Eastern Bay samples incubated at 0 ~ and 5~ (Tables 6 and 7). Acinetobacter spp. and a Coryneform were isolated at 10~ Subtle differences were observed for bacteria from the same inoculum cultured in oil salts versus Bay water at the same temperature. Oil salts supported growth of a Pseudomonas sp. and Vibrio sp. from Colgate Creek water, but Bay water supported only a Pseudomonas sp. at O~ An inoculum from Eastern Bay sediment yielded growth of an unidentified isolate and a Vibrio sp. when cultured in oil salts and a Pseudomonas sp. and Vibrio sp. when cultured in Bay water at 0~ With Eastern Bay water as the inoculum, the oil-salts medium supported growth of a Vibrio sp. and Pseudomonas sp., but the Bay water medium, at 0~ supported growth of a Pseudomonas sp. and two unidentified isolates.

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82

J.D. Walker and R.R. Colweli

Similar variations in flora composition were observed for cultures at 5~ but the most striking differences were observed for cultures incubated at 10~ especially those inocula from Colgate Creek and Eastern Bay sediment. The inoculum from Colgate Creek sediment contained a Vibrio sp. and an unidentified isolate, when cultured in oil salts. When the same inoculum was cultured in Bay water, an Acinetobacter sp., a Vibrio sp., Aeromonas spp., and two unidentified isolates were observed. In the presence of fungizone the same inoculum yielded an Aeromonas sp. and a Vibrio sp., whereas in the presence of streptomycin, Vibrio spp., a Pseudomonas sp., and an unidentified isolate were observed. A Coryneform, an Aeromonas sp., and two unidentified isolates were observed when Eastern Bay sediment was cultured in oil salts. Culturing the same inoculum in Bay water yielded Aeromonas spp., Acinetobacter spp., a Vibrio sp., and an unidentified isolate. Differences were also observed when inocula from the same sampling station were cultured at 0 ~ 5~ and 10~ in the same media. Again, the most noticeable change was the appearance of representatives of a greater number of

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Fig. 8. Growth of indigenous bacteria (closed figures) and yeast (semi-closed figures) from Colgate Creek sediment (circles), Colgate Creek water (squares), and Eastern Bay sediment (triangles) culture in Chesapeake Bay water with model petroleum at I 0~

Microbial Degradation of Petroleum

83

genera at IO~ e.g., an Acinetobacter sp. and Aeromonas spp. from the Colgate Creek sediment inoculum and a Coryneform, Aeromonas spp., and Acinetobacter spp. from the Eastern Bay sediment inoculum. In addition to the observed oscillations in the total populations of Colgate Creek and Eastern Bay populations at IO~ (Figs. 7 ~ 10), a succession of the individual isolates was also noted (Figs. 15 and 16). The initial stationary phase in the Eastern Bay culture (Fig. 15) resulted from growth of an Aeromonas sp. which slowly died off. Most of the growth after the initial stationary phase was that of two unidentified strains and a Coryneform. In the Colgate Creek culture (Fig. 16), the initial stationary phase resulted from growth of an unidentified isolate, and further growtfi to a second stationary phase resulted from growth of a Vibrio sp. and an Aeromonas sp. This second stationary phase was accompanied by a decline in another Aeromonas sp. and two unidentified isolates. Discussion As part of an on-going program in microbial ecology, two stations in Chesapeake Bay are routinely sampled for petroleum-degrading microorganisms

'~n IO

Ill Io ~

i /1

~*~.~"~-'~_. ~

,~

I

0

7

14 21

28

55 42 49

56 63 70 77

T i m e (days)

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84

J.D. Walker and R.R. Colwell

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J.D. Walker and R.R. Colwell

Table 8 Some

Characteristics Growing

229 230 231 232 233 234 235 236 237 238 239 240 241 242 244 245 246 247 248 249 250 251 252 253 254 262 263 264 265 266

aCommon

name.

