Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H₂ plus CO₂, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day⁻¹ for methanol, 0.38 mM day⁻¹ for acetate, 0.90 mM day⁻¹ for H₂, and 1.85 mM day⁻¹ for formate. Substrate consumption and CH₄ production during all tests suggested that at least three different physiologic types of methanogens were present: H₂ plus CO₂ or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H₂ and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO₂-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO₄²⁻ reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day⁻¹ for sulfate reduction and from 0.13 to 0.60 day⁻¹ for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Microbial Diversity in a Hydrocarbon- and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.     

Potential of Bioaugmentation and Biostimulation for Enhancing Intrinsic Biodegradation in Oil Hydrocarbon-Contaminated Soil

Studies were conducted using a 10-chamber Micro-Oxymax (Columbus, OH, USA) respirometer to determine the effect of bioaugmentation and biostimulation (by diverse ways of O₂ supply) on enhancing biodegradation of oil hydrocarbons to reduce risk at a former military airport in Kluczewo, Poland. Indigenous or exogenous bacteria bioaugmentation was used to degrade hydrocarbons. Aerated water and/or aqueous solutions of H₂O₂ or KMnO₄ were used to supply O₂. The intrinsic and enhanced biodegradation was evaluated by the O₂ uptake and CO₂ production rates obtained using a linear regression of the cumulative O₂ uptake and CO₂ production curves. Generally, in all cases biodegradation rates enhanced by bioaugmentation were two to four times higher than the rates of intrinsic biodegradation. Moreover, application of indigenous bacteria was more efficient in comparison to the exogenous consortia. The highest CO₂ production rates were achieved when aqueous solution of KMnO₄ was applied, as the increase of CO₂ production rates were about 71% to 97% higher compared to a control. The aqueous solution of H₂O₂ did not cause any significant improvement of the biodegradation rates. Compared to a control, the addition of aerated water resulted in a decrease of CO₂ production rates. Most probably the excessive soil moisture could reduce the air-filled porosity and, consequently, the oxygen contents in soil.     

Effect of Hydrologic and Geochemical Conditions on Oxygen-Enhanced Bioremediation in a Gasoline-Contaminated Aquifer

Oxygen addition to enhance bioremediation of gasoline-contaminated ground water was performed in two locations of a shallow aquifer in South Carolina characterized by benzene, toluene, and methyl tert-butyl ether (MTBE) at concentrations greater than 1 mg/L, respectively. Oxygen addition with an oxygen-release compound (a proprietary form of magnesium peroxide [MgO₂]) produced markedly different results with respect to dissolved oxygen (DO) generation and contaminant decrease in the two locations. Oxygen-release compound injected at the former underground storage tank (UST) source area did not significantly change measured concentrations of DO, benzene, toluene, or MTBE. Conversely, oxygen-release compound injected 200 m downgradient of the former UST source area rapidly increased DO levels, and benzene, toluene, and MTBE concentrations decreased substantially. The different results can be related to differences in hydrologic and geochemical conditions that characterized the two locations prior to oxygen addition. For example, the contaminated aquifer downgradient of the former UST source area was anoxic, but frequently received oxygen-saturated recharge during rainfall events. As such, the aquifer was characterized by low concentrations of reduced species (such as ferrous iron (Fe²⁺), as well as relatively high numbers of aerobic heterotrophic bacteria (as most probable number [MPN] per milliliter). In contrast, recharge does not occur in the paved, former UST source area. The anoxic aquifer was characterized by higher concentrations of Fe²⁺ that exerted a significant chemical oxygen demand on the oxygen injected, and much lower numbers of aerobic heterotrophic bacteria. The results of this investigation indicate the important role that pre-existing hydrologic, geochemical, and microbiologic conditions have on the outcome of oxygen-based remediation strategies, and suggest that these properties should be evaluated prior to the implementation of oxygen-based remedial strategies.     

Synthesis of the Neurotoxin Quinolinic Acid in Apoptotic Tissue from Suberites domuncula: Cell Biological, Molecular Biological, and Chemical Analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue. Apoptosis was induced by exposing the sponges to 2,2'-dipyridyl or by maintaining them under nonaeration conditions. After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue. Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity. However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity. The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight). The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized. The expression of this gene is up-regulated in apoptotic tissue. These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts.     

