Download Effects of Nitrogen Fixing Bacteria on Algal Growth

Effects of Nitrogen
Fixing Bacteria on Algal
Growth
Noah Donnenberg
Central Catholic High School
Grade 11
Purpose
The purpose of this experiment is to
examine and quantify the influence of
nitrogen fixing bacteria on the uptake of
nitrogen, derived from ammonium
phosphate, and consequently the
proliferation of an algal model.
Summary of Previous Results
•Chlamydomonas:
• Grew continuously in the absence of ammonium phosphate.
• Early in culture with intermediate doses the stages of eutrophication
were observed.
• Later in culture growth was decreased as compared to the no (NH4)3PO4
control.
• At the highest dose (%2) almost all of the algae were dead.
• Euglena:
• In the absence of (NH4)3PO4 Euglena number decreased with time in
culture.
• Addition of (NH4)3PO4 improved cell growth at all but the highest dose.
Background
•Algal models describing
eutrophication are incomplete
because they fail to take into
account biological requirements
for nitrogen uptake
•Most plants require NO-3 for use
•Fertilizers are mostly NH3 based
•Nitrifying bacteria are required
Hypothesis
The hypothesis is that the presence of
nitrogen fixing bacteria will significantly
increase the uptake of nitrogenous
compounds, derived from ammonium
phosphate, and therefore increase algal
proliferation.
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Improvements In Experimental Design
•Problem: Euglena did not grow well under baseline
conditions
•Solution: Euglena and Chlamydomonas were expanded in
species specific growth medium
•Problem: Algal cultures may have contained endogenous
nitrogen fixing bacteria
•Solution: Sterile algal cultures were used and sterility was
maintained
•A combination of two species of nitrogen fixing bacteria
were investigated
•Antibiotics were used to evaluate specificity of bacterial
effects
Materials
Reagents
• Chlamydomonas reinhardtii
• Euglena gracilis
• Sterilized pond water
• Nitobacter
• Nitrosomonas
• (NH4)3PO4 20% stock solution in deionized water.
• Penicillin
• Gentamicin
Nitrosomonas
Nitrobacter
Materials (cont.)
• Supplies
• Carolina Digital Spectrophotometer Test Tube Cuvettes (Cat. No. 653342)
Instruments
• Neubauer Hemacytometer
• Spectrophotometer- Carolina Digital Spectrophotometer (Cat. No. 653303)
• Microscopy - Nikon Labophot microscope
• Photography- Spot Imaging Software, Insight Color Mosaic camera (model 18.2,
Diagnostic Instruments), Canon EOS camera
Methods
•Time
course / dose response experiment on four algal
cultures:
#1 Negative control: algae + no nitrifying bacteria + no
antibiotic
#2 Test group 1: algae + bacteria + antibiotics
#3 Test group 2: algae + no bacteria + antibiotics
#4 Test group 3: algae + bacteria + no antibiotics.
Methods (cont.)
•Cultures were be subjected to ammonium phosphate
concentrations, one of which being a 0% control. The
following dilutions were proceed from 2, 1, 0.5, 0.25,
0.125, 0.06 to 0.03%.
• Triplicate determinations for each condition
• Algal growth was evaluated with a spectrophotometer by
the chlorophyll absorbance at 540nm.
•Statistical analysis was performed on data collected from
163 test tubes on day 6, 9 and 13.
