Ronald M. Atlas

Oil Biodegradation and Bioremediation: A Tale of the Two Worst Spills in U.S. History

The devastating environmental impacts of the Exxon Valdez spill in 1989 and its media notoriety made it a frequent comparison to the BP Deepwater Horizon spill in the popular press in 2010, even though the nature of the two spills and the environments impacted were vastly different. Fortunately, unlike higher organisms that are adversely impacted by oil spills, microorganisms are able to consume petroleum hydrocarbons. These oil degrading indigenous microorganisms played a significant role in reducing the overall environmental impact of both the Exxon Valdez and BP Deepwater Horizon oil spills.

Hydrocarbon Biodegradation and Oil Spill Bioremediation

Much of the early work on the microbial utilization of petroleum hydrocarbons, conducted in the 1950s and 1960s, was done with the goal of using hydrocarbons as substrates for producing microbial biomass (Shennan, 1984; Champagnat, 1964; Champagnat and Llewelyn, 1962; Cooney et al., 1980; Ballerini, 1978). Petroleum was viewed as an inexpensive carbon source and single cell protein (microbial biomass) was considered as a possible solution to the perceived impending world food shortage for the predicted global population explosion. Applied studies focused on optimizing microbial growth on low- to middle-molecular-weight hydrocarbons. These studies developed fermentor designs for large-scale single cell protein production with agitation and aeration systems that permitted high rates of microbial growth on soluble and highly emulsified hydrocarbon substrates. High-speed impellers (>800 rpm) were used to mix the hydrocarbon substrates and high rates of forced aeration with baffles within the fermentors were used to supply the molecular oxygen necessary for the microbial utilization of hydrocarbons (Hatch, 1975; Prokop and Sobotka, 1975). Optimized microbial growth in these fermentors consumes as much as 100,000 g hydrocarbon/m3 per day (Kanazawa, 1975).

Bioremediation of petroleum pollutants

Hydrocarbon-degrading microorganisms are ubiquitously distributed in soil and aquatic environments. Populations of hydrocarbon-degraders normally constitute less than 1% of the total microbial communities, but when oil pollutants are present these hydrocarbon-degrading populations increase, typically to 10% of the community. With regard to rates of natural degradation, these typically have been found to be low and limited by environmental factors. Rates reported for pristine marine waters typically are less than 0·03 g/m3/day. In adapted communities rates of hydrocarbon degradation of 0·5–50 g/m3/day have been reported. Bioremediation tries to raise the rates of degradation found naturally to significantly higher rates. The two general approaches that have been tested for the bioremediation of marine oil spills are the application of fertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria and the addition of naturally occurring adapted microbial hydrocarbon-degraders by seeding. Bioremediation, accomplished by the application of fertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria, was successfully applied for the treatment of the 1989 Alaskan oil spill in Prince William Sound, Alaska. Seeding with adapted nonindigenous microbial hydrocarbon degraders was tested on smaller spills in Texas—such as the Mega Borg spill—but bioremediation to remove petroleum pollutants by seeding has yet to be demonstrated as efficacious in field trials. The spill of more than 200,000 barrels of crude oil from the oil tanker Exxon Valdez in Prince William Sound, Alaska, as well as smaller spills in Texas—such as the Mega Borg spill—have been treated by bioremediation to remove petroleum pollutants. The Exxon Valdez spill formed the basis for a major study on bioremediation through fertilizer application and the largest application of this emerging technology. Three types of nutrient supplementation were tested: water-soluble (23:2 N:P garden fertilizer formulation): slow-release (Customblen); and oleophilic (Inipol EAP 22). Each fertilizer was tested in laboratory simulations and in field demonstration plots to determine the efficacy of nutrient supplementation. The use of Inipol EAP22 (oleophilic microemulsion with urea as a nitrogen source, laureth phosphate as a phosphate source, and oleic acid as a carbon source) and Customblen (slow-release calcium phosphate, ammonium phosphate, and ammonium nitrate within a polymerized vegetable oil coating) was approved for shoreline treatment and was used as a major part of the cleanup effort. Multiple regression models showed that nitrogen applications were effective in stimulating the rates of biodegradation.

