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Biostimulation of Oil Sands Process-Affected Water with Phosphate Yields Removal of Sulphur-Containing Organics and Detoxification Dean Michael Quesnel, Thomas B.P. Oldenburg, Stephen R. Larter, Lisa M Gieg, and Gordon Chua Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01391 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 8, 2015

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Environmental Science & Technology

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Biostimulation of Oil Sands Process-Affected Water with Phosphate Yields Removal of

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Sulphur-Containing Organics and Detoxification

3 4

Dean M. Quesnel,§ Thomas B.P. Oldenburg,ψ Stephen R. Larter,ψ Lisa M. Gieg,§ and Gordon

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Chua,*, §

6 7

§

8

Alberta, Canada T2N 1N4

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary,

9 10

ψ

11

University Drive NW, Calgary, Alberta, Canada T2N 1N4

Petroleum Reservoir Group (PRG), Department of Geosciences, University of Calgary, 2500

12 13

1 ACS Paragon Plus Environment


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ABSTRACT

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The ability to mitigate toxicity of oil sands process-affected water (OSPW) for return into the

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environment is an important issue for effective tailings management in Alberta, Canada. OSPW

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toxicity has been linked to classical naphthenic acids (NAs) but the toxic contribution of other

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acid-extractable organics (AEOs) remains unknown. Here, we examine the potential for in situ

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bioremediation of OSPW AEOs by indigenous algae. Phosphate biostimulation was performed in

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OSPW to promote the growth of indigenous photosynthetic microorganisms and subsequent

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toxicity and chemical changes were determined. After 12 weeks, the AEO fraction of phosphate-

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biostimulated OSPW was significantly less toxic to the fission yeast Schizosaccharomyces

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pombe than unstimulated OSPW. Fourier transform ion cyclotron resonance mass spectrometry

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(FTICR-MS) analysis of the AEO fraction in phosphate-biostimulated OSPW showed decreased

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levels of SO3 class compounds, including a subset that may represent linear arylsulfonates. A

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screen with S. pombe transcription factor mutant strains for growth sensitivity to the AEO

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fraction or sodium dodecylbenzenesulfonate revealed a mode of toxic action consistent with

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oxidative stress and detrimental effects on cellular membranes. These findings demonstrate a

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potential algal-based in situ bioremediation strategy for OSPW AEOs and uncover a link

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between toxicity and AEOs other than classical NAs.

31 32

INTRODUCTION

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The Alberta oil sands represent the third largest oil reserve in the world containing ~168

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billion barrels1. Surface mining of the oil sands to extract bitumen requires substantial amounts

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of water from the Athabasca River (117 million m3 in 2012)2. This process generates vast

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amounts of toxic OSPW that have to be stored in settling ponds due to a zero-discharge policy.


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Lack of a cost-effective method to treat OSPW and reduce toxicity for eventual return into the

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environment has resulted in large tailings ponds2. The accelerated accumulation of OSPW as oil

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sands operations ramp up poses a serious threat to the future ecological and commercial

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sustainability of the region.

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The OSPW constituents from the extraction process include residual bitumen, suspended

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clay and sand particles, salts, heavy metals and a complex mixture of naturally-occurring organic

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acids (also known as acid-extractable organics (AEOs). The most characterized group of AEOs

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is the naphthenic acids (NAs) which are classically defined as cyclic carboxylic acids abiding by

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the molecular formula Cn2n+ZO2, wherein “n” and “Z’ indicate the number of carbon atoms and

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hydrogen deficiency due to ring cyclization, respectively. Recent advances in mass spectrometry

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such as Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and negative

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ion electrospray ionization mass spectrometry (ESI-MS) reveal that a substantial proportion of

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AEOs are non-classical NAs including dicarboxylic NAs, hydroxy NAs and acidic hydrocarbons

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containing nitrogen and sulphur3-5.

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Classical NAs are a main contributor of OSPW toxicity as they have been extensively

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demonstrated to be acutely and chronically detrimental to aquatic and terrestrial organisms6,7.

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For example, OSPW and NA exposure have been linked to impaired reproduction and embryonic

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development of various species of fish, frog and phytoplankton8-13. Detrimental effects linked to

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OSPW and NAs have also been observed in native plant species such as cattail, aspen and jack

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pine and the Arabidopsis plant model system14-17. With the recent revelations of the increased

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complexity of compounds present in the AEO fraction of OSPW, it remains unclear as to which

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compounds besides classical NAs are responsible for toxicity. In addition, the mode of toxic

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action of these bioactive compounds is not well understood.



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The fission yeast Schizosaccharomyces pombe is an ideal model system to elucidate the

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mode of toxic action and detoxification of various chemicals. A large collection of mutant

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strains, each containing a deletion of a unique gene is available to rapidly screen for

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hypersensitive growth to a toxicant of interest. The hypersensitive growth of mutant strains

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identifies genes that can provide clues about mode of toxic action and detoxification of the

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toxicant in the cell. Moreover, these studies have the potential to apply in multicellular

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organisms since numerous S. pombe genes are conserved in all eukaryotes18. This approach,

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known as chemical-genetic profiling, has been utilized successfully in the toxicological

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assessment of several heavy metals and genotoxicants in S. pombe19-21.

