The diversity of bacteria isolated from creosote- contaminated soils in the United States, Norway, and Germany was determined by comparing their ability to degrade polycyclic aromatic hydrocarbons (PAHs), their phospholipid ester-linked fatty acid (GC-FAME) profiles, sole carbon source utilization patterns (Biolog™ assays (Use of trade names or specific products does not imply endorsement by the U.S. EPA.), and 16S rRNA sequences. Bacteria were initially obtained by enrichment with phenanthrene and fluoranthene. Many were capable of degrading a broad range of the PAHs found in creosote. Phenanthrene- or fluoranthene- degraders were abundant in most of the soils tested. Several of the fluoranthene-degrading isolates clustered with Sphingomonas (formerly Pseudomonas) paucimobilis strain EPA505 in the GC-FAME and Biolog™ analyses and three of the isolates examined by 16S rRNA sequence comparisons showed a close relationship with Sphingomonas. In addition, the Sphingomonas strains showed the most extensive degradation of 4- & 5-ring PAHs in creosote. Burkholderia cepacia strains isolated on phenanthrene from PAH-contaminated soils had limited ability to attack higher molecular weight PAHs either individually or in creosote. Thus, PAH degradation capabilities appeared to be associated with members of certain taxa, independent of the origin of the soils from which the bacteria were isolated.

Anaerobic biodegradation of an artificial mixture ofpolycyclic aromatic hydrocarbons (PAHs), which simulates the PAH component ofcreosote, was examined under methanogenic, sulfidogenic, and nitrate-reducingconditions using creosote-contaminated sediment as the source of inoculum.PAH degradation, CH4 formation and ion reduction were monitoredfor up to one year. Despite demonstrating active methanogenic andnitrate-reducing anaerobic bacterial communities, only limited degradation ofa few PAHs was observed. Under methanogenic conditions limited degradation ofall bicyclic (naphthalene, 1-and 2-methylnaphthalene, biphenyl, and2,6-dimethylnaphthalene) and one tricyclic PAH, anthraquinone, was detected.2-Methylanthracene was apparently degraded under nitrate-reducing conditions.Anthraquinone declined in sulfate enrichments, but this decline was notdependent upon sulfate reduction. None of the 4- or 5-ring PAHs were degradedunder any of the enrichment conditions. These data indicate that under theanaerobic conditions tested there is only a limited potential to degrade PAHswhich must be considered when proposing bioremediation technologies forPAH-contaminated sites, especially if high-molecular-weight PAHs are present.

Sphingomonas paucimobilis strain EPA505 is capable of utilizing many components of coal tar creosote as sole sources of carbon and energy for bacterial growth, including fluoranthene and other polycyclic aromatic hydrocarbons (PAH). During several bioremediation studies, however, we observed that the fluoranthene degradative activity of strain EPA505 was inhibited by the presence of undefined creosote constituents. In practice, integration of a pretreatment step prior to inoculation with strain EPA505 was necessary to facilitate the biodegradation of high molecular weight (HMW) PAHs. Experiments were thus initiated to determine which compound classes in creosote inhibited fluoranthene metabolism by strain EPA505. Creosote was fractionated by solvent extraction at various pH, and three chemical classes were examined: acid (phenolics), base (N-heterocyclics), and neutral (PAH). The mineralization rate of C-14-labeled fluoranthene and cell viability were examined in the presence of these creosote fractions at a range of concentrations. These studies confirm that strain EPA505 has differing susceptibility to the effects of the three classes of creosote constituents. The observed order of toxicity/inhibition was basic fraction > acidic fraction > neutral fraction. These studies provide engineering guidelines and define contamination ranges under which strain EPA505 can be used most effectively as a catalyst in bioremediation (Figure 4).

Chemical analyses and biological response data were used to assess the efficacy of a field-scale hyperfiltration unit in the removal of polycyclic aromatic hydrocarbons (PAHs) and other organic compounds from creosote- and pentachlorophenol (PCP)-contaminated ground water. The hyperfiltration unit consisted of four modules containing porous stainless steel tubes which were coated with a formed-in-place zirconium hydrous oxide-polyacrylic acid (ZOPA) membrane. A fivefold concentration of the feed water (80% volume reduction) with up to 97% removal of high molecular weight PAHs was achieved during pre-demonstration and field-demonstration runs of the hyperfiltration unit. Approximately 68% of PCP was removed by the unit. Removal of phenolics averaged 27% and 36%, respectively, for the two runs.

A two-stage, sequential inoculation bioreactor strategy for the bioremediation of groundwater contaminated with creosote and pentachlorophenol (PCP) was evaluated at bench scale (1.2 L) and pilot scale (454 L). Bioreactor performance using specially selected microorganisms was assessed according to chemical analyses of system influent, effluent, and bioreactor residues, a chemical mass balance evaluation, and comparative biological toxicity and teratogenicity measurements. During pilot-scale operations, the concentration of creosote constituents was reduced from ca. 1000 ppm in the groundwater feed (flow rate 114 L/day) to <9 ppm in the system effluent (total removal efficiency of >99%). Notably, the cumulative concentration of carcinogenic polycyclic aromatic hydrocarbons was reduced from 368 ppm in the feed to 5.2 ppm in the system effluent. Moreover, the toxicity and teratogenicity of the bioreactor effluent were significantly reduced. In general, field data correlated well with those obtained from bench-scale studies.

