Fertilizer and Acid Rain Effects on Algal Growth science/PJAS/PJAS 15 pdf... · Fertilizer and Acid Rain Effects on Algal Growth ... on a table under two lamps providing equal light

Fertilizer and Acid Rain

Effects on Algal GrowthJoshua Egan

9th Grade, Pittsburgh Central Catholic High School, 1st

Year in P.J.A.S.

Algal Importance

Base of the aquatic food chain

Bio-indicators of most stimuli present in marine environments

Studies upon this model may apply to other forms of life and provide vital information for protecting life

Chlamydomonas reinhardtii

Species of green algae

Tenfold increase in cells per day

Used as bio-indicator

Found in stagnant water, damp

soil, freshwater, seawater, and

even in snow as “snow algae”

Contains two flagella and large,

crescent shaped chloroplasts

Euglena gracilis

Genus of single-celled flagellate

protists, members of the class

Euglenoidea

Performs mitosis/reproduces

quicker in warm surroundings

Contain flagella and chloroplasts

Found in nutrient-rich freshwater

and significantly affected by

acidity

Runoff

Surface water movement and how

it goes through the water cycle

over land

Water neither evaporates nor

penetrates the surface

Creates floods and “watersheds”

with excess precipitation

Fertilizers and other chemicals

may be transported through the

cycle and in turn pollute water

sources

EutrophicationCaused by an overabundance of nutrients

in an ecosystem

No limiting factor on algal populations

Uncontrollable growth takes up resources

necessary for other organisms’ oxygen

Limits biodiversity

Can occur naturally

Occurs today by fertilizer runoff

Miracle-Gro Active Ingredients include:

Urea, Ammonium Phosphate, Urea Phosphate,

Potassium Chloride, Boric Acid, Copper Sulfate, Iron

EDTA, Manganese EDTA, Sodium Molybdate, Zinc

Sulfate, and Nuisance Dust

Phosphates: important for cellular processes including

the synthesis of ATP and ADP

Nitrogen: found in ammonia contained in fertilizers,

which may be a part of surface runoff; causes

eutrophication

Only aids plant growth at intended

concentrations

Acid Precipitation

Worldwide environmental

problem

Results from pollution by sulfur

dioxide and nitrogen oxide

Originates from smokestacks,

vehicle exhaust, and burning

fossil fuels

General pH is 5.6 (acidic)

Reduces biodiversity

Sulfuric Acid as a Pollutant

Common form of acid rain

Popular product in chemical industry

Factories produce and release the

substance

Emitted into the atmosphere; contaminates

precipitation and harms life as a result

Sulfur oxides can combine with water in the

atmosphere to create acid rain

Affects organisms such as algae in marine

environments

Spectrophotometry

Quantitative study of electromagnetic spectra

Visible light, near-ultraviolet, and near-infrared

and the use of a spectrophotometer: can measure

intensity as a function of color or light absorption

Important features include the spectral

bandwidth and linear range

Commonly used in scientific fields such as

chemistry, biochemistry, and molecular biology

Purpose

To determine if miracle-gro

fertilizer and acid rain (sulfuric

acid) have a significant effect on

algal growth

Either individually or

synergistically

Hypotheses

Null Hypothesis- Neither miracle-gro

fertilizer nor the sulfuric acid (acid rain)

will have significant effects on the algal

survivorship alone or synergistically

Alternate Hypothesis- Both the miracle-

gro fertilizer and the sulfuric acid (acid

rain) will have a significant synergistic

effect on the algal survivorship

Materials Sulfuric acid (concentration 10-1 molar)

Miracle-Gro fertilizer (concentration 10x)

X= recommended concentration for soil application

Chlamydomonas reinhardtii and Euglena gracilis algae

73 tubes (one with water for blanking the

spectrophotometer)