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The potential application of bioremediation to detoxify OSPW has been investigated, but

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not been implemented in situ on a large scale22,23. Non-photosynthetic aerobic bacteria

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indigenous to tailings ponds have been shown to biodegrade NAs and potentially decrease the

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toxicity of OSPW in laboratory experiments24-30. However, the biodegradation of NAs found

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naturally in OSPW by bacteria was shown to be considerably less efficient than for model and

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commercially-available NAs25,29. Recently, biofilms of indigenous aerobic microorganisms

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grown in bioreactors have been shown to remove model and OSPW NAs31-35. Photosynthetic

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microorganisms such as algae and cyanobacteria also hold considerable potential for the

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bioremediation of OSPW because they are able to grow quickly with minimal nutrient inputs in

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extreme environments. Only a few papers have reported the ability of certain algal species to

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degrade classical NAs36-38. Our previous study showed that the marine alga Dunaliella tertiolecta

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could rapidly degrade several single-ringed model and OSPW NAs to undetectable levels within

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45 days36. However, the main limitation of these studies is that none involved algal strains

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indigenous to the oil sands tailings ponds. Ultimately, the use of indigenous algae and


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photosynthetic bacteria would be important for any in situ remediation scheme as these

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microorganisms are most likely able to proliferate and adapt to the toxicity of OSPW.

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Here, we examined the potential for an in situ bioremediation strategy of AEOs within

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OSPW involving indigenous photosynthetic species. We discovered that biostimulation with

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phosphate in OSPW and light exposure substantially promotes the growth of indigenous

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photosynthetic microorganisms and reduces toxicity. A class of AEOs other than classical NAs

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was inferred to contribute to OSPW toxicity from FTICR-MS analysis. In addition, we identify

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the fission yeast Schizosaccharomyces pombe as a useful model organism for acute toxicity

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testing of AEOs and determining their mode of toxic action.

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METHODS AND MATERIALS

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OSPW Samples and Chemicals.

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Eighty liters of OSPW were collected from a closed and active tailings pond in October

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2010. The closed pond had not received fresh tailings for over a year while the active pond

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received fresh tailings from the primary bitumen extraction process. The aged and fresh OSPW

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were stored in 20 L plastic buckets in a 4˚C cold room. Biostimulation experiments of OSPW

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were initiated in July 2011 and AEO extractions of fresh OSPW for the yeast toxicological

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assays and FTICR-MS analysis were performed within a six month period that ended in

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December 2012. Dichloromethane, toluene, dimethyl sulfoxide (DMSO), K2HPO4, KH2PO4 and

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sodium dodecylbenzenesulfonate (SDBS) were purchased from Sigma-Aldrich (St. Louis, MO).

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Stock solutions of SDBS (20 g/L), K2HPO4 (7.5 g/L) and KH2PO4 (17.5 g/L) were prepared in

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ddH2O and were sterilized by autoclaving or using 0.22 µm filters.

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Schizosaccharomyces pombe Strains, Media and General Methods.

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S. pombe strains were grown and maintained in yeast extract with supplements (YES)

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medium or Edinburgh minimal medium (EMM) at 30˚C39. Yeast toxicological assays for

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phosphate-biostimulated and unstimulated OSPW were conducted in liquid EMM medium with

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the wild-type (972h-) strain. Growth sensitivity assays involving AEO fractions and SDBS were

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performed on solid EMM medium with an array of 91 transcription factor haploid deletion

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strains and a wild-type control. Each array strain contained a unique S. pombe transcription

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factor open reading frame that was deleted and replaced with the KanMX6 cassette. A list of S.

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pombe transcription factors and details describing construction of the deletion strains have

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previously been reported40.

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Biostimulation of OSPW with Phosphate and Light Exposure.

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Algal Bold’s Minimal Medium (BMM) was added at various dilutions to 50 mL of

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OSPW in 125 mL Erlenmeyer flasks capped with sterile foam stoppers. The incubations were

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established in triplicate at room temperature under 48 inch F40 fluorescent bulbs (1900

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lumens/bulb) (General Electric, Fairfield, CT) with an 8 hour light: 16 hour dark cycle for 4

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weeks. Growth of photosynthetic microorganisms in unstimulated and biostimulated aged OSPW

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was monitored daily via optical density at 680 nm using a Spectramax Plus microplate reader

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(Molecular Devices, Sunnyvale, CA). In fresh OSPW cultures, growth of photosynthetic

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microorganisms could only be determined qualitatively as cell flocculation and suspension of

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fine tailings particles prevented accurate OD680 readings. When the lowest concentration of

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BMM (1/8) that could promote substantial growth of photosynthetic microorganisms in OSPW

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during a four week period was determined, individual constituents were then added separately at


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that concentration (10.5 mg/L NaNO3, 3.5 mg/L CaCl2, 10.5 mg/L MgSO4, 3.5 mg/L NaCl, 10.5

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mg/L K2HPO4 or 24.5 mg/L KH2PO4) to identify the key nutrient(s) required for biostimulation.

