Department of Microbiology, University of Maryland, College Park, Maryland 20742 ... A total of 99 strains of petroleum-degrading bacteria isolated from Chesa-.
APPIED AND ENVIRONMENTAL MICROBIOLOGY, July 1977, P. 60-68 Copyright 0 1977 American Society for Microbiology
Vol. 34, No. 1 Printed in U.S.A.
Numerical Taxonomy and Ecology of Petroleum-Degrading Bacteria B. AUSTIN, J. J. CALOMIRIS,1 J. D. WALKER,2 AND R. R. COLWELL* Department of Microbiology, University of Maryland, College Park, Maryland 20742
Received for publication 15 February 1977
A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 ofthe petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.
Despite numerous studies undertaken to examine the structure and metabolism of petroleum-degrading bacteria, the taxonomy of these organisms has been comparatively neglected. Petroleum-degrading bacteria occur extensively in the aquatic environment and have been found in sediment and seawater collected from temperate, tropical, and arctic zones (32). Microbial degradation of petroleum is influenced by a number of factors, including season, history of previous exposure of the given environment to oil, temperature, sediment type, and medium used for the isolation of the organisms (4, 7). The method used for isolation of petroleum-degrading bacteria has a strong influence on the number and types of bacteria recovered; for example, Sohngen (25), in 1913, observed colonies of mycobacteria on mineral agar exposed to hydrocarbon vapor, whereas in liquid culture designed to isolate hydrocarbon-utilizing bacteria, pseudomonads are generally found (4, 28). In the study reported here, petroleum-degrading bacteria isolated from samples of oil-polluted and unpolluted water and sediment have been classified by using numerical taxonomic procedures. The results of the analyses have proved useful in
elucidating ecological aspects of petroleum degradation in the estuarine environment. MATERIALS AND METHODS Isolation and maintenance of strains. Bacterial strains used in this study were isolated from sediment and water samples collected in Colgate Creek in Baltimore Harbor, an oil-contaminated site, and from Eastern Bay and Poole's Island, both nonpolluted areas of Chesapeake Bay. Water temperatures at the time of samplings were inthe range 14 to 25°C. The samples were collected aseptically, using a Niskin sterile bag sampler (General Oceanics Inc., Miami, Fla.) and a Ponar grab sampler (Wildlife Supply Co., Saginaw, Mich.) for water and sediment, respectively. The samples were diluted and inoculated into petroleum media immediately after collection. Liquid enrichment cultures were prepared by inoculating 100 ml of a sterile salts solution supplemented with nitrate and phosphate (29, 31) with 1.0 ml of a 10-2 dilution of sediment or with 1.0 ml of water, and overlaying it with 1.0 ml of a 20weight motor oil. Direct plating of the same sediment and water samples was accomplished by using this liquid salts medium to which agar (Difco Laboratories, Detroit, Mich.) was added to a final concentration of 2% (wt/vol). The oil medium and its preparation have been described elsewhere (29, 30). After incubation at 15°C for 21 days, colonies appearing on the oil agar and 0.1 ml of appropriate dilutions of the liquid cultures were streaked onto GTYEA (glucose-tryptone-yeast extract agar, Difco). After incubation at 15°C for 21 -days, the GTYEA plates were examined, and colonies were
I Present address: Mail Stop 236-5, NASA/AMES, Moffett Field, CA 94035. 2 Present address: Environment Technology Center, Martin Marietta Corp., Baltimore, MD 21227.
