Waste to Water: Biodegrading Naphthenic Acids using Novel Sand Bioreactors


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During bitumen extraction from the oil sands, a mixture called oil sands tailings is produced. Naphthenic acids (NAs) are the primary toxic component of oil sands tailings [1] which resist biodegradation, presenting a long-term environmental hazard. By 2025, the total volume of accumulated tailings is expected to equal one billion m3[2].

‘Slow’ sand filters (SSFs) are a water treatment process invented in 1804 [3]. These filters rely on the development of a biofilm on a sand bed for filtration. Despite their ability to promote biological growth, the ability of SSFs to biodegrade NAs has not been reported in any literature.

I investigated the previously undiscovered potential of SSFs when used as bioreactors for the purpose of biodegrading the toxic NAs found in oil sands tailings. The effectiveness of bench-scale SSF bioreactors was assessed by comparing them to planktonic batch culture (PBC) bioreactors.  


Visual observations and epifluorescence microscopy indicated the formation of biofilms around the sand particles in these bioreactors. In one week, the SSF bioreactors reduced the NA concentration from 100 mg/L to 7.67 mg/L - 92.33% were removed on average while the PBC bioreactors only removed 37.55%.


Based on my results, 400 Olympic swimming pool-sized SSF bioreactors could potentially bioremediate the NAs in all oil sands tailings free water  (as of 2025 [1]) in less than 20 years (14 times faster than PBC bioreactors). The significance of these results is the discovery of a sustainable way to decrease the detoxification of tailings ponds from centuries to decades.


About me

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Before watching Al Gore’s “An inconvenient Truth” at the age of ten, I never imagined the possibility that humans could significantly affect the climate.  The zeal for environmentalism that the film inspired drove me to address environmental issues in my science fair projects over the next six years.

I made my first career decision during this whirlwind of projects on topics ranging from atmospheric haze to solar energy.  In eighth grade, I stayed up late to research environmental issues while my parents slept.  At that time of night, I should have been tired and weary; instead I felt invigorated.  In high school I realized the significance of my scientific passion.  I was meant to become a scientist.  After all, I couldn’t imagine pursuing a better path than one that constantly fascinated me.

Unfortunately, my career decisions weren’t finished because I still didn’t know which scientific discipline to choose.  Yet again, I discovered the answer through science fair.  After spending hundreds of hours working on this project in a microbiology lab, I was still fighting the urge to skip down the hallways.  A few months into my research, I realized I’d never enjoyed anything more.  Although neither of my parents work in this field, my passion for science has motivated me to rise to the challenge.  My future academic goals involve the PhD degree program after working alongside PhD students in the lab. Winning a scholarship would unlock a world of learning opportunities, allowing my dream career to become a reality.


Question / Proposal

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Despite their ability to promote biofilm growth, SSFs have only ever been used for the treatment of surface or ground water for human consumption. In this project, I investigated the previously undiscovered potential of SSFs when used as bioreactors for the unconventional purpose of biodegrading toxic NAs found in oil sands tailings ponds. The implications of this original scientific research could possibly reveal a new way to treat the NAs in the constantly expanding volume of oil sands tailings. 


To design, construct, and investigate the use of SSFs newly applied as novel aerobic bioreactors to the microbial degradation of NAs.

  • Specifically, the effectiveness of bench-scale SSF bioreactors will be assessed relative to equivalent planktonic batch culture (PBC) bioreactors by evaluating their:
    • Success at promoting the microbial growth of three separate indigenous tailings bacterial isolates.
    • Efficiency at reducing the concentrations of six increasingly-complex NAs through biodegradation.



If SSFs and PBCs are used as bioreactors to degrade NAs, then the NAs in the SSF bioreactors will consistently undergo the most biodegradation due to the formation of biofilms on the sand particles.