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For the remediation experiments, only fresh OSPW was used because the aged OSPW

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had been used up in another study. Two concentrations of KH2PO4 (17.5 mg/L or 175 mg/L)

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were used to determine whether the level of biostimulation had an effect on remediation of fresh

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OSPW. The lower concentration of KH2PO4 was the minimum amount required to promote

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substantial growth of photosynthetic microorganisms in fresh OSPW while the higher KH2PO4

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concentration was used to determine if the effectiveness of AEO remediation and detoxification

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could be increased. All biostimulation experiments and an unstimulated control (no phosphate)

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were performed in triplicate (n=3) with 1 L of fresh OSPW in 2 L Erlenmeyer flasks capped with

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sterile foam stoppers and incubated at room temperature with light exposure as described above

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for 12 weeks.

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Preparation of the AEO Fraction from OSPW for Toxicological Assays.

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Only fresh OSPW was used in the toxicological assays with S. pombe. AEOs were

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extracted from three independent replicates of the initial OSPW stored at 4°C in the dark (time =

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0), as well as phosphate-biostimulated (17.5 mg/L or 175 mg/L KH2PO4) and unstimulated

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OSPW incubated with light for 6 and 12 weeks. For each extraction, cellular material from 50 ml

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of OSPW was removed using a 0.22 µm filter. The clarified OSPW was acidified to a pH of 2.0

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using 5.2 M HCl and extracted three times with 1/3 volume of dichloromethane in a separatory

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funnel. The dichloromethane from each sample was then removed by evaporation using a

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Rotovapor R II rotary evaporator (BÜCHI Labortechnik AG, Flawil, St. Gallen, Switzerland).

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The remaining extract was dried overnight at room temperature to remove any traces of



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dichloromethane. The extract from 50 ml of OSPW was then resuspended in 50 µL of DMSO,

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thereby concentrating the AEOs in relation to the OSPW volume by approximately 1000-fold.

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The variation between organic extractions was determined by comparing the mass of AEOs in

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three replicates of a single OSPW sample and found to have a standard deviation of ± 3.97%.

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Therefore, the organic extractions were consistent between samples.

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The initial OSPW stored at 4˚C in the dark was used to determine the minimum

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inhibitory concentration (MIC) of AEOs in S. pombe. The extracts were spiked in S. pombe

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cultures at one to four times the actual concentration of AEOs in the initial OSPW sample. The

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MIC was defined as the relative concentration of AEOs in relation to initial OSPW volumes that

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caused complete growth inhibition in S. pombe cultures after 24 hours (see below). Once the

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MIC was determined, the AEO fractions from the OSPW incubations with and without

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phosphate were subsequently spiked into S. pombe cultures at that concentration to determine

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relative toxicity changes caused by biostimulation. Relative concentrations of AEOs in relation

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to OSPW volumes were primarily used rather than gravimetric concentrations as biostimulation

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could result in overall changes to AEO concentrations. If this occurred, comparison of equivalent

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gravimetric quantities would reflect different volumes of AEOs. Therefore, we designed

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experiments to compare equivalent volumes of OSPW in order to address changes to the AEO

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fraction and also circumvent the difficulty in quantitative characterization of the complex

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mixture of compounds in AEOs.

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S. pombe Toxicological Assays.

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Wild-type S. pombe cells (972h-) were streaked onto solid EMM medium and incubated

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at 30˚C for 24 hours. Cells were then inoculated into 50 mL liquid EMM medium and grown


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overnight at 30˚C in a New Brunswick Innova 43 Incubator Shaker to a final concentration of 1.4

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x 107 - 3.5 x 107 cells/mL (OD600 = 0.2 - 0.5). 5 x 106 cells from the overnight culture were

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aliquoted to 1.5 mL tubes and centrifuged at 3000 rpm for 1 minute. The supernatant was

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removed and cells were resuspended in 100 µL of liquid EMM medium. The cell suspension was

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transferred to glass tubes containing 5 mL of liquid EMM medium and then spiked with the

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proper concentration of AEOs as described above. Equivalent volumes of DMSO used to

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dissolve the AEO fraction were also spiked into S. pombe control cultures to rule out growth

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effects from the carrier solvent. Cultures were incubated at 30˚C under shaking conditions

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described above and growth was monitored by OD600 at 0, 4, 8, 12, 24 and 48 hours using a

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Spectramax Plus plate reader (Molecular Devices, Sunnyvale, CA). The growth of these cultures

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was compared after 24 hours to determine the relative toxicity of the AEO fraction of the OSPW

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with and without phosphate biostimulation.

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FTICR-MS Analysis.

