THE EFFECT OF ON THE MICROBIAL POPULATIONS OF OILED BEACHES IN , Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1993/1/469/2357220/2169-3358-1993-1-469.pdf by guest on 25 September 2021

Roger C. Prince, Richard E. Bare, Graham N. George, Copper E. Haith, Matthew J. Grossman, James R. Lute, David L. Elmendorf, Vera Minak-Bernero, James D. Senius, Lois G. Keim, Russell R. Chianelli, Stephen M. Hinton Research and Engineering Company Annandale, New Jersey 08801

Andrew R. Teal Box 1020 Edmonton, Alberta T5J 2Ml, Canada

ABSTRACT: Bioremediation, the stimulation of the natural process of likely limiting nutrients are nitrogen and phosphorus; so the bio- biodégradation, played an important role in the cleanup of the remediation strategy was to supply these nutrients in the form of from the in Prince William Sound, Alaska. Since there carefully chosen fertilizers.14,25,27 The success of this strategy was were already substantial indigenous populations of oil-degrading mi- assessed in several intensely monitored test sites by microbiological, crobes in the area, it was apparent that degradation was likely to be ecological, and chemical techniques.8,22,26,27 Here we report the results nutrient—notmicrobial—limited. Bioremediation therefore involved the of microbiological measurements in a survey that extended throughout application of carefully selected fertilizers to provide assimilable nitro- Prince William Sound and the Gulf of Alaska,19 and show that the gen and phosphorus to the indigenous organisms, with the intent to bioremediation strategy was successful at increasing the number of stimulate their activity and enhance their numbers. We show here that heterotrophic and oil-degrading bacteria in the beach sediments. We the indigenous microbial populations were indeed substantially in- also report the initial results of a taxonomic study aimed at identifying creased, throughout the sound, approximately one month after wide- the bacteria present in this environment. Our results also indicate that spread fertilizer applications in both 1989 and 1990. Furthermore, while the structure of the microbial ecosystem was returning to pre-spill oil-degrading bacteria made up a significant fraction of the microbial conditions by the summer of 1990, just one year after the spill. populations on contaminated beaches in September and October 1989, they had declined to less than 1 percent by the summer of 1990, suggest- ing that the microbial populations on the shorelines were returning to their pre-spill conditions. Methods

The samples analyzed here were collected as part of an extensive monitoring of 27 sites beginning in September 1989, after cleanup operations were suspended because of winter weather, and ending The grounding of the Exxon Valdez on Bligh Reef on , September 1990. Seventeen of the sites were within Prince William 1989, released approximately 11 million gallons (U.S.) of North Slope Sound and 10 in the Gulf of Alaska (Figure 1, Table 1). They were crude oil into the waters of Prince William Sound. A major storm a few chosen to cover the range of wave exposures (high, medium, and low days later spread the oil onto the shores of numerous small islands in energy shorelines) and to include areas that received washing alone, the western part of the sound, and out into the Gulf of Alaska. The washing plus bioremediation (fertilizer), bioremediation alone, and no subsequent cleanup washed much of the oil back to the water so that it treatment.19 All sampling sites had been oiled by the spill; no samples could be removed by skimmers. This was followed by the application of were collected from the unaffected parts of the sound. (Less than 15 fertilizers to the shorelines to stimulate the biodégradation of the percent of the shoreline was affected by the spill.) Samples were remaining oil by indigenous microbial populations.1416'23,25,27 collected from the surface and subsurface (10 to 20 cm) of upper, The logic of applying fertilizer as the bioremediation strategy was middle, and lower intertidal zones, and shipped to New Jersey on ice, that under pre-spill conditions the number of oil-degrading bacteria but not frozen, for analysis. was limited by the availability of oil as a carbon source. Prince William The indigenous microbes were washed off the sediment, and oil- Sound and the Gulf of Alaska have particularly abundant and diverse degrading and heterotrophic bacterial populations assessed by most- populations of oil-degrading bacteria, which have been attributed to probable-number techniques. Oil-degrading microbes were assessed the presence of natural marine oil seeps and terrestrial input of hydro- by the "sheen-screen" method,9 and the general heterotrophic popula- carbons from the conifers of the temperate rain forest.13 Nevertheless, tion, presumably including the oil-degrading population, was assessed the background level of hydrocarbons and hydrocarbon-degrading by growth on a nutrient marine broth in a similar fashion.22 The oil we bacteria in this environment is low. After the spill, oil-degrading bacteria used in the sheen-screen tests had been heated to 521° F to remove the were no longer limited by the availability of oil, and their numbers volatile components that would have evaporated from the spilled oil increased dramatically until they were limited by other factors. prior to its arrival at the shoreline. Twenty grams of sediment were In the highly porous aerobic sediments typical of this area, the most shaken in 80 mL of saline Bushnell-Haas medium11 for 20 minutes at 469 470 1993 OIL SPILL CONFERENCE