on

of Bacteria Isolated Model

Unidentified Unidentified

Petroleum

from

at

Cultures

10~

- - -

+ +

-

++-

+ +

+ +

+ +

Aeromonas

- -

+

-

+-

+

+

+

Coryneform a

-_

+

-

+ - _

+

+

Aeromo,~as

- -

+

+

+

+

+

Aeromonas

-

+

+

+

+

+

+

+

+-

-

Vibrio

+

Unidentified

+

+-

+

+

+

Vibrio

+

+-

+

+

+

Unidentified

+

+

+

+

+

Aeromonas

+

+

+

+

+

Aeromonas

+

+

+

+

+

Vibrio

+

+

+

+-

+-

Unidentified

+

+

+

Aeromonas

+

+

+

+

Vibrio

+

+

+

+

+

Unidentified

+

+

+

+

+

Vibrio

+

+

+

+

+

Vibrio

+

+

+

+

+

Pseudomonas

+

+

+

+

+

Vibrio

+

+

+

+

+

Vibrio

-

+-

-

-

+

+

+

+

Vibrio

-

+-

-

-

+

+

+

-

Pseudomonas

-

+-

-

+-

+

+

+

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-

+-

-

+-

+

+

+

Unidentified

- -

-+

+

+

+

Acinetobacter

-

-

+

+

+

Acinetobacter

-

-+

+

+

+

+

Acinetobaeter

+

+

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+

+

+

+

+

++

- -

-

+

+

+

Microbial

Degradation

87

of Petroleum

=

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+

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+

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+

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+

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+

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--

88

J.D. Walkerand R.R. Colwell

(Fig. 2). Eastern Bay, a relatively unpolluted and commercially productive region, has been shown from previous studies to contain stable bacterial populations in the water and sediment [6]. Colgate Creek in Baltimore Harbor, in contrast, is subjected to oil pollution from tank washings and accidental spills, which probably accounts for the greater amount of benzene-extractable material observed in this study, compared with that in Eastern Bay (Table 2). The percent benzene-extractables from Colgate Creek sediment on April 3, 1973 was four to five times lower than normally observed [5]. This observation can be explained by the occurrence of a heavy rainstorm on the previous day. Results given in this paper, describing the type and amount of benzene-extractable compounds, as well as the number of petroleum-utilizing microorganisms, indicate that the choice of Eastern Bay as the control site for comparison with the oil-polluted site, Colgate Creek, was appropriate. In order to assess the ability of Chesapeake Bay microorganisms to degrade petroleum at low, i.e., winter temperatures, water and sediment samples were collected in February and April 1973, when the water temperatures for the

A c

E 0 r

=. r

kt. r

0

714

Zt

~

35

4t~ d~

~

6~

7Q 7"t"

Time (days)

Fig. 10. Growth of indigenous bacteria (open figures) and yeast (semi-closed figures) from Colgate Creek sediment and Cladosporium resinae (closed figures) cultured in Chesapeake Bay water plus model petroleum supplemented with 0.5 /~g (circles), 5.0 /ag (squares), and 50.0 btg (triangles) streptomycin per milliliter medium at 10~

Microbial Degradation of Petroleum

89

Colgate Creek and Eastern Bay areas were 2.0 ~ - 11.2~ and 0.7 ~ - 10.2~C, respectively (Table 2). A salts solution supplemented with nitrate and phosphate (salts medium) was used to provide optimum conditions for petroleum utilization, whereas Chesapeake Bay water was used to simulate the natural environmental conditions. The lag phase observed for the Eastern Bay samples most likely represents either a time period required for adaptation of the mixed culture to the model petroleum or inhibition of microbial growth by nonvolatile components, as observed by Atlas and Bartha [1]. The latter alternative is supported by the fact

Fig. 11. Plates illustrating colonies of Cladosporium resinae and yeasts from cultures inoculated with Colgate Creek sediment and cultured in Bay water with model petroleum at 10OC.

