Abstract 2017-271
In recent years, diluted bitumen (or dilbit) has become an important source of hydrocarbon-based fuel. While information on the degradation of crude oils has been well researched, dilbit degradation has been studied at a much lesser extent. The objective of this study was to compare biodegradation of dilbit with a conventional crude oil (CCO) under various conditions. Two different microcosm experiments were set up, one containing a mixed culture acclimated to dilbit (Kalamazoo River Enrichment, KRC) and the other having a mixed culture enriched on soil contaminated with hydrocarbons (Anderson Ferry Enrichment, AFC). The microcosms were run for 60 d at 25 °C and for 72 days at 5 °C in flasks containing sterile Bushnell Hass broth and naturally dispersed oil. Each flask was inoculated with the KRC and AFC mixed cultures, and rotated on an orbital shaker (200 rpm) at the above stated temperatures. On each sampling day, triplicates were sacrificed to determine the residual hydrocarbon concentration. Additionally, some samples were used to determine the bacterial composition using 16S rRNA gene sequencing analysis. Hydrocarbon analysis (alkanes and PAHs) was performed by gas chromatography/mass spectrometry (GC/MS/MS). Higher degradation rates were achieved at 25 °C as compared to 5 °C. All the enrichments metabolized CCO as well dilbit, but the nature and extent of the degradation was distinct. KRC meso culture was the most effective among all, as it completely removed alkanes and most of the PAHs. AFC enrichment performed differently at the two temperatures; an acclimation period (8 d) was observed at 5 °C while there was no lag at 25 °C. KRC cryo culture as well as AFC culture at both temperatures degraded alkanes completely while they were not able to metabolize heavier fractions of the oil (C2–4 homologues of 3- and 4-ring compounds). All cultures showed the presence of diverse oil degrading bacteria and the differences in their compositions affected the biodegradation. Although dilbit was biodegraded, for all the treatments except AFC at 5 °C, the rate of degradation and the extent of degradation was greater for CCO owing to the higher concentrations of lighter hydrocarbons.
INTRODUCTION
The production and usage of unconventional fuels (oil sands, shale gas etc.) within the oil market has grown in the last decades. For example, the import of petroleum products derived from Canadian oil sands into the United Sates has sharply increased in the past 40 years. Oil sands yield bitumen, the heaviest form of crude oil which at room temperature is a solid petroleum deposit. To be transported through a pipeline, bitumen must meet specifications such as a density of approximately 940 kg/m3 and a viscosity of 350 cSt at the pipeline reference temperature (7.5 – 18.5 °C). Thus bitumen is either upgraded by coking or hydrocracking to make synthetic crude oil. Bitumen may also be blended with synthetic crude oil in 1:1 proportion to produce synthetic bitumen (i.e., synbit), or with diluents like naphtha, natural gas condensate, or light hydrocarbons to produce diluted bitumen (i.e., dilbit), generally as ~75% of bitumen and ~25% of diluent (Canadian Energy Pipelines Association (CEPA), 2013; Government of Canada, 2013).
Dilbit is classified as a heavy crude oil; its physical and chemical properties are quite different to a conventional crude oil. Dilbit has higher concentrations of heavier fractions like resins and asphaltenes, and therefore it is denser and more viscous than conventional crude oil. Dilbit has high sulfur, metal content and total acid number (Meyer et al., 2007; National Academies of Sciences, Engineering, 2016).
Due to sizeable dilbit spills in 2010 (Kalamazoo, MI) and 2013 (Mayflower, AR) and the ongoing proposal for pipeline expansions within the US, understanding the degradation properties of dilbit has recently gained considerable attention. In contrast to numerous studies reporting the biodegradation of conventional crude oil (Atlas and Bartha, 1972; Cao et al., 2009; Margesin and Schinner, 2001; Rojo, 2009), limited and inconsistent information is available regarding biodegradability of diluted bitumen. Cobanli et al. (2015) and King et al. (2014) reported biodegradation of dilbit, while Crosby S et al. (2013) and Yang et al. (2011) suggested that dilbit cannot be degraded further as bitumen is formed due to the extensive biodegradation of conventional crude. In another study only 25% decrease of total petroleum hydrocarbon concentration was observed after 28 d, suggesting that dilbit may not be completely biodegradable (USEPA, 2013).
