Direct Biofilm Culturing for Alberta Oil Sands Tailings Pond Water Remediation

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1 Direct Biofilm Culturing for Alberta Oil Sands Tailings Pond Water Remediation Raymond J. Turner, PhD Professor; Biochemistry and Microbiology Microbiology Research Cluster Chair Department of Biological Sciences, Faculty of Science, University of Calgary Calgary, Alberta, Canada CPANS; April 2012

2 Oil Sands Alberta has one of the largest oil sand deposits in the world (a mixture of bitumen, clay and sand) Athabasca Basin has over 1.7 trillion barrels of bitumen In order to obtain crude oil, this bitumen must be extracted from the sands

3 Extraction Process For every 1 m 3 of oil sands extracted, the process requires ~3 m 3 of water (Athabasca river) Results in 3 4 m 3 of oil sands tailings

4 Naphthenic Acids During Bitumen Extraction Bitumen is extracted from oil sands using an alkaline hot water process Naphthenic acids are deprotonated creating salt naphthenates Removing them from the organic portion of bitumen As they are water soluble they land up in the tailings of albertaoil sands production could increase co2 by/

5 Tailings Ponds Tailings Sand, clay, water, residual bitumen, metals, organics (PAH), Also include settling additives and are quite alkaline Tailings are collected in large settling basins known as tailings ponds ada/edmonton/story/2 008/05/01/ducksthursfollo.html (Quagraine, Peterson, & Headley, 2005)

6 Tailings Ponds are very toxic Tailings Ponds are very toxic Main organic pollutant of toxicity is organic acids known as Naphthenic Acids (NAs) ( mg/l) But also metal ions Hg, Cd, Zn, Cu, V, Pb, and others Clean up Reclamation but NO Remediation

7 Naphthenic Acid Formation Aliphatic or alicyclic carboxylic acids C n H 2n+z O 2 n = no. of carbons Z = hydrogen deficiency

8 Environmental Remediation Remediation is the removal of pollutants from aquatic or terrestrial systems. Reclamation is to return the land to a useful state; not the same state Bioremediation is the use of biological systems for the removal of pollutants from aquatic or terrestrial systems. It is based on the extremely diverse metabolic potential of natural microbial, fungal and plant communities.

9 Microbe Cycle Biofilms are a major form of bacterial growth in their environment

10 Experimental Goals Culture mixed species biofilms using tailings pond water and fine tailings material as an inoculant(s) sources for a tailings pond processed water treatment bioreactor. Why? Biofilms are more tolerant to environmental stress (ph, toxins, temperature). Biofilms contain high density of cells so more metabolic activity Natural state so culture closer representation. Culturing provides control over nutrients and growth conditions.

11 Bioreactor = Water treatment plant NA degrading bacteria exist in Ponds. Can we help these bacteria out to make a step in the treatment process for the water from the tailings ponds? Use in bioreactors for water treatment

12 Calgary Biofilm Device Tailings ponds mixed species biofilms were grown directly on the Calgary Biofilm Device

13 How does the microbial community look Denaturing Gradient Gel Electrophoresis (DGGE) Aerobic, 96 hrs, 25 No medium R2B Aerobic, 14 days, 4 R2B TSB No medium Anaerobic, 10 days, 25 No medium General Anaerobic medium Golby et al. (2012), FEMS Micro Ecol 79;240

14 Sequence analysis Growth on CBD has good representation from inoculants sludge but considerable differences in the minor populations inoculum (sludge) biofilms; aerobic with R2B (A+) aerobic no medium (A ) Anaerobic with medium (An+) anaerobic no medium (An ). Golby et al. (2012), FEMS Micro Ecol 79;240

15 With the culture in hand now test for different activities CHCA Simple Model NA CHAA 2χNA = 50:50 mix of CHCA and CHAA (as sodium naphthenates) What kind of biostimulation is required; nutrients and Carbon source for co metabolism

16 Mixed Species Biofilm Growth Results Abbreviations are as follows: TSB = Trypticase soy broth, BH = Bushnell Haas, 2χNA = 50:50 mixture of Cyclohexane carboxylic acid and Cyclohexane acetic acid