  • Biofilms have frequently been more metabolically efficient than planktonic cells when found in other biological and medical contexts [4].
  • The schmutzdecke (German for 'dirt covering') that SSFs traditionally develop is a biofilm composed of microorganisms derived from the water supply.
    • Therefore, the SSF bioreactors may promote similar potentially advantageous biofilm growth using the indigenous tailings bacteria.


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Tailings ponds are an environmental concern currently facing the rapidly expanding oil sands industry. During bitumen extraction, an aqueous mixture of fine silts, hydrocarbons, salts, and soluble organic compounds called oil sands tailings is produced. By 2025, the total volume of accumulated tailings is expected to equal one billion m3 [2]. This large volume of tailings is stored in outdoor reservoirs where natural consolidation into a trafficable surface would take hundreds of years [1].  During this time, the tailings are acutely toxic to mammals, fish, plants, and all but the most resilient bacteria [5]. In the tailings free water zone, 76% of the acute toxicity is caused by organic compounds called NAs [1]. NAs are a persistent mixture of mono- and polycycloalkane carboxylic acids with aliphatic side chains that are especially difficult to break down due to their hydrophilic and hydrophobic moieties [6]. As a result, they resist biodegradation and present a long-term environmental hazard.


Since the NAs in oil sands tailings occur as a variable and uncharacterized mixture, they cannot be effectively treated using physical or chemical methods [7]. The alternative, biodegradation, is the biologically-catalyzed alteration of the chemical structure of pollutants that results in less toxic metabolites [8]. Accelerated biodegradation can be accomplished in a bioreactor (an apparatus used to carry out any kind of biological process) [9]. The primary biodegradation of NAs occurs via β-oxidation of the carboxylic acid functional group [5]. Biological methods are often safe and effective because they involve the chemical breakdown of contaminants instead of storing, evaporating, or diluting them. However, previous technologies involving biodegradation (e.g., constructed wetlands) have been impractical or ineffective at removing NAs [2].

Invented in 1804, SSFs were the first modern water treatment process [3]. These filters produce potable water by developing a biofilm called a schmutzdecke on top of a sand bed which removes any contaminants. A biofilm is the accretion of bacteria embedded in an extracellular polymeric matrix attached to a solid surface [10]. SSF technology has been proven effective over the last two hundred years, with many large European cities still relying on this to clean municipal water [3]. Despite their ability to promote biofilm growth, the potential of SSFs to biodegrade NAs has not been reported in any literature. Instead, SSFs have only ever been used for the treatment of surface or ground water for human consumption.  



Method / Testing and Redesign

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  • Type of bioreactor (SSF or PBC).


  • Naphthenic acid (NA) concentrations using gas chromatography.
  • Absorbance using UV-Vis spectrophotometry at a 600 nm wavelength.
  • Visual evidence of biofilm growth.
  • Epifluorescence microscopy of cake layer (sand surface) samples.


  • Several control sets (sterile with NAs, bacteria without NAs, and sterile without NAs).


Nine sets of PBC bioreactors containing one of seven bacterial isolates were created in flasks. I determined which bacteria demonstrated the greatest amount of growth after a week (despite the toxic NAs) using UV-Vis spectrophotometry. Isolates of the bacteria were subjected to DNA extraction, amplification, and purification before being sent for independent Sanger sequencing. The three bacterial  isolates that thrived were identified as Acidovorax sp., Pseudomonas sp., and Xanthobacter sp.

An original bench-scale SSF bioreactor system was designed, constructed, and tested. Glass 50 mL syringes filled with fine aquarium sand were used as SSF bioreactors. The SSF bioreactors were rinsed with the Modified Bushnell-Haas (MBH) salts medium and seeded with the selected bacteria before a solution of the MBH salts medium and the simplest NA [cyclohexanecarboxylic acid (CHCA)] was trickled through them daily for three weeks.

