Phillip M. Fedorak

The effects of phenol and some alkyl phenolics on batch anaerobic methanogenesis

Abstract Phenol and seven alkylphenols (o-, m- and p-cresol, 2.5-, 2.6-, 3.4- and 3,5-dimethylphenol) were added at various concentrations to aliquots of domestic anaerobic sludge in Hungate serum bottles and these were incubated at 37°C. The concentration of methane in the headspace gas was monitored to determine if the phenolics were fermented to methane or if they inhibited the anaerobic process. Only phenol and p-cresol were fermented to methane. At 500 mg l−1 (but not at 300 mg l−1) 2,5-, 3,4- and 3,5-dimethylphenol reduced the rate and the amount of methane produced. The cresols were inhibitory at 1000 mg l−1 but not at 400 mg l−1. In cultures supplemented with acetate and propionate (VOA), and in unsupplemented cultures, phenol at concentrations up to 500 mg l−1 was fermented to methane. Between 800 and 1200 mg l−1 phenol, methane production was neither enhanced nor inhibited relative to control cultures containing no phenol. Inhibition of methane production was evident when phenol was present at ⩾ 2000 mg l−1. Thus the methanogens are less susceptible to phenol inhibition than are the phenol-degrading acid formers. In similar experiments with p-cresol: enhanced methane production was observed at concentrations of ⩽ 400 mg l−1; no enhancement or inhibition was observed at 600 mg l−1; and inhibition was noted when p-cresol was present at ⩾ 1000 mg l−1.

Naphthenic acids and surrogate naphthenic acids in methanogenic microcosms.

Naphthenic acids (NAs) are a complex mixture of naturally occurring acyclic and cyclic aliphatic carboxylic acids in petroleum. In the Athabasca oil sands. NAs have been identified as the largest component of dissolved organic matter in the tailings waters from oils sands extraction processes. They are the major contributor to the acute toxicity of the fine tailings wastewaters at the oil sands extraction plants in northeastern Alberta, Canada. In this study, three sources of NAs were studied, including commercially available NAs, those extracted from oil sands process-affected waters, and individual naphthenic-like surrogate compounds. Analysis by gas chromatography-mass spectrometry demonstrated differences between the commercial and extracted NAs. The NAs derived from the process-affected waters showed a short-term inhibition of methanogenesis from H2 or acetate, but with time the populations resumed methane production. It has been postulated that microbial metabolism of the carboxylated side chains of NAs would lead to methane production. The two NA mixtures failed to stimulate methanogenesis in microcosms that contained either oil sands fine tailings or domestic sewage sludge. However, in microcosms with sewage sludge, methanogenesis was stimulated by some surrogate NAs including 3-cyclohexylpropanoic acid at 400-800 mg/L, 5-cyclohexylpentanoic acid at 200 mg/L or 6-phenylhexanoic acid at 200 and 400 mg/L. When added at 200 mg/L to methanogenic microcosms containing fine tailings, 3-cyclohexylpropanoic and 4-cyclohexylbutanoic acids produced methane yields that suggested mineralization of the side chain and the ring.

Ozonation of Oil Sands Process-Affected Water Accelerates Microbial Bioremediation

Ozonation can degrade toxic naphthenic acids (NAs) in oil sands process-affected water (OSPW), but even after extensive treatment a residual NA fraction remains. Here we hypothesized that mild ozonation would selectively oxidize the most biopersistent NA fraction, thereby accelerating subsequent NA biodegradation and toxicity removal by indigenous microbes. OSPW was ozonated to achieve approximately 50% and 75% NA degradation, and the major ozonation byproducts included oxidized NAs (i.e., hydroxy- or keto-NAs). However, oxidized NAs are already present in untreated OSPW and were shown to be formed during the microbial biodegradation of NAs. Ozonation alone did not affect OSPW toxicity, based on Microtox; however, there was a significant acceleration of toxicity removal in ozonated OSPW following inoculation with native microbes. Furthermore, all residual NAs biodegraded significantly faster in ozonated OSPW. The opposite trend was found for ozonated commercial NAs, which are known to contain no significant biopersistent fraction. Thus, we suggest that ozonation preferentially degraded the most biopersistent OSPW NA fraction, and that ozonation is complementary to the biodegradation capacity of microbial populations in OSPW. The toxicity of ozonated OSPW to higher organisms needs to be assessed, but there is promise that this technique could be applied to accelerate the bioremediation of large volumes of OSPW in Northern Alberta, Canada.