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Figure 1. Location of the sampling sites in Prince William Sound, Alaska—the indicated extent of oil loading reflects the initial oiling in 1989; the area was essentially free of oil by 1992.

100 rpm prior to sequential dilution in 24-well plates. The plates were populations shown in Figure 2 vary over 7 orders of magnitude.) The incubated at 15° C for 7 (heterotrophic) or 14 (sheen-screen) days. large number of samples necessitated only a few being measured in Experiments using higher ionic strength and/or dilute triplicate; these showed excellent reproducibility, indicating that the gave no higher estimate of the numbers of organisms. Samples from tremendous range shown in Figure 2 reflects the true heterogeneity of the different beaches had different sediment sizes, but we estimate that the bacterial populations. the maximal difference in surface area would amount to less than 2 Preliminary identification of microbial isolates was made using clas- orders of magnitude difference. (Note that the estimates of microbial sical morphological criteria, and the Biolog identification system.7 BIOREMEDIATION 471

Table 1. Sampling sites samples represent a particularly challenging matrix for enumeration, since many of the organisms are intimately associated with the oil, which itself adheres to the sediment. To assess the efficacy of bio-

Site Oiling^wave energy Treatment2 remediation from a microbiological perspective, we have focused on determining the total number of heterotrophic microorganisms (ones Prince William Sound that require complex organic substances for growth) and the number of N.E. Smith Is. Heavy oil/high FF/HP/BR these that are capable of growth on weathered crude oil. The methods 18 energy we have used, three-tube most-probable-number techniques, al- N.W. Smith Is. Heavy oil/high FF/HP lowed us to process the large number of samples collected in our surveys in a timely fashion. While the numbers quoted here may energy 18 N.E. Little Smith Is. Heavy oil/high BR represent a systematic underestimate of the true populations, the energy comparison of the populations on the fertilized and unfertilized Foul Passage Heavy oil/low energy FF/HP/BR* beaches, rather than their absolute numbers, is most germane here. Passage Point Heavy oil/moderate FF/HP/BR Figure 2 shows the frequency distributions of the microbial popula- energy tions on fertilized and unfertilized beaches in Prince William Sound, Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1993/1/469/2357220/2169-3358-1993-1-469.pdf by guest on 25 September 2021 E. Herring Bay Heavy oil/low energy set aside while Figure 3 presents the median populations of oil-degrading and E. Green Is. Heavy oil/moderate BR heterotrophic bacteria in these samples, together with the ratio of energy these medians. The data include all tidal zones and both surface and W. Green Is. Heavy oil/high FF/HP/BR subsurface samples. Those shorelines that received fertilizer were energy treated (arrows in Figure 3) prior to the first sampling (August and Point Helen MR/FF/HP/BR early September 1989) and after the sampling in June 1990. Statistical Heavy oil/high 32 energy analysis using the nonparametric Mann-Whitney test indicates that N.E. Latouche Is. Heavy oil/high set aside the populations of both oil-degrading and heterotrophic bacteria were energy greater in samples collected from fertilized shorelines than in those N.E. Latouche Is. Heavy oil/high MR/FF/HP/BR from unfertilized areas, in both September 1989 and September 1990 energy (p < 0.05). Bioremediation increased the populations of both oil- Sleepy Bay Heavy oil/high MR/HP/BR degrading and heterotrophic microbes approximately 10- to 100-fold. energy This effect was not, however, seen beyond the first sampling after N.W. Latouche Is. Heavy oil/moderate HP/BR fertilizer addition; the populations on fertilized and unfertilized energy beaches were not significantly different at the other sampling times, N. Evans Is. Moderate oil/moder- MR/FF/HP/BR* with one exception that we shall discuss below. This implies that the ate energy effect of fertilizer addition lasted for at least a month, but not as long as W. Ellington Is. set aside two months. Similar conclusions were drawn from other monitoring Moderate oil/high 26 energy studies, and so the bioremediation strategy in 1990 and 1991 was to W. Ellington Is. Moderate oil/low BR reapply fertilizer on a monthly basis. energy The one exception to this simple picture is that samples collected in W. Ellington Is. Moderate oil/low MR/HP/BR May 1990 showed more heterotrophic bacteria (p < 0.05) in samples energy collected from sites fertilized in 1989 than in samples from unfertilized Gulf of Alaska sites. We believe that this