1. Effects of Nitrogen Fixing Bacteria on Algal Growth Noah Donnenberg Central Catholic High School Grade 11

2. 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

3. 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 (NH 4 ) 3 PO 4 control. At the highest dose (%2) almost all of the algae were dead. Euglena: In the absence of (NH 4 ) 3 PO 4 Euglena number decreased with time in culture. Addition of (NH 4 ) 3 PO 4 improved cell growth at all but the highest dose.

4. 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

5. 5 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

6. 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

7. Reagents Chlamydomonas reinhardtii Euglena gracilis Sterilized pond water Nitobacter Nitrosomonas (NH 4 ) 3 PO 4 20% stock solution in deionized water. Penicillin Gentamicin Materials Nitrobacter Nitrosomonas

8. 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.)

9. 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

10. 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.)

11. None LowMid High 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)

12. 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)

13. None LowMid High 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)

14. 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)

15. 15 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.

16. 16 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.

17. 17 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

18. 18 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