Combined Effect Combined Effect of Ferric Chloride of Ferric Chloride and Mercuric and Mercuric Chloride on the Chloride on the Growth of Growth of Cyanobacteria Cyanobacteria AnacystisAnacystis nidulansnidulans

By Ted Handler, Farrell By Ted Handler, Farrell Parker, Alex Lewis, and Nick Parker, Alex Lewis, and Nick

ReedReed

Dr. Lee H. LeeDr. Lee H. Lee

Background Background InformationInformation

Industry spills massive Industry spills massive amounts of heavy metals such amounts of heavy metals such as mercury, selenium, ferric as mercury, selenium, ferric chloride, and mercuric chloride, and mercuric chloride in our water supply. chloride in our water supply. This study tests the effects of This study tests the effects of ferric and mercuric chloride ferric and mercuric chloride on the growth of on the growth of AnacystisAnacystis nidulansnidulans as an indicator of life as an indicator of life in our waters.in our waters.

AnacystisAnacystis nidulansnidulans

SmallSmall SensitiveSensitive PhotosyntheticPhotosynthetic SimpleSimple Provide an Provide an

indication of heavy indication of heavy metal pollution on metal pollution on metabolic activities metabolic activities of higher organisms of higher organisms in the ecosystem.in the ecosystem.

Toxicity of MercuryToxicity of Mercury

Mercury in any form is toxic. Toxic effects can result from vapor

inhalation, ingestion, injection, or absorption through the skin.

Hg accumulates mostly in the kidney, causing significant renal damage.

Chronic exposure leads to accumulation within the brain, causing neurological symptoms and birth defects.

Iron as anIron as anEssential MicronutrientEssential Micronutrient

Mineral needed in small amounts Mineral needed in small amounts for body to stay healthy.for body to stay healthy.

Found in meat, fish, and poultry.Found in meat, fish, and poultry. Functions in the transport and Functions in the transport and

storage of oxygen.storage of oxygen. Involved in DNA synthesis and Involved in DNA synthesis and

catecholamine metabolism.catecholamine metabolism.

Toxicity of IronToxicity of Iron

Toxicity most common in Toxicity most common in children.children.

Toxicity occurs when free iron Toxicity occurs when free iron levels exceed the iron binding levels exceed the iron binding capacity of transferrin in blood.capacity of transferrin in blood.

Free iron damages many organs Free iron damages many organs by direct cellular toxicity.by direct cellular toxicity.

MaterialsMaterials 4 Flasks4 Flasks 4 Cuvettes4 Cuvettes Disposable PipettesDisposable Pipettes 5 mL sterile pipettes5 mL sterile pipettes CottonCotton Cloth (breathable)Cloth (breathable) FoilFoil TapeTape 3M Medium3M Medium Cadmium and Ferric Cadmium and Ferric

chloridechloride Hand Tally CountersHand Tally Counters MicroscopeMicroscope Micropipettes Micropipettes

ProcedureProcedure We made a 3M medium for cell growth, using pipettes We made a 3M medium for cell growth, using pipettes

and micropipettes, and ten compounds.and micropipettes, and ten compounds. We adjusted the pH to 7.95.We adjusted the pH to 7.95. We sterilized it by autoclaving the medium at 121We sterilized it by autoclaving the medium at 121°° C. C. We mixed the We mixed the AnacystisAnacystis nidulansnidulans with the medium. with the medium.

Then distributed it into 4 flasks. Each flask with Then distributed it into 4 flasks. Each flask with 100mL of 100mL of AnacystisAnacystis nidulansnidulans culture.culture.

11stst flask had no heavy metals, this was our control. flask had no heavy metals, this was our control. 22ndnd flask has 0.5 mg/L of HgCl flask has 0.5 mg/L of HgCl22 and 50 mg/L of FeCl and 50 mg/L of FeCl33.. 33rdrd flask has 1 mg/L of HgCl flask has 1 mg/L of HgCl22 and 50 mg/L of FeCl and 50 mg/L of FeCl33.. 44thth flask has 5 mg/L of HgCl flask has 5 mg/L of HgCl22 and 50 mg/L of FeCl and 50 mg/L of FeCl33..

Procedure (cont’d