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FTICR-MS analysis was performed on one of the three AEO fractions from the same

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OSPW incubations (6 and 12 weeks) as the toxicological assays. These incubations contained:

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(1) KH2PO4-biostimulated OSPW with light exposure; (2) unstimulated OSPW with light

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exposure and; (3) the initial OSPW sample stored at 4˚C in the dark. The AEO fraction from

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OSPW treated with 175 mg/L KH2PO4 was chosen for the FTICR-MS analysis because this

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concentration showed the largest magnitude of detoxification compared to 17.5 mg/L KH2PO4 in

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the S. pombe toxicological assays. Details of the FTICR-MS methods are described in

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Supporting Information and in a previous report41.

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Growth Sensitivity Assays of S. pombe Transcription Factor Mutant Strains.

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A collection of 91 S. pombe haploid mutant strains, each containing a deletion of a

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unique transcription factor (TF) gene was screened for reduced or sensitive growth relative to

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wild type to the AEO extract from fresh OSPW or the surfactant SDBS (Sigma-Aldrich, St.

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Louis, MO). The construction and phenotypic description of these TF mutant strains have been

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described elsewhere40,42. The TF mutant strains and five replicates of the wild-type strain (972h-)

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were arrayed in 96 well microtiter plates containing 200 µL of EMM medium and 25% glycerol

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per well and stored at -80˚C until needed. Prior to the growth-sensitivity experiments, the

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microtiter plates were thawed at room temperature and the cells were transferred to solid YES

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medium by replica plating using a 96 solid pin multi-blot replicator (V&P Scientific Inc., San

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Diego, CA). The strains were incubated at 30˚C for 48 hours and replica plated onto YES solid

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medium. These plates were then incubated for another 24 hours at 30˚C to ensure that cells were

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in log phase prior to the growth-sensitivity experiments. The strains were then transferred into 96

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well microtiter plates containing 200 µL of EMM per well using the multi-blot replicator and

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shaken for 3 hours at 30˚C. After shaking, the strains were replica plated onto EMM plates

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containing a range of concentrations of the AEO fraction from fresh OSPW or SDBS. These

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plates were incubated with an untreated control at 30˚C for 48 to 96 hours. Sensitive TF mutant

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strains were visually identified by decreased growth relative to wild type on the treated plates.

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Putative TF mutant strains sensitive to the AEO fraction or SDBS from the primary

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screen were confirmed by spot dilutions. The TF mutant strains and wild type were grown

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overnight in EMM liquid medium to a final concentration of 1.4 x 107 - 3.5 x 107 cells/mL

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(OD600 = 0.2 – 0.5). 1.4 x 107 cells from each culture were transferred into microcentrifuge

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tubes, centrifuged at 3000 rpm for 1 minute and the cell pellet was resuspended in 1 mL of liquid


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EMM medium. Each cell suspension (200 µL) was placed in a 96 well microtiter plate, serially

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diluted with liquid EMM medium by a factor of 10 to a final dilution factor of 10,000 and then

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replica plated with a multi-blot replicator onto solid EMM medium containing 1.125 X

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concentration of the AEO fraction from fresh OSPW or 1.7 mg/L SDBS. These concentrations

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were selected because they had negligible effects on growth of the wild-type strain. TF mutant

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strains and wild type were grown for 48 to 96 hours at 30˚C and then their growth rate captured

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with a SPImager (S&P Robotics Inc., Toronto, ON).

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RESULTS AND DISCUSSION

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Growth of Indigenous Photosynthetic Microorganisms by Phosphate Biostimulation of

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OSPW.

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Although some algae have previously been shown to biodegrade both model and OSPW

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NAs, these studies were performed in synthetic media and involved non-indigenous species36,37.

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Any effective in situ algal-based bioremediation scheme would require substantial growth of

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indigenous photosynthetic species with biodegradation activities of AEOs in OSPW. Indigenous

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algal communities have been found in OSPW, but they exist at very low concentrations13,43.

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Therefore, we wanted to determine whether OSPW could be biostimulated to induce significant

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growth of indigenous algae and photosynthetic bacteria. A standard microalgal medium (BMM)

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was diluted at several concentrations in aged OSPW and assayed for growth of photosynthetic

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microorganisms in the presence of light. The lowest amount of BMM that could substantially

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promote the growth of photosynthetic microorganisms in aged OSPW was 1/8 concentration.

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Each BMM constituent was then added separately at 1/8 concentration in aged OSPW to

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determine the limiting nutrient for enhanced growth of photosynthetic microorganisms. Only the



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phosphate-containing BMM constituents (KH2PO4 or K2HPO4) were required to promote the

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growth of indigenous photosynthetic microorganisms in aged OSPW (Figure S1). Maximum cell

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density of photosynthetic microorganisms in the aged OSPW was attained approximately two

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weeks after supplementation with 24.5 mg/L KH2PO4 and was 3-fold higher than biostimulation

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with 10.5 mg/L K2HPO4. For fresh OSPW, the minimum amount of KH2PO4 that could

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substantially promote the growth of indigenous photosynthetic microorganisms was 17.5 mg/L.

