Enhanced Degradation of a Model Naphthenic Acid ... - · PDF fileEnhanced Degradation of a...
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Enhanced Degradation of a Model Naphthenic Acid
Compound in Bioreactors
Janice Paslawski, P.Eng.University of Saskatchewan
(Dr. G. Hill and Dr. M. Nemati)National Water Research Institute
(Dr. John Headley)
October 25, 2007
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Outline• Brief Overview of Oilsands• Review of NAs and
Background• Objectives of this Study• Materials and Methods• Results• Discussion and Direction
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Background1995 Goal: 1 million barrels/day by 2020Between 1995 and 2004, production rates increased to 1.1 million barrels/dayCurrent rates projected to 2015:2.7 million barrels/dayBy 2030: 5 million barrels/day
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“Given these projections, the question of how to manage the associated
environmental impacts becomes even more urgent.
The magnitude of the risks and opportunities is unprecedented in the
history of Canadian energy production.”
- Pembina Report
August 23, 2006
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Clean-up approach still deemed a “mess” – Edmonton Journal
Photo by the Edmonton Journal, July 28, 2006
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Background (cont’d)Naphthenic acids – natural organic compoundsCorrosive to refinery processesCaustic soda extraction method for extraction of naphthenic acids
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Background (cont’d)Naphthenic acids are toxic to aquatic algae and other micro organismsNAs are acutely toxic to aquatic organismsNAs have shown cellular respiratory inhibition in mammalian evaluations (Headley et. al, 2002)
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Naphthenic Acids: Review• Natural components found in most
petroleum sources including the Athabasca oil sands
• Released from the oil sands during aqueous extraction at elevated pH
• General chemical formula: CnH2n+zO2
• n indicates the carbon number• z indicates hydrogen deficiency number
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Structure of NAs
Z = 0O
OH( )n
Z = -2
Z = -4
Z = -6
O
OH( )n
O
OH( )n
O
OH( )n
O
OH( )n
R
RR
O
OH( )n
R
O
OH( )n
R
O
OH( )n
R
O
OH( )n
R
O
OH( )n
RO
OH( )n
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Naphthenic Acids (cont’d)• Eventual biodegradation of petroleum
leads to the formation of naphthenic acids• Industrial uses: manufacture of tires and
paints• Used as lubricants, fuel additives and
corrosion inhibitors• Naphthates have been used as ground
contact wood preservativesBiryukova et al. 2007
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Naphthenic Acids (cont’d)• Oil Sand is 10 – 12% Bitumen (Clemente
et. al. 2005)• Average naphthenic acid concentration
200 mg/kg ore (Syncrude)• Syncrude processes approx. 500 000
tonnes of ore/day• 100 tonnes of naphthenic acid from ore
each day!
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What do we know?• We can achieve microbial growth in both
commercial NAs and tailings pond water• Microbial growth varies within the range of
NA molecular structures• There has been no separation and
identification of individual NA compounds (Scott et al., 2005)
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What do we know?• Some success with biodegradation of
standard NAs• Biodegradation of tailings NAs is
considerably slower (Scott et al., 2005)• Low molecular mass NAs (<C18) are more
readily biodegraded than high molecular mass NAs (Scott et al., 2005)
• Toxicity is dominant in lower molecular weight NAs (<C22) (MacKinnon, 2001)
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What we need to do?• Understand and quantify the growth
kinetics of biodegradation for individual compounds as a basis for evaluating and engineering enhanced biodegradation systems
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Objectives• To develop a microbial consortium
capable of biodegrading a NA model compound
• To experimentally determine the biokinetic parameters associated with biodegradation of the model compound
• To study the biodegradation of a model NA in bioreactors with freely suspended cells
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Selection of a Model Compound
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Trans 4-methyl-1-cyclohexane carboxylic acid
CH3C6H10CO2HMolecular Weight
142.20
CAS Number13064-83-0
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Trans 4-methyl-1-cyclohexane carboxylic acid
- Sigma-Aldrich
CH3C6H10CO2H
Molecular Weight142.20
CAS Number13064-83-0
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Why 4MCHCA trans?• Readily soluble similar to NAs in tailings
water• Trans isomers seem to be more
consistently degradable (Tanapat, 2002)• n=8 (C8) outside the range of strong
sorption to soils (Janfada, 2006)• Within the range of NA compounds that
are more toxic to fish (<C22)• Timely and direct quantitative analyses
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Materials and Methods• A timely and reliable analytical method• A consortium capable of degrading the
model compound• A consistent means of measuring the
microbial concentration• Bioreactors• Toxicity reduction evaluation