17 Multispecies Biofilm Population Enumeration 16S rrna gene copies/peg TSB BH BH-2χNA BH-Y BH-Y 2χNA BH-G BH-G 2χNA TPW BiofilmGrowth Condition TSB BH BH-2χNA BH-Y BH-Y 2χNA BH-G BH-G 2χNA N=3

18 Summary of Findings Tailings pond water mixed species biofilms are capable of growing on the CBD in the presence of 2χNAs Addition of NA selects for slightly less numerous communities

19 Cultures degrade 2 NA Abundance by Integration the model naphthenic acids completely Mature TPW mixed biofilms 0 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 2 NA Abundance by Integration BH-Y BH-G Number of Days Mature TPW Biofilms Were Incubated With 2 NA Demeter et al., in preparation 2 NA Abundance by Integration Juvenile mixed biofilms Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Number of Days TPW Biofilms Were Incubated With 2 NA Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 BH-Y BH-G Mature biofilms pre conditioned to 2χNA BH-G BH-Y Number of Days Mature TPW Biofilms Were Incubated With 2 NA

20 Summary of Findings Tailings pond water mixed species biofilms are capable of growing on the CBD in the presence of 2χNAs Addition of NA selects for slightly less numerous communities TPW mixed species biofilms are capable of degrading CHCA and CHAA (2χNA) Pre conditioning and Carbon source augmentation can improve the degradation timeframe

21 How does the community of bacteria compare individual Isolates Y1 Attempts to grow individual isolates for TPW led to only 6 different species. Y2 Y3

22 Degradation of Model NAs G1 G2 G3 BH-G Sterile BH-Y Day 0 Y1 Y2 Y3 BH-Y Sterile CHCA CHAA Takes a Village of Bacteria to degrade the multiple compounds! TPW Isolate BH-G Day 0 NA Abundance by Integration

23 Move towards a bioreactor A) B) Treatment Control Treatment Control TSB Day 3 BHy Day 5 BH Day 11 Lemire et al; in preparation

24 I) Control Treatment Control Treatment B) 7 Day Incubation A) Grape Inoculation II) Outside A) B) Inside Edge Middle Edge Middle

25 Summary of Findings Tailings pond water mixed species biofilms are capable of growing on the CBD in the presence of 2χNAs Addition of NA selects for slightly less numerous communities TPW mixed species biofilms are capable of degrading CHCA and CHAA (2χNA) Pre conditioning and Carbon source augmentation can improve the degradation timeframe Biofilms can be grown on water treatment plant support materials, and transfer to other supports to generate a stable inoculant reactor.

26 Pollutants types to think about Metals & Organics Can be separate problems or as co contaminants Problem of co contamination presence of metals results in a attenuation of organic pollutant bioremediation.

27 Copper Exposure to Tailings pond biofilm cultures Exposure to 1000 µg/ml Cu(II) 24 hrs Control Copper exposed EDAX indicates crystals are Cu Golby et al; submitted

28 Future Considerations Use a mixture of NAs of increased complexity and those found in different TPW Weaning organisms off supplementary additional carbon sources or use simpler cheaper carbon source additives Evaluate the limits of tolerance to NA and heavy metal mixtures Scale up bioreactors Collaboration with University of Alberta Engineers

29 Oil Sands Water Remediation - Acknowledgements Labs of Dr. Howard Ceri & Raymond J. Turner Susanne Golby (MSc) Marc Demeter (MSc Student) Dr. Joe Lemire (PDF) Iain George (undergrad) Carol A. Stremick (Senior Technician) Laura Kaufman (Research assistant) Collaborations Environmental Engineering (University of Alberta) Dr. Tong Yu Dr. Yang Liu AHFMR HydroQual Laboratories Dr. Lyriam Marques Metabolomics Group; University of Calgary Prof. Hans Vogel & Dr. Aalim Welji Sean Booth (MSc Student) Jing Wen & Dr. Rustem Shaykhutdinov Petroleum Microbiology Group; Calgary Dr. Lisa Gieg Dr. Gerrit Vourdoow

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