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However, their growth rate could only be determined qualitatively since flocculation of the

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culture and suspension of clay fines prevented proper photospectrometric analysis. Microscopic

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visualization of the biostimulated fresh OSPW indicated that the photosynthetic microorganisms

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were predominantly algae and not cyanobacteria (Figure S2). These results indicate that

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substantial growth of indigenous photosynthetic microorganisms in these aged and fresh OSPW

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samples can simply be achieved by light exposure and biostimulation with KH2PO4. Indeed, the

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abundance of phosphorus in these OSPW samples (0.04 mg/L and 0.08 mg/L total phosphorus

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for fresh and aged OSPW, respectively) was approximately 50 - 70 times lower than utilized in

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the biostimulation experiments, and therefore, was likely limiting for growth of indigenous

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photosynthetic microorganisms.

260 261

Effect of Phosphate Biostimulation of OSPW on AEO Toxicity of S. pombe Cells.

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We next determined whether the toxicity of OSPW from the AEO fraction could be

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reduced following KH2PO4 biostimulation using the fission yeast S. pombe. We decided to utilize

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this model organism for AEO toxicity because of its advanced molecular genetics and genomic

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tools that are potentially useful in elucidating the mode of toxic action and detoxification

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mechanisms of various compounds20,44. Moreover, this eukaryotic organism is easily cultivated


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in the lab and a fairly rapid and simple toxicological assay can be implemented using growth rate

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as a toxicity indicator. S. pombe cells were first tested for their sensitivity to the AEO fraction of

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fresh OSPW to establish a baseline for toxicity. Fresh OSPW was selected because there was

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likely a greater AEO concentration and toxicity, but also the aged OSPW was used up in another

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study36. Growth of S. pombe cells was initially inhibited after 24 hours at 3.5 times the AEO

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concentration of fresh OSPW, which corresponded to the minimum inhibitory concentration for

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that exposure time (Figure 1A). In contrast, 2.5 times the AEO concentration of fresh OSPW

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inhibited growth of S. pombe cells only slightly after 24 hours (Figure 1A) while the AEO

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concentration corresponding to the OSPW sample (1X) exhibited growth rates similar to the

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DMSO control (Figure S3). Interestingly, the complete growth inhibition of S. pombe cells by

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3.5 times the AEO concentration of fresh OSPW only occurred up to 24 hours. Proliferation

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resumed after 24 hours, and a cell density comparable to the DMSO control was attained at 48

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hours (Figure S3). These results indicate that S. pombe cells exhibit differential growth

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sensitivity to AEOs at the optimum time of 24 hours in a concentration range suitable for an

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effective toxicological assay.

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Toxicological assays with S. pombe cells were next performed to determine if KH2PO4

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biostimulation could reduce AEO toxicity of the fresh OSPW sample. OSPW was biostimulated

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in the presence of light with either 17.5 mg/L KH2PO4, which was the minimum concentration

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required to promote substantial growth of indigenous photosynthetic microorganisms or a 10-

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fold increase in KH2PO4 concentration (175 mg/L) to determine if detoxification of AEOs can be

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enhanced. We found that OSPW biostimulated with both concentrations of KH2PO4 and

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incubated for 12 weeks with light reduced AEO toxicity compared to unstimulated cultures

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under the same conditions (Figure 1B). The reduction of AEO toxicity was directly proportional



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to the amount of KH2PO4 biostimulation. S. pombe cultures spiked with the AEO fraction of

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biostimulated OSPW with 17.5 mg/L and 175 mg/L KH2PO4 attained cell densities of 66.7% and

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83.3%, respectively, relative to a DMSO-treated control after 24 hours (Figure 1B). In contrast,

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S. pombe cultures failed to grow for 24 hours after spiked with the AEO fractions from the initial

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OSPW stored at 4˚C in the dark or unstimulated OSPW after 12 weeks with light exposure

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(Figure 1B). These data suggest that in situ detoxification of AEOs can be accelerated simply by

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augmenting OSPW with phosphate and light, and that the detoxification is associated with the

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enhanced growth of indigenous photosynthetic microorganisms.

298

Moreover, we attempted to roughly measure the mass of AEOs extracted from the

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phosphate-stimulated and unstimulated OSPW incubations after 12 weeks with light exposure.

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There was very little change in overall mass (40.0 mg/L and 39.0 mg/L for phosphate-stimulated

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and unstimulated OSPW, respectively). This suggests that minor changes in the chemical

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composition of AEOs between these two OSPW incubations could have a large effect on toxicity

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and the compounds that exhibit these changes would be toxic at very low levels, at least a couple

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orders of magnitude lower than the weight of the total extract.

305 306

FTICR-MS Profiling of the AEO Fraction from Unstimulated and Biostimulated OSPW.

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Our previous results revealed that KH2PO4-biostimulated OSPW from an active tailings

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pond could decrease the AEO toxicity after 12 weeks. The next step was to investigate the

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underlying cause for the changes in AEO toxicity observed in the S. pombe toxicological assays.