A review of the occurrence, toxicity,and biodegradation of condensed thiophenes found in petroleum

Condensed thiophenes comprise a significant portion of the organosulfur compounds in petroleum and in other products from fossil fuels. Dibenzothiophene (DBT) has served as a model compound in biodegradation studies for over two decades. However, until quite recently, few other organosulfur compounds were studied, and their fates in petroleum-contaminated environments are largely unknown. This paper presents a review of the types of organosulfur compounds found in petroleum and summarizes the scant literature on toxicity studies with condensed thiophenes. Reports on the biodegradation of benzothiophene, alkylbenzothiophenes, DBT, alkylDBTs, and naphthothiophenes are reviewed with a focus on the identification of metabolites detected in laboratory cultures. In addition, recent reports on quantitative studies with DBT and naphtho[2,1-b]thiophene indicate the existence of polar sulfur-containing metabolites that have escaped detection and identification.

A statistical comparison of naphthenic acids characterized by gas chromatography–mass spectrometry

Naphthenic acids are complex mixtures of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula C(n)H(2n+z)O(2), where n is the carbon number and Z specifies a homologous family. These acids have a variety of commercial uses, including being used as wood preservatives. They are found in conventional and heavy oils, and in the oil sands of northeastern Alberta, Canada. Naphthenic acids are major contributors to the toxicity of tailings waters that result from the oil sands extraction process. Eight naphthenic acids preparations (four from commercial sources and four from the oil sands operations) were derivatized and analyzed by gas chromatography-mass spectrometry. The composition of each mixture was summarized as a three-dimensional plot of the abundance of specific ions (corresponding to naphthenic acids) versus carbon number (ranging from 5 to 33) and Z family (ranging from 0 to -12). The data in these plots were divided into three groups according to carbon number (group 1 contained carbon numbers 5-14, group 2 contained carbon numbers 15-21, and group 3 contained carbon numbers 22-33). A t-test, using arcsine-transformed data, was applied to compare corresponding groups in samples from various sources. Results of the statistical analyses showed differences between various commercial naphthenic acids preparations, and between naphthenic acids from different oil sands ores and tailings ponds. This statistical approach can be applied to data collected by other mass spectrometry methods.

Fathead minnow (Pimephales promelas) reproduction is impaired in aged oil sands process-affected waters.

Large volumes of fluid tailings are generated during the extraction of bitumen from oil sands. As part of their reclamation plan, oil sands operators in Alberta propose to transfer these fluid tailings to end pit lakes and, over time, these are expected to develop lake habitats with productive capabilities comparable to natural lakes in the region. This study evaluates the potential impact of various oil sands process-affected waters (OSPW) on the reproduction of adult fathead minnow (Pimephales promelas) under laboratory conditions. Two separate assays with aged OPSW (>15 years) from the experimental ponds at Syncrude Canada Ltd. showed that water containing high concentrations of naphthenic acids (NAs; >25 mg/l) and elevated conductivity (>2000 μS/cm) completely inhibited spawning of fathead minnows and reduced male secondary sexual characteristics. Measurement of plasma sex steroid levels showed that male fathead minnows had lower concentrations of testosterone and 11-ketotestosterone whereas females had lower concentrations of 17β-estradiol. In a third assay, fathead minnows were first acclimated to the higher salinity conditions typical of OSPW for several weeks and then exposed to aged OSPW from Suncor Energy Inc. (NAs ∼40 mg/l and conductivity ∼2000 μS/cm). Spawning was significantly reduced in fathead minnows held in this effluent and male fathead minnows had lower concentrations of testosterone and 11-ketotestosterone. Collectively, these studies demonstrate that aged OSPW has the potential to negatively affect the reproductive physiology of fathead minnows and suggest that aquatic habitats with high NAs concentrations (>25 mg/l) and conductivities (>2000 μS/cm) would not be conducive for successful fish reproduction.

