Tailings Ponds Tailings consist of 5 : • Water Sand Clay Metals (As, Al, Cr, Cu, Pb, Fe, Ni, Zn) • Contaminants 6 Naphthenic acids • Unrecovered hydrocarbons Polycyclic aromaGc hydrocarbons What else can be done: Remedia5on We are invesGgaGng the microbial communiGes present in the Oil Sands Process Water (OSPW) for the potenGal applicaGon in bioremediaGon. Specifically aim to harnessing the microbes in biofilms to use in bioreactors. Evalua&ng the Metal Tolerance Capacity of Microbial Communi&es Isolated from the Alberta Oil Sands Process Water The Alberta Oil Sands are one of the largest oil sand deposits in the world. It took unGl 2003 to be considered economically viable. The oil sands have been known as “tar sands” because the oil is found in a tarlike form mixed with sand, making it more difficult and costly to extract 8 . The Extrac5on Process Oil sands are mined via open pit mining. In the 1990s new methods improving efficiency 7 : • Trucks loaded with the oil sands, sent to extracGon plant • Hot causGc water used to separate crude oil from sand • Tailings/wastewater sent to tailings ponds to se\le, overlying water can be recycled http://www.cbc.ca/calgary/features/oilsands/ What is being done: Reclama5on Goal – to return the landscape to natural, sustainable form *Contaminants sGll present in underlying tailings* Why biofilms? Mixed species environmental biofilms have a compeGGve edge 3 : 1. CooperaGve community effort to degrade complex organic molecules 2. ProtecGon from metals present in OSPW 3. Diffusion limitaGons within biofilm create different nutrient tensions 4. Highly adapGve consorGa (Cunningham et al. 2011) FRANKEL, Mathew L., DEMETER, Marc, LEMIRE, Joe, TURNER, Raymond J., University of Calgary, Calgary AB, Canada Methods • Bacteria grown under metal stresss using the Calgary Biofilm Device and 96well microGter plates • Organisms tested: • OSPW mixed species community • C. metallidurans (model organism) • Confocal Laser Scanning Microscopy (CLSM) • StaGsGcal analysis and correlaGons with physiochemical parameters 1. CBC. (2013). CBC Calgary | Features | Oilsands 2020 retrieved on 18 Oct, 2013 from h\p://www.cbc.ca/calgary/features/oilsands/ 2. A. B. Cunningham et al. (2011) Biofilms: The Hypertextbook: Chapter 7 Controlling Biofilms, SecGon 4 Mechanisms of anGmicrobial tolerance in biofilms 3. M. Demeter. (2013) Harnessing oil sands microbial__ communiGes for use in exsitu naphthenic acid bioremediaGon [PowerPoint PresentaGon]. 4. Government of Alberta (2013). Retrieved on 18 Oct 2013 from h\p://oilsands.alberta.ca/FactSheets/Tailings_FSht_Mar_2013_Dm_1.pdf 5. M. D. MacKinnon et al. (2001). "Water Quality Issues Associated with Composite Tailings (CT) Technology for Managing Oil Sands Tailings," Interna9onal Journal of Surface Mining, Reclama9on and Environment 15, no. 4. 6. Allen, E. (2008). ”Process water treatment in Canada’s oil sands industry: I. Target pollutants and treatment objecGves," Journal of Environmental Engineering and Science 7, no. 2. 7. Oil Shale and Tar Sands ProgrammaGc EIS. (2012). Tar Sands Basics. Retrieved on 18 Oct, 2013 from h\p://ostseis.anl.gov/guide/tarsands/ 8. Woldwatch InsGtute. (2013). Oil Sands: The Costs of Alberta's "Black Gold" | Worldwatch InsGtute . retrieved on 18 Oct, 2013 from h\p://www.worldwatch.org/node/4222 References Results Conclusions Metal toxicity Som acids > borderline acids > hard acids Consistent correla&ons with ΔE 0 ,X m ,σ p , pK sp ReflecGon of a metal’s affinity for electrons and ligands Single versus mul& species Stronger correlaGons were observed with the C. metallidurans (except pK sp ) Tolerance values grouped closer by media augment than growth form Highlights importance of growth condiGons when invesGgaGng metalmicrobe interacGons (in vitro vs in situ) [email protected] MIC Y MBIC Y MBIC G MIC G MBIC G MIC G MBIC Y MIC Y −5 5 Value Color Key Ag Te Cd Ni Co Ga Zn Se Mo Fe V Al Pb Mn Ba W Ca Sr Li As Mg Cu Metal ! " # # # # # # # # Rela@ve Toxicity + F HSAB " SoH Acid ! Borderline Acid # Hard Acid OSPW C. metallidurans ! ! ! " ! ! Rela5ve toxicity of metals -20 0 20 40 60 -5 0 5 10 pK sp Log MBIC Y -4 -3 -2 -1 0 1 -10 -5 0 5 10 ΔE 0 Log MBIC Y 4 6 8 10 -10 -5 0 5 10 I 1 Log MBIC Y 1.0 1.5 2.0 -10 -5 0 5 10 X m Log MBIC Y 0.0 0.1 0.2 0.3 -10 -5 0 5 10 σ p Log MBIC Y 0 5 10 15 -10 -5 0 5 10 |Log K OH | Log MBIC Y Typical OSPW consor5a correla5ve profile Metalsulfide solubility product (pK sp ) ElectronegaGvity (X m ) Standard redox potenGals (ΔE 0 ) Pearson’s somness index (σ p ) First ionizaGon energy (I 1 ) Log of the first hydrolysis constants (|Log K OH |) CLSM – OSPW biofilm growth A B C D E F G H BH G BH Y [As] mM 0 2.5 25 250 I J K L M N O P [Cu] mM 0 0.0023 0.023 0.23 Correla5onal disparity between OSPW and single species cultures Introduc5on -20 0 20 40 60 -5 0 5 10 Log OSPW MIC Y -20 0 20 40 60 -5 0 5 10 pK sp Log C. metallidurans MIC Y Fig. 2. Heat map showing relaGve toxicity of metals tested on the OSPW mixed species consorGa and C. metallidurans. The heat map colors represent averages based on values obtained from two to nine trials, where red reflects the most toxic metals and green represents the least toxic. MIC G : Minimum inhibitory concentraGon (MIC) with glucose amendment; MIC Y : MIC with yeast extract amended; MBIC G : minimum biofilm inhibitory concentraGon (MBIC), glucose amended; MBIC Y : MBIC, yeast extract amended. Fig. 1. CLSM 3D images of OSPW biofilms under two metal stressors: one that the consorGa exhibited a relaGvely high tolerance (As, 125 – 250 mM), and another to which a lower tolerance was evident (Cu, 0.4 – 1.6 mM). Contrasts can be seen in biofilm microcolony formaGon from unstressed to inhibitory concentraGons of metal stresses, over several orders of magnitude. Biofilms were grown using the Calgary Biofilm Device (CBD) aerobically (25°C 125 rpm) for six days, with a tailings pond water inoculant, using a minimal salts media (BH) amended with 1gL 1 yeast extract (BH Y ) or glucose (BH G ). Fig. 3. Typical trends seen between physiochemical parameters and OSPW mixed species community metal suscepGbility (MBIC Y shown). Trend lines and 95% confidence bands (do\ed lines) shown on linear regressions that correlate with significance. Fig. 4. Comparison of pK sp vs. MIC Y , where significant correlaGon is evident with the OSPW community (top), and absent with C. metallidurans (bo\om). Filling Stage Intermediate Stage Far Future Stage Cap Water (Athabasca River water, runoff, process water, precipitaGon) Som tailings ConsolidaGng Som tailings ConsolidaGng Som tailings Monitoring Modify management and design as needed Typical lake straGficaGon and ecology hIp://www.pembina.org/images/oilsands/endpitlakecema.png
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Tailings Ponds Tailings consist of5: • Water � Sand � Clay Metals (As, Al, Cr, Cu, Pb, Fe, Ni, Zn) • Contaminants6 Naphthenic acids • Unrecovered hydrocarbons Polycyclic aromaGc hydrocarbons
What else can be done: Remedia5on We are invesGgaGng the microbial communiGes present in the Oil Sands Process Water (OSPW) for the potenGal applicaGon in bioremediaGon. Specifically aim to harnessing the
microbes in biofilms to use in bioreactors.