During the first trial design, fountain pumps, modified plastic valves, and a digital power bar were used to automate the hydraulic loading of the sand syringes. However, rapid draining of the supernatant and turbulent disturbance of the cake layer hindered schmutzdecke formation. Despite significant troubleshooting, little bacterial growth or biodegradation occurred.

The second trial design utilized passive gravity-driven IV bags and binder clips for hydraulic loading. A diatomaceous earth precoat layer and a U-bend design in the outflow tubing were also added to increase hydraulic head loss and successfully maintain the supernatant. After being exposed to the simple NA for three weeks, visible biofilm development and high microbial growth levels (measured via UV-Vis spectrophotometry) indicated that the second trial design was a success and ready to be used.

Biodegradation was assessed using 100 mg/L in total of the following NAs: CHCA, cyclohexaneacetic acid (CHAA), cyclohexanebutyric acid (CHBA), cyclohexanepentanoic acid (CHPeA), 1-adamantanecarboxylic acid (AdCA), and 5,6,7,8-tetrahydro-2-naphthoic acid (THNA). The five remaining acids were added to the established second trial SSF bioreactors. Certain existing PBC bioreactors from the bacteria selection process were used since they already contained all six acids.

After the new NA solution had been processed by the bioreactors for a week, samples were taken and used for NA concentration measurements via gas chromatography. The bioreactor triplicate sets were distinguished based on their bacterial content - Acidovorax sp., Pseudomonas sp., Xanthobacter sp., all three (combination), or none (sterile controls).

After probing the cake layer in the SSF bioreactors with the tip of a pipette, small samples were removed. These cake layer samples were subjected to epifluorescence microscopy using DAPI (blue), SYTO 9 (green), and propidium iodide (red) dyes to assess biofilm development. 



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The pudding-like substances mixed in with the cake layer sand in the SSF  bioreactors were probably schmutzdeckes.

The white net-like biological structures observed in the SSF bioreactors were identified as streamer biofilms [11].

  Brightfield Merged
Acidovorax sp.
Pseudomonas sp.
Xanthobacter sp.

Epifluorescence microscopy revealed that there were high levels of STYO 9 (green) and DAPI (blue) dyes where sand particles were located in the brightfield micrographs.

  • Since these two dyes bind to nucleic acids and there was a lack of propidium iodide (red) dye (indicating individual cells) in these locations, this strongly suggests that the dyes were identifying the DNA found in the extracellular polymeric matrix of a schmutzdecke on the sand particles.

After one week, the arithmetic mean total NA concentration in the PBC bioreactors was reduced from 100 mg/L to 62.45 mg/L. The SSF bioreactors reduced the arithmetic mean total NA concentrations to 7.67 mg/L.

  • To put this in perspective, total NA concentrations below 5.00 mg/L are no longer acutely toxic to fish [5].

The arithmetic mean rate of individual NA removal in the PBC bioreactors was 3.51% per day.

  • In the SSF bioreactors, this mean rate was 15.79% per day (five times faster).

The PBC bioreactor P and X sets were unable to biodegrade certain NAs (e.g., CHAA), while the SSF bioreactor P and X sets accomplished notable biodegradation.

  • However, heteroscedastic two-tailed t-tests indicated that only some of the differences in biodegradation rates were statistically significant (indicated by an "*" in the graph).
    • It is possible that the conversion from planktonic to biofilm form selectively enhanced certain metabolic capabilities [4].

This graph operates on the assumptions that the upper three metres of the average 45 metre-deep tailings pond is free water [1], one billion m3 of tailings will be produced by 2025 [2], and that the biodegradation rates observed in this experiment would be similar to the reaction rates involving the uncharacterized NA mixture in oil sands tailings. Based on these assumptions, large PBC bioreactors would take an arithmetic mean time of 264.02 years while the large SSF bioreactors would take 19.17 years. These results indicate that  400 Olympic swimming pool-sized SSF bioreactors could potentially bioremediate the NAs in all oil sands tailings free water (as of 2025 [1]) in less than 20 years (14 times faster than the PBC bioreactors).