Response Characteristics between Sulfate-Reducing Bacteria and Environmental Factors in Petroleum Hydrocarbon-Contaminated Field of Northeast China

Lithology samples were collected from six sites of a petroleum hydrocarbon-contaminated site in northeast China along a contamination plume. The sulfate-reducing bacteria (SRB) diversity of all samples was analyzed by PCR-DGGE technology. The Shannon-Wiener indexes (1.004–3.665), Simpson indexes (0.516–0.907) and Pielou indexes (0.996–1.004) of all samples were used to characterize the abundances, advantages and evenness of the microbial communities. Additionally, Canoco for Windows 4.5 was utilized to analyze the correlation between dominant SRB and environmental factors. The results showed that the abundance, advantages and evenness of the sulfate-reducing bacterial community changed regularly along the contamination plume direction. The microbial homology of the samples was not high (0.25–0.80), and the dominant bacteria exhibited heteroplasmy. Additionally, the dominant bacteria were identified as uncultured bacteria. Canonical correspondence analysis (CCA) analysis showed that the distribution and structure of SRB communities were not obviously correlated with the concentration of total petroleum hydrocarbons (TPH), dissolved oxygen (DO) and other environmental factors. The results presented herein provide evidence of natural bioremediation in petroleum hydrocarbon-contaminated fields and information that will be useful to bioremediation of other contaminated fields.     

Kinetics of Sulfate Reduction in a Coastal Aquifer Contaminated with Petroleum Hydrocarbons

An integrated field and laboratory study was conducted to quantify the effect of environmental determinants on the activity of sulfate reducers in a freshwater aquifer contaminated with petroleum hydrocarbons (PHC). Within the contaminated zone, PHC-supported in␣situ sulfate reduction rates varied from 11.58±3.12 to 636±53 nmol cm⁻³ d⁻¹ and a linear increase (R ²=0.98) in reduction rate was observed with increasing in situ sulfate concentrations suggesting sulfate limitation. Half-saturation concentration (K _s) for sulfate reduction coupled to PHC mineralization was determined for the first time. At two different sites within the␣aquifer, maximum sulfate reduction rate under␣non-limiting conditions (R _max) was 5,000 nmol cm⁻³ d⁻¹, whereas the retrieved K _s values were 3.5 and 7.5 mM, respectively. The K _s values are the highest ever reported from a natural environment. Furthermore, the K _s values were significantly higher than in situ sulfate concentrations confirming sulfate limited growth. On addition of lactate and formate, sulfate reduction rate increased indicating that reactivity and bioavailability of organic substrate may also have played a role in rate inhibition in certain parts of the aquifer. Experiments with sulfide amendments show statistically minor decrease in sulfate reduction rates on addition of sulfide and analogous increase in sulfide toxicity with increasing sulfide concentrations (0.5–10 mM) was not apparent.     

The Bioremediation Potential of Hydrocarbonoclastic Bacteria Isolated From a Mangrove Contaminated by Petroleum Hydrocarbons on the Cilacap Coast,Indonesia

The main purpose of the study was to isolate strains of bacteria capable of degrading hydrocarbons from contaminated mangroves and to investigate the ability of the isolated bacteria to degrade total petroleum hydrocarbons (TPH) in a microcosm model of an oily sludge. The potential use of these bacteria strains as environmental clean-up agents was tested by culturing them with six different polyaromatic hydrocarbon (PAH) compounds (phenothiazine, fluorene, fluoranthene, dibenzothiophene, phenanthrene, and pyrene). Six viable and culturable bacteria were isolated, and the 16S rDNA sequence for each was amplified using the primers 9F and 1510R. Sequence results were compared using the National Center for Biotechnology Information (NCBI) BLAST program and, combined with phenotypic and phylogenetic data, were used to identify three strains that belonged to the Bacillus genus and were most closely related (98–99%) to Bacillus aquimaris, Bacillus megaterium, and Bacillus pumilus. The other three strains were closely related (98–100%) to Flexibacteraceae bacterium, Halobacilus trueperi, and Rhodobacteraceae bacterium. Two isolates, BA-PZN and BM-PFFP, which were related to Bacillus aquimaris and Bacillus megaterium, respectively, were further characterized and showed great potential for the removal of more complex hydrocarbon compounds in the oily microcosm model.     

