Vol. 54, No. 3 MICROBIOLOGICAL REVIEWS, Sept. 1990, p. 305-315 0146-0749/90/030305-11$02.00/0 Copyright C 1990, American Society for Microbiology Microbial Degradation of Hydrocarbons in the Environment JOSEPH G. LEAHY AND RITA R. COLWELL* Department of Microbiology, University of Maryland, College Park, Maryland 20742 INTRODUCTION ...................................................................... 305 PHYSICAL AND CHEMICAL FACTORS AFFECTING THE BIODEGRADATION OF HYDROCARBONS ...................................................................... 305 Chemical Composition of the Oil or Hydrocarbons .................................................................... 305 Physical State of the Oil or Hydrocarbons ...................................................................... 306 Concentration of the Oil or Hydrocarbons ...................................................................... 306 Temperature ...................................................................... 307 Oxygen ...................................................................... 307 Nutrients ...................................................................... 307 Salinity ...................................................................... 307 Pressure ...................................................................... 308 Water Activity ...................................................................... 308 pH ...................................................................... 308 BIOLOGICAL FACTORS AFFECTING THE BIODEGRADATION OF HYDROCARBONS ............... 308 Hydrocarbon Degradation by Bacteria, Fungi, and Other Microorganisms ..................................... 308 Adaptation-Effect of Prior Exposure ...................................................................... 309 Adaptation by Alteration of the Genetic Composition of the Microbial Community .......................... 310 Role of Plasmids in Adaptation ...................................................................... 310 Seeding ...................................................................... 310 CONCLUSIONS ...................................................................... 311 ACKNOWLEDGMENTS ...................................................................... 312 LITERATURE CITED ...................................................................... 312 INTRODUCTION The recent spill of more than 200,000 barrels of crude oil from the oil tanker Exxon Valdez in Prince William Sound, Alaska (65), as well as smaller spills in Texas, Rhode Island, and the Delaware Bay (5), has refocused attention on the problem of hydrocarbon contamination in the environment. It is estimated that the annual global input of petroleum is between 1.7 and 8.8 million metric tons, the majority of which is derived from anthropogenic sources (95). Biodeg- radation of hydrocarbons by natural populations of microor- ganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants are eliminated from the environment (95). The effects of environmental parameters on the microbial degradation of hydrocarbons, the elucidation of metabolic pathways and genetic bases for hydrocarbon dissimilation by microorganisms, and the ef- fects of hydrocarbon contamination on microorganisms and microbial communities have been areas of intense interest and the subjects of several reviews (7, 9, 44, 95). The intent of the present review is to present a broad and updated overview of the microbial ecology of hydrocarbon degradation, emphasizing both environmental and biological factors which are involved in determining the rate at which and extent to which hydrocarbons are removed from the environment by biodegradation. Aspects of biodegradation of petroleum and individual hydrocarbons in marine, fresh- water, and soil ecosystems are presented. It should be noted that the majority of studies have been concerned with degradation of oil in the marine environment, and this is necessarily reflected, to a certain extent, in this review. * Corresponding author. Applications of relatively recent advances in molecular biological techniques, such as the isolation of plasmid DNA and the construction of DNA probes, to the study of hydrocarbon degradation by microbial communities will be discussed, as well as the use of natural or genetically engineered microorganisms as seeds to increase rates of biodegradation of hydrocarbon pollutants in the environ- ment. PHYSICAL AND CHEMICAL FACTORS AFFECTING THE BIODEGRADATION OF HYDROCARBONS Chemical Composition of the Oil or Hydrocarbons Petroleum hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty acids, ketones, esters, and porphyrins), and the resins (py- ridines, quinolines, carbazoles, sulfoxides, and amides) (44). Hydrocarbons differ in their susceptibility to microbial at- tack and, in the past, have generally been ranked in the following order of decreasing susceptibility: n-alkanes branched alkanes low-molecular-weight aromatics cy- clic alkanes (104). Biodegradation rates have been shown to be highest for the saturates, followed by the light aromatics, with high-molecular-weight aromatics and polar compounds exhibiting extremely low rates of degradation (59, 76, 145). This pattern is not universal, however, as Cooney et al. (46) reported greater degradation losses of naphthalene than of hexadecane in water-sediment mixtures from a freshwater lake and Jones et al. (78) observed extensive biodegradation of alkylaromatics in marine sediments prior to detectable changes in the n-alkane profile of the crude oil tested. Fedorak and Westlake (53) also reported a more rapid attack of aromatic hydrocarbons during the degradation of crude oil 305

