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Mining Mystery: The effect of
acid mining (sulfur compounds) on Bacillus Mycoides found on the stalk of wild rice
(Zizania palustris)
Cassandra Roy Grade 12
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• Wild Rice, Manoomin, is a vital part of the Anishinaabe diet – Ojibwee people have
harvested wild rice (Zizania palustris) for over 2,000 years
• But Ojibwe people are now concerned for their wild rice beds http://o.aolcdn.com/photo-hub/news_gallery/
7/1/711797/1302866984619.JPEG
Introduction
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Introduction • The largest deposit of copper
and nickel has been discovered in northern Minnesota – Copper and nickel are
extracted from tons of sulfur-bearing rock
– Creating sulfuric acid • Leaches heavy metals
– Mercury and arsenic – Ground water, rivers, and
lakes – Where wild rice beds may
be located (Kraker, 2012; Marcotty, 2011)
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Introduction • Minnesota Department of
Natural Resources analysis – Natural wild rice stands – Sulfate levels in 2000
Minnesota bodies of water – Over thirty years
• Predominantly – Sulfate levels above 10 mg/
L – No natural and self-
perpetuating wild rice stands exists in Minnesota (Maccabee, 2011)
http://www.nature.org/ourinitiatives/habitats/riverslakes/explore/explore-the-mississippi-river-day-1-afternoon-mother-natures-sacred-gift.xml
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Question/Hypothesis
• What is the effect of acid mining (sulfur compounds) on Bacillus mycoides found on the stock of wild rice stalk (Zizania palustris)?
• If Bacillus mycoides cultured from wild rice stalk (Zizania palustris) is exposed to acid mine drainage (sulfur compounds), then colony growth will be negatively affected.
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Method Table
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Nutrient Agar (only SO4) Serial dilutions of 0, 10, 50, 100, and 200 mg/L of sulfate (S04).
• Bacillus mycoides was inoculated on nutrient agar plates – Three plates per concentration – Twenty one trials – Three control plates not inoculated
with bacteria and three without sulfate
– Incubated at room temperature for three days
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Nutrient Agar (only SO4)
• Placed under a quadrant counter – Percentage of bacteria
per plate was recorded
• As the amount of sulfate increased the percentage of bacteria increased
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Plain Agar (SO4) Effects of just sulfur compounds minus the nutrient affect • Serial dilutions 10, 100 and 300 mg/
L (SO4) • Sixty agar plates were inoculated
with Bacillus mycoides • No growth occurred after three days
of incubation at room temperature • Possibly the Bacillus mycoides culture
was dead.
The above procedure was again repeated with sixty agar plates, again, no growth occurred
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Plain Agar With Wild Rice Stalks
• Five plain agar plates • Five grams (two
centimeters) of a sterile wild rice stalk
• Inoculated with Bacillus mycoides
• The agar plates did not visually show any growth
• Minimal growth of Bacillus mycoides was indicated microscopically.
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Agar 5% Glucose (Carbon Energy SO4, SO3, and S2 ) • 10, 100 and 300 mg/L)
of the three sulfur compounds
• Plain agar and five percent glucose (carbon energy)
• Sixty agar plates were inoculated
• After a three day incubation, again no growth occurred
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Agar 5% nutrient (Never Jelled)
• Previous procedure was repeated – Five percent nutrient, and
only nutrient agar – Added to 100 milliliters of
sterile water
• Growth was apparent, but the agar did not gel and bacterial growth could not be measured
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95% Agar, 5% Nutrient Agar
• Previous procedure was repeated – 95% plain agar with
5% nutrient agar • Finally, measureable
growth occurred • Sixty plates was held up to
a light • Maximum growth was
measured – Across the agar plates
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• The amount of bacterial growth per treatment was compared for statistical difference using a computer program called SPSS
• A Univariate Analysis of Variance (ANOVA) was done to determine if there was a difference in bacterial colony growth due to the sulfur compounds or just due to chance. A probability or p value of p<.05 was used to determine significance.