latter observation is unrelated to the applica- tion of fertilizer the previous summer; we note that its occurrence (1 in Pervalnie Point Moderate oil/low FF/BR* 16 comparisons) is approximately the statistical uncertainty (p < 0.05; energy i.e., 1 chance in 20 that we reject the null hypothesis that there is no Black Cape Light oil/moderate difference between the populations when there is in fact no differ- energy ence). Cape Nukshak Light oil/moderate Unfortunately the monitoring program did not start until after the energy application of fertilizers in 1989, and it might be argued that the Cape Kubugakli Moderate oil/high MR differences seen in September 1989 reflect a fundamental difference energy between the different shorelines rather than the effect of fertilizer. The Ushagat Island Heavy oil/high MR/BR fact that the differences between fertilized and unfertilized shorelines energy seen in September 1989 had disappeared by October 1989 argues Pony Cove Moderate oil/moder- HP/BR* strongly that the difference was indeed due to the application of the ate energy bioremediation fertilizers prior to the September sampling. As shown Gore Point Moderate oil/high MR in Figure 3, the differences between the populations of oil-degrading energy organisms on the two classes of beaches did not reappear until after the N. Windy Bay Moderate oil/high HP/BR application of fertilizers in 1990. Similar trends of an increase in energy microbial numbers following fertilizer application were detected on Badger Cove Moderate oil/moder- MR three beaches monitored intensively from June to August 1990,22 ate energy although with this rather smaller data set the differences were usually Chugach Bay Moderate oil/moder- MR/BR not statistically different at p < 0.05. ate energy The trends seen in Figure 3 were seen in both surface and subsurface sediments, and in samples from upper, middle, and lower intertidal 1. Description of initial oiling in 1989 zones. The samples from the Gulf of Alaska did not include enough 2. FF—flooding and flushing; HP—high pressure washing; MR— from fertilized beaches for an independent analysis; but when the Gulf manual removal; BR—bioremediation in 1989 and 1990; BR*— of Alaska samples were included in the analysis of Figure 3, the bioremediation only in 1990 conclusions, and their statistical validity, were essentially unchanged; with the exception that the increase in heterotrophic organisms in May 1990 was no longer statistically significant. Figure 4 indicates that oil-degrading microorganisms made up a Results substantial proportion of the heterotrophic population in late 1989, but that this fraction decreased substantially during the winter of 1989-1990 until it was less than 1 percent by September 1990. Microbial enumeration. Microbes are ubiquitous, and the enumera- Microbial identification. The microbial guilds of estuarine and tion of their populations may at first appear to be a relatively simple marine environments have been widely studied; and there is reason- matter. Ideally the counting method should include all the viable cells able agreement about the most common genera in these environ- in an environment; but this is rarely, if ever, achieved.18 Oiled sediment ments.1-4'61015'20'21'30 Our preliminary studies (Table 2) are in broad 472 1993 OIL SPILL CONFERENCE

Oil degrading microorganisms Heterotrophic microorganisms

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Sep. 90

3 4 § 1 0-10Wf w'-10* e10$-10 7 0-10 w -io w*-id io -io Figure 2. Frequency distributions of the microbial populations in Prince William Sound—The x-axis is the number of organisms per gram of sediment; the y-axis the frequency of populations of that number. Solid bars are from fertilized beaches, open bars from unfertilized ones. Note that the sum of the frequencies for each equals 1. The number of samples for each month was 54 to 82 from fertilized beaches, 14 to 30 from unfertilized ones, with the exception of November and December 1989, when 0 and 6 samples were collected, respectively, from unfertilized areas. BIOREMEDIATION 473

Oil degrading microorganisms Heterotrophic microorganisms

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Figure 3. Median populations of oil-degrading and heterotrophic bacteria on fertilized and unfertilized beaches—the lower panel in each section is the ratio of median population on fertilized to unfertilized beaches. The arrows indicate the approximate date of bioremediation treatment. Since the sheen-screen detects the growth of organisms that produce to disrupt the floating sheen of oil, rather than the growth of organisms per se, it is possible that the decrease in number of oil-degrading organisms, with time, reflects a change in their type, as well as their number.