306 LEAHY AND COLWELL by marine microbial populations from a pristine site and a commercial harbor. Horowitz and Atlas (71), using an in situ continuous-flow system in a study of biodegradation in Arctic coastal waters, and Bertrand et al. (26), using a continuous-culture fermen- tor and a mixed culture of marine bacteria, observed degra- dation of all fractions of crude oil at similar rates, in marked contrast to the results of most other studies. In the latter investigation, experimental conditions were optimized and extensive losses of resins (52%) and asphaltenes (74%) were observed. The microbial degradation of these fractions, which have previously been considered relatively recalci- trant to biodegradation (143), can be ascribed to cooxidation, in which non-growth hydrocarbons are oxidized in the presence of hydrocarbons which can serve as growth sub- strates (103). Evidence for cooxidation of asphaltenes was provided by Rontani et al. (114), who reported degradation of asphaltenic compounds in mixed bacterial cultures to be dependent upon the presence of n-alkanes 12 to 18 carbon atoms in length. Compositional heterogeneity among different crude oils and refined products influences the overall rate of biodegra- dation both of the oil and of its component fractions. Walker et al. (147) compared the degradation of two crude and two fuel oils by a mixed culture of estuarine bacteria. Low- sulfur, high-saturate South Louisiana crude oil was the most susceptible to microbial degradation, and high-sulfur, high- aromatic Bunker C fuel oil was the least susceptible. Percent losses of saturated, aromatic, resinous, and asphaltenic hydrocarbons were highly variable among the four oils. Similarly, Jobson et al. (76) observed a greater degree of degradation of "high-quality" North Cantal crude oil than of Lost Horse Hill crude oil, which contained higher levels of sulfur, aromatics, asphaltenes, and resins, when a mixed culture enriched with the North Cantal oil was used. More extensive biodegradation of the Lost Horse Hill oil occurred when a mixed culture enriched with the same oil was used. Physical State of the Oil or Hydrocarbons Oil spilled in water tends to spread and form a slick (25). As a result of wind and wave action, oil-in-water or water- in-oil ("mousse") emulsions may form (45). Dispersion of hydrocarbons in the water column in the form of oil-in-water emulsions increases the surface area of the oil and thus its availability for microbial attack. However, large masses (or plates) of mousse establish unfavorably low surface-to- volume ratios, inhibiting biodegradation (49). Tarballs, which are large aggregates of weathered and undegraded oil, also restrict access by microorganisms because of their limited surface area (43). The formation of emulsions through the microbial produc- tion and release of biosurfactants is an important process in the uptake of hydrocarbons by bacteria and fungi (125). Broderick and Cooney (32) reported that 96% of hydrocar- bon-utilizing bacteria isolated from freshwater lakes were able to emulsify kerosene, and it has been observed that mixed cultures of marine (108) and soil (77) bacteria which effectively degrade crude oil also exhibit strong emulsifying activity. Artificial dispersants have been studied as a means of increasing the surface area and hence the biodegradability of oil slicks. Dispersant formulations used in the 1960s were highly toxic, and their application to oiled intertidal areas following the Torrey Canyon spill resulted in widespread mortality of flora and fauna (48, 126). More recently devel- oped dispersants, such as Corexit, are considerably less toxic (52), but still have been shown to inhibit microbial processes (63). The effectiveness of dispersants in enhancing the biodegradation of oil has been shown to be extremely variable and to be dependent on the chemical formulation of the dispersant, its concentration, and the dispersant/oil application ratio. Studies with different dispersants have reported increases (112), decreases (56, 93), and transitory or slight increases (56, 93, 136) in the rates of microbial degradation of crude oil and individual hydrocarbons. The key differences between petroleum biodegradation in soil and aquatic ecosystems following an oil spill, discussed by Bossert and Bartha (29), are related to the movement and distribution of the oil and the presence of particulate matter, each of which affects the physical and chemical nature of the oil and hence its susceptibility to microbial degradation. Terrestrial oil spills are characterized primarily by vertical movement of the oil into the soil, rather than the horizontal spreading associated with slick formation. Infiltration of oil into the soil prevents evaporative losses of volatile hydro- carbons, which can be toxic to microorganisms. Particulate matter can reduce, by absorption, the effective toxicity of the components of petroleum, but absorption and adsorption of hydrocarbons to humic substances probably contribute to the formation of persistent residues. Concentration of the Oil or Hydrocarbons The rates of uptake and mineralization of many organic compounds by microbial populations in the aquatic environ- ment are proportional to the concentration of the compound, generally conforming to Michaelis-Menten kinetics (28, 105). Michaelian kinetics have been demonstrated for the micro- bial uptake and oxidation of toluene (35, 111), a low- molecular-weight aromatic hydrocarbon of relatively high water solubility, but may not apply to the more insoluble hydrocarbons. The rates of mineralization of the higher- molecular-weight aromatic hydrocarbons, such as naphtha- lene and phenanthrene, are related to aqueous solubilities rather than total substrate concentrations (135, 152, 153). The microbial degradation of long (.C12) alkanes, for which solubilities are less than 0.01 mg/liter (23), occurs at rates which exceed the rates of hydrocarbon dissolution (135, 156) and are a function of the hydrocarbon surface area available for emulsification or physical attachment by cells (55, 94, 148). Biodegradation rates for many hydrocarbons, there- fore, will not display the dependence on concentration which is typically observed with more soluble organic substrates. High concentrations of hydrocarbons can be associated with heavy, undispersed oil slicks in water, causing inhibi- tion of biodegradation by nutrient or oxygen limitation or through toxic effects exerted by volatile hydrocarbons (see below). Fusey and Oudot (59) reported that contamination of seashore sediments with crude oil above a threshold con- centration prevented biodegradation of the oil because of oxygen and/or nutrient limitation. It is likely that high concentrations of oil have similarly negative effects on biodegradation rates following oil spills in other quiescent, low-energy environments such as beaches, harbors, and small lakes or ponds, in which the oil is relatively protected from dispersion by wind and wave action. Rashid (109), for example, observed that the lowest rates of degradation of crude oil spilled from an oil tanker occurred in protected bays and the highest rates occurred in the areas of greatest wave energy. The concept of a maximum or threshold concentration for MICROBIOL. REV.