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Materials and Methods• A timely and reliable analytical method• A consortium capable of degrading the
model compound
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Development of a Microbial Consortium
• Pseudomonas putida (ATCC 17484)• Culture developed using tailings pond
water • Culture developed using commercial NAs
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Microbial Identification
=Variovorax paradoxus(Alcaligenes paradoxus)
Pseudomonas putida
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Mixed Culture
Psuedomonas
http://www-micro.msb.le.ac.ukSEM x 10,000
• Gram-negative• Rod shaped• Approximately 1 to 3
microns in length
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Materials and Methods• A timely and reliable analytical method• A consortium capable of degrading the
model compound• A consistent means of measuring the
microbial concentration
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Measurement of Biological Growth
• Spectrophotometer (optical density)• Most probable number (MPN)• Plate counts
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Materials and Methods• A timely and reliable analytical method• A consortium capable of degrading the
model compound• A consistent means of measuring the
microbial concentration• Bioreactors (batch; continuous;
immobilized cell)
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Continuous Stirred Tank Reactor
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Immobilized Cell System
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Results
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Quantifying Growth and Biodegradation Kinetics
• Microbial growth • Specific growth rate• Substrate utilization • Removal rate of model NA• Yield
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Yield v. Initial Substrate Concentration
Yield = -0.0003(So) + 0.348
0
0.2
0.4
0.6
0.8
1
0 100 200 300 400 500 600 700 800
Initial Substrate Conc. (mg/L)
Yiel
d (m
g bi
omas
s/m
g su
bstra
te)
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Volumetric Reaction RateImmobilized Cell System
(based on theoretica l reactor vo lume)
0
1000
2000
3000
4000
5000
6000
0 5 10 15 20 25
Dilu tio n Rate (d -1)
(Co-
Cf)
x D
VRR (500 mg/L)VRR (250 mg/L)
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Immobilized Cell Reactor(Residence Times)
(based on theoretical reactor volume)
0
1000
2000
3000
4000
5000
6000
0 5 10 15 20 25 30 35 40
Residence Time (h)
(Co-
Cf)
x D
VRR (500 mg/L)VRR (250 mg/L)
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Summary• Yield for microbial degradation was 0.3 mg
biomass/mg substrate• The growth of this consortium on the
model NA compound is up to 5 times slower than that of other environmental contaminants
• The maximum degradation rate occurredin the immobilized cell system
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Discussion• The observed degradation rates increased
significantly from batch systems to a continuously stirred tank reactor and an immobilized cell system
• The most efficient residence time decreased from 40 to 2.4 hours between the CSTR and the immobilized cell system (17 times more efficient)
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Future DirectionContinued evaluation of biokinetics in an immobilized cell system (biofilms in an immobilized cell system) Evaluation of the substrate capacity of the immobilized cell system Evaluation of the biokinetics of an immobilized cell system and continuous reactor in varying environmental conditions
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Acknowledgements• University of Saskatchewan• National Water Research Institute• Natural Science and Engineering
Research Council of Canada• Canadian Council of Professional
Engineers
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Acknowledgements
• Dr. Hill and Dr. Nemati, Chemical Engineering, U of S
• Dr. Headley, NWRI
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Comments and Questions?
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Specific Growth Rate v. So
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400 500 600 700 800
So (mg/L)
μ (d
ay-1
)
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Volumetric Reaction RateImmobilized Cell System
500 mg/L(based on final measured reactor volume)
0
5000
10000
15000
20000
25000
0 20 40 60 80 100 120
Dilution Rate (d-1)
(Co-
Cf)
x D
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Materials and Methods• A timely and reliable analytical method• A consortium capable of degrading the
model compound• A consistent means of measuring the
microbial concentration• Bioreactors (batch; continuous;
immobilized cell)• Toxicity reduction evaluation
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Toxicity Reduction Evaluation• Acceptable toxicity bioassays
– EUB Guide 50: MicrotoxR EC50; Daphniamagna (LC50; EC50)
– Artemia salina toxicity test
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Toxicity Reduction EvaluationResults
• Daphnia magna (LC50)• MicrotoxR (EC50)• Artemia salina toxicity reduction test
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Daphnia magna 48 hour Acute Toxicity Test (LC50)
Treated Effluent
Stock Solution(500 mg/L)
79.5% 35.4%
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MicrotoxR Toxicity Test (EC50)
Treated Effluent
Stock Solution(500 mg/L)
>82% 14%