agreement with this earlier body of work. Many of these genera are known, or have been suggested, to contain species capable of growth on hydrocarbons; but the definitive taxonomy of these organisms is relatively undefined, and studies are still in progress to identify the organisms involved in oil degradation. 50 í "\ I • fertilized Discussion I 40 + o unfertilized 1 v J Bioremediation was an important part of the cleanup of the spill .o from the Exxon Valdez, especially in 1990 and 1991. This was the first 30 + time that this technology was used on a large scale in a marine environ- ment; and its use was accompanied by several independent monitoring rA efforts.8,22'26'27 The biodégradation of oil is readily demonstrated, in 20 the laboratory, to be dependent on the presence of nitrogenous nutri- ents;14 it proved rather more of a challenge to demonstrate effective- ness in the field. This has now been achieved by monitoring the 10 systematic changes in oil chemistry relative to hopane, a non- 12 \ biodegradable hydrocarbon. Multivariate analysis shows that bio- remediation was effective, and dependent on the amount of nitrogen J 1 O M— _^ 8 delivered per unit of oil. 1 Sept 89 Jan.90 May 90 Sept 90 The work reported here provided the first statistically significant demonstration of the success of fertilizer application in Prince William Figure 4. The percentage of the total heterotrophic microbial popula- Sound, based on microbiological monitoring. Figure 3 shows that there tion detected as oil-degrading microorganisms with the screen- were approximately 10- to 100-fold more bacteria, both heterotrophic sheen9—the arrows indicate the approximate dates of bioremediation and oil-degrading, on sediment on beaches that had received fertilizer treatment. 474 1993 OIL SPILL CONFERENCE

