1. The Physiological Effects
of
Nickel Chloride Hexahydrate
on
Aquatic Microbial Biofilm
By: Desiree’ Shaw
Background at http://faculty.uca.edu/sentrekin/

2. Content
• Background
• Hypothesis and Question
• Methods
• Results
• Conclusion
• Future Work
• Acknowledgments

3. Background
Nickel
• Occurrences
– Natural at low levels
• Earth’s crust
• Volcanos
– Contamination of ground and surface water
• Industrial waste enter environment through water cycle
• Natural gas hydraulic fracturing drilling

4. Background
Environment
• Varied levels of nickel in streams in the Scott
Henderson Gulf Mountain Wildlife
Management Area in Fayetteville Shale Gas
Region Highlighted portion
is Fayetteville Shale
Gas Region.
Marked spot is the
Scott Henderson
Gulf Mountain
WMA
http://www.searcychamber.com/economi
c-development/fayetteville-shale

5. Background
Biofilm
• Biofilms
– Complex communities of aquatic microorganisms
attached to surfaces
– Bottom of aquatic food chain

6. Hypothesis and Question
• As nickel concentrations increase:
– the microbial community would be adversely
affected
– less oxygen would be consumed by the aquatic
microbial biofilm
• Does nickel have a physiological effect on the
aquatic microbial community when measured
by respiration?

7. Methods
Sweet Gum Leaf Preparation
• Cut 105 Sweet Gum (Liquidambar styraciflua)
leaves into 2 cm by 2 cm squares
• Leeched leaves in hot tap water for 12 hours

8. Methods
Culturing of Aquatic Biofilm
• Incubated in enriched water
– 1400 μg/L Nitrate
– 140 μg/L Phosphorus
– 2 L of distilled water
• Leaves placed in mesh bag in plastic tank for
containment
• Bubblier added to provide oxygen
• 25 days of incubation at room temperature
– Enriched water changed once per week

9. Leaves in incubation
tank after 25 days
Leaves after incubation
period (biofilm are the
visible dark spots)

10. Methods
Nickel Concentration Determination
Concentration of
Classification Criteria
Nickel (mg/L)
Blank No nickel nor leaves 0.00
Control No nickel 0.00
EPA Chronic EPA assignment 0.05
Low Black Fork Stream 0.20
Medium Clifty Stream 0.70
EPA Acceptable Allowed value under EPA standards 1.10
High Sunny Side Stream 1.60
Elevated Comparison value 3.00

11. Methods
Dilution of Nickel Concentrations
• Prepared concentrated nickel chloride solution
– 0.0122 g of nickel chloride hexahydrate dissolved in 1 L of
enriched water to formulate 3 mg Ni2+/L
• Varied concentrations of nickel were created by
diluting the concentrated form based on the table
below into 500 mL beakers
Concentration of Volume Nickel for dilution Volume Nutrient water for
Classification
Nickel (mg/L) into 500 mL solution (mL) dilution to 500 mL solution (mL)
Blank 0.00 0.00 500.00
Control 0.00 0.00 500.00
EPA Chronic 0.05 8.33 491.67
Low 0.20 33.33 466.67
Medium 0.70 116.67 383.33
EPA
1.10 183.33 316.67
Acceptable
High 1.60 266.67 233.33
Extreme 3.00 500.00 0.00

12. Methods
Initial Dissolved Oxygen
• Calibrated Dissolved Oxygen (DO) probe
according to the operation manual
• Placed probe inside the 500 mL beakers
• Recorded DO levels in mg/L and percent
saturation after the probe had stabilized

13. Methods
Incubation of Leaves in Nickel
• Placed 15 leaves in each of the 500 mL
beakers
• Allowed leaves to incubate for 24 hours in the
dark

14. Methods
Preparation of Samples
• 24 sterile 50 mL centrifuge tubes were utilized
• 3 centrifuge tubes were labeled and 5
incubated leaves were inserted from their
corresponding beaker
• Filled the tubes with their corresponding
concentrations of nickel solution until a
meniscus formed over the tube

15. Methods
Final Dissolved Oxygen
• The leaves incubated in the dark for three
hours at room temperature
• Placed DO probe into each centrifuge tube
• Recorded the DO levels in mg/L and percent
saturation after the probe had stabilized

16. Methods
Weighing the Microbes
• Recorded weights for 24 pre-weighed
weighing-tins
• Placed leaves from centrifuge tubes into
weigh-tins
• Dry weight and Ash weight were taken

17. Data
• Average of 3 replicates
Nickel Chloride
Classification %Saturation/g Stdev (%/g) Stderror (%/g)
Concentration (mg/L)
Control 0.00 86.65 18.95 10.94
EPA Chronic 0.05 62.54 13.47 7.78
Low 0.20 170.94 42.83 24.73
Medium 0.70 44.14 46.07 26.60
EPA Acceptable 1.10 86.69 34.85 20.12
High 1.60 76.20 9.78 5.65
Extreme 3.00 9.59 9.55 5.51

18. Results
Change in Dissolved Oxygen
250.00
0.02
200.00 mg Ni/L
Control
% Saturation/ gram
150.00 EPA Chronic
Low
0.00 1.10 Medium
mg Ni/L 1.60
100.00 0.05 0.70 mg Ni/L EPA Acceptable
mg Ni/L
mg Ni/L mg Ni/L High
Extreme
50.00 3.00
mg Ni/L
0.00
Nickel Concentrations
• Relationship of concentrations of nickel in terms
of change in dissolved oxygen per gram
• ± Standard Error

19. Discussion
• Hypothesis was affirmed
– Increased respiratory functions from trace
amounts of nickel
– As nickel concentrations increased, biofilm
respiration decreased

20. Future Work
• Repeat the experiment:
– Add more replicates per sample
– Use nickel as the cation for 2 solutions
• Nickel Chloride
• Nickel Nitrate

21. Acknowledgments and References
• Dr. Entrekin: Assistant Professor of Biology at the
University of Central Arkansas (UCA)
• Dr. Mauldin: Chair of Chemistry Department at UCA
• Dr. Desrochers: Professor of Chemistry at UCA
• Adam Musto: Graduate Student in the Biology
Department at UCA
• Davey, M, and G O'toole. "Microbial Biofilms: from
Ecology to Molecular Genetics." Microbiology and
Molecular Biology Reviews. 64.4 (2000): 847-867. Web.
3 Sept 2012.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9901
6/>.