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The relative abundance of 19 classes of AEO compounds in biostimulated and unstimulated

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OSPW incubated with light, as well as the initial OSPW stored at 4˚C in the dark was obtained

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by FTICR-MS and investigated for possible changes (Figure 2; Tables S1 and S3). Classical


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NAs belonging to the O2 class were the most abundant representing approximately 38% of the

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AEO fraction in the initial OSPW stored at 4˚C in the dark. The abundance of the O2 class

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decreased about 13% after 12 weeks of incubation with light exposure, however this decrease

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was observed in both KH2PO4-biostimulated and unstimulated OSPW samples (Figure 2; Tables

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S1 and S3). These results suggested that the decrease in classical NAs after 12 weeks was not

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related to the reduction in AEO toxicity of biostimulated OSPW observed with the S. pombe

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toxicological assays. When the O2 class distribution was examined in terms of double bond

320

equivalency (DBE), we observed that the level of compounds with a DBE of one (DBE 1)

321

showed the greatest reduction in the biostimulated OSPW sample compared to the initial OSPW

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stored at 4˚C in the dark (Figure 2 inset). This class of compounds would likely include mainly

323

fatty acids, which are readily degradable and pose little environmental risk. The abundance of

324

DBE 1 pseudohomologs also decreased in the unstimulated OSPW after 12 weeks, albeit less

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than the biostimulated OSPW. These results suggest that the unstimulated OSPW incubated with

326

light at room temperature for 12 weeks also contained indigenous microorganisms capable of

327

biodegrading DBE 1 pseudohomologs. Classical NAs are traditionally defined as cyclic or

328

polycyclic acids which would have a DBE of two or greater. These compounds, primarily those

329

with lower molecular weights, have been shown to be biodegraded by both eukaryotic and

330

prokaryotic microorganisms13,24-26,28,36,38,45-51.

331

decreases in compounds classified as classical NAs under these biostimulation conditions

332

relative to unstimulated OSPW.

However, we did not observe any substantial

333

When the remainder of the 18 classes of AEO compounds were examined, few

334

differences between the three OSPW samples were observed. It was not until the classes were

335

resolved in terms of DBE and carbon numbers that significant changes occurring between the



Page 16 of 36

336

three OSPW samples were specifically identified within the SO3 class (Figure 3; Tables S2 and

337

S4). The SO3 class corresponded to approximately 13% of the AEO fraction and the DBE 4

338

pseudohomologs were the most abundant comprising 45% of this class. Among the SO3 class,

339

only DBE 4 pseudohomologs appeared to follow trends observed with the toxicological assays

340

performed on the same OSPW samples (Figure 3A; Tables S2 and S4). The initial OSPW stored

341

at 4˚C in the dark, which was the most toxic of the three samples, contained the highest relative

342

abundance of SO3 DBE 4 pseudohomologs. The unstimulated OSPW showed a 15.8% decrease

343

in SO3 DBE 4 abundance after 12 weeks of incubation with light. This OSPW sample was

344

previously shown to be slightly less toxic than the initial sample in our S. pombe toxicological

345

assays. This result would be expected as the unstimulated OSPW sample likely contained

346

indigenous populations of microorganisms that could attenuate organic contaminants, albeit

347

more slowly23. Finally, the greatest reduction of SO3 DBE 4 pseudohomologs (29.0% relative to

348

initial OSPW) was observed in the OSPW sample augmented with KH2PO4. After 12 weeks,

349

both toxicity and specific SO3 compounds decreased suggesting that these changes were related

350

and a result of KH2PO4 biostimulation of OSPW. Similar trends were also observed after 6

351

weeks, but the magnitude of changes between the OSPW samples were less (data not shown).

352

Further analysis of the SO3 DBE 4 pseudohomologs in the three OSPW samples revealed

353

that the changes in abundance were primarily occurring to constituents containing 17 to 19

354

carbons (Figure 3B). SO3 DBE 4 compounds have been previously found in the water soluble

355

fractions of crude oil samples at fairly high levels and are believed to be linear arylsulfonates

356

because of their highly biodegraded source material and solubility52. Collectively, these results

357

suggest that the reduced toxicity in the KH2PO4-biostimulated OSPW sample is due to the

358

removal of SO3 DBE 4 pseudohomologs which a subset may be linear arylsulfonates (see


Page 17 of 36


359

below). The substantial enhancement of photosynthetic microbial growth in the KH2PO4-

360

biostimulated OSPW sample indicates that indigenous algae and/or cyanobacteria are

361

contributing to the decrease in the SO3 DBE 4 pseudohomologs. However, similar activities from

362

non-photosynthetic bacteria that may also be present cannot be ruled out. Moreover, it is possible

363

that other compounds in OSPW could also contribute to toxicity reduction from biostimulation,

364

but were not detected by ESI negative-ion mode.

365 366

Cellular Response of S. pombe to AEO or SDBS Exposure.

