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Microbial degradation of petroleum at low temperature
was supported in part by the Delaware River Basin Authority and NOAA, Sea Giant, College of Marine Studies, University of Delaware. WAYNE LEATHEM
PBT~R K ~ E R DON MAURER ROBERT BIGGS WILLIAM TREASURE
Field Station and Campus, College of Marine Studies, University of Delaware, Lewes and Newark, Delay,are, USA. Cronin, L. E., Biggs, R. B., Flemer, D. A., Pfitzenmeyer, H. T., Goodwyn, F., Dovel, W. L. & Ritchie, D. E. (1970). Gross physical and biological effects of overboard spoil disposal in upper Chesapeake Bay. Nat. Res. Inst. Spec. Rept. 3, Univ. of Maryland, 66 pp. Environmental Protection Agency (1971). Dissolved oxygen criteria, division of water quality standards, 10 pp. Jenkinson, I. R. (1972). Sludge dumping and benthic communities. Mar. Poll. Bull., 3 (7): 102-105. Lie, U. (1969). The logarithmic series and the lognormal distri-
bution applied to benthic infauna from Puget Sound, Washington, USA. Fisk. Div. Skr. Ser. Hav. Unders., 15: 234-245. Manning, J. H. (1957). The Maryland soft-shell clam industry and its effect on fide water resources. Md. Dept. Res. & Educ. C.B.L. Rept. No. 11, 25 pp. Maurer, D., Biggs, R., Leathern, W., Kinner, P., Treasure, W., Otley, M., Watling, L. & Klemas, V. (1973). Effect of spoil disposal on benthic communities near mouth of Delaware Bay. College of Marine Studies, NOAA. Sea Grant Report, Univ. of Del. (in press). Postma, H. (1967). Sediment transport and sedimentation in the estuarine environment, pp. 158-184. In: Estuaries. Ed. George H. Lauff. Pub. No. 83 Amer. Assoc. Adv. Sci. Wash. DC. Salia, S. B., Pratt, S. D. & Polgar, T. T. (1972). Dredge spoil disposal in Rhode Island Sound. Mar. Tech. Rept. No. 2, Univ. Rhode Island, 48 pp. Sherk, J. A. (1971a). The effects of suspended and deposited sediments on estuarine organisms: literature summary and research needs. Natural Resources Institute, Univ. Maryland, Contrib. No. 443, 73 pp. Sherk, J. A. & O'Connor, J. M. (1971b). The effects of suspended and deposited sediments on estuarine organisms, phase lI. Ann. Rept. to Coastal Engineering Res. Center, US Corps of Engineers. Strom, R. N. (1972). Sediment distribution in southwestern Delaware Bay. Master's Thesis, 116 pp. Univ. of Delaware.
Microbial Degradation of Petroleum at Low Temperature Bacteria were isolated from littoral sediments collected in Chedabucto Bay, Nova Scotia, and from oil-contaminated soil adjacent to a natural oil seep at Cape Simpson, A l a s ~ . Data are presente~ on the range of hydrocarbon utilization and growth temperature of two bacteria, which suggest that bacteria existing in these environments play a significant role in the bindegradation of pollutant hydrocarbons. Oil pollution of the marine environment must be regarded as at least a possibility wherever offshore oil fields are developed (Cundell, 1972; Sander, 1972). The discovery of natural gas and condensate in the E-48 wildcat well Mobil Oil on Tetco Sable Island, 100 miles southeast of Cape Canso, Nova Scotia in 1971 was the first announced strike on the Atlantic seaboard (Anon, 1971). Little is known about the environmental effects of petroleum hydrocarbons in waters originating from the cold Labrador current. Similarly the estimated production from Prudhoe Bay on the coastal plain of Alaska's North Slope will be over two million barrels per day by 1980 (McMinn & Golden, 1973). This represents an enormous potential for pollution in the arctic environment.
(Midget et al., 1969; Kator et al., 1971). The documentation of the presence of microorganisms in marine environments capable of degrading branch-chain alkanes, cycloalkanes and polynuclear aromatic hydrocarbons should be given high priority (Friede et al., 1972).