Petroleum biodegradation and oil spill bioremediation

Abstract Hydrocarbon-utilizing microorganisms are ubiquitously distributed in the marine environment following oil spills. These microorganisms naturally biodegrade numerous contaminating petroleum hydrocarbons, thereby cleansing the oceans of oil pollutants. Bioremediation, which is accomplished by adding exogenous microbial populations or stimulating indigenous ones, attempts to raise the rates of degradation found naturally to significantly higher rates. Seeding with oil degraders has not been demonstrated to be effective, but addition of nitrogenous fertilizers has been shown to increase rates of petroleum biodegradation. In the case of the Exxon Valdez spill, the largest and most thoroughly studied application of bioremediation, the application of fertilizer (slow release or oleophilic) increased rates of biodegradation 3–5 times. Because of the patchiness of oil, an internally conserved compound, hopane, was critical for demonstrating the efficacy of bioremediation. Multiple regression models showed that the effectiveness of bioremediation depended upon the amount of nitrogen delivered, the concentration of oil, and time.

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

Response of Microbial Populations to Environmental Disturbance

Taxonomic and genetic diversities of microbial communities disturbed by chemical pollutants were lower than in undisturbed reference communities. The dominant populations within the disturbed communities had enhanced physiological tolerances and substrate utilization capabilities, indicating that generalized physiological versatility is an adaptive characteristic of populations that successfully compete within disturbed communities.

The Microbiology of Aquatic Oil Spills

Publisher Summary This chapter discusses the microbiology of aquatic oil spills. Immediately upon spilling, oil begins to undergo a series of physical and chemical changes. The processes causing these changes include spreading, emulsification, dissolution, evaporation, sedimentation, and adsorption. Collectively, the oil is weathered by these processes. The weathering of oil depends on the amount and type of oil spilled, and on environmental conditions. Petroleum hydrocarbons have only a very limited solubility in water. Therefore, most oil spillages initially form a surface slick. The surface slick can be moved by wind, wave, and current action. A surface oil slick immediately begins to spread, initially owing to gravitational forces, resulting in a thinner layer of oil covering a larger area. The viscosity of the spilled oil will, to some extent, influence the rate of spreading and, as viscosity is temperature dependent, water temperature will also influence the area covered by a surface slick. The chapter illustrates the effects of petroleum hydrocarbons on microorganisms, microbial emulsification and degradation of petroleum, and the microorganisms and oil pollution abatement.

Bioremediation of Petroleum Pollutants Diversity and environmental aspects of hydrocarbon biodegradation

The manufacture, transportation, and distribution of petroleum and chemical products during the last century has resulted in hydrocarbon-contamination becoming a major environmental problem. Most of the environmental inputs of petroleum are accommodated largely due to the capacities of microorganisms to biodegrade hydrocarbons. Bioremediation has gained significant political support among competing technologies for in situ cleanup of pollutants. In addition to its potential for local cleanup of contaminated water and soil, bioremediation may have a future role in solving problems on a global scale, including removal of greenhouse gases from the atmosphere. This article examines in detail the following topics: bacterial and fungal metabolism of hydrocarbons; bioremediation of marine oil spills; site remediation; performance and regulatory oversight. In summary diverse microorganisms using diverse metabolic pathways have the capacities for degrading a wide spectrum of hydrocarbon structures in petroleum. Some pathways lead to detoxification and destruction of the pollutants whereas others activate potentially harmful compounds. In most cases of detoxification, environmental modification is used to stimulate the biodegradative activities of indigenous organisms. Biodegradation is emerging as an important cost-effective treatment for marine oil spills and contaminated sites. 13 refs., 2 figs.

Diversity of microbial communities

As used by microbiologists, the term diversity has various meanings, often describing qualitative morphological or physiological variances among microorganisms (Starr and Skerman, 1965; Belser, 1979; Hamada and Farrand, 1980; Hanson, 1980; Stanley and Schmidt, 1981; Walker, 1978; Yeh and Ornston, 1980). Microbial populations indeed exhibit great heterogeneity or diversity in their morphological, physiological, and ultimately genetic characteristics. An extensive list of diversifying factors that act to establish differentiating characteristics between microbial species has been discussed by Starr and Schmidt (1981). Some examples of these diversifying features are listed in Table I. These diversifying features have traditionally been employed by bacteriologists as the criteria for differentiating species. Often, the ability to recognize and distinguish species of microorganisms is difficult, but it is essential for assessing diversity.

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.