367

We previously found that the AEO fraction of fresh OSPW from an active tailings pond

368

inhibits the growth of S. pombe cells (Figure 1A). However, the mode of toxic action of AEOs at

369

the cellular level has not been well studied. To elucidate the cellular toxicity of AEOs, we

370

screened a collection of 91 S. pombe mutant strains, each with a unique transcription factor (TF)

371

gene deleted, for growth sensitivity to the AEO fraction from fresh OSPW. This mutant strain

372

collection represents almost 90% of TF genes in S. pombe and encompasses most of the major

373

cellular processes53. Seven TF genes (pap1+, prr1+, esc1+, ste11+, prt1+, grt1+ and sre1+), when

374

deleted, exhibited growth sensitivity relative to wild type to AEOs at a concentration similar to

375

the initial OSPW stored at 4°C in the dark (Figure 4). The Pap1 and Prr1 TFs are known to

376

protect the cell from oxidative stress by activation of antioxidant genes54,55. This observation

377

indicates that AEO exposure causes oxidative stress in S. pombe cells, and therefore, loss of

378

these two TFs causes sensitivity by preventing activation of antioxidant genes. Ste11 and Esc1

379

TFs have primary roles in mating and sexual differentiation56,57. Although the sensitive growth

380

phenotypes of the esc1 and ste11 mutants to AEOs are not initially obvious, genetic analysis of

381

prr1+ suggests that sexual development in S. pombe may be regulated by oxidative stress58.



Page 18 of 36

382

Altogether, these findings are consistent with recent studies linking the oxidative stress response

383

to aquatic organisms exposed to OSPW59-61. In addition, AEO exposure may disrupt lipid

384

membrane structure and organization. The Sre1 TF is a major positive regulator of ergosterol

385

biosynthesis genes in S. pombe62. Ergosterols function to increase rigidity of the plasma

386

membrane in yeasts63. The sensitivity of the sre1 mutant could be caused by a failure to maintain

387

the integrity of the plasma membrane when its structure is compromised by AEOs. Furthermore,

388

putative detoxifying mechanisms of AEOs were uncovered from the isolation of prt1+ in our

389

hypersensitivity screen. The Prt1 TF has been shown to transcriptionally activate several genes

390

encoding MFS and ABC transporters which function as drug efflux pumps64. These transporters

391

may be able to extrude certain AEOs from the cell, thereby reducing cytotoxicity. If this is the

392

case, the absence of these transporters in the prt1 mutant would result in sensitivity to AEOs.

393

Finally, the Grt1 TF appears to function in metaphase65, however, its role in the response of AEO

394

exposure in S. pombe cells remains unclear.

395

Our FTICR-MS analysis suggests that the toxicity of the AEO fraction of OSPW may be

396

caused by anionic surfactants such as linear arylsulfonates. To further test this hypothesis, we

397

proceeded to screen the TF mutant strains for sensitive growth to the model arylsulfonate sodium

398

dodecylbenzenesulfonate (SDBS), a SO3 DBE 4 compound containing 18 carbons. If linear

399

arylsulfonates contributed to AEO toxicity in the OSPW sample, then we might expect similar

400

sensitivities of TF mutant strains to the AEO fraction and SDBS. Indeed, all of the TF mutant

401

strains sensitive to the AEO fraction with the exception of esc1 exhibited reduced growth on

402

SDBS (Figure 4). Consistent with our studies, treatment of the budding yeast Saccharomyces

403

cerevisiae with linear arylsulfonates causes oxidative stress and upregulation of antioxidant

404

genes66. In addition, anionic surfactants like SDBS are able to partition into biological


Page 19 of 36


405

membranes and perturb membrane function by increasing fluidity and permeability67,68.

406

Altogether, these results indicate that linear arylsulfonates may contribute to the AEO toxicity of

407

the OSPW sample.

408 409

ENVIRONMENTAL IMPLICATIONS

410

Indigenous aerobic bacteria have been shown to degrade OSPW NAs and reduce

411

toxicity28,29, but similar activities have not been reported for indigenous algae. The results

412

presented here show considerable promise for an in situ algal-based bioremediation scheme for

413

AEOs within OSPW. However, since OSPW also contains other components such as metals and

414

salts that can also have toxic effects, it will be important to understand the toxicity of OSPW as a

415

whole before applying bioremediation strategies. In addition, our finding that SO3 class

416

compounds contribute to AEO toxicity reveals the importance of not just considering classical

417

NAs in toxicological studies of OSPW. Only one OSPW sample was utilized here to investigate

418

the potential of indigenous algae to reduce AEO toxicity. Because OSPW chemistry and

419

indigenous microorganisms are likely to differ among tailings ponds, the effectiveness of

420

phosphate biostimulation to reduce toxicity of AEOs may vary. We have also identified S. pombe

421

as a new potential toxicity indicator for AEOs and perhaps additionally for whole OSPW.

422

Toxicological assays with other organisms will be required to properly evaluate the effectiveness

423

of this bioremediation scheme since different outcomes may be observed among multiple test

424

organisms. Furthermore, it would be useful to determine the relevant species of indigenous

425

photosynthetic microorganisms that are degrading the AEO compounds responsible for OSPW

426

toxicity. These important issues need to be addressed in order to advance this in situ

427

bioremediation approach to eventual field studies.