Two test tube racks

Micro-pipettes and tips

Soil water

Spring water

Spectrophotometer

Wax paper

Two lamps for elevated light source

Procedure 1. Two racks with a total of 72 tubes were set up

on a table under two lamps providing equal light

for all of the algae

2. The following ingredients were inserted into

the tubes to create different experimental

variable concentrations of miracle-gro fertilizer

and sulfuric acid

Each combination was made up four times each for

both types of algae, creating 72 tubes

Tube Concentration Table

0 0.1x (L) 1x(H)

0 00 0L 0H

pH=7

10-5 (L) L0

pH=6.5

LL LH

10-3 (H) H0

pH=5

HL HH

pH= 7.5

Miracle-gro Fertilizer

Sulf

uri

c A

cid

Tube Concentration Table

Control:

0x/0

0x/10-5 0x/10-3 0.1x/0 0.1x/10-5 0.1x/10-3 1x/0 1x/10-5 1x/10-3

Algae 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

Soil

water

1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

Spring

water

3 mL 2.995

mL

2.95 mL 2.995

mL

2.945

mL

2.945

mL

2.95 mL 2.9 mL 2.9 mL

Fertilizer

(10x)0 mL 0 mL 0 mL 5 µL 5 µL 5 µL 50 µL 50 µL 50 µL

Sulfuric

acid (10-1

molar)

0 mL 5 µL 50 µL 0 mL 0.05 mL 50 µL 0 mL 0.05 mL 50 µL

Total

volume

5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL

Procedure (continued) 3. The tubes were kept in the two racks under

constant, equal elevated lamp lighting for 14 days

4. The top of each tube was covered with wax

paper and mixed by inversion before each reading

5. Every two days, the tubes were measured in

the spectrophotometer at 430 nm (wavelength)

for 14 days

6. After the experiment, the results were

analyzed with stats and graphs

0x/10-5

0x/10-3

0x/0

0.1x/0

0.1x/10-5

0.1x/10-3

1x/0

1x/10-5

1x/10-3

Fert-

0.571

SA-

0.001

Synergy-

0.002

Fert-

0.107

SA-

0.000002

Synergy-

0.043

Fert-

0.013

SA-

0.00005

Synergy-

0.001

0x/0

0x/10-5

0x/10-3

0.1x/0

0.1x/10-5

0.1x/10-3

1x/0

1x/10-5

1x/10-3

Fert-

0.082

SA-

0.008

Synergy-

0.973

Fert-

0.142

SA-

0.003

Synergy-

0.853

Fert-

0.804

SA-

0.000006

Synergy-

0.201

P-Value Table

Chlamydomonas reinhardtii Euglena gracilis

Day 2: Fertilizer- 0.571

Sulfuric acid- 0.001

Synergy- 0.002

Fertilizer- 0.082


Synergy- 0.973



Synergy- 0.043

Fertilizer- 0.142


Synergy- 0.853



Synergy- 0.001

Fertilizer- 0.804


Synergy- 0.201

Statistical Analysis (ANOVAS) for

Chlamydomonas reinhardtii

Varying fertilizer concentration effects:

Not significant: p-values >0.05 accept null

hypothesis

Varying sulfuric acid effects:


hypothesis

Synergistic/interactive effects:

Significant: p-values <0.05 reject null

hypothesis

Statistical Analysis (ANOVAS) for Euglena

gracilis

Varying fertilizer effects:


hypothesis

Varying sulfuric acid effects:

Significant: p-values <0.05 reject null

hypothesis

Synergistic/interactive effects:


hypothesis

Limitations

Lighting inefficiencies

Algal health?

Living/thriving cells during study?