Table 2. Bacterial genera isolated from sediments References in Prince William Sound Atlas, R. M., 1981. Microbial degradation of hydrocar- bons: an environmental perspective. Microbiological Reviews, Description Genera v45, ppl80-209 Gram-negative aerobic Pseudomonas, Xanthomonas, Morax- Austin, B. J., J. J. Calomiris, J. D. Walker, and R. R. Colwell, rods and cocci ella, Acinetobacter, Kingella, Al- 1977a. Numerical taxonomy and ecology of petroleum-degrading caligenes, Psychrobacter, bacteria. Applied and Environmental Microbiology, v34, pp60-68 Flavobacterium Austin, B. J., R. R. Colwell, J. D. Walker, and J. J. Calomiris, Gram-negative aerobic Aquaspirillum, Spirillum, Ocean- 1977b. The application of numerical taxonomy to the study of and microaerophilic ospirillum petroleum-degrading bacteria isolated from the aquatic environ- helical bacteria ment. Developments in Industrial Microbiology, vl8, pp685-695 Gram-negative facul- Enterobacter, Klebsiella, Aeromonas, Austin, B. J., S. Garges, B. Conrad, E. E. Harding, R. R. tatively anaerobic rods Vibrio, Shewanella Colwell, U. Simidu, and N. Taga, 1979. Comparative study of the Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1993/1/469/2357220/2169-3358-1993-1-469.pdf by guest on 25 September 2021 Gram-positive endo- Bacillus aerobic heterotrophic bacterial flora of Chesapeake Bay and spore-forming rods and Tokyo Bay. Applied and Environmental Microbiology, v37, cocci pp704-714 Gram-positive cocci Micrococcus Baker, J. M., R. B. Clark, P.F. Kingston, and R. H. Jenkins, 1990. Gram-positive nonspor- Cory neb acterium Natural recovery of cold water marine environments after an oil ing rods and cocci spill. Proceedings of the 13th Arctic and Marine Oil Spill Program Technical Seminar, Environment Canada, Ottawa, Ontario, ppl73-177 Bartha, R. and R. M. Atlas, 1977. The microbiology of aquatic oil spills. Advances in Applied Microbiology, v22, pp225-266 Bochner, B. R., 1989. Sleuthing out bacterial identities. Nature, v339, ppl57-158 Bragg, J. R., R. C. Prince, R. M. Atlas, and E. J. Harner, 1993 within the previous one to two months, and that this difference disap- (this volume). Bioremediation effectiveness following the Exxon peared after two months. Such results are entirely consistent with Valdez spill. Proceedings of the 1993 International Oil Spill Confer- expectations that fertilizer would stimulate the biodégradation of the ence, American Petroleum Institute, Washington, D.C. spilled oil and increase microbial biomass in the sediment, and that Brown, E. J. and J. F. Braddock, 1990. Sheen-screen, a min- when the fertilizer nutrient levels returned to near background levels iaturized most-probable-number method for enumeration of oil- this increase would disappear. The fertilizer applications were de- degrading microorganisms. Applied and Environmental Micro- signed to provide nutrients for a reasonable period, but the release biology, v56, pp3895-3896 28 characteristics of the fertilizers are such that monthly reapplication 10 Buckley, E. N., R. B. Jonas, and F. R. Pfaender, 1976. Character- was expected to be necessary. The results presented here, and else- ization of microbial isolates from an estuarine ecosystem: relation- 8 26 where, ' confirm that this was indeed appropriate. ship of hydrocarbon utilization to ambient hydrocarbon concen- The preliminary characterization of the microbial genera found on trations. Applied and Environmental Microbiology, v32, oiled sediments from Prince William Sound (Table 2) is fully consistent pp232-237 with the work of others who have studied similar environ- 11 Bushnell, L. D. and H. F. Haas, 1941. The utilization of certain ments.1-4'6'10'15 <17'20-21'30 The identified genera are widespread, and hydrocarbons by microorganisms. Journal of Bacteriology, v41, individual species of many of them have been shown to be capable of pp653-673 growth on petroleum hydrocarbons. We have not yet examined in 12 Butler, E. L., G. S. Douglas, W. S. Steinhauer, R. C. Prince, T. detail the role of fungi in the biodégradation of oil in Prince William Aczel, C. S. Hsu, M. T. Bronson, J. R. Clark, and J. E. Lin- Sound, although we have isolated Trichosporon from some samples.14 dstrom, 1991. Hopane, a new chemical tool for measuring oil Snellman, Collins, and Cooke29 provide a list of fungi isolated from tar biodégradation, in On-site Reclamation—Processes for Xenobio- balls collected at sea, some of which are capable of using oil as their tic and Hydrocarbon Treatment, R. E. Hinchee and R. F. Olfen- sole carbon source. buttel, eds. Butterworth-Heinemann, Boston, pp515-521 The scientific understanding of the food web in intertidal zones—the 13 Button, D. K., B. R. Robertson, and K. S. Craig, 1981. Dissolved complex ecological interactions between primary consumers, grazers, hydrocarbons and related microflora in a fjordal seaport: sources, and predators—is still mainly in the descriptive stage.24 In the absence sinks and kinetics. Applied and Environmental Microbiology, v42, of petroleum hydrocarbons, hydrocarbon utilizing microorganisms pp708-719 generally constitute <0.1 % of the microbial population, and it has 14. Chianelli, R. R., T. Aczel, R. E. Bare, G. N. George, M. W. been proposed that the degree of elevation above this level is a reason- Genowitz, M. J. Grossman, CE. Haith, F. J. Kaiser, R. R. able indicator of the extent of hydrocarbon contamination.31 As petro- Lessard, R. Liotta, R. L. Mastracchio, V. Minak-Bernero, R. C. leum hydrocarbons are removed from an ecosystem, the original food Prince, W. K. Robbins, E. I. Stiefel, J. B. Wilkinson, S. M. web will be re-established, and the levels of oil-degrading bacteria will Hinton, J. R. Bragg, S. J. McMillen, and R. M. Atlas, 1991. decline. Figure 4 thus suggests that the shorelines impacted by the oil Bioremediation technology development and application to the spill from the Exxon Valdez were well on the way to recovery, at least as Alaskan spill. Proceedings of the 1991 International Oil Spill Con- far as the overall microbial populations were concerned, by the sum- ference, American Petroleum Institute, Washington, D.C, mer of 1990.5 pp549-558 15. Foght, J. M., P. M. Fedorak, and D. W. S. Westlake, 1990. Mineralization of (14C)hexadecane and (14C)phenanthrene in crude oil: specificity among bacterial isolates. Canadian Journal of Summary Microbiology, v36, pp 169-175 16. Harrison, O. R., 1991. An overview of the Exxon Valdez oil spill. Our data indicate that the bioremediation strategy used in Prince Proceedings of the 1991 International Oil Spill Conference, Ameri- William Sound and the Gulf of Alaska was successful at increasing the can Petroleum Institute, Washington, D.C, pp313-319 number of oil-degrading bacteria in oiled sediments for approximately 17. Hauxhurst, J. D., M. I. Krichevsky, and R. M. Atlas, 1980. one to two months following fertilizer application. This would certainly Numerical taxonomy of bacteria from the Gulf of Alaska. Journal be expected to increase the rate of oil biodégradation, and we show of General Microbiology, vl20, ppl31-148 elsewhere in this volume8 that this was indeed evident in samples 18 Herbert, R. A., 1990. Methods for enumerating microorganisms collected during the Exxon/U.S. Environmental Protection Agency/ and determining biomass in natural environments, in Methods in Alaska Department of Environmental Protection Monitoring Program Microbiology 22, R. Grigorova and J. R. Norris, eds. Academic in 1990. Press, London, ppl-39 BIOREMEDIATION 475