Effects of Low Temperature
Since about three-quarters of the world's surface is covered by oceanic water, the bulk of which rarely reaches a temperature of 5°C, the majority of marine microorganisms are probably adapted to low temperatures (psychronphiles). In a review of the microbial degradation of oil, Professor C. E. Zobell of the Scripps Institute of Oceanography (Zobell, 1969) stressed that the contribution of psychrophilic marine bacteria to the degradation of crude oil and petroleum products was unknown, but he recently reported the capacity for aliphatic hydrocarbon degradation in seawater samples at temperatures as low as 0-2°C (Zobell, 1972). Atlas & Bartha (1972) have found that the biodegradation of petroleum hydrocarbons by marine bacteria is temperature-dependent. Fresh Sweden crude oil (1 per cent w/v) was added to seawater collected from the New Jersey coast in September when the water temperature Hydrocarbon-degrading Microorganisms Hydrocarbon-degrading microorganisms have been was 17.5°C and in December when it was 7.5°C. The demonstrated in water and sediment samples collected seawater was supplemented with nitrogen and phosin harbours, bays and estuaries in areas of known pollu- phorus and the percentage of added oil degraded to CO2 tion (Poliakova, 1962; Zobell & Prokop, 1966; Ahem after 60 days incubation at 5, 10, 15 and 20°C was 8, et al., 1971; Walker & Colwell, 1972). It is generally 31, 46 and 46 per cent respectively for the September thought that aliphatic hydrocarbons are more readily water sample and 20, 37, 46 and 46 per cent in the degraded than aromatic hydrocarbons by marine December sample. These results suggest a seasonal inbacteria (Zobell et al., 1943; Konovaltschikoff-Mazoyer crease in psychrophilic hydrocarbon-degrading bacteria & Senez, 1956) and evidence has been advanced for the in the December seawater. The seasonal selection of preferential degradation of saturated paraffan in crude estuarine bacteria by water temperature has been reoil under laboratory and simulated field conditions cently investigated by Sieburth (1967). 125
Experimental Procedure Hydrocarbon-degrading bacteria were isolated from littoral sediments taken at Arichat on Isle de Madame, Nova Scotia, which was heavily polluted with No. 6 fuel oil from the Arrow spill, and oil-contaminated soil adjacent to a natural oil seep at Cape Simpson, Alaska (McCown, Brown & Murramann, 1971) by the enrichment culture technique using Nos. 1 and 6 fuel oils and naphthalene as enrichment substrates. The environmental temperatures at these two sampling sites were 16°C and 8°C respectively. The enrichment flasks were incubated at 0, 8, 16 and 24°C. The majority of bacteria were isolated from the 16 and 24°C enrichment flasks and were members of the genera Pseudomonas, Arthrobacter, Corynebacterium, Vibrio, Achromobacter, and Brevibacterium. Representative isolates were streaked onto Rila salts agar containing n-dodecane, n-hexylbenzene, decalin, tetralin, naphthalene, and phenanthracene or on basal medium plates stored in cans saturated with xylene or methylcyclohexane. The streak inoculated plates were incubated at 0, 8, 16 and 24°C to determine the growth temperature of the organisms on the respective hydrocarbons. A detailed description of the isolation and screening procedures has been published recently by Cundell & Traxler (1973). As a preliminary report we will present data on the range of hydrocarbon utilization of two organisms. Coryneform bacterium number 6-A7-24-1 was isolated from Chedabucto Bay, Nova Scotia, and a pseudomonad number R-N-24-3 was isolated from Cape Simpson, Alaska. Isolate 6-A7-24-1, which was tentatively assigned to the genus Arthrobacter, had the following characteristics: Gram positive; motile in broth culture; pleomorphic rods on nutrient seawater agar plates which form coccoid cells on prolonged incubation; no acid formation in glucose under aerobic and anaerobic conditions; nitrate reduced; positive lipase activity; hydrolysed starch and gelatin; and cytochrome oxidase positive. Isolate R-N-24-3, which was assigned to the genus Pseudomonas, had the following characteristics: Gram negative; motile; short rods; acid produced in glucose under aerobic conditions; nitrate reduced; positive lipase activity, hydrolysed starch but not gelatin; and cytochrome oxidase positive. Using the growth on solid media containing the hydrocarbons at 16°C as the criteria, a wide range of hydrocarbon utilization by the two organisms was evident. Isolates 6-A7-25-1 and R-N-24-3 grew at the expense of dodecane, hexylbenzene, naphthalene, phenanthracene, decalin, tetralin and methylcyclohexane but not xylene. Growth occurred between 8 ° and 24°C within 14 days of incubation suggesting the bacteria are tolerant of a range of temperatures.