Only two concentrations of sulfuric acid and fertilizer

Miracle-gro had buffering effect on sulfuric acid

Extensions

Test other chemical effects

More varying variable

concentrations

Use other living species

Do the study at a larger scale

Longer experiment length

Bibliography http://www.ruf.rice.edu/~bioslabs/studies/in

vertebrates/chlamydomonas.html

http://toxics.usgs.gov/definitions/eutrophication.html

https://www.google.com/search?q=encyclopedia&gws_rd=ssl

http://www.all-science-fair-projects.com/print_project_1235_122

http://homeguides.sfgate.com/fertilizers-pollutants-78452.html

Mark Krotec, PTEI

http://www.ruf.rice.edu/~bioslabs/studies/invertebrates/chlamydomonas.html

http://toxics.usgs.gov/definitions/eutrophication.html

https://www.google.com/search?q=encyclopedia&gws_rd=ssl

http://www.all-science-fair-projects.com/print_project_1235_122

http://homeguides.sfgate.com/fertilizers-pollutants-78452.html

ANOVA Statistical test that allows for the comparison of

means of different groups, to determine significant variation

Two factor ANOVA will determine where the significant effect is: either through one of the variables or both in synergy with each other

Utilize p-values as measure of significance

Confidence interval of the likelihood of seeing the same affect produced by the same variable with replication