Solute Sources in Stream Water during Consecutive Fall Storms in a Northern Hardwood Forest Watershed: A Combined Hydrological, Chemical and Isotopic Approach

Understanding the effects of climate change including precipitation patterns has important implications for evaluating the biogeochemical responses of watersheds. We focused on four storms in late summer and early fall that occurred after an exceptionally dry period in 2002. We analyzed not only the influence of these storms on episodic chemistry and the role of different water sources in affecting surface water chemistry, but also the relative contributions of these storms to annual biogeochemical mass balances. The study site was a well studied 135-ha watershed in the Adirondack Park of New York State (USA). Our analyses integrated measurements on hydrology, solute chemistry and the isotopic composition of NO₃⁻(δ¹⁵N and δ¹⁸O) and SO₄²⁻(δ³⁴S and δ¹⁸O) to evaluate how these storms affected surface water chemistry. Precipitation amounts varied among the storms (Storm 1: Sept. 14–18, 18.5 mm; Storm 2: Sept. 21–24, 33 mm; Storm 3: Sept. 27–29, 42.9 mm; Storm 4: Oct. 16–21, 67.6 mm). Among the four storms, there was an increase in water yields from 2 to 14%. These water yields were much less than in studies of storms in previous years at this same watershed when antecedent moisture conditions were higher. In the current study, early storms resulted in relatively small changes in water chemistry. With progressive storms the changes in water chemistry became more marked with particularly major changes in C_b (sum of base cations), Si, NO₃⁻, and SO₄²⁻, DOC and pH. Analyses of the relationships between Si, DOC, discharge and water table height clearly indicated that there was a decrease in ground water contributions (i.e., lower Si concentrations and higher DOC concentrations) as the watershed wetness increased with storm succession. The marked changes in chemistry were also reflected in changes in the isotopic composition of SO₄²⁻ and NO₃⁻. There was a strong inverse relationship between SO₄²⁻ concentrations and δ³⁴S values suggesting the importance of S biogeochemical redox processes in contributing to SO₄²⁻ export. The isotopic composition of NO₃⁻ in stream water indicated that this N had been microbially processed. Linkages between SO₄²⁻ and DOC concentrations suggest that wetlands were major sources of these solutes to drainage waters while the chemical and isotopic response of NO₃⁻ suggested that upland sources were more important. Although these late summer and fall storms did not play a major role in the overall annual mass balances of solutes for this watershed, these events had distinctive chemistry including depressed pH and therefore have important consequences to watershed processes such as episodic acidification, and the linkage of these processes to climate change.     

Bioremediation of Soil Artificially Contaminated with Petroleum Hydrocarbon Oil Mixtures: Evaluation of the Use of Animal Manure and Chemical Fertilizer

The search for cheaper and environmentally friendly options of enhancing petroleum hydrocarbon degradation has continued to elicit research interest. One of such options is the use of animal manure as biostimulating agents. A combination of treatments consisting of the application of poultry manure, piggery manure, goat manure, and chemical fertilizer was evaluated in situ during a period of 4 weeks of remediation. Each treatment contained petroleum hydrocarbon mixture (kerosene, diesel oil, and gasoline mixtures) (10% w/w) in soil as a sole source of carbon and energy. After 4 weeks of remediation, the results showed that poultry manure, piggery manure, goat manure, and NPK (nitrogen, phosphorous, and potash [potassium]) fertilizer exhibited 73%, 63%, 50%, and 39% total petroleum hydrocarbon degradation, respectively. Thus, all the biostimulating treatment strategies showed the ability to enhance petroleum hydrocarbon microbial degradation. However, poultry manure, piggery manure, and goat manure treatments showed greater petroleum hydrocarbon reductions than NPK fertilizer treatment. A first-order kinetic equation was fitted to the biodegradation data and the specific degradation rate constant (k) values obtained showed that the order of effectiveness of these biostimulating strategies in the cleanup of soil contaminated with petroleum hydrocarbon mixtures (mixture of kerosene, diesel oil, and gasoline) is NPK fertilizer < goat manure < piggery manure < poultry manure. Therefore, this present work has indicated that the application of poultry manure, piggery manure, goat manure, and chemical fertilizer could enhance petroleum hydrocarbon degradation with poultry manure, showing a greater effectiveness and thus could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil.     