Discussion This report suggests that hydrocarbon-degrading bacteria are present in low temperature marine and coastal environments. These organisms possess the ability to degrade low molecular weight alkanes, cycloalkanes and mono- and dinuclear aromatic hydrocarbons. Although these bacteria can grow at reduced temperatures their proliferation will be curtailed at freezing point. Nutrient availability, especially the lack 126
of nitrogen and phosphorus, is limiting in marine environments (Atlas & Bartha, 1972). Certain components of crude oil and petroleum products such as long-chain alkanes, polynuclear aromatic hydrocarbons and asphalt are apparently refractory to microbial degradation. Analyses of oil polluted marine sediments should reveal which petroleum hydrocarbons are accumulating in marine environments (Blumer & Sass, 1972). On identification of these persistent petroleum hydrocarbons attempts should be made to confirm the inability of marine microorganisms to degrade them. The aliphatic hydrocarbons contained in crude oil are readily utilized by marine bacteria. Cooxidation of aromatic and cylic hydrocarbons by bacteria growing on hexadecane have been extensively reported (Raymond & Jamison, 1971). The significance of the cooxidation of petroleum hydrocarbons in marine environments needs investigation. The combination of high pressure and low temperatures may mean that petroleum hydrocarbons reaching deep sea sediments will remain essentially unchanged. This possibility was dramatically demonstrated by recovery of almost perfectly preserved food in a waterfilled lunchbox from the salvaged research submarine Alvin of Woods Hole Oceanographic Institute after a year below 1,500 m of seawater.
Future Work A full report on the characterization of the hydrocarbon-degrading bacteria from the Attic oil seep will appear at a future date. The authors are investigating the enrichment of hydrocarbon-degrading bacteria in the water column and on an oil-contaminated beach in Upper Narragansett Bay following the liberation of approximately 90,000 gallons of No. 6 fuel oil when the tanker Pennant hit an obstruction off Popasquash Point on the night of April 10, 1973. This oil spill in our backyard gives us an excellent opportunity to study the microbial degradation of a residual fuel oil and identify the bacteria which are most prominent in the biodegradation of acute levels of petroleum hydrocarbons. A . M . CUNDELL R. W. TRAXLER
Department of Plant Pathology, University of Rhode Island, Kingston, RI 02881, USA. Ahearn, D. G., Meyers, S. P. & Standard, P. G. (1971). The role of yeasts in the decomposition of oils in the marine environment. Develop. Ind. Microbiol., 12: 126-134. Anonymous (1971). Offshore East Canada has first discovery. World Oil 173; August 1971. Atlas, R. M. & Bartha, R. (1972). Degradation and mineralization of petroleum: limitation by nitrogen and phosphorus. Biotech. Bioeng., 41: 309-318. Atlas, R. M. & Bartha, R. (1972). Biodegradation of petroleum in seawater at low temperatures. Can. J. Microbiol., 18: 1,851-1,855. Blumer, M. & Sass, J. (1972). Oil pollution: Persistence and degradation of spilled fuel oil. Science, 176: 1,120-1,122. Cundell, A. M. (1972). Oil pollution and offshore field development. J.N.Z. Inst. Chem., 36: 184-187. Cundell, A. M. & Traxler, R. W. (1973). The isolation and characterization of hydrocarbon-utilizing bacteria from Chedabucto Bay, Nova Scotia. pp. 421-426. In: Proceedings of joint conference on prevention and control of oil spills. American Petroleum Institute, New York 1973. Friede, I , Guire, P., Gholson, R. K., Gaudy, E. & Gaudy, A. F. (1972). Assessment of biodegradation potential for controlling oil spills on the high seas. United States Coast Guard Report 4110.1/3.1. Kator, H. I., Oppenheimer, C. H. & Miget, R. J. (1971). Micro-
bial degradation of a Louisiana crude oil in closed flasks under simulated field conditions, pp. 287-298. In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1971. Konovaltschikoff-Mazoyer, M. & Senez, J. (1956). Degradation bacterienne des hydrocarbures paraffiniques. Ann. Inst. Pasteur, 91: 60-67. McCown, B. H., Brown, J. & Murrmann, R. P. (1971). Effect of oil seepages and spills on the ecology and biochemistry in cold dominated environments. 1971 Annual Report, US Army Cold Regions Research and Engineering Laboratory, Hanover, New Hampshire. McMinn, T. J. & Golden, P. (1971). Behavioral characteristics and cleanup techniques of North Slope crude oil in an arctic winter environment, pp. 263-276. In: Proceedings of joint conference on prevention and control of oil spills. American Petroleum Institute, New York, 1973. Miget, R. J., Oppenheimer, C. H. & Kator, H. I. (1969). Microbial degradation of normal paraffin hydrocarbons in crude oils. pp. 327-331. In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1969. Poliakov, I. N. (1962). Distribution of microorganisms of oxidizing hydrocarbons in the water of Neva Inlet. and
Mikrobiol., 13: 1,076-1,081. Raymond, R. L. & Jamison, V. W. (1971). Biochemical activities of Nocardia. Adv. Appl. Microbiol., 14: 93-122. Sanders, N. (1972). North sea oil: can the technology cope. New Scientist, 16: 380-382. Sieburth, J. McN. (1967). Seasonal selection of planktonic estuarine bacteria by water temperature. J. Exp. Mar. Biol. Ecol., 1: 98-121. Walker, J. D. & Colwell, R. R. (1973). Microbial ecology of petroleum utilization in Chesapeake Bay. pp. 685-690. In: Proceedings of joint conference on preventio,n and control of oil spills, American Petroleum Institute, New York, 1973. Zobell, C. E. (1969). Microbial modification of crude oil in the sea. pp. 317-326, In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1969. Zobell, C. E. ~1972). Bacterial degradation of oil pollutants. A Workshop, Georgia State University, December 4-6, 1972 preprint. Zobell, C. E., Grant, C. W. & Haas, H. F. (1943). Marine microorganisms which oxidize petroleum hydrocarbons. Bull. Am. Assn. Pet. Geol., 27: 1,175-1,193. Zobell, C. E. & Prokop, J. F. (1966). Microbial oxidation of mineral oils in Barataria Bay bottom deposits. Z. Allg. Microbiol., 6: 143-162.
