Effects of Ammonium Phosphate on Algal Populations

Noah Donnenberg

Central catholic High School

!2

Eutrophication

• The addition of nitrates to water bodies results in eutrophication:

• Increased biomass of phytoplankton, zooplankton, and algae

• Dissolved oxygen depletion • Large marine life kills • Bacterial blooms

Chlamydomonas

Abs

orba

nce

(430

nm

)

0.000

0.100

0.200

0.300

0.400

Day

0 2 5 7 9

Control1% ammonium phosphate2% ammonium phosphate

Euglena

Abs

orba

nce

(430

nm

)

0.00

0.09

0.18

0.26

0.35

Day

0 2 5 7 9

Rationale Inconclusive class results on the effects of ammonium phosphate on algae.

• Ammonium phosphate can enter the environment as runoff from fertilized farmland. How it can effect algae present in the environment is of concern.

• To determine the effects of ammonium phosphate ((NH4)3PO4 ) on algal populations.

• Ammonium phosphate dose, time of exposure and potential sources of experimental variation will be measured.

Purpose

Specific Objectives • Determine effects of ammonium phosphate on 2 algal species • Determine whether absorbance is a good measure of cell count

Hypothesis: Addition of ammonium phosphate will increase population growth in Euglina gracillis and Chlamydomonas reinhardtii.

Hypothesis

Reagents • Chlamydomonas reinhardtii, Euglena gracillis cultures were purhased from Carolina

Biological Supply) and passaged once in sterile pond water (Figure 2) • Sterilized pond water was collected from a local pond and filter sterilized (0.22 µ

filter) • (NH4)3PO4 , 20% stock solution in deionized water Supplies • Carolina Digital Spectrophotometer Test Tube Cuvettes (Cat. No. 653342) • 10 mL Pippette with Drummond Pipette-Aid • Gilson Pipetteman with disposable tips 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 digital camera

Procedure: Materials

Figure 2. Photomicrographs of algal cultures at the beginning of the experiment. Note the difference in the size of the two species.

Procedure: Methods• Culture conditions- Cell cultures were distributed in equal amount into 18

Test Tube Cuvettes (Carolina Digital #653342) and brought to 5 mL with sterile pond water. Ammonium phosphate (0%, 0.125%, 0.25%, 0.5%, 1%, 2% final concentration) was added as shown in DAY 0 setup (Figure 3). The tubes were held in racks and placed on a south-facing window sill.

• Spectrophotometry- Prior to each session the spectrophotometer was set to 320 nm and calibrated with a water blank. Each sample was vortexed (Vortex Genie) gently to distribute the cells evenly and read in triplicate, waiting each time for the reading to stabilize.

• Hemacytometer readings- Cells were diluted in pond water as shown in Figure 4. After vortexing, cells were removed from the tube with a micropipettor and carefully loaded into the hemacytometer. Because the cells were highly motile, counting was performed on photographic images (10 x objective). Raw counts in 64 large squares were multiplied by 2.5 x 103 to get cell counts in cells/mL.

• Statistical Methods- Statistical calculations were performed in Systat (Systat Inand Microsoft Excel. Non-linear 2D (wavelenghth experiment) and 3D (response by time and dose) curvefitting was performed by distance weighted least squares. Linear fitting (AU versus cell count) was performed using linear regression. Readings and replicates of day 0 data were compared by analysis of variance (ANOVA).

Figure 3. DAY 0 setup. Algae and (NH4)3PO4 dose response over time, triplicate spectrophotometer readings of triplicate determinations.

Algae+ 0 (NH4)3PO4

Algae+ 0.25%

(NH4)3PO4

Algae+ 0.125%

(NH4)3PO4

Algae+ 0.5 %

(NH4)3PO4

Algae+ 2.0%

(NH4)3PO4

Algae+ 1.0% (NH4)3PO4

Triplicate readings for each tube

Figure 4. Serial dilutions of algae for counting correlation experiment, shown in Figure 6.

1 : 1 Undiluted (count determined on

hemocytometer)

1 : 4 1 : 8 1 : 16

2 x 5 mL Algae 5 mL Algae 1:2 5mL water

5 mL Algae 5mL water

5 mL Algae 1:4 5mL water

5 mL Algae 1:8 5mL water

1 : 2 Count

calculated (Undiluted count/2)

Side experiment: What is the optimal wavelength for chlorophyll absorption? On day 0 the absorbance of a cuvette tube containing Chlamydomonas was measured over the range of the spectrophotometer Result: The highest absorbance was measured at a wavelength (Lambda) of 320 nm. This wavelength was used in all experiments (Figure 5).

Results

Figure 5. Absorbance spectrum of Chlamydomonas.

Side experiment: Is spectophotometry a good surrogate for cell counts? On day 0, the concentration of cells for each organism was determined using a hemacytometer. Cells were serially diluted in pond water (2-fold dilutions), and absorbance was measured at 320 nm. The hemacytometer counts in the diluted tubes were calculated from the count of the undiluted tube. Result: A plot of AU versus Count showed a linear relationship, allowing the estimation of cell count from absorbance. The slope was greater for Euglena, perhaps due to their larger size (Figure 2). Calculated cell count (cells/mL) was used for subsequent analyses.   

CHLAM EUG

Figure 6. Correlation of absorbance with cell counts. Regression equations are shown.

CV= 2.0% CV=2.9%

Results: ANOVA of triplicate Day 0 readings

Euglena

1 2 342,836.0

47,630.5

52,425.0

57,219.5

62,014.0

Chlamydomonas

1 2 3281,203

307,026

332,849

358,672

384,495

CE

LLS

_ML

Replicate Reading Replicate Reading

Figure 7. Variation in replicate absorbance readings (absorbance was converted to cells/mL using the equations shown in Figure 6).