Quantification of Compositional Changes of Petroleum Hydrocarbons by GC/FID and GC/MS during a Long-Term Bioremediation Experiment

Samples from a long-term bioremediation experiment contaminated with two crude oils, Arabian Heavy and Gullfax, was used to analyze the compositional change of petroleum hydrocarbons. A time course of five different homologous series of petroleum hydrocarbons were analysed by GC/FID and GC/MS. The homologous series were n-alkanes, acyclic isoprenoids, alkylated naphthalenes, alkylated phenanthrenes, and alkylated dibenzothiophenes. Several biomarker compounds were monitored during the experiment to evaluate the possible use as conserved reference compounds for the quantification of other oil compounds, that is, nor-hopanes, hopanes, methyl-hopanes, steranes, mono- og triaromatic steranes. The 17α(H),21β(H)-hopane was found to be stable toward biodegradation and was used as reference compound. The internal standard quantification method was used to quantify changes of the homologous series of oil compounds, and a graphic presentation was used to compare the decrease of the individual compounds. This was found to be an easy way of comparing relative changes in oil. The disappearance of the compounds was extensive and in 6 to 7 months less than 6% remained. The decrease of the n-alkanes (>C15) and acyclic isoprenoids was almost uniform within each homologous series and thus independent of physical-chemical characteristics. Evaporation affected compounds with boiling points lower than n-C15. The alkylated aromatic and sulfur-aromatic compounds decreased according to the degree of alkylation and the decrease showed to be delayed by 10 to 20% by each additional alkyl group. The lack of isomeric-specific degradation of most of the aromatic and sulfur-aromatic compounds, until extensive decrease in concentration had occurred, suggests these compounds have to be dissolved, before any biodegradation occurs.     

On the Need to Develop Guidelines for Characterizing and Reporting Intrinsic Disorder in Proteins

Since the early 2000s, numerous computational tools have been created and used to predict intrinsic disorder in proteins. At present, the output from these algorithms is difficult to interpret in the absence of standards or references for comparison. There are many reasons to establish a set of standard‐based guidelines to evaluate computational protein disorder predictions. This viewpoint explores a handful of these reasons, including standardizing nomenclature to improve communication, rigor and reproducibility, and making it easier for newcomers to enter the field. An approach for reporting predicted disorder in single proteins with respect to whole proteomes is discussed. The suggestions are not intended to be formulaic; they should be viewed as a starting point to establish guidelines for interpreting and reporting computational protein disorder predictions.     

Studies on the Lipid Components of Endotoxin II. Chemical Analyses and Biological Properties of Neutral,Polar-I and Polar-II Subfractions

Lipid components obtained from Salmonella typhosa O-901 endotoxin by acid hydrolysis were separated into neutral, polar-I and polar-II lipid fractions by silica gel column chromatography. These lipids were further separated by silica gel column and/or thin-layer chromatography. The subfractions were analyzed by thin-layer chromatography, gas chromatography and infrared spectrophotometry. Seven subfractions obtained from the neutral lipid fraction contained lauric, myristic, palmitic, 3-OH-myristic acid, artificial products of 3-OH-myristic acid, or a small amount of two unidentified fatty acids. These fatty acids and glucosamine were commonly detected in six subfractions obtained from the polar-I lipid fraction. Fatty acids, glucosamine, and O-phosphorylethanolamine were detected in all of the 13 subfractions obtained from the polar-II lipid fraction. Chick embryo lethal activity, rabbit pyrogenicity and in vitro interferon inducing activity were found in three polar-I lipid subfractions and five polar-II lipid subfractions, but not in neutral lipids. The activities were highest in a polar-II lipid subfraction, which contained smaller amounts of O-phosphorylethanolamine and glucosamine than the other subfractions. However, no particular chemical constituent (s) related to the biological activities could be found. Prolonged acid hydrolysis of the polar-II lipids gave rise to neutral and polar-I lipids. Chemical and biological aspects of the lipid constituents of endotoxin are discussed.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.     

Treatability Testing for Weathered Hydrocarbons in Soils: Bioremediation,Soil Washing,Chemical Oxidation,and Thermal Desorption

Soils previously treated with landfarming to reduce petroleum hydrocarbon concentrations are often left with a less biodegradable residual fraction that can present challenges for additional treatment. Four possible polishing technologies were tested on the bench scale for weathered hydrocarbons present in fine-grain soils obtained from a previously landfarmed area at an active oil refinery. The technologies included additional bioremediation (both biostimulation and bioaugmentation tested), soil washing, chemical oxidation, and low-temperature thermal desorption. Multiple parameters were tested separately for each technology to identify possible factors that were relevant across technologies. Extractable hydrocarbons comprised only approximately 35% of the organic carbon in the soils, and this component was considerably less affected by biological, surfactant, and oxidant treatment than organic materials that are not quantified by the TEH analysis. Treatment testing of thermal desorption indicated removal of large quantities of extractable hydrocarbons despite the presence of high organic matter. The additional demand to the system would likely result in considerably large timeframes (biological treatment), reagent quantities (soil washing and oxidation), or energy input (thermal desorption) for treatment of target hydrocarbons on a full scale.