Fig. 12. Photomicrograph or bacteria in oil globule from Eastern Bay sediment cultured in salts solution with model petroleum at 10~ The marker indicates 10/am.

90

J.D. Walker and R.R. ColweU

Fig. 13. Photomicrograph of yeasts surrounding oil globule from Colgate Creek sediment cultured in salts solution with model petroleum at 10~ The marker indicates 10/~m. (a)

(b)

Fig. 14. Scanning electron micrographs of Pullularia sp. and bacteria from Eastern Bay sediment cultured in salts medium plus model petroleum. (a) Microorganisms surrounding model petroleum at 5000X; (b) microorganisms surrounding model petroleum at 10,000• The markers indicate 2.0/aml

Microbial Degradation of Petroleum

91

that only a negligible loss of components was noted for the weathered samples incubated at 0 ~ and at 5~ coupled with the absence of a lag phase for cultures held at 5~ (Figs. 5 and 6).

14

i

i

i

!

~

i

i

i

E

~

42

49

~

~

i

.1~ ~

/{

~

$

o

7

~4

21

28

~

Time

7O

77

(days)

Fig. 15. Growth of the mixed bacterial culture (open circles) and isolates 229 (closed squares), 230 (triangles), 231 (open squares), and 232 (closed circles) from Eastern Bay sediment cultured in salts medium with model petroleum at IO~

5

IXI

0

?

~4

21

28

35

42

49

~

63

70

7T

Time (days) Fig. 16. Growth of the mixed bacterial culture (open circles) and isolates 238 (closed circles), 239 (closed squares), 240 (closed triangles), 241 (open triangles), and 242 (open squares) from Colgate Creek sediment cultured in Chesapeake Bay water with model petroleum at I0~

92

J.D. Walker and R.R. Colwell

In general, increased utilization of the model petroleum was observed for cultures incubated at 0~ Because of a lower solubility at 0~ certain hydrocarbons may be less toxic at the lower temperature, hence the increased degree of utilization. On the other hand, selection of a different microbial population can occur at lower temperatures. It is obvious that experiments should be undertaken to examine the role of obligate psychrophiles in model petroleum degradation. Such research is in progress. Greater utilization of petroleum in the presence of added nitrate and phosphate has been reported previously for marine bacteria [2]. However, in our studies, a significant degradation of model petroleum was observed in samples of water and sediment inoculated into media prepared with Chesapeake Bay water, especially in the case of microorganisms in the Colgate Creek and Eastern Bay water samples. Nutrients present in the Chesapeake Bay water may have been preferentially utilized by the Eastern Bay water samples, since a higher concentration and greater qualitative distribution of the components of the model petroleum remained at the termination of the experiment. Growth of bacteria was noted for samples cultured in Chesapeake Bay water, without petroleum added; however, greater yields were obtained when model petroleum was added. Antibiotics provided a means for estimating the utilization of model petroleum by bacteria and by the yeasts and fungi. Bacteria accounted for all the model petroleum utilization at 0 ~ and 5~ However, at IO~ yeasts were also responsible for model petroleum utilization. Since no fungi grew in Bay water with model petroleum added and inoculated with Colgate Creek sediment, Cladosporium resinae ATCC 22711 was used as the inoculum to examine fungal growth. Cladosporium resinae was selected because of its possible role in petroleum degradation [14] and because it is the only hydrocarbon-utilizing fungus for which the type of hydrocarbons degraded and pathway of hydrocarbon oxidation have been reported [3, 15, 16]. The growth of C resinae in the presence of 1.0 gg/ml fungizone, coupled with lack of growth of C. resinae at a concentration of 10.0/~g/ml fungizone, indicated C resinae to be responsible for between 20 and 40% of the total utilization of each of the hydrocarbons in the model petroleum (Table 5). Although streptomycin was ineffective in inhibiting growth of bacteria, data obtained recently in our laboratory suggest the use of 50.0 tag/ml of streptomycin and tetracycline to inhibit growth and degradation of model petroleum in the case of Chesapeake Bay bacteria. The observation of microorganisms associated with oil globules may be related to emulsification. Colgate Creek microorganisms cultured in oil-salts medium emulsified model petroleum and were found adhered on the outside of the globules. Observations of Eastern Bay microorganisms revealed their presence on the inside of the globules and they did not emulsify the model petroleum.