Results (Euglena)
ORGANISM = Euglena BACTERIA = 1
ANTIBIOTICS = 0
ORGANISM = Euglena BACTERIA = 0
ANTIBIOTICS = 1
High
Mid
Low
None
ORGANISM = Euglena BACTERIA = 0
ANTIBIOTICS = 0
ORGANISM = Euglena BACTERIA = 1
ANTIBIOTICS = 1
Results (Euglena)
F-Ratio
p-Value
+
11.8405063
0.0006545
126.6455519
0.0000000
ANTIBIOTICS
+ at Low, ++ at Mid,
negative at High
-
4.8803030
0.0278556
DAY_GROUP
NS
2.5950983
0.1081565
BACTERIA*AP_GROUP
NS
0.5811322
0.6277635
BACTERIA*ANTIBIOTICS
Antibiotics negative only 6.0380397
when bacteria present
0.0145180
BACTERIA*DAY_GROUP
Bacteria + when day is >6 5.7983088
0.0165931
Source
BACTERIA
AP_GROUP
Effect (change in AU)
AP_GROUP$*ANTIBIOTICS
NS
2.4902528
0.0602334
AP_GROUP$*DAY_GROUP
NS
0.9897798
0.3977592
ANTIBIOTICS*DAY_GROUP
NS
0.0000047
0.9982770
BACTERIA*AP_GROUP$*ANTIBIOTICS
NS
1.8646589
0.1353715
BACTERIA*AP_GROUP$*DAY_GROUP
NS
0.0845438
0.9684484
BACTERIA*ANTIBIOTICS*DAY_GROUP
NS
0.4265441
0.5141471
AP_GROUP$*ANTIBIOTICS*DAY_GROUP
NS
0.0516258
0.9844931
BACTERIA*AP_GROUP$*ANTIBIOTICS*DAY_GROUP
NS
1.4331693
0.2329612
Results (Chlamydomonas)
ORGANISM = Chlamydo BACTERIA = 0
ANTIBIOTICS = 1
High
Mid
Low
None
ORGANISM = Chlamydo BACTERIA = 0
ANTIBIOTICS = 0
ORGANISM = Chlamydo BACTERIA = 1
ANTIBIOTICS = 0
ORGANISM = Chlamydo BACTERIA = 1
ANTIBIOTICS = 1
Results (Chlamydomonas)
Source
BACTERIA
AP_GROUP
ANTIBIOTICS
Effect (change in AU)
NS
F-Ratio
p-Value
0.3068719
0.5799836
No effect at Low, ++ at Mid, no 27.8258945
effect at High
Very negative (lethal)
149.3360630
0.0000000
0.0000000
++ at Day >6
14.7197751
0.0001496
Bacteria + at High AP dose
6.8517958
0.0001724
BACTERIA*ANTIBIOTICS
NS
1.6372302
0.2016100
BACTERIA*DAY_GROUP
NS
0.9645897
0.3267571
DAY_GROUP
BACTERIA*AP_GROUP
AP_GROUP$*ANTIBIOTICS
++ at High AP dose (AP rescued 43.1813896
from antibiotic toxicity
0.0000000
AP_GROUP$*DAY_GROUP
++ at Day >6
4.6155679
0.0035314
ANTIBIOTICS*DAY_GROUP
Negative (antibiotics were
more toxic at Day >6)
Antibiotic toxicity results in
multiple interactions
NS
76.5007204
0.0000000
6.6339048
0.0002314
2.0874967
0.1016999
Antibiotic toxicity results in
multiple interactions
Antibiotic toxicity results in
multiple interactions
Antibiotic toxicity results in
multiple interactions
2.8818459
0.0905317
3.8929779
0.0093370
3.3086966
0.0204036
BACTERIA*AP_GROUP$*ANTIBIOTICS
BACTERIA*AP_GROUP$*DAY_GROUP
BACTERIA*ANTIBIOTICS*DAY_GROUP
AP_GROUP$*ANTIBIOTICS*DAY_GROUP
BACTERIA*AP_GROUP$*ANTIBIOTICS*DAY_GROUP
Conclusions
•Preculture in growth medium allowed both species to continue to grow in
sterile pond water in the absence of ammonium phosphate.
•Both organisms had increased proliferation in response to low and
intermediate doses of ammonium phosphate indicating conclusively that
nitrogen fixing bacteria are not required to utilize ammonium phosphate as a
nitrogen source.
•Addition of nitrogen fixing bacteria exerted a small, but statistically significant
positive influence on algal growth.
•In Euglena, the bacterial effect was abrogated by addition of penicillin and
gentamicin.
•In Chlamydomonas, antibiotics (gentamycin) were toxic.
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Conclusions and Implications
•Ammonia-based fertilizers (such as ammonium phosphate) are
expected to increase algal growth in aquatic ecosystems.
•Nitrogen fixing bacteria, which are ubiquitous in the
environment, significantly enhance this effect.
•Introduction of exogenous nitrogenous compounds through
fertilizer runoff can be expected to promote algal growth in
aquatic ecosystems, and therefore initiate eutrophication.
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Extensions
•Laboratory cultures serve as incomplete surrogates for complex
biomes
•Data collected in laboratory models should be validated by field
work in areas effected by fertilizer runoff
•Various types of fertilizers and pesticides influence the
interaction between farmland and aquatic ecosystems
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Acknowledgements
Thank you to Mr. Mark Krotec for his guidance and support over
the three years of this project
and
Thank you to Drs. Albert and Vera Donnenberg for their helpful
discussions and a critical reading of this presentation
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