Biotransformation of the cyanobacterial hepatotoxin microcystin-LR, as determined by HPLC and protein phosphatase bioassay

Microcystin-LR (MCLR) is a cyclic hepatotoxin and tumor promotor produced by some freshwater cyanobacteria. The occurrence of microcystin-containing blooms in freshwater has been implicated in animal and human poisonings worldwide. To assess the fate of MCLR in a heterotrophic microbial population, purified MCLR was incubated with a microbial community orginating from the Edmonton wastewater treatment plant. After 10 days of aerobic incubation, a significant decrease in MCLR concentration was observed. Analysis of the culture supernatant by highperformance liquid chromatograph, monitoring absorbance at 210 and 238 nm, suggested that the heptapeptide ring of MCLR and its Adda group had been modified, respectively

The influence of nitrate on microbial processes in oil industry production waters

Sulfide accumulation due to bacterial sulfate reduction is responsible for a number of serious problems in the oil industry. Among the strategies to control the activity of sulfate-reducing bacteria (SRB) is the use of nitrate, which can exhibit a variety of effects. We investigated the relevance of this approach to souring oil fields in Oklahoma and Alberta in which water flooding is used to enhance oil recovery. SRB and nitrate-reducing bacteria (NRB) were enumerated in produced waters from both oil fields. In the Oklahoma field, the rates of sulfate reduction ranged from 0.05 to 0.16 μM S day−1 at the wellheads, and an order of magnitude higher at the oil–water separator. Sulfide production was greatest in the water storage tanks in the Alberta field. Microbial counts alone did not accurately reflect the potential for microbial activities. The majority of the sulfide production appeared to occur after the oil was pumped aboveground, rather than in the reservoir. Laboratory experiments showed that adding 5 and 10 mM nitrate to produced waters from the Oklahoma and Alberta oil fields, respectively, decreased the sulfide content to negligible levels and increased the numbers of NRB. This work suggests that sulfate reduction control measures can be concentrated on aboveground facilities, which will decrease the amount of sulfide reinjected into reservoirs during the disposal of oil field production waters. Journal of Industrial Microbiology & Biotechnology (2001) 27, 80–86.

Structure-reactivity of naphthenic acids in the ozonation process.

Large volumes of oil sands process-affected water (OSPW) are produced in northern Alberta by the surface mining oil sands industry. Naphthenic acids (NAs) are a complex mixture of persistent organic acids that are believed to contribute to the toxicity of OSPW. In situ microbial biodegradation strategies are slow and not effective at eliminating chronic aquatic toxicity, thus there is a need to examine alternative remediation techniques. NAs with multiple rings and alkyl branching are most recalcitrant to microbial biodegradation, but here we hypothesized that these same structural features may lead to preferential degradation in the ozonation process. Total NA degradation increased with increasing pH for commercial NA solutions, suggesting a hydroxyl radical mechanism and that naturally alkaline OSPW would unlikely require pH adjustment prior to treatment. For commercial NAs and OSPW, NAs with more rings and more carbon (and more H atoms) were depleted most rapidly in the process. Relative rate measurements with binary mixtures of model NA compounds not only confirmed this structure reactivity but also indicated that alkyl branching patterns were an additional factor determining NA reactivity. The results demonstrate that ozonation is complementary to microbial biodegradation, and the process remains a promising water reclamation strategy for the oil sands industry.

Fathead minnow (Pimephales promelas) reproduction is impaired when exposed to a naphthenic acid extract

Previous studies have demonstrated that oil sands process-affected water (OSPW) impairs the reproduction of fish and that naphthenic acids (NAs), a natural constituent of oil sands, are suspected of being responsible. This study evaluates the potential impact of NAs on the reproduction of adult fathead minnows (Pimephales promelas) under laboratory conditions. Fathead minnows exposed to a 10 mg/l naphthenic acid extract (NAE) for 21 days spawned fewer eggs and males had reduced expression of secondary sexual characteristics. Male fathead minnows exposed to a 5 mg/l NAE had lower plasma levels of 11-ketotestosterone whereas those exposed to a 10 mg/l NAE had lower concentrations of both testosterone and 11-ketotestosterone. Since OSPW also contains high concentrations of salts, this study also investigated whether they modify the toxicity of NAEs. Spawning was significantly reduced in fathead minnows exposed to a 10 mg/l NAE alone and in combination with NaHCO₃ (700 mg/l), typical of concentrations in OSPW(.) Interestingly, the addition of NaHCO₃ reduced the inhibitory effects of the NAE on the numbers of reproductive tubercles and plasma testosterone levels. Further studies showed that NaHCO₃ acted by reducing the uptake of the NAE to the fish. NaHCO₃ but not NaCl or Na₂SO₄ reduced the acute toxic effects of the NAE on fathead minnow embryo and larvae mortality. Collectively, these studies show that the NAs in OSPW have the potential to negatively affect reproduction in fathead minnows and that HCO₃⁻ reduces the acute and chronic toxicity of NAs.