A Gram-negative bacterium,Pseudomonas sp. strain SR3, was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basal salts medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39–40 μg PCP/ml to 0.0006 μg PCP/ml was observed within 120 h of incubation in the presence of PCP-induced cells ofPseudomonas sp. strain SR3. The initial cell density in these cultures was 6 x 106 cfu/ml. Microtox® 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39–40 p μ/ml were toxic but that final biodegraded samples, 0.0006 μ/g PCP/ml were nontoxic. However, bioassays with embryonic inland silversides,Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing added PCP at concentrations up to 30 times higher than measured in the final biodegraded samples was less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process or undegraded impurities in PCP were toxic or teratogenic. Thus, theM. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Performance data on slurry-phase bioremediation of pentachlorophenol- (PCP-) and creosote-contaminated sediment and surface soil were generated at the bench-scale level. Aqueous slurries were prepared from sediment and surface soil freshly obtained from the American Creosote Works Superfund site at Pensacola, FL. Slurries (1.1 L) were incubated for 30 days in separate, 1.5-L bioreactors operated in the batch mode at 28.5-degrees-C with continuous mixing (300 rpm), DO = 90% and pH = 7.0. Samples removed with time from each reactor were extracted and analyzed by gas chromatography for PCP and 42 monitored creosote constituents to delineate the activity of indigenous microorganisms. Changes in microbial biomass were also recorded. Excluding PCP, benzo[b]fluoranthene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene, slurry-phase bioremediation of highly contaminated sediment (pH adjusted) resulted in rapid and extensive biodegradation (3-5 days to biodegrade > 50% of targeted compounds) of monitored constituents. Conversely, microbial activity in surface soil slurries was slower and generally confined to the more readily biodegradable, lower molecular weight compounds. These data suggest that slurry-phase bioremediation strategies can be effectively employed to remediate creosote-contaminated materials.

Bench-scale biotreatability studies were performed to determine the most effective of two bioremediation application strategies to ameliorate creosote- and pentachlorophenol (PCP)-contaminated soils present at the American Creosote Works Superfund site, Pensacola, Florida: solid-phase bioremediation or slurry-phase bioremediation. When indigenous microorganisms were employed as biocatalysts, solid-phase bioremediation was slow and ineffective (8-12 weeks required to biodegrade >50% of resident organics). Biodegradation was limited to lower-molecular-weight constituents rather than the more hazardous, higher-molecular-weight (HMW) compounds; PCP and HMW polycyclic aromatic hydrocarbons (PAHs) containing 4 or more fused rings resisted biological attach. Moreover, supplementation with aqueous solution of inorganic nutrients had little effect on the overall effectiveness of the treatment strategy. Alternatively, slurry-phase bioremediation was much more effective: >50% of targeted organics were biodegraded in 14 days. Again, however, more persistent contaminants, such as PCP and HMW PAHs, were not extensively degraded when subjected to the action of indigenous microorganisms.

Chemical analyses revealed that polycyclic aromatic hydrocarbons (PAHs) and other organic compounds were present in a perennial freshwater stream that flowed through the abandoned American Creosote Works and into Pensacola Bay, Florida. Moreover, groundwater pumped from a well depth of 21 m at a location adjacent to the site was heavily contaminated with PAHs and other organics.

Shake flask studies examined the rate and extent of biodegradation of pentachlorophenol (PCP) and 42 components of coal-tar creosote present in contaminated groundwater recovered from the American Creosote Works Superfund site, Pensacola, Fla. The ability of indigenous soil microorganisms to remove these contaminants from aqueous solutions was determined by gas chromatographic analysis of organic extracts of biotreated groundwater. Changes in potential environmental and human health hazards associated with the biodegradation of this material were determined at intervals by Microtox assays and fish toxicity and teratogenicity tests. After 14 days of incubation at 30-degrees-C, indigenous microorganisms effectively removed 100, 99, 94, 88, and 87% of measured phenolic and lower-molecular-weight polycyclic aromatic hydrocarbons (PAHs) and S-heterocyclic, N-heterocyclic, and O-heterocyclic constituents of creosote, respectively. However, only 53% of the higher-molecular-weight PAHs were degraded; PCP was not removed. Despite the removal of a majority of the organic contaminants through biotreatment, only a slight decrease in the toxicity and teratogenicity of biotreated groundwater was observed. Data suggest that toxicity and teratogenicity are associated with compounds difficult to treat biologically and that one may not necessarily rely on indigenous microorganisms to effectively remove these compounds in a reasonable time span; to this end, alternative or supplemental approaches may be necessary. Similar measures of the toxicity and teratogenicity of treated material may offer a simple, yet important, guide to bioremediation effectiveness.