Spread of an Introduced Ascidian to Ireland In the last twenty years Styela clava (=S. mammiculata Carlisle), an introduced ascidian from the western Pacific (Millar, 1970), has been noted at Plymouth (Carlisle, 1954), Langstone Harbour, Hampshire (Houghton & Millar, 1960) and is now abundant in the Solent. In 1971-72 we found that this species is well established in areas of full salinity in Cork Harbour, Republic of Ireland. Subsequent investigations have shown that this species is common on small stones in most muddy, sheltered areas within the outer Harbour. It has been shown that the Solent area in Hampshire and Cork Harbour are centres of primary infection for some recently introduced species: a barnacle, Elminius modestus Darwin (Crisp, 1958; Crisp & Southward, 1959) and a serpulid tubeworm Mercierella enigmatica Fauvel (Guiry & Guiry, 1973), both of which are known to be transported via shipping. In addition, two species of Grateloupia (Rhodophyceae) (Farnham & Irvine, 1968, 1973) and Sargassurn muticum (Yendo) Fensholt, from the Pacific (Farnham et al., 1973) have been noted in the Solent. In Cork Harbour a species of Cryptonemia (Rhodophyceae) new to the Atlantic is well established (Guiry et al., 1973). In view of the appearance of Styela clava in Ireland many of the species already introduced into Britain may be expected to spread there also, particularly into Cork Harbour. Several oyster pests are now well established in Britain and the slipper limpet, Crepidula fornicata (L.) is particularly common in the Solent. In Ireland only a single collection of this species has been
Pollution Information Pollution: Sources of Information: (ed. K. Henderson). Proceedings of a 1-day conference held on 27 October, 1971, at the Library Association, London. Aslib, London. (1972). iii+101 pp. Price £2.50.
recorded (McMillan, 1970) and oyster beds there are, at present, remarkably free from pests. G. M. GUIRY M. D. GUIRY
Department of Biology and Geology, Polytechnic of North London:, Holloway Road, London, N7 8DB. Carlisle, D. B. (1954). Styela mammiculata n. sp., a new species of ascidian from the Plymouth area. J. mar. biol. Ass. UK, 33: 329-334. Crisp, D. J. (1958). The spread of Elrninius modestns Darwin in north-west Europe. J. mar. biol. Ass. UK, 37: 483-520. Crisp, D. l. & Southward, A. J. (1959). The further spread of Elminius modestus in the British Isles to 1959. J. mar. biol. Ass. UK, 38: 429-437. Farnham, W. F. & Irvine, L. M. (1968). Occurrence of unusually large plants of Grateloupia in the vicinity of Portsmouth, Nature, Lond., 219: 744-746. Farnham, W. F. & Irvine, L. M. (1973). The addition of a foliose species of Grateloupia (Rhodophyceae) to the British marine flora. Br. phycol. J.: abstract (in press). Farnham, W. F., Fletcher, R. L. & Irvin, L. M. (1973). Attached sargassum found in Britain. Nature, 243: 231-232. Guiry, G. M. & Guiry, M. D. (1973). Mercierella enigmatica Fauvel (Polychaeta, Serpulidae) from Cork Harbour. Ir. Nat. J. : (in press). Guiry, M. D., Irvine, L. M. & Farnham, W. F. (1973). A species of Cryptonemia new to Europe. Br. phycol. J.: abstract (in press). Houghton, D. R. & Millar, R. H. (1960). Spread of the ascidian, Styela rnammieulata Carlisle. Nature Lond., 185: 862. McMillan, N. F. (1970). British Shells. Wart:e, London and New York. Millar, R. H. (1970). British Ascidians . . . Synopses of the British Fauna (New Series) No. 1. Academic Press, London and New York.