CV=4.6% CV=8.0 %

Results: ANOVA of triplicate Day 0 cultures

Clamydomonas

1 2 3

Replicate Culture

278,796

314,043

349,290

384,537

CE

LLS

_ML

Euglena

1 2 3

Replicate Culture

42,775

46,734

50,693

54,652

58,611

62,570

Figure 8. Variation in replicate cultures.

Results: Control culture growth time course (raw and Δ from mean)

EUGCHLAM

ORGANISM$

0 4 8 12 16

Day in Culture

-2

-1

0

1

2

3

Cells

/mL (S

tandard

ized)

Δ fro

m m

ean

(SD

)

Day in Culture

EUGCHLAM

ORGANISM$

0 4 8 12 16

Day in Culture

0

100,000

200,000

300,000

400,000

500,000

600,000

Cells

/mL

Cel

ls /

mL.

Day in Culture

Figure 9. Raw and normalized cell count data of algal cultures in the absence of added ammonium phosphate.

Time Course (normalized to no ammonium phosphate control)

210.50.250.1250

DOSE_PCT_AP

0 4 8 12 16

Day in Culture

-20,000

-10,000

0

10,000

20,000

30,000

Change in

Cells

/mL

Δ Ce

lls /

mL

Day in Culture

EUG

210.50.250.1250

DOSE_PCT_AP

0 4 8 12 16

Day in Culture

-400,000

-300,000

-200,000

-100,000

0

100,000

Change in

Cells

/mL

CHLAM

Δ Ce

lls /

mL

Day in Culture

Figure 10. Change in cell number with time, relative to 0 AP control. AP doses are shown as separate lines.

1511831

DAY

0.1 1.0

[AP]%

-400,000

-300,000

-200,000

-100,000

0

100,000

Change in

Cells

/mL

1511831

DAY

0.1 1.0

[AP]%

-20,000

-10,000

0

10,000

20,000

30,000

Change in

Cells

/mL

CHLAM EUG

Results: Dose response (normalized data)

Δ Ce

lls /

mL.

[(NH4)3PO4]%

Δ Ce

lls /

mL.

[(NH4)3PO4]%

Figure 11. Change in cell number with AP dose, relative to 0 AP control. Times in culture are shown as separate lines.

Euglena DAY 15 (end of experiment)

2% Ammonium phosphate

No Treatment

Results

Figure 12. Photomicrographs of Euglena after 15 days in culture.

Chlamydomonas DAY 15 (end of experiment)

No Treatment2% Ammonium Phosphate

Figure 12. Photomicrographs of Chlamydomonas after 15 days in culture.

Conclusions

The hypothesis that addition of ammonium phosphate to algal cultures would mimic the stages of eutrophication was only partially supported by the data. The data (AU, algal counts) collected in this experiment showed a more complex relationship between algal growth and ammonium phosphate concentration.

Conclusions

• Chlamydomonas: • Grew continuously in the absence of ammonium phosphate. • Early in culture (days 1 and 3) with intermediate doses ((0.25%, 0.5% (NH4)3PO4 ) the stages of eutrophication were observed. • Later in culture (days 5-14) growth was decreased as compared to the no (NH4)3PO4 control. • At the highest dose (2% (NH4)3PO4) almost all of the algae were dead by day15 (Figure 12).

• 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. • Even at the highest dose (2%), cultures remained viable at day 15 (Figure 11).

Limitations and Extensions

1) The growth conditions of Chlamydomonas and Euglena were not the same. This may have been due to the fact that the sunny growth conditions favored one organism over the other. 2) The two organisms behaved differently in the absence of added (NH4)3PO4. This may also have been due to the different starting concentrations and the growth conditions. Future experiments would standardize the cell number in the starting cultures, and examine the effects of high and low sunlight.

Annotated Bibliography

• Cramer M, Myers J. Growth and photosynthetic characteristics of euglena gracilis. Archiv fur Mikrobiologie 1952;17(1-4):384-402. Used Euglena Gracillis as a model for photosynthesis and developed a growth medium which contained 0.1% (NH4)2HPO4.

• Abe K, Imamaki A, Hirano M. Removal of nitrate, nitrite, ammonium and phosphate ions from water by the aerial microalga Trentepohlia aurea. Journal of Applied Phycology 2002;14(2):129-134. Interested in study of removal of compounds associated with eutrophication. Study found algae T-Aurea “able to remove nitrogenous and phosphate compounds for growth.”

• Dawes CJ, Chen CP, Jewett-Smith J, Marsh A, Watts SA. Effect of phosphate and ammonium levels on photosynthetic and respiratory responses of the red alga Gracilaria verrucosa. Marine Biology 1984;78(3):325-328. Measured effect of (NH4)3PO4 enriched medium on photosynthesis. Enriched medium yielded enhanced amounts of chlorophyll and accelerated photosynthesis rates.

• Jo JH, Lee DS, Park JM. Modeling and Optimization of Photosynthetic Hydrogen Gas Production by Green Alga Chlamydomonas reinhardtii in Sulfur-Deprived Circumstance. Biotechnology Progress 2006;22(2):431-437. Studied optimization of photosynthesis production by Chlamydomonas. Found optimum NH4

+ to be 8 mM. And PO4

-3 to be 1.11 mM. • Mohler WA, Charlton CA, Blau HM. Spectrophotometric quantitation of tissue culture

cell number in any medium. Biotechniques 1996;21(2):260-266. Showed spectrophotometry to be effective for cell counting.