Microbial De~adation of Petroleum

93

As was expected, microorganisms from Colgate Creek utilized alicyclic and aromatic hydrocarbons to a greater extent than microorganisms from Eastern Bay. Increased utilization of these compounds by microorganisms from Colgate Creek water, compared with the sediment samples, may be due to the continuous exposure of the microbial populations in these samples to the compounds. Significant utilization of naphthalene by Colgate Creek water and sediment microorganisms was noted, confirming earlier reports that 98.2 and 84.6% respectively, of the aerobic heterotrophic bacterial population of water and sediment of this area in Chesapeake Bay utilizes naphthalene [5]. At 10~ a Pullularia sp. was observed in the Eastern Bay sediment sample cultured in oil-salts medium. It probably accounts for the greater utilization of normal and branched paraffins, compared with the Colgate Creek sample. Alkanes were degraded as would have been predicted from previously published studies. The incomplete degradation observed in this study very likely reflected nutrient limitation. The partial degradation of pristane, which is sometimes considered refractory, reflects the petroleum-degrading ability of bacterial populations or of insufficient concentration of hydrocarbons necessary to repress degradation (M. P. Pirnik, R. M. Atlas, and R. Bartha. 1973. Abstracts of the Annual Meeting of the American Society of Microbiologists, p. 170). The microbial degradation of PAH's has been reported by others [10]. However, to our knowledge, no reports have been published concerning the fate of these compounds during microbial degradation of crude oil. We are currently examining the fate of these and other hydrocarbons during the process of microbial degradation of crude oil. The changes in the microbial composition, i.e., representative of the bacterial genera, observed at different temperatures most certainly reflect the bacteriological potential for petroleum degradation in the geographical areas examined during different seasons of the year. The potential for petroleum degradation of each of the isolates is being examined, using pure cultures. In summary, it can be concluded from the results of this study that the microbial potential for petroleum degradation in samples collected from nature is a function of the numbers and types of microorganisms present in the natural substrate and the seasonal flux in these populations. Low temperatures clearly do not block or completely inhibit the autochthonous microbial degradation of oil. However, a selection for specific members of the indigenous microbial population capable of carrying out microbial degradation at low temperatures does occur. An extension of this study is in progress, which aims at the assessment of microbial degradation of model petroleum at warm temperatures, i.e., > 10~

94

J.D. Walker and R.R. Colwell Acknowledgments

We are grateful to Dr. W. R. Taylor, Chesapeake Bay Institute, Johns Hopkins University for assistance in obtaining access to facilities for the field work. Ship time on the R/V Ridgely Warfield was provided through NSF Grant GD-31707. We also acknowledge the very kind cooperation of Dr. Richard L. Raymond, Research and Development Division, Sun Oil Co., Marcus Hook, Pa. in obtaining pure samples of 1,2-benzanthracene, perylene, and pyrene. The excellent technical assistance of Jon Calomiris with the numerical taxonomy and Mrs. Lily Wan with the photomicroscopy is gratefully acknowledged. We also thank Dr. M. J. Marcinkowski and M. E. Taylor of the Department of Mechanical Engineering for use of the Cambridge Mark II Scanning Electron Microscope. This work was supported by Contract No. N00014-69-A-0220-006 between the Office of Naval Research and the University of Maryland. J. D. Walker acknowledges partial support from an Environmental Conservation Postdoctoral Fellowship from the American Petroleum Institute and National Wildlife Federation.

References 1.

Atlas, R. M., and Bartha, R. 1972. Biodegradation of petroleum in seawater at low temperatures. Can. J. Microbiol. 18:1851-1855.

2.