Evalua&ng the Metal Tolerance Capacity of Microbial Communi&es Isolated from the Alberta Oil Sands Process Water
The Alberta Oil Sands are one of the largest oil sand deposits in the world. It took unGl 2003 to be considered economically viable. The oil sands have been known as “tar sands” because the oil is found in a tar-‐like form mixed with sand, making it more difficult and costly to extract8.
The Extrac5on Process
Oil sands are mined via open pit mining. In the 1990s new methods improving efficiency7: • Trucks loaded with the oil sands, sent to extracGon plant • Hot causGc water used to separate crude oil from sand • Tailings/wastewater sent to tailings ponds to se\le, overlying water can be recycled
http://www.cbc.ca/calgary/features/oilsands/
What is being done: Reclama5on
Goal – to return the landscape to natural, sustainable form
*Contaminants sGll present in underlying tailings*
Why biofilms? Mixed species environmental biofilms have a compeGGve edge3:
1. CooperaGve community effort to degrade complex organic molecules
2. ProtecGon from metals present in OSPW 3. Diffusion limitaGons within biofilm create different nutrient
tensions 4. Highly adapGve consorGa
(Cunningham et al. 2011)
FRANKEL, Mathew L., DEMETER, Marc, LEMIRE, Joe, TURNER, Raymond J., University of Calgary, Calgary AB, Canada
Methods • Bacteria grown under metal stresss using the Calgary Biofilm Device and 96-‐well microGter plates • Organisms tested:
• OSPW mixed species community • C. metallidurans (model organism)
• Confocal Laser Scanning Microscopy (CLSM) • StaGsGcal analysis and correlaGons with physiochemical parameters
1. CBC. (2013). CBC Calgary | Features | Oilsands 2020 retrieved on 18 Oct, 2013 from h\p://www.cbc.ca/calgary/features/oilsands/ 2. A. B. Cunningham et al. (2011) Biofilms: The Hypertextbook: Chapter 7 Controlling Biofilms, SecGon 4 Mechanisms of anGmicrobial
tolerance in biofilms 3. M. Demeter. (2013) Harnessing oil sands microbial__ communiGes for use in ex-‐situ naphthenic acid bioremediaGon [PowerPoint
PresentaGon]. 4. Government of Alberta (2013). Retrieved on 18 Oct 2013 from h\p://oilsands.alberta.ca/FactSheets/Tailings_FSht_Mar_2013_Dm_1.pdf 5. M. D. MacKinnon et al. (2001). "Water Quality Issues Associated with Composite Tailings (CT) Technology for Managing Oil Sands
Tailings," Interna9onal Journal of Surface Mining, Reclama9on and Environment 15, no. 4. 6. Allen, E. (2008). ”Process water treatment in Canada’s oil sands industry: I. Target pollutants and treatment objecGves," Journal of
Environmental Engineering and Science 7, no. 2. 7. Oil Shale and Tar Sands ProgrammaGc EIS. (2012). Tar Sands Basics. Retrieved on 18 Oct, 2013 from
h\p://ostseis.anl.gov/guide/tarsands/ 8. Woldwatch InsGtute. (2013). Oil Sands: The Costs of Alberta's "Black Gold" | Worldwatch InsGtute . retrieved on 18 Oct, 2013 from
h\p://www.worldwatch.org/node/4222
References
Results
Conclusions Metal toxicity Ø Som acids > borderline acids > hard acids Consistent correla&ons with ΔE0, Xm, σp, pKsp Ø ReflecGon of a metal’s affinity for electrons and ligands Single versus mul& species Ø Stronger correlaGons were observed with the C. metallidurans
(except pKsp) Tolerance values grouped closer by media augment than growth form Ø Highlights importance of growth condiGons when invesGgaGng
metal-‐microbe interacGons (in vitro vs in situ)
!MICY!!MBICY!MBICG!!MICG!!MBICG!!MICG!MBICY!!MICY!
AgTeCdNiCuCoGaZnSeMoFeVAlPbMnBaWCaSrLiAsMg
3 1 2 4 6 8 5 7
−55
Value
Color Key
Ag!Te!Cd!Ni!
Co!Ga!Zn!Se!Mo!Fe!V!Al!Pb!Mn!Ba!W!Ca!Sr!Li!As!Mg!