p>0.05: not significant

p<0.05: significant

Day 2 ANOVAs

Anova: Two-Factor With Replication

SUMMARY 0 10(-5) 10(-3) Total

0x

Count 4 4 4 12

Sum 0.481 0.566 0.554 1.601

Average 0.12025 0.1415 0.1385 0.133417

Variance 2.96E-05 7E-06 0.000239 0.000171

0.1x

Count 4 4 4 12

Sum 0.597 0.389 0.524 1.51

Average 0.14925 0.09725 0.131 0.125833

Variance 0.00039 4.23E-05 0.000441 0.000745

1x

Count 4 4 4 12

Sum 0.567 0.396 0.579 1.542

Average 0.14175 0.099 0.14475 0.1285

Variance 0.000183 4.73E-05 0.001417 0.000926

Total

Count 12 12 12

Sum 1.645 1.351 1.657

Average 0.137083 0.112583 0.138083

Variance 0.000329 0.000483 0.000606

ANOVA

Source of VariationSS df MS F P-value F crit

Sample 0.000355 2 0.000178 0.571501 0.57136 3.354131

Columns 0.005006 2 0.002503 8.055186 0.001805 3.354131

Interaction 0.00686 4 0.001715 5.519101 0.002219 2.727765

Within 0.00839 27 0.000311

Total 0.020611 35



0x

Count 4 4 4 12

Sum 0.676 0.81 0.72 2.206

Average 0.169 0.2025 0.18 0.183833

Variance 0.000417 4.83E-05 0.000931 0.000593

0.1x

Count 4 4 4 12

Sum 0.775 0.924 0.791 2.49

Average 0.19375 0.231 0.19775 0.2075

Variance 0.000243 0.001794 0.001448 0.001254

1x

Count 4 4 4 12

Sum 0.737 0.839 0.724 2.3

Average 0.18425 0.20975 0.181 0.191667

Variance 0.000198 0.000362 0.000278 0.000409

Total

Count 12 12 12

Sum 2.188 2.573 2.235

Average 0.182333 0.214417 0.18625

Variance 0.000348 0.000761 0.000797

ANOVA


Sample 0.003489 2 0.001744 2.7449 0.082191 3.354131

Columns 0.007352 2 0.003676 5.784721 0.008112 3.354131

Interaction 0.000309 4 7.73E-05 0.121627 0.973531 2.727765

Within 0.017158 27 0.000635

Total 0.028308 35

Chlamydomonas

reinhardtiiEuglena gracilis

Day 4 ANOVAs



0x

Count 4 4 4 12

Sum 0.633 0.618 0.808 2.059

Average 0.15825 0.1545 0.202 0.171583

Variance 0.000146 0.000184 0.000233 0.000661

0.1x

Count 4 4 4 12

Sum 0.72 0.439 0.811 1.97

Average 0.18 0.10975 0.20275 0.164167

Variance 4.47E-05 0.000993 0.000595 0.002155

1x

Count 4 4 4 12

Sum 0.677 0.468 0.658 1.803

Average 0.16925 0.117 0.1645 0.15025

Variance 0.000679 0.000227 0.002103 0.001428

Total

Count 12 12 12

Sum 2.03 1.525 2.277

Average 0.169167 0.127083 0.18975

Variance 0.000323 0.000802 0.001147

ANOVA


Sample 0.002815 2 0.001408 2.434485 0.106645 3.354131

Columns 0.024487 2 0.012244 21.17589 2.95E-06 3.354131

Interaction 0.006573 4 0.001643 2.841938 0.043533 2.727765

Within 0.015611 27 0.000578

Total 0.049486 35



0x

Count 4 4 4 12

Sum 0.806 0.956 0.854 2.616

Average 0.2015 0.239 0.2135 0.218

Variance 0.000942 6.13E-05 0.001353 0.000909

0.1x

Count 4 4 4 12

Sum 0.84 1.077 0.946 2.863

Average 0.21 0.26925 0.2365 0.238583

Variance 0.002065 0.001412 0.001852 0.002094

1x

Count 4 4 4 12

Sum 0.798 0.982 0.782 2.562

Average 0.1995 0.2455 0.1955 0.2135

Variance 0.00098 0.000228 0.000327 0.00098

Total

Count 12 12 12

Sum 2.444 3.015 2.582

Average 0.203667 0.25125 0.215167

Variance 0.00111 0.000648 0.00127

ANOVA


Sample 0.004292 2 0.002146 2.09523 0.142599 3.354131

Columns 0.014794 2 0.007397 7.221212 0.003075 3.354131

Interaction 0.001363 4 0.000341 0.332767 0.853461 2.727765

Within 0.027657 27 0.001024

Total 0.048106 35

Chlamydomonas

reinhardtiiEuglena gracilis

Day 14 ANOVAs



0x

Count 4 4 4 12

Sum 0.769 0.828 1.027 2.624

Average 0.19225 0.207 0.25675 0.218667

Variance 0.000158 3.27E-05 0.004402 0.002083

0.1x

Count 4 4 4 12

Sum 1.344 0.579 1.488 3.411

Average 0.336 0.14475 0.372 0.28425

Variance 0.004909 0.000442 0.002259 0.012926

1x

Count 4 4 4 12

Sum 1.386 0.563 0.613 2.562

Average 0.3465 0.14075 0.15325 0.2135

Variance 0.005872 0.000399 0.014749 0.01541

Total

Count 12 12 12

Sum 3.499 1.97 3.128

Average 0.291583 0.164167 0.260667

Variance 0.008385 0.001242 0.014548

ANOVA


Sample 0.037334 2 0.018667 5.056905 0.013636 3.354131

Columns 0.106012 2 0.053006 14.35953 5.65E-05 3.354131

Interaction 0.128925 4 0.032231 8.73152 0.000117 2.727765

Within 0.099667 27 0.003691

Total 0.371938 35



0x

Count 4 4 4 12

Sum 0.959 1.24 0.979 3.178

Average 0.23975 0.31 0.24475 0.264833

Variance 0.001014 0.000149 0.001005 0.001708

0.1x

Count 4 4 4 12

Sum 0.947 1.338 0.977 3.262

Average 0.23675 0.3345 0.24425 0.271833

Variance 0.002425 0.001486 0.001218 0.003551

1x

Count 4 4 4 12

Sum 1.082 1.176 0.911 3.169

Average 0.2705 0.294 0.22775 0.264083

Variance 0.000838 0.000624 0.000254 0.001288

Total

Count 12 12 12

Sum 2.988 3.754 2.867

Average 0.249 0.312833 0.238917

Variance 0.00142 0.000919 0.000743

ANOVA


Sample 0.000439 2 0.000219 0.218976 0.804753 3.354131

Columns 0.03856 2 0.01928 19.25601 6.35E-06 3.354131

Interaction 0.00643 4 0.001608 1.605576 0.201609 2.727765

Within 0.027034 27 0.001001

Total 0.072463 35

Chlamydomonas

reinhardtii Euglena gracilis

Fertilizer and Acid Rain Effects on Algal Growth science/PJAS/PJAS 15 pdf... · Fertilizer and Acid Rain Effects on Algal Growth ... on a table under two lamps providing equal light

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