  • The proposed surface area (0.007 km2) would be 4/100,000ths of the total surface area currently occupied by all oil sands tailings ponds [12].

Based on the graph above, the SSF bioreactors encouraged higher planktonic bacterial growth.

The steeper downward trend in the absorbance values of the SSF bioreactor A and X sets indicates that there was a negative correlation between planktonic bacterial growth and biodegradation rates. The SSF bioreactor P and C sets had a smaller upward slope than their equivalent PBC bioreactor sets (not shown). This can be explained by lower planktonic bacterial growth (found on the left end of the x-axis) potentially indicating metabolically efficient biofilm growth (thus reducing the slope).


Conclusion / Report

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The results of this experiment supported my hypothesis that the NAs in the novel SSF bioreactors would undergo the most biodegradation due to the formation of biofilms on the sand particles.

  • Epifluorescence microscopy identifying the presence of biofilms, combined with the correlation between biofilm growth and higher reaction rates, provided support for the hypothesis that the SSF bioreactors may promote metabolically efficient biofilm growth.
  • In one week, the total NA concentration of 100 mg/L was reduced to 7.67 mg/L by the SSF bioreactors (92.33% removed) on average.
  • The average biodegradation rates in the SSF bioreactors were five times faster than the PBC bioreactors.
  • Certain NAs might never be biodegraded by the planktonic cells in PBC bioreactors; however, all the NAs tested were broken down by the biofilms in the SSF bioreactors (likely due to enhanced metabolic capabilities).
  • According to my results, a sizeable set of SSF bioreactors could potentially bioremediate the NAs in all oil sands tailings free water (as of 2025 [1]) in less than 20 years on average (14 times faster than equivalent PBC bioreactors).

Future Research:

  • Expansion of this bench-scale system to create bioreactors with a larger sand surface to support the formation of a larger schmutzdecke.
  • Improvements and alternate designs for the bench-scale influent system.
  • Investigating the construction of pre-treatment filters to remove suspended particles from the tailings water could allow additional processing of fluid fine tailings [13].
  • A final outcome of this research would be a year-long pilot study involving small on-site SSF bioreactors processing raw tailings free water.


The significance of the SSF bioreactors' efficiency at biodegrading NAs is the discovery of a sustainable and easily implementable way to reduce the toxicity of the constantly expanding volume of oil sands tailings. These SSF bioreactors use gravity instead of electricity, do not introduce potentially invasive species, can be constructed outdoors, require little maintenance, are made with natural or recycled material, are relatively low-cost, and are based on existing historical technology that has been proven effective over centuries. Despite their simple design, SSFs have been used for reliably processing large volumes of water in conventional applications for centuries [3] so SSF bioreactors would likely have similar processing capabilities.


The clean water recovered from the tailings ponds could be reused to reduce the overall freshwater footprint of the oil sands industry. Three m3 of water are required and four m3 of tailings are generated for every one m3 of surface mined oil sands ore that is processed [2]. The free water in tailings ponds is already recycled several times until it can no longer be efficiently used for the extraction of bitumen. Removing the NAs that have become concentrated in the free water would allow this 'lost' water to re-enter the recycling process. Most importantly, with oil sands development expected to accelerate in the near future, this passive and sustainable technology could decrease the detoxification of the free water covering tailings ponds from centuries to decades. 



Bibliography, References and Acknowledgements

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A special thanks to Dr. Lisa Gieg, an Assistant Professor at the University of Calgary, and Lindsay Clothier, a Masters Candidate working in the lab, for donating and supervising the use of various materials and lab equipment used for my original project idea.

Also, thanks to Dr. Shawn Lewenza, an Associate Professor at the University of Calgary, and Dr. Mike Wilton, a Postdoctoral Fellow, for helping with the epifluorescence microscopy after the experiment concluded.


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