ELSEVIER International Biodeterioration & Biodegradation (1995) 311-321 Copyright 0 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 09&l-8305/95/$9.50+.00 0964-8305(95)00030-5 Bioremediation of Petroleum Pollutants Ronald M. Atlas Department of Biology, University of Louisville, Louisville, KY 40292, USA ABSTRACT Hydrocarbon-degrading microorganisms are ubiquitously distributed in soil and aquatic environments. Populat
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  ELSEVIER International Biodeterioration Biodegradation (1995) 311-321 Copyright 0 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 09&l-8305/95/ 9.50+.00 0964-8305(95)00030-5 Bioremediation of Petroleum Pollutants Ronald M. Atlas Department of Biology, University of Louisville, Louisville, KY 40292, USA ABSTRACT 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 degra- dation, these typically have been found to be low and limited by environ- mental actors. Rates reported for pristine marine waters typically are less than 0=03g/m3/day. In adapted communities rates of hydrocarbon degra- dation of 0.5-50g/m3jday 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 ertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria and the addition of naturally occurring adapted microbial hydrocarbon-degraders by seeding. Bior- emediation, 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 infield 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 petro- leum pollutants. The Exxon Valdez spill ormed the basis or 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- 317  3 8 R. M. Atlas release (Customblen); and oleophilic (Inipol EAP 22). Each fertilizer was tested in laboratory simulations and in field demonstration plots to deter- mine 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. INTRODUCTION Bioremediation is an acceleration of the natural fate of oil pollutants and hence a natural or ‘green solution’ to the problem of oil pollutants that causes minimal (if any) ecological effects. There are two approaches used for bioremediation. The first relies upon the metabolic capacities of the indigenous microbial populations. Bioremediation is accomplished by environmental modification, for example through aeration or fertilizer application, to overcome factors that limit the rates of hydrocarbon biodegradation by the indigenous microbial populations. In the second approach, exogenous microbial populations are added. The seed cultures are selected for their hydrocarbon-degradation activities. Bioremediation has not yet been shown to be effective for the treatment of open water oil following a spill, but is a cost-effective approach to oil spill cleanup of oiled shoreline environments. To demonstrate that a bioremediation technology is potentially useful, it is important that the ability to enhance the rates of hydrocarbon biodegradation be demonstrated under controlled conditions. This gener- ally requires both laboratory and in situ experiments to demonstrate the efficacy of a bioremediation treatment. The evaluation of hydrocarbon biodegradation in situ is far more difficult than in laboratory studies. The analysis of residual hydrocarbons is especially complicated since the distribution of oil in the environment is typically patchy, and therefore, a high number of replicate samples must be obtained in order to obtain statistically valid results. Because of the problems with quantitation of hydrocarbon recovery from field sites, ratios of hydrocarbons within the complex hydrocarbon mixture have been used to assess the degree of biodegradation. In parti- cular, the fact that hydrocarbon-degrading microorganisms usually degrade pristane and phytane at much lower rates than n-alkanes has permitted the use of pristane or phytane as internal recovery standards  Bioremediation of petroleum pollutants 319 (Atlas, 1981). These measurements assume that pristane and phytane remain undegraded and, therefore, by determining the ratio of straight- chain alkanes to these highly branched alkanes, it is possible to estimate the extent to which microorganisms have attacked the hydrocarbons in the petroleum mixture. However, in situations where pristane or phytane is degraded at similar rates to straight-chain alkanes, this assumption is invalid and alternative internal standards, such as hopanes, are required. METHODOLOGIES FOR BIOREMEDIATION Seeding involves the introduction of microorganisms into the natural environment for the purpose of increasing the rate or extent, or both, of biodegradation of pollutants. The rationale for this approach is that indi- genous microbial populations may not be capable of degrading the wide range of potential substrates present in such complex mixtures as petro- leum. By adding a large biomass of hydrocarbon-degraders, the rates of hydrocarbon biodegradation can be increased if the added cultures are able to survive and express their hydrocarbon-degradation activities in the environments to which they are added. This assumes that they can outcompete the indigenous microbial populations and that they have superior capacities. Seeding with specific cultures of oil-degrading bacteria will fail to enhance the hydrocarbon degradation capability of natural environments if they do not survive or if they are displaced by indigenous hydrocarbon-degrading microorganisms. The National Environmental Technology Assessment Corporation (NETAC) has been established to establish protocols to test the safety and efficacy of such seed cultures. To be effective, seeding would have to be applied early if the purpose was to reduce the lag period before indigenous populations began to attack the oil. This would require actively growing cultures and a huge biomass. Seeding could also be used to extend the range of compounds that would be attacked. Indigenous populations generally fail to attack asphaltics and polynuclear aromatics with four or more rings. Seed cultures could be found to attack such compounds. However, this may not be required since asphaltics have minimal solubilities and effects on biota and most crude oils have very limited concentrations of higher molecular weight polynuclear aromatics. Since microorganisms require nitrogen, phosphorus and other mineral nutrients for incorporation into biomass, the availability of these nutrients within the area of hydrocarbon degradation is critical. Concentrations of available nitrogen and phosphorus in seawater generally are severely limiting to microbial hydrocarbon degradation (Atlas, 1981; Leahy &  320 R. M. Atlas Colwell, 1990). Laboratory experiments with an olephilic fertilizer have demonstrated significant enhancement of oil biodegradation (Bragg et al. 1993a; Chianelli et al. 1991); in some experiments 60% of added oil was biodegraded in fertilized flasks compared to 38% in unfertilized ones within 60 days (LaDousse & Tramier, 1991). CASE STUDIES Exxon Valdez seeding trials In the initial effort to identify cultures that might be applied to the cleanup effort in Prince William Sound following the Exxon VaZdez acci- dental oil spillage, products from 10 companies were selected for labora- tory phase testing by the USEPA (Venosa, 1991; Venosa et al. 1991a,b). Some products delayed biodegradation. Most degradation, when it occurred, started after a 3-5 day lag period and reached significant levels after 20-30 days. Of the products tested, two were selected for further field testing in Prince William Sound on shorelines impacted by the Exxon Valdez spill. In the field trials, four small plots were used to assess the effectiveness of seeding (Venosa et al. 1991c, in press). These field trials failed to demon- strate enhanced oil biodegradation by these products. There were no significant differences between the four plots during a 27-day trial period. It must be noted, however, that the oil was already highly degraded by the time these field trials were conducted and that environmental variability makes it difficult to observe statistically significant differences between experimental and reference sites when relatively few samples are collected and analyzed. Mega Borg spill seeding trials Biotreatment of the Mega Borg spill off the Texas coast consisted of applying a seed culture produced by Alpha Corporation. The Texas General Land Office has reported that the use of the Alpha culture on the Mega Borg spill and also on a spillage from the Apex barge that impacted the Marrow marsh was effective at removing significant amounts of oil (Mauro, 1990a,b; Mauro & Wynne, 1990). Independent observations indicated that treated oil changed in physical appearance and may have been emulsified as a result of addition of the Alpha product; chemical analyses on samples from impacted and reference sites failed to demon- strate that treatment with the Alpha product enhanced rates of petroleum
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