It should be noted that most dilbit degradation studies have been conducted for less than 30 days and therefore, information on its biodegradability beyond these short-term incubation times is lacking. To address some of these research gaps, the objective of the present work was to assess biodegradability of diluted bitumen and a conventional crude oil under prolonged conditions. Specifically, experiments were set up at 5 and 25 °C for 60–72 days with microbial consortia obtained from two different hydrocarbon impacted areas. The bacteria implicated in degradation of hydrocarbon fractions were identified by using DNA-based techniques.
MATERIALS AND METHODS
Microcosm experiments were setup to assess the biodegradation of Western Canadian Select dilbit and a conventional crude oil, Prudhoe Bay crude (CCO). The experimental layout is summarized in Table 1. The microcosms were prepared with Bushnell Hass broth (Bushnell and Hass, 1941) and inoculated with mixed cultures that were originally enriched from samples collected from two different sites impacted by hydrocarbons. Kalamazoo River culture (KRC) was enriched on dilbit at 5 (cryo) and 25 (meso) °C whereas Anderson Ferry culture (AFC) was obtained from Ohio River and grown on Alaskan North Slope crude oil (ANS521) at only 25 °C.
Fresh oil (0.07 g) and an aliquot of each mixed culture (0.5 mL) were added to flasks containing 100 mL media (Figure 1-A). Flasks were rotated at 200 rpm in temperature controlled rooms (Figure 1-B). On each sampling day, three microcosms were sacrificed per treatment (Figure 1-C). The oil was extracted with dichloromethane (Figure 1-D) and analyzed for the presence hydrocarbons by gas chromatography-mass spectroscopy as described by Campo et al. (2013). The bacterial composition of the samples used as inoculum was determined analyzing Illumina MiSeq 250bp pair-ended 16S rRNA gene libraries as described elsewhere (Kapoor, et al., 2016).
RESULTS AND DISCUSSION
Oil Characterization
The two oils exhibited different hydrocarbon profiles, with CCO comprising of higher concentrations of lighter hydrocarbons such as alkanes and naphthalene, while dilbit was rich in heavier PAHs (Figure 2). Alkane content of CCO was greater than its PAH content, whereas dilbit had almost equal proportions of alkanes and PAHs. Overall dilbit exhibited lower concentrations of aliphatics and aromatics as compared to CCO. For CCO, pristane and phytane concentrations were lower than C17 and C18 n-alkanes, but in dilbit these branched alkanes were in higher quantities. The PAH distribution for both oils was considerably different. The naphthalene content was 58% in CCO while it was only 38% in dilbit. Furthermore, dilbit contained higher fractions of three and four ring compounds than the CCO. Overall, the quantity of all alkanes and PAHs monitored were significantly lower in dilbit.
Biodegradation of Hydrocarbons
In all the treatments, alkanes were almost completely degraded and residuals mainly comprised of branched alkanes. At higher temperatures, alkanes were much rapidly metabolized. At 25 °C, the rates of alkane degradation were significantly higher for CCO by both AFC and KRC enrichments (p < 0.001). The degradation rate for alkanes in dilbit was slightly lower than CCO in KRC cryo treatment. Nevertheless, the difference was not statistically significant (p = 0.6332). At 5 °C, the AFC consortium consumed alkanes faster for treatment involving dilbit as compared to CCO.