Statistics
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Conclusion • The original hypothesis was if
Bacillus mycoides cultured from wild rice stalk (Zizania palustris) was exposed to acid mine drainage (sulfur compounds), then colony growth will be negatively affected
• The hypothesis was partially supported depending on the nutrient situation
• As the sulfur compound concentrations increased
– Increased in high nutrient – Bacterial growth significantly
decreased (p<0.017) low nutrients
• Not for the 10 mg/L sulfur compound concentrations (p<0.301)
http://thetyee.ca/News/2011/05/23/MiningMess/
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Conclusion • Minnesota (1973) water quality
ruling – Limiting sulfates to 10
milligrams per liter (mg/L) in wild rice waters.
(Maccabee, 2011)
• Bacillus mycoides cultured from wild rice stalks – Similar growth rates – Significantly reduced growth
above 10 mg/L in wild rice waters
• There appears to be a relationship between Sulfur compound levels, Bacillus mycoides growth and that of Wild Rice in Minnesota Lakes
• Supporting the idea that B. mycoides is the unknown bacterial in Pastor, Walker, and Dewey (2011)
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• Pastor, Walker, and Dewey (2011) found four year cycling of wild rice stand growth
• B. mycoides could be a key player in the cycling
• In the presence of a unlimited nutrients like
– Large biomass wild rice/Nutrient agar plate
– The growth of B. mycoides increases
– Breaking down wild rice liter – High carbon to nitrogen ratio – Diverting nitrogen from the
water
Conclusion
C:N
• Yet when in a low nutrient agar – Sulfur compounds appeared to have a
negative affect on bacterial growth. – Subsequent low nitrogen levels leads to
wild rice biomass oscillations
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C:N
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Productivity of Wild Rice (Zizania palustris L.) in Northern Minnesota. Ecosystems, 13, 458-498. • Eule-Nashoba, A. R., Biesboer, D. D., & Newman, R. M. (2012). Seed size in lacustrine and riverine population of wild rice in northern
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density in Zizania palustris. Canadian Journal of Botany, 80(12), 1283-1294. doi: 10.1139/b02-118 • Leonard, E. N., Mattson, V. R., Benoit, D. A., Hoke, R. A., & Ankley, G. T. (1993). Seasonal variation of acid volatile sulfide
concentration in sediment cores from three northeastern Minnesota lakes. Hydrobiologia, 271, 87-95. • Maccabee, P. (2011, March). Wild Rice and the Sulfate Standard. Water Legacy. Retrieved September, 2012, from www.waterlegacy.org • Magnuson, J. (2012). The good grain. Christian Century, 10-11. • Marcotty, J. (2011). Minnesota's mining boom: New riches or new threat. Star Tribune. Retrieved December 26, 2012. • Mercury Poisoning. (n.d.). MedicineNet.com. Retrieved December 1, 2012. • Minnesota Pollution Control Agency (MPCA) (2011). Wild Rice/Sulfate Protocol Development Discussion Document, 1-29. • MPCA Response to Comments Received Regarding Hydroponic Experiments. (2012). MPCA Response to Comments, 1-5. • Myrbo, D. (2011). Wild rice-sulfate 2011 preliminary field study. Preliminary Field Study, 1-7. Retrieved December 26, 2012. • Nakamura, L. K. (1998). Bacillus pseudomycoides sp. nov. International Journal of Systematic Bacteriology, 48, 1031-1035. • P. (n.d.). Guardians of manomin: Aboriginal self-management of wild rice harvesting. Alternatives, 19.3, 29. • Sims, L., Pastor, J., Lee, T., & Dewey, B. (2012). Nitrogen, Phosphorus, and light effects o reproduction and fitness of wild rice. Botany,
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Acknowledgements
• I would like to thank: • Dr. Cynthia Welsh for six years of support
and mentoring • Michael Gillespie biochemistry professor
at Fond du Lac Tribal College for his supervision and the use of his lab
• Holly Pellerin for supporting my project and travels