Atlas, R. M., and Bartha, Ro 1972. Degradation and mineralization of petroleum in seawater: Limitation by nitrogen and phosphorous. Biotechnol. Bioeng. 14: 309-318.

3.

Cofone, L., Jr., Walker, J. D., and Cooney, J. J. 1973. Utilization of hydrocarbons by Cladosporium resinae. J. Gen. Microbiol. 76: 243-246.

4.

Colwell, R. R. 1970. Poly~hasic taxonomy of the genus Vibrio: Numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus and realted Vibrio species. J. Bacteriol. 104: 410-433.

5.

Colwell, R. R., Walker, J. D., and Nelson, Jr., J. D. 1973. Microbial ecology and the problem of petroleum degradation in Chesapeake Bay. ln: The Microbial Degradation of Oil Pollutants. D. G. Ahearn and S. P. Meyers, editors. Publication No. LSU-SG-73-01, Center for Wetland Resources, Louisiana State University, Baton Rouge, La. pp. 185-197.

6.

Lovelace, T. E., Tubiash, H., and Colwell, R. R. 1968. Quantitative and qualitative commensal bacterial flora of Crassotrea virginica in Chesapeake Bay. Proc. Nat'L Shellfish Assoc. 58: 82-87.

7.

Massmann, W. H. 1971. The significance of an estuary on the biology of aquatic organisms of the middle Atlantic region. In: A Symposium on the Biological Significance of Estuaries. Sport Fishing Institute, Washington, D. C. pp. 96109.

8.

Niskin, S. J. 1962. A water sampler for microbiological studies. Deep-Sea Res. 9: 501-503.

9.

Schubel, J. R. 1972. The physical and chemical conditions of the Chesapeake Bay. J. Wash. Acad. Sci. 62: 56-87.

10.

Sisler, F. D., and ZoBell, C. E. 1947. Microbial utilization of carcinogenic hydrocarbons. Science 106: 521-522.

Microbial Degradation of Petroleum

95

11.

Traxler, R. W. 1973. Bacterial degradation of petroleum materials in low temperature marine environments. In: The Microbial Degradation of Oil Pollutants. D. G. Aheaxn and S. P. Meyers, editors. Publication No. LSU-SG-73-01, Center for Wetland Resources, Louisiana State University, Baton Rouge, La. pp. 163-170.

12.

Walker, J. D., and Colwell, R. R. 1973. Microbial ecology of petroleum-utilization in Chesapeake Bay. In: API/EPA/USCG Conference on Prevention and Control of Oil Spills. American Petroleum Institute, Washington, D. C. pp. 685-690.

13.

Walker, J. D., and Colwell, R. R. 1973. Mercury resistant bacteria and petroleum degradation. Appl. Microbiol. 27: 285-287.

14.

Walker, J. D., Cofone, Jr., L., and Cooney, J. J. 1973. Microbial petroleum degradation: The role of Cladosporium resinae. In: API/EPA/USCG Conference on Prevention and Control of Oil Spills. American Petroleum Institute, Washington, D. C. pp. 685-691.

15.

Walker, J. D., and Cooney, J. J. 1973. Pathway of n-alkane oxidation in Cladosporium resinae. J. Bacteriol. 115: 635-639.

16.

Walker, J. D., and Cooney, J. J. 1973. Oxidation of n-alkanes by Cladosporium resinae. Can. J. Microbiol. 19: 1325-1330.

17.

ZoBell, C. E. 1969. Microbial modification of crude oil in the sea. In: API]FWPCA Conference on Prevention and Control of Oil Spills. American Petroleum Institute, Washington, D. C. pp. 317-326.

18.

ZoBell, C. E. 1973. Bacterial degradation of mineral oils at low temperatures. In: The Microbial Degradation of Oil Pollutants. D. G. Ahearn and S. P. Meyers, editors. Publication No. LSU-SG-73-O1, Center for Wetland Resources, Louisiana State University, Baton Rouge, La. pp. 153-161.