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selected for further study. Isolates were examined for purity by alternatively transferring inocula into ESWYE broth (estuarine salts solution [4] containing 0.15% [wt/vol] yeast extract) and onto GTYEA. This procedure was repeated three times. Organisms were stored at 5°C on GTYEA slants overlaid with sterile mineral oil. The sources of the petroleum-degrading strains used in this study are listed in Table 1. Bacterial strains isolated from Chesapeake Bay during October 1972 through September 1973 and reference strains were examined for ability to utilize mixed hydrocarbon substrate (29). After purification, each isolate was transferred to basal medium broth. After incubation for 4 to 8 h at 15°C, ca. 102 to 103 cells (final concentration) were added to a 5-ml oil-salts broth in a 16- by 125-mm test tube. Each culture, as well as uninoculated controls (triplicate), was overlayered with sterile 1% (wt/vol) mixed hydrocarbon substrate. The test culture and control tubes were incubated, without shaking, at 25°C for 30 days. On day 30, each culture was mixed thoroughly with a Vortex mixer and allowed to stand for several minutes to stabilize the culture, after which 3 ml was transferred to a cuvette to measure absorbance (at 600 nm) and pH. The transfer pipette was rinsed by drawing up 2 ml of n-hexane and depositing the n-hexane in the original culture tube. The 2 ml of nhexane was removed, and the original culture was extracted twice more, each time with 2 ml of nhexane. After absorbancy and pH were measured, the pH meter probe was rinsed by pipetting 2 ml of n-hexane into the cuvette. The n-hexane was removed, and the culture in the cuvette was extracted with an additional 2 ml of n-hexane. All portions of n-hexane were combined, and any remaining water was removed by drying over Na2SO4. The anhydrous extracts were transferred to a vial and stored under N2 at -20°C until gas-liquid chromatography analysis was accomplished. Before gas-liquid chromatography analysis, 0.1 ml of 1-dodecanol and/or 0.1 ml of indan was added to each extract, serving as the internal standard. Chromatographs were obtained on a Shimadzu model GC-4BMPF gas chromatograph equipped with a single-flame ionization detector. A glass column (3 by 1,500 mm) packed with 3% OV-1 on 80/ 100-mesh Shimalite was used. The carrier gas, nitrogen, was run through the column at a rate of 40 ml/min. Temperature was programmed from 50 to 300°C at 5°C/min, and individual peak areas were quantified by using a Hewlett-Packard model 3373B
TABLE 1. Sources of petroleum-degrading strains included in this study No. of strains isolated from:
Sample type Polluted waUnpolluted water ter (Colgate Eastern Bay Poole's Island Creek) 2 0 16 Water 54 18 9 Sediment
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integrator. Column efficiency and detector response, measured with a mixture of C14-C20 saturated and unsaturated hydrocarbons (Applied Science mixture no. 19251), yielded a relative error of less than 2.5% for all components. Percent hydrocarbon remaining was calculated using the following formula, 100 x [(peak area of hydrocarbon X for culture Y/peak area of hydrocarbon X for control)/(peak area of internal standard for culture Y/peak area of internal standard for control)], where X represents one of the 15 hydrocarbons. The internal standard was 1-dodecanol and/or indan. The ability of the cultures to degrade petroleum was confirmed as follows. The strains were inoculated into 5-ml portions of oil-salts overlaid with 1% (wt/vol) mixed hydrocarbon substrate (29, 30). After incubation at 25°C for 28 days, petroleum degradation was recorded as the presence of growth in the oil layer and turbidity in the liquid phase. In addition, growth in crude oil or the hydrocarbon mixture was recorded by measuring protein in the liquid culture by the modified Folin method of Lowry et al. (19). Positive tubes were those containing at least 50 ,ug/ml, a protein concentration significantly higher than that of the inoculum. Utilization of the crude oil or hydrocarbon mixture was determined by measuring weight loss (in milligrams), compared with sterile controls. Thus, utilization of the substrate was measured, as well as cell yield, to verify the degradation of hydrocarbon. The cultures were also examined for lipolytic activity. The results of the growth and utilization studies are being prepared for publication separately. Reference strains. In addition to the freshly isolated strains of petroleum-degrading bacteria, 33 cultures representing a diverse range of taxa were included in the study, serving as reference cultures. These included: Acinetobacter calcoaceticus (ATCC 15308); A. iwoffi (ATCC 15309); Bacillus cereus subsp. mycoides (ATCC 6462); B. subtilis (ATCC 6051); Corynebacterium poinsettiae (ATCC 9069); Enterobacter aerogenes (ATCC 13048); Erwinia herbicola (ATCC 12287); Escherichia coli (ATCC 11775); Flavobacterium spp., group llb (NCTC 10795) and group llf (NCTC 10798); Klebsiella aerogenes (NCTC 8172); Leucothrix mucor (ATCC 25107); Micrococcus luteus (ATCC 4698); M. roseus (ATCC 418); Moraxelha osloensis (ATCCQ 19976); Nocardia asteroides (ATCC 14759); N. corallina (ATCC 4273); N. otitidis-caviarum (ATCC 14629); Pseudomonas acidovorans (ATCC 17438); P. aeruginosa (ATCC 10145); P. fluorescens (ATCC 13525); P. maltophilia (ATCC 13637); P. pseudoalcaligenes (ATCC 17440); P. putida (ATCC 12633); Rhizobium leguminosarum (ATCC 10004); R. meliloti (ATCC 4399); Serratia marcescens (ATCC 13880); Sphaerotilus natans (ATCC 15291); Staphylococcus epidermidis (ATCC 14990); S. lactis (ATCC 15306); Staphylococcus spp. (ATCC 15365); Streptomyces griseus (ATCC 23345); and Vibrio parahaemolyticus (ATCC 17082). Numerical taxonomy. Each strain was examined for 48-unit characters. All media were inoculated with cultures incubated for 5 days at 15°C on
