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

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 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 (NH 4 ) 3 PO 4 20% stock solution in deionized water. Penicillin Gentamicin Materials Nitrobacter Nitrosomonas

Supplies Carolina Digital Spectrophotometer Test Tube Cuvettes (Cat. No ) Instruments Neubauer Hemacytometer Spectrophotometer- Carolina Digital Spectrophotometer (Cat. No ) 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.)

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)

Source Effect (change in AU)F-Ratio p-Value BACTERIA AP_GROUP + at Low, ++ at Mid, negative at High ANTIBIOTICS DAY_GROUP NS BACTERIA*AP_GROUP NS BACTERIA*ANTIBIOTICS Antibiotics negative only when bacteria present BACTERIA*DAY_GROUP Bacteria + when day is > AP_GROUP$*ANTIBIOTICS NS AP_GROUP$*DAY_GROUP NS ANTIBIOTICS*DAY_GROUP NS BACTERIA*AP_GROUP$*ANTIBIOTICS NS BACTERIA*AP_GROUP$*DAY_GROUP NS BACTERIA*ANTIBIOTICS*DAY_GROUP NS AP_GROUP$*ANTIBIOTICS*DAY_GROUP NS BACTERIA*AP_GROUP$*ANTIBIOTICS*DAY_GROUP NS Results (Euglena)

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)

Source Effect (change in AU)F-Ratio p-Value BACTERIA NS AP_GROUP No effect at Low, ++ at Mid, no effect at High ANTIBIOTICS Very negative (lethal) DAY_GROUP ++ at Day > BACTERIA*AP_GROUP Bacteria + at High AP dose BACTERIA*ANTIBIOTICS NS BACTERIA*DAY_GROUP NS AP_GROUP$*ANTIBIOTICS ++ at High AP dose (AP rescued from antibiotic toxicity AP_GROUP$*DAY_GROUP ++ at Day > ANTIBIOTICS*DAY_GROUP Negative (antibiotics were more toxic at Day >6) BACTERIA*AP_GROUP$*ANTIBIOTICS Antibiotic toxicity results in multiple interactions BACTERIA*AP_GROUP$*DAY_GROUP NS BACTERIA*ANTIBIOTICS*DAY_GROUP Antibiotic toxicity results in multiple interactions AP_GROUP$*ANTIBIOTICS*DAY_GROUP Antibiotic toxicity results in multiple interactions BACTERIA*AP_GROUP$*ANTIBIOTICS*DAY_GROUP Antibiotic toxicity results in multiple interactions Results (Chlamydomonas)

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 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 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 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