Effects of Nitrogen Fixing Bacteria on Algal

GrowthNoah Donnenberg

Central Catholic High SchoolGrade 11

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.

Purpose

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

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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.

Hypothesis

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

Reagents• Chlamydomonas reinhardtii• Euglena gracilis• Sterilized pond water• Nitobacter• Nitrosomonas• (NH4)3PO4 20% stock solution in deionized water.• Penicillin• Gentamicin

Materials

Nitrobacter

Nitrosomonas

• 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

Materials (cont.)

•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

•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.

Methods (cont.)

Non

e

Low

Mid

Hig

h

ORGANISM = Euglena BACTERIA = 0 ANTIBIOTICS = 0

ORGANISM = Euglena BACTERIA = 0 ANTIBIOTICS = 1

ORGANISM = Euglena BACTERIA = 1 ANTIBIOTICS = 0

ORGANISM = Euglena BACTERIA = 1 ANTIBIOTICS = 1

Results (Euglena)

Source Effect (change in AU) F-Ratio p-Value

BACTERIA + 11.8405063 0.0006545

AP_GROUP + at Low, ++ at Mid, negative at High

126.6455519 0.0000000

ANTIBIOTICS - 4.8803030 0.0278556

DAY_GROUP NS 2.5950983 0.1081565

BACTERIA*AP_GROUP NS 0.5811322 0.6277635

BACTERIA*ANTIBIOTICS Antibiotics negative only when bacteria present

6.0380397 0.0145180

BACTERIA*DAY_GROUP Bacteria + when day is >6 5.7983088 0.0165931

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 (Euglena)

Non

e

Low

Mid

Hig

h

ORGANISM = Chlamydo BACTERIA = 0 ANTIBIOTICS = 0

ORGANISM = Chlamydo BACTERIA = 0 ANTIBIOTICS = 1

ORGANISM = Chlamydo BACTERIA = 1 ANTIBIOTICS = 0

ORGANISM = Chlamydo BACTERIA = 1 ANTIBIOTICS = 1

Results (Chlamydomonas)

Source Effect (change in AU) F-Ratio p-Value

BACTERIA NS 0.3068719 0.5799836

AP_GROUP No effect at Low, ++ at Mid, no effect at High

27.8258945 0.0000000

ANTIBIOTICS Very negative (lethal) 149.3360630 0.0000000

DAY_GROUP ++ at Day >6 14.7197751 0.0001496

BACTERIA*AP_GROUP Bacteria + at High AP dose 6.8517958 0.0001724

BACTERIA*ANTIBIOTICS NS 1.6372302 0.2016100

BACTERIA*DAY_GROUP NS 0.9645897 0.3267571

AP_GROUP$*ANTIBIOTICS ++ at High AP dose (AP rescued from antibiotic toxicity

43.1813896 0.0000000

AP_GROUP$*DAY_GROUP ++ at Day >6 4.6155679 0.0035314

ANTIBIOTICS*DAY_GROUP Negative (antibiotics were more toxic at Day >6)

76.5007204 0.0000000

BACTERIA*AP_GROUP$*ANTIBIOTICS Antibiotic toxicity results in multiple interactions

6.6339048 0.0002314

BACTERIA*AP_GROUP$*DAY_GROUP NS 2.0874967 0.1016999

BACTERIA*ANTIBIOTICS*DAY_GROUP Antibiotic toxicity results in multiple interactions

2.8818459 0.0905317

AP_GROUP$*ANTIBIOTICS*DAY_GROUP Antibiotic toxicity results in multiple interactions

3.8929779 0.0093370

BACTERIA*AP_GROUP$*ANTIBIOTICS*DAY_GROUP

Antibiotic toxicity results in multiple interactions

3.3086966 0.0204036

Results (Chlamydomonas)

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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