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GTYEA. The taxonomic tests were repeated when inconclusive results were obtained. Micromorphology and staining reactions. Isolates, grown in ESWYE broth, were examined after 18 h. Motility was determined by examination of wet-mount preparations under a phase-contrast microscope. Gram reaction and cell morphology were recorded from observations of heat-fixed smears stained by the Gram reaction (2). The presence of soluble, nonsoluble, and fluorescent pigments was determined by examining 24- to 48-h cultures in daylight and under ultraviolet light. Biochemical characters. Where possible, tests were performed by using the multipoint inoculation procedure (18). Catalase activity was determined by the addition of hydrogen peroxide to 24-h cultures, and a positive response was recorded when effervescence of oxygen resulted. Oxidase activity was detected by using the method of Kovacs (13). Fermentative and oxidative metabolism of glucose was determined by using the method of Hugh and Leifson (12). The tubes were examined for acid and gas production at 1, 2, 7, and 14 days after inoculation. Nitrate and nitrite reduction was recorded from nitrate broth (Difco), using the method described by Cowan and Steel (8). Methyl red and Voges-Proskauer tests were performed by using Difco medium amended with NaCl (1%, wt/vol) and examined after 5 days by the method described by Cowan and Steel (8). Substrate utilization. Casein hydrolysis was determined by using GTYEA medium with skim milk (5%, wt/vol), following the procedure of Sizemore and Stevenson (23). Gelatin, starch, and Tween 20 and Tween 80 hydrolysis were measured according to the methods of Frazier (10), Cowan and Steel (8), and Sierra (22), respectively. Tubes containing urea broth (Difco), to which NaCl (1%, wt/vol) was added, were examined 28 days after inoculation for an alkaline reaction, indicative of urea hydrolysis. Utilization of substrates as sole carbon sources. The ability to utilize D-arabinose, ethanol, n-fructose, D-galactose, lactose, maltose, sucrose, and D(+ )-xylose was tested, using basal oxidation-fermentation medium (Difco) to which the carbon sources were added to a final concentration of 1% (wt/vol). Acid production was scored as positive. Utilization of substrate as sole nitrogen source. The utilization of NH4H2PO4 (1%, wt/vol) was scored by the presence of an acid reaction. Coding of data. The characters were coded "1" for positive or present, "0" for negative or absent, and "9" for noncomparable or not applicable. The final n x t matrix contained 132 strains and 48 characters. Computer analyses. The data were analyzed by using the simple matching coefficient (26), which includes both positive and negative matches, and the Jaccard coefficient (24), which excludes negative matches. Clustering was achieved by means of the unweighted average linkage method (26), and a sorted similarity matrix and dendrogram were constructed. Programs used included the GTP2 and UMDTAXON 3 programs, written for the IBM 370/ 165 and the Univac 1108 computers, respectively.
APPL. ENVIRON. MICROBIOL.
RESULTS Clustering of strains. A total of 85 (85%) of the petroleum-degrading bacteria and 4 reference cultures, K. aerogenes, N. asteroides, N. corallina, and S. natans, were recovered in the 12 clusters defined at the 80 to 85% similarity level (Fig. 1 and 2). Five clusters (1, 2, 3, 4, and 10) comprised 54 strains (54% of the total), with the remaining 31 strains distributed among 8 smaller clusters. Cluster 4 was divided into two subgroups (4a and 4b) at the 90% similarity level (Fig. 1). The composition and shared characters of these groups are given in Table 2. All of the clusters defined by using the SSM (simple matrix) coefficient (Fig. 1) were recognizable in the analysis prepared with the SJ (Jaccard) coefficient (Fig. 2), but at a lower similarity level. It is important when comparing a wide spectrum of bacterial types, such as will occur in the natural environment, to be aware of the possibility of negative matches resulting in high similarity values. Thus, the SJ coefficient proved to be more useful in discriminating clusters of related strains when ecological conclusions were intended. Since the outcome of the Sj analysis was similar to that obtained by using the SsmI coefficient, the full results for both analyses are not presented here; instead, a shaded diagram, representing a sorted similarity matrix obtained from the analysis with the SSM coefficient (Fig. 1), and a simplified dendrogram of the analysis with the SJ coefficient (Fig. 2) are presented. Identification and description of the phenetic groups. Relationships among the groups of petroleum-degrading bacteria can be seen in Fig. 2. The characters of the groups are listed in Table 2. Four phena were readily identified, since the reference cultures were included in the groups. These were K. aerogenes (phenon 7), N. asteroides (phenon 9), N. corallina (phenon 10), and S. natans (phenon 8). The remaining eight phenetic groups were identified by matching their descriptions with those provided in Bergey's Manual of Determinative Bacteriology (3) (Table 3). The most numerous of the petroleum-degrading bacteria were the pseudomonads, which were recovered in two phena (4a, 4b, and 5) and accounted for 19% of the strains. However, the intergroup similarity was low (