Cu!
Metal!
!"!
#!
#!
#!
#!#!
#!
#!#!
Rela@ve!Toxicity!
Ag Te Cd Ni
Cu Co Mo Se Ga Zn Al
Fe V Pb Mn Ba Ca Sr W Li As Mg
1243
−5 5Value
Color Key!+!!!!!!!!!!!F!
HSAB!"!!SoH!Acid!!!!Borderline!Acid!!#!!!Hard!Acid!
AgTeCdNiCuCoGaZnSeMoFeVAlPbMnBaWCaSrLiAsMg
3 1 2 4 6 8 5 7
−5Value
Color K
ey
OSPW! C.#metallidurans#
!
!!
"!
!!
Rela5ve toxicity of metals
-20 0 20 40 60-5
0
5
10
pKsp
Log
MB
ICY
-4 -3 -2 -1 0 1-10
-5
0
5
10
ΔE0
Log
MB
ICY
4 6 8 10-10
-5
0
5
10
I1
Log
MB
ICY
1.0 1.5 2.0-10
-5
0
5
10
Xm
Log
MB
ICY
0.0 0.1 0.2 0.3-10
-5
0
5
10
σp
Log
MB
ICY
0 5 10 15-10
-5
0
5
10
|Log KOH|
Log
MB
ICY
"typical" OSPW toxicity trends
Typical OSPW consor5a correla5ve profile
Metal-‐sulfide solubility product (pKsp) ElectronegaGvity (Xm) Standard redox potenGals (ΔE0) Pearson’s somness index (σp) First ionizaGon energy (I1) Log of the first hydrolysis constants (|Log KOH|)
CLSM – OSPW biofilm growth
A$ B$
C$ D$
E$ F$
G$ H$
BHG$BHY$
[As]$m
M$
0$2.5$
25$
250$
I" J"
K" L"
M" N"
O" P"
BHG"BHY"
[Cu]"m
M"
0"0.0023"
0.023"
0.23"
OSPW"
Correla5onal disparity between OSPW and single species cultures
Introduc5on
-20 0 20 40 60-5
0
5
10
pKsp
Log
OSP
W M
ICY
-20 0 20 40 60-5
0
5
10
pKsp
Log
C. m
etal
lidur
ans
MIC
Y
pksp OSPW vs model (big diff in corrolations)
Fig. 2. Heat map showing relaGve toxicity of metals tested on the OSPW mixed species consorGa and C. metallidurans. The heat map colors represent averages based on values obtained from two to nine trials, where red reflects the most toxic metals and green represents the least toxic. MICG: Minimum inhibitory concentraGon (MIC) with glucose amendment; MICY: MIC with yeast extract amended; MBICG: minimum biofilm inhibitory concentraGon (MBIC), glucose amended; MBICY: MBIC, yeast extract amended.
Fig. 1. CLSM 3D images of OSPW biofilms under two metal stressors: one that the consorGa exhibited a relaGvely high tolerance (As, 125 – 250 mM), and another to which a lower tolerance was evident (Cu, 0.4 – 1.6 mM). Contrasts can be seen in biofilm microcolony formaGon from unstressed to inhibitory concentraGons of metal stresses, over several orders of magnitude. Biofilms were grown using the Calgary Biofilm Device (CBD) aerobically (25°C 125 rpm) for six days, with a tailings pond water inoculant, using a minimal salts media (BH) amended with 1 g L-‐1 yeast extract (BHY) or glucose (BHG).
Fig. 3. Typical trends seen between physiochemical parameters and OSPW mixed species community metal suscepGbility (MBICY shown). Trend lines and 95% confidence bands (do\ed lines) shown on linear regressions that correlate with significance.
Fig. 4. Comparison of pKsp vs. MICY, where significant correlaGon is evident with the OSPW community (top), and absent with C. metallidurans (bo\om).
Filling Stage Intermediate Stage Far Future Stage
Cap Water (Athabasca River water, runoff, process water, precipitaGon)
Som tailings ConsolidaGng Som tailings
ConsolidaGng Som tailings
Monitoring Modify
management and design as
needed
Typical lake straGficaGon and
ecology
hIp://www.pembina.org/images/oil-‐sands/end-‐pit-‐lake-‐cema.png
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