PAH degradation trends were distinct for each treatment. Similar to alkanes, PAH removal was much faster at 25 °C. For all the treatments, PAHs in CCO were reduced to greater extent. As mentioned above, naphthalenes were the primary PAHs present in CCO and all the enrichments were able to metabolize these two-ring aromatics along with their alkylated homologues. Residual PAHs included pyrene, chrysene as well as C3–4 homologues of phenanthrene and fluorene. These PAHs were abundant in dilbit, hence dilbit degradation was less efficient. The rate of degradation of PAHs in dilbit was lower for all treatments except AFC at 5 °C but the differences in the rates were not statistically significant. Day 0 and killed control samples showed comparable concentrations, strongly indication that the degradation was entirely due to bacterial activity.
Studies by King et al. (2014) and Cobanli et al. (2015) reported dilbit biodegradation but their observed alkane and PAH degradation rates were much lower than the rates reported in this study. Differences in the experimental set up may explain some the contrasting results. For example, King et al. (2014) and Cobanli et al. (2015) conducted their experiments with non-amended sea water and non-enriched indigenous bacteria as the inoculum. In contrast, in our study we used synthetic freshwater media inoculated with mixed cultures that were previously enriched on oil.
Experiments were carried out at 5 and 25 °C which mimics biodegradation in winter and summer conditions, respectively. The intensity of biodegradation was much lower at the colder temperature since the rate and extent of degradation was significantly lower than at 25 °C as previously reported (Atlas,1975; Atlas and Bartha, 1972; Campo et al., 2013; Margesin and Schinner, 2001). Extended acclimation period and slower rates of degradation at lower temperatures can be due to decrease in solubility, crystallization of hydrocarbons, and lower metabolic rates.
Bacterial Composition of Mixed Cultures
The composition of the KRC and AFC cultures used in this study was determined by sequence analysis of the 16S rRNA gene (Table 3). At the phylum level, Proteobacteria was the dominating phylum in all the cultures, with Actinobacteria and Bacteroidetes also present the KRC cultures. The dominant genera were markedly different between the KRC and AFC cultures. Specifically, Acinetobacter (72%) was the dominant genus in AFC microbial community, whereas a higher abundance of Pseudomonas (13%, 17%), Rhodococcus (22%, 26.5%), and Hydrogenophaga (15%, 12%) was observed in KRC cryo and meso consortia respectively. Although several other genera were present in these enrichments, their abundance was very low as compared to the dominant genera.
Along with the temperature, microbial enrichment composition may have a substantial influence on the degradation of both crude oils. Significant differences were noted in the nature and extent of hydrocarbon reduction in the three cultures. All the cultures were able to degrade alkanes, while KRC cryo culture was less effective than KRC meso culture in degrading PAHs. Additionally, the AFC enrichment behaved differently at 5 and 25 °C. At the colder temperature, the AFC culture had a lag period of about 8 d before metabolizing the hydrocarbons. The differences in microbial activities may be explained by noted differences in their composition as well as the conditions under which they were initially enriched. Diversity in the composition may be influenced by their origin (Kalamazoo River vs Ohio River) and the carbon source on which they were enriched (dilbit vs ANS 521). Although several studies have reported degradation of crude oils by the predominant bacteria seen in this study (Cao et al., 2009; Jurelevicius et al., 2013; Margesin and Schinner, 2001; Rojo, 2009), AF was less competent, perhaps due to its low diversity of hydrocarbon degrading bacteria genera identified.
In conclusion, this study showed that dilbit can be biodegraded, but under similar conditions, conventional crude oil was eliminated more effectively due to the higher content of lighter hydrocarbons. The potential of microbial enrichment to degrade crude oil was highly influenced by temperature as well as the composition. Well-known oil degraders metabolized both oils but their performance varied.
ACKNOWLEDGMENTS
Funding for this research was provided in part by EPA, National Risk Management Research Laboratory (NRMRL), Cincinnati OH, under Pegasus Technical Services Inc. Contract No. EP-C-15-010. We are grateful to Yu Zhang and Michael Elk for technical assistance.