This book deals with the problem of disseminating information about pollution. To this end two 'consumers' and four 'suppliers' were invited to talk on pollution. The first 'consumer', Gerald Leach, the 127
PBT~R K ~ E R DON MAURER ROBERT BIGGS WILLIAM TREASURE
Field Station and Campus, College of Marine Studies, University of Delaware, Lewes and Newark, Delay,are, USA. Cronin, L. E., Biggs, R. B., Flemer, D. A., Pfitzenmeyer, H. T., Goodwyn, F., Dovel, W. L. & Ritchie, D. E. (1970). Gross physical and biological effects of overboard spoil disposal in upper Chesapeake Bay. Nat. Res. Inst. Spec. Rept. 3, Univ. of Maryland, 66 pp. Environmental Protection Agency (1971). Dissolved oxygen criteria, division of water quality standards, 10 pp. Jenkinson, I. R. (1972). Sludge dumping and benthic communities. Mar. Poll. Bull., 3 (7): 102-105. Lie, U. (1969). The logarithmic series and the lognormal distri-
bution applied to benthic infauna from Puget Sound, Washington, USA. Fisk. Div. Skr. Ser. Hav. Unders., 15: 234-245. Manning, J. H. (1957). The Maryland soft-shell clam industry and its effect on fide water resources. Md. Dept. Res. & Educ. C.B.L. Rept. No. 11, 25 pp. Maurer, D., Biggs, R., Leathern, W., Kinner, P., Treasure, W., Otley, M., Watling, L. & Klemas, V. (1973). Effect of spoil disposal on benthic communities near mouth of Delaware Bay. College of Marine Studies, NOAA. Sea Grant Report, Univ. of Del. (in press). Postma, H. (1967). Sediment transport and sedimentation in the estuarine environment, pp. 158-184. In: Estuaries. Ed. George H. Lauff. Pub. No. 83 Amer. Assoc. Adv. Sci. Wash. DC. Salia, S. B., Pratt, S. D. & Polgar, T. T. (1972). Dredge spoil disposal in Rhode Island Sound. Mar. Tech. Rept. No. 2, Univ. Rhode Island, 48 pp. Sherk, J. A. (1971a). The effects of suspended and deposited sediments on estuarine organisms: literature summary and research needs. Natural Resources Institute, Univ. Maryland, Contrib. No. 443, 73 pp. Sherk, J. A. & O'Connor, J. M. (1971b). The effects of suspended and deposited sediments on estuarine organisms, phase lI. Ann. Rept. to Coastal Engineering Res. Center, US Corps of Engineers. Strom, R. N. (1972). Sediment distribution in southwestern Delaware Bay. Master's Thesis, 116 pp. Univ. of Delaware.
Microbial Degradation of Petroleum at Low Temperature Bacteria were isolated from littoral sediments collected in Chedabucto Bay, Nova Scotia, and from oil-contaminated soil adjacent to a natural oil seep at Cape Simpson, A l a s ~ . Data are presente~ on the range of hydrocarbon utilization and growth temperature of two bacteria, which suggest that bacteria existing in these environments play a significant role in the bindegradation of pollutant hydrocarbons. Oil pollution of the marine environment must be regarded as at least a possibility wherever offshore oil fields are developed (Cundell, 1972; Sander, 1972). The discovery of natural gas and condensate in the E-48 wildcat well Mobil Oil on Tetco Sable Island, 100 miles southeast of Cape Canso, Nova Scotia in 1971 was the first announced strike on the Atlantic seaboard (Anon, 1971). Little is known about the environmental effects of petroleum hydrocarbons in waters originating from the cold Labrador current. Similarly the estimated production from Prudhoe Bay on the coastal plain of Alaska's North Slope will be over two million barrels per day by 1980 (McMinn & Golden, 1973). This represents an enormous potential for pollution in the arctic environment.
(Midget et al., 1969; Kator et al., 1971). The documentation of the presence of microorganisms in marine environments capable of degrading branch-chain alkanes, cycloalkanes and polynuclear aromatic hydrocarbons should be given high priority (Friede et al., 1972).