Page 20 of 36

428 429

ASSOCIATED CONTENT

430

Supporting Information

431

A detailed description of FTICR-MS procedures and data, microscope images of algal cells, as

432

well as additional results for phosphate biostimulation experiments and S. pombe toxicological

433

assays are provided.

434 435

AUTHOR INFORMATION

436

Corresponding Author

437

*

438

Notes

439

The authors declare no competing financial interest.

Phone: (403)-220-7769; fax: (403) 210-8655; email: [email protected]

440 441

ACKNOWLEDGEMENTS

442

This work was supported by the following companies from the Canada’s Oil Sands Innovation

443

Alliance (COSIA): Suncor Energy Inc., Shell Canada Energy, Syncrude Canada Ltd., Imperial

444

Oil Resources Ltd. and Total E&P Canada Ltd. We would like to thank Lindsay Clothier and

445

Chu Chu for conducting some of the yeast toxicity assays in this study and Dr. Douglas Muench

446

for the use of his microscope in acquiring the images of the native algal cells in KH2PO4-

447

biostimulated OSPW.

448


Page 21 of 36


A

1.2 DMSO

OD 600 nm

1

2.5X OSPW

0.8

3X OSPW

0.6

3.5X OSPW

0.4

4X OSPW

0.2 0 0

20 Hours

B 1.2

DMSO

OD600 nm

1 3.5X OSPW (No KH₂PO₄) W12

0.8 0.6

3.5X OSPW (17.5 mg/L KH₂PO₄) W12

0.4

3.5X OSPW (175 mg/L KH₂PO₄) W12

0.2

3.5X Initial OSPW

0 0

20 Hours

449 450 451

Figure 1. Toxicological assays with S. pombe of AEO fractions from biostimulated and

452

unstimulated OSPW from an active tailings pond. (A) A minimum inhibitory concentration of

453

approximately 3.5 times the AEO concentration of the initial OSPW sample stored at 4°C in the

454

dark was required to completely suppress the growth of S. pombe cells. (B) Cultures of S. pombe

455

treated with the AEO fraction of KH2PO4-biostimulated OSPW (17.5 mg/L and 175 mg/L) for

456

12 weeks in the presence of light showed recovery of growth. In contrast, AEOs extracted from

457

unstimulated OSPW exposed to light for 12 weeks (No KH2PO4) and OSPW stored at 4°C in the

458

dark (Initial) inhibited growth of S. pombe cells for 24 hours. AEOs were spiked into the S.

459

pombe cultures at 3.5 times concentration in relation to the OSPW volume. The growth rate was

460

monitored by OD600 for 24 hours at 30˚C after the AEO fractions and a DMSO control were 21 ACS Paragon Plus Environment


Page 22 of 36

461

spiked into S. pombe cultures. The graphs represent the average of three biological replicates

462

with standard deviations.

463


Page 23 of 36


O2 class

464 465 466

Figure 2. Relative abundances of 19 families of AEO compounds from OSPW of an active

467

tailings pond determined via FTICR-MS analysis. Comparisons were made between: (1) OSPW

468

stored at 4°C in the dark (Initial); (2) OSPW exposed to light for 12 weeks without phosphate

469

biostimulation (12W Untreated) and; (3) OSPW exposed to light for 12 weeks and biostimulated

470

with 175 mg/L KH2PO4 (12W KH2PO4 Treated). Inset panel depicts the relative abundances of

471

O2 class compounds that partially represent the classical NAs. FTICR-MS unprocessed and

472

processed data are contained in Supporting Information (Tables S1 and S3).



Page 24 of 36

O3S class

O3S class DBE 4

473 474 475

Figure 3. Upper panel: The relative abundances of all SO3 class compounds in the AEO fraction

476

of the OSPW samples as determined by FTICR-MS analysis. Comparisons were made between:

477

(1) OSPW stored at 4°C in the dark (Initial); (2) OSPW exposed to light for 12 weeks without

478

phosphate biostimulation (12W Untreated) and; (3) OSPW exposed to light for 12 weeks and

479

biostimulated with 175 mg/L KH2PO4 (12W KH2PO4 Treated). The most significant changes

480

resulting from KH2PO4 biostimulation of OSPW occurred with compounds having a DBE of 4.

481

Lower panel: Abundances of specific SO3 DBE 4 pseudohomologs in the AEO fraction of the

482

same OSPW samples. Pseudohomologs with 17 to 19 carbons appeared to decrease the most in

483

response to KH2PO4 biostimulation of OSPW. FTICR-MS unprocessed and processed data are

484

contained in Supporting Information (Tables S2 and S4).


Page 25 of 36


485 486 487

Figure 4. Growth sensitivity of S. pombe TF mutant strains to the AEO fraction of OSPW from

488

an active pond and sodium dodecylbenzenesulfonate (SDBS). Mutant strains and a wild-type

489

control were diluted 1/10 fold from left to right starting at 1.4 x 107 cells/ml and then pinned on

490

solid EMM medium in the absence and presence of AEOs and SDBS. Images were obtained

491

after growth for 48 to 96 hours at 30˚C.

492



Page 26 of 36

493

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