19. Jahns, H. O., J. R. Bragg, L. C. Dash, and E. H. Owens, 1991. 26. Prince, R. C., J. R. Clark, and J. E. Lindstrom, 1990. Bioremedia- Natural cleaning of shorelines following the Exxon Valdez oil spill. tion Monitoring Program. Joint report of Exxon, U.S. Environ- Proceedings of the 1991 International Oil Spill Conference, Ameri- mental Protection Agency, and the Alaska Department of Envi- can Petroleum Institute, Washington, D.C., pp 167-176 ronmental Conservation, Anchorage, Alaska 20. Kaneko, T., M. I. Krichevsky, and R. M. Atlas, 1979. Numerical 27. Pritchard, P. H. and C. F. Costa, 1991. EPA's Alaska oil spill taxonomy of bacteria from the Beaufort Sea. Journal of General bioremediation project. Environmental Science and Technology, Microbiology, vllO, pplll-125 v25, pp372-379 21. Leahy, J. G. and R. R. Colwell, 1990. Microbial degradation of 28. Safferman, S. L, 1991. Selection of nutrients to enhance biodé- hydrocarbons in the environment. Microbiological Reviews, v54, gradation for the remediation of oil spilled on beaches. Proceed- pp303-315 ings of the 1991 International Oil Spill Conference, American 22. Lindstrom, J. E., R. C. Prince, J. R. Clark, M. J. Grossman,T. R. Petroleum Institute, Washington, D.C., pp571-576 Yeager, J. F. Braddock, and E. J. Brown, 1991. Microbial popula- 29. Snellnam, E. A., R. P. Collins, and J. C. Cooke, 1988. Utilization tions and hydrocarbon biodégradation potentials in fertilized of fuel oils by fungi isolated from oceanic tarballs. Letters in shoreline sediments impacted by the T/V Exxon Valdez oil spill. Applied Microbiology, v6, ppl05-107 Applied and Environmental Microbiology, v57, pp2514-2522 30. Venkateswaran, K., T. Iwabuchi, Y. Matsui, H. Toki, E. Ham- Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1993/1/469/2357220/2169-3358-1993-1-469.pdf by guest on 25 September 2021 23. Nauman, S. A., 1991. Shoreline cleanup: equipment and opera- ada, and H. Tanaka, 1991. Distribution and biodégradation poten- tions. Proceedings of the 1991 International Oil Spill Conference, tial of oil-degrading bacteria in north-eastern Japanese coastal American Petroleum Institute, Washington, D.C., ppl41-148 waters. FEMS Microbiological Ecology, v86, ppl 13-122 24. Pimm, S. L., J. H. Lawton, and J. E. Cohen, 1991. Food web 31. Walker, J. D. and R. R. Colwell, 1976. Enumeration of petro- patterns and their consequences. Nature, v350, pp669-674 leum-degrading microorganisms. Applied and Environmental Mi- 25. Prince, R. C, 1992. Bioremediation of oil spills, with particular crobiology, v31, ppl98-207 reference to the spill from the Exxon Valdez. in Microbial Control 32. Zar, J. H., 1984. Biostatistical Analysis. Prentice-Hall, En- of Pollution, J. C. Fry, G. M. Gadd, R. A. Herbert, C. W. Jones, glewood Cliffs, New Jersey and LA. Watson-Craik, eds. Society for General Microbiology, Symposium 48. Cambridge University Press, ppl9-34 Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1993/1/469/2357220/2169-3358-1993-1-469.pdf by guest on 25 September 2021