Effects of Low Temperature
Since about three-quarters of the world's surface is covered by oceanic water, the bulk of which rarely reaches a temperature of 5°C, the majority of marine microorganisms are probably adapted to low temperatures (psychronphiles). In a review of the microbial degradation of oil, Professor C. E. Zobell of the Scripps Institute of Oceanography (Zobell, 1969) stressed that the contribution of psychrophilic marine bacteria to the degradation of crude oil and petroleum products was unknown, but he recently reported the capacity for aliphatic hydrocarbon degradation in seawater samples at temperatures as low as 0-2°C (Zobell, 1972). Atlas & Bartha (1972) have found that the biodegradation of petroleum hydrocarbons by marine bacteria is temperature-dependent. Fresh Sweden crude oil (1 per cent w/v) was added to seawater collected from the New Jersey coast in September when the water temperature Hydrocarbon-degrading Microorganisms Hydrocarbon-degrading microorganisms have been was 17.5°C and in December when it was 7.5°C. The demonstrated in water and sediment samples collected seawater was supplemented with nitrogen and phosin harbours, bays and estuaries in areas of known pollu- phorus and the percentage of added oil degraded to CO2 tion (Poliakova, 1962; Zobell & Prokop, 1966; Ahem after 60 days incubation at 5, 10, 15 and 20°C was 8, et al., 1971; Walker & Colwell, 1972). It is generally 31, 46 and 46 per cent respectively for the September thought that aliphatic hydrocarbons are more readily water sample and 20, 37, 46 and 46 per cent in the degraded than aromatic hydrocarbons by marine December sample. These results suggest a seasonal inbacteria (Zobell et al., 1943; Konovaltschikoff-Mazoyer crease in psychrophilic hydrocarbon-degrading bacteria & Senez, 1956) and evidence has been advanced for the in the December seawater. The seasonal selection of preferential degradation of saturated paraffan in crude estuarine bacteria by water temperature has been reoil under laboratory and simulated field conditions cently investigated by Sieburth (1967). 125
Experimental Procedure Hydrocarbon-degrading bacteria were isolated from littoral sediments taken at Arichat on Isle de Madame, Nova Scotia, which was heavily polluted with No. 6 fuel oil from the Arrow spill, and oil-contaminated soil adjacent to a natural oil seep at Cape Simpson, Alaska (McCown, Brown & Murramann, 1971) by the enrichment culture technique using Nos. 1 and 6 fuel oils and naphthalene as enrichment substrates. The environmental temperatures at these two sampling sites were 16°C and 8°C respectively. The enrichment flasks were incubated at 0, 8, 16 and 24°C. The majority of bacteria were isolated from the 16 and 24°C enrichment flasks and were members of the genera Pseudomonas, Arthrobacter, Corynebacterium, Vibrio, Achromobacter, and Brevibacterium. Representative isolates were streaked onto Rila salts agar containing n-dodecane, n-hexylbenzene, decalin, tetralin, naphthalene, and phenanthracene or on basal medium plates stored in cans saturated with xylene or methylcyclohexane. The streak inoculated plates were incubated at 0, 8, 16 and 24°C to determine the growth temperature of the organisms on the respective hydrocarbons. A detailed description of the isolation and screening procedures has been published recently by Cundell & Traxler (1973). As a preliminary report we will present data on the range of hydrocarbon utilization of two organisms. Coryneform bacterium number 6-A7-24-1 was isolated from Chedabucto Bay, Nova Scotia, and a pseudomonad number R-N-24-3 was isolated from Cape Simpson, Alaska. Isolate 6-A7-24-1, which was tentatively assigned to the genus Arthrobacter, had the following characteristics: Gram positive; motile in broth culture; pleomorphic rods on nutrient seawater agar plates which form coccoid cells on prolonged incubation; no acid formation in glucose under aerobic and anaerobic conditions; nitrate reduced; positive lipase activity; hydrolysed starch and gelatin; and cytochrome oxidase positive. Isolate R-N-24-3, which was assigned to the genus Pseudomonas, had the following characteristics: Gram negative; motile; short rods; acid produced in glucose under aerobic conditions; nitrate reduced; positive lipase activity, hydrolysed starch but not gelatin; and cytochrome oxidase positive. Using the growth on solid media containing the hydrocarbons at 16°C as the criteria, a wide range of hydrocarbon utilization by the two organisms was evident. Isolates 6-A7-25-1 and R-N-24-3 grew at the expense of dodecane, hexylbenzene, naphthalene, phenanthracene, decalin, tetralin and methylcyclohexane but not xylene. Growth occurred between 8 ° and 24°C within 14 days of incubation suggesting the bacteria are tolerant of a range of temperatures.
Discussion This report suggests that hydrocarbon-degrading bacteria are present in low temperature marine and coastal environments. These organisms possess the ability to degrade low molecular weight alkanes, cycloalkanes and mono- and dinuclear aromatic hydrocarbons. Although these bacteria can grow at reduced temperatures their proliferation will be curtailed at freezing point. Nutrient availability, especially the lack 126
of nitrogen and phosphorus, is limiting in marine environments (Atlas & Bartha, 1972). Certain components of crude oil and petroleum products such as long-chain alkanes, polynuclear aromatic hydrocarbons and asphalt are apparently refractory to microbial degradation. Analyses of oil polluted marine sediments should reveal which petroleum hydrocarbons are accumulating in marine environments (Blumer & Sass, 1972). On identification of these persistent petroleum hydrocarbons attempts should be made to confirm the inability of marine microorganisms to degrade them. The aliphatic hydrocarbons contained in crude oil are readily utilized by marine bacteria. Cooxidation of aromatic and cylic hydrocarbons by bacteria growing on hexadecane have been extensively reported (Raymond & Jamison, 1971). The significance of the cooxidation of petroleum hydrocarbons in marine environments needs investigation. The combination of high pressure and low temperatures may mean that petroleum hydrocarbons reaching deep sea sediments will remain essentially unchanged. This possibility was dramatically demonstrated by recovery of almost perfectly preserved food in a waterfilled lunchbox from the salvaged research submarine Alvin of Woods Hole Oceanographic Institute after a year below 1,500 m of seawater.
Future Work A full report on the characterization of the hydrocarbon-degrading bacteria from the Attic oil seep will appear at a future date. The authors are investigating the enrichment of hydrocarbon-degrading bacteria in the water column and on an oil-contaminated beach in Upper Narragansett Bay following the liberation of approximately 90,000 gallons of No. 6 fuel oil when the tanker Pennant hit an obstruction off Popasquash Point on the night of April 10, 1973. This oil spill in our backyard gives us an excellent opportunity to study the microbial degradation of a residual fuel oil and identify the bacteria which are most prominent in the biodegradation of acute levels of petroleum hydrocarbons. A . M . CUNDELL R. W. TRAXLER
Department of Plant Pathology, University of Rhode Island, Kingston, RI 02881, USA. Ahearn, D. G., Meyers, S. P. & Standard, P. G. (1971). The role of yeasts in the decomposition of oils in the marine environment. Develop. Ind. Microbiol., 12: 126-134. Anonymous (1971). Offshore East Canada has first discovery. World Oil 173; August 1971. Atlas, R. M. & Bartha, R. (1972). Degradation and mineralization of petroleum: limitation by nitrogen and phosphorus. Biotech. Bioeng., 41: 309-318. Atlas, R. M. & Bartha, R. (1972). Biodegradation of petroleum in seawater at low temperatures. Can. J. Microbiol., 18: 1,851-1,855. Blumer, M. & Sass, J. (1972). Oil pollution: Persistence and degradation of spilled fuel oil. Science, 176: 1,120-1,122. Cundell, A. M. (1972). Oil pollution and offshore field development. J.N.Z. Inst. Chem., 36: 184-187. Cundell, A. M. & Traxler, R. W. (1973). The isolation and characterization of hydrocarbon-utilizing bacteria from Chedabucto Bay, Nova Scotia. pp. 421-426. In: Proceedings of joint conference on prevention and control of oil spills. American Petroleum Institute, New York 1973. Friede, I , Guire, P., Gholson, R. K., Gaudy, E. & Gaudy, A. F. (1972). Assessment of biodegradation potential for controlling oil spills on the high seas. United States Coast Guard Report 4110.1/3.1. Kator, H. I., Oppenheimer, C. H. & Miget, R. J. (1971). Micro-
bial degradation of a Louisiana crude oil in closed flasks under simulated field conditions, pp. 287-298. In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1971. Konovaltschikoff-Mazoyer, M. & Senez, J. (1956). Degradation bacterienne des hydrocarbures paraffiniques. Ann. Inst. Pasteur, 91: 60-67. McCown, B. H., Brown, J. & Murrmann, R. P. (1971). Effect of oil seepages and spills on the ecology and biochemistry in cold dominated environments. 1971 Annual Report, US Army Cold Regions Research and Engineering Laboratory, Hanover, New Hampshire. McMinn, T. J. & Golden, P. (1971). Behavioral characteristics and cleanup techniques of North Slope crude oil in an arctic winter environment, pp. 263-276. In: Proceedings of joint conference on prevention and control of oil spills. American Petroleum Institute, New York, 1973. Miget, R. J., Oppenheimer, C. H. & Kator, H. I. (1969). Microbial degradation of normal paraffin hydrocarbons in crude oils. pp. 327-331. In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1969. Poliakov, I. N. (1962). Distribution of microorganisms of oxidizing hydrocarbons in the water of Neva Inlet. and
Mikrobiol., 13: 1,076-1,081. Raymond, R. L. & Jamison, V. W. (1971). Biochemical activities of Nocardia. Adv. Appl. Microbiol., 14: 93-122. Sanders, N. (1972). North sea oil: can the technology cope. New Scientist, 16: 380-382. Sieburth, J. McN. (1967). Seasonal selection of planktonic estuarine bacteria by water temperature. J. Exp. Mar. Biol. Ecol., 1: 98-121. Walker, J. D. & Colwell, R. R. (1973). Microbial ecology of petroleum utilization in Chesapeake Bay. pp. 685-690. In: Proceedings of joint conference on preventio,n and control of oil spills, American Petroleum Institute, New York, 1973. Zobell, C. E. (1969). Microbial modification of crude oil in the sea. pp. 317-326, In: Proceedings of joint conference on prevention and control of oil spills, American Petroleum Institute, New York 1969. Zobell, C. E. ~1972). Bacterial degradation of oil pollutants. A Workshop, Georgia State University, December 4-6, 1972 preprint. Zobell, C. E., Grant, C. W. & Haas, H. F. (1943). Marine microorganisms which oxidize petroleum hydrocarbons. Bull. Am. Assn. Pet. Geol., 27: 1,175-1,193. Zobell, C. E. & Prokop, J. F. (1966). Microbial oxidation of mineral oils in Barataria Bay bottom deposits. Z. Allg. Microbiol., 6: 143-162.
Spread of an Introduced Ascidian to Ireland In the last twenty years Styela clava (=S. mammiculata Carlisle), an introduced ascidian from the western Pacific (Millar, 1970), has been noted at Plymouth (Carlisle, 1954), Langstone Harbour, Hampshire (Houghton & Millar, 1960) and is now abundant in the Solent. In 1971-72 we found that this species is well established in areas of full salinity in Cork Harbour, Republic of Ireland. Subsequent investigations have shown that this species is common on small stones in most muddy, sheltered areas within the outer Harbour. It has been shown that the Solent area in Hampshire and Cork Harbour are centres of primary infection for some recently introduced species: a barnacle, Elminius modestus Darwin (Crisp, 1958; Crisp & Southward, 1959) and a serpulid tubeworm Mercierella enigmatica Fauvel (Guiry & Guiry, 1973), both of which are known to be transported via shipping. In addition, two species of Grateloupia (Rhodophyceae) (Farnham & Irvine, 1968, 1973) and Sargassurn muticum (Yendo) Fensholt, from the Pacific (Farnham et al., 1973) have been noted in the Solent. In Cork Harbour a species of Cryptonemia (Rhodophyceae) new to the Atlantic is well established (Guiry et al., 1973). In view of the appearance of Styela clava in Ireland many of the species already introduced into Britain may be expected to spread there also, particularly into Cork Harbour. Several oyster pests are now well established in Britain and the slipper limpet, Crepidula fornicata (L.) is particularly common in the Solent. In Ireland only a single collection of this species has been
Pollution Information Pollution: Sources of Information: (ed. K. Henderson). Proceedings of a 1-day conference held on 27 October, 1971, at the Library Association, London. Aslib, London. (1972). iii+101 pp. Price £2.50.
recorded (McMillan, 1970) and oyster beds there are, at present, remarkably free from pests. G. M. GUIRY M. D. GUIRY
Department of Biology and Geology, Polytechnic of North London:, Holloway Road, London, N7 8DB. Carlisle, D. B. (1954). Styela mammiculata n. sp., a new species of ascidian from the Plymouth area. J. mar. biol. Ass. UK, 33: 329-334. Crisp, D. J. (1958). The spread of Elrninius modestns Darwin in north-west Europe. J. mar. biol. Ass. UK, 37: 483-520. Crisp, D. l. & Southward, A. J. (1959). The further spread of Elminius modestus in the British Isles to 1959. J. mar. biol. Ass. UK, 38: 429-437. Farnham, W. F. & Irvine, L. M. (1968). Occurrence of unusually large plants of Grateloupia in the vicinity of Portsmouth, Nature, Lond., 219: 744-746. Farnham, W. F. & Irvine, L. M. (1973). The addition of a foliose species of Grateloupia (Rhodophyceae) to the British marine flora. Br. phycol. J.: abstract (in press). Farnham, W. F., Fletcher, R. L. & Irvin, L. M. (1973). Attached sargassum found in Britain. Nature, 243: 231-232. Guiry, G. M. & Guiry, M. D. (1973). Mercierella enigmatica Fauvel (Polychaeta, Serpulidae) from Cork Harbour. Ir. Nat. J. : (in press). Guiry, M. D., Irvine, L. M. & Farnham, W. F. (1973). A species of Cryptonemia new to Europe. Br. phycol. J.: abstract (in press). Houghton, D. R. & Millar, R. H. (1960). Spread of the ascidian, Styela rnammieulata Carlisle. Nature Lond., 185: 862. McMillan, N. F. (1970). British Shells. Wart:e, London and New York. Millar, R. H. (1970). British Ascidians . . . Synopses of the British Fauna (New Series) No. 1. Academic Press, London and New York.
This book deals with the problem of disseminating information about pollution. To this end two 'consumers' and four 'suppliers' were invited to talk on pollution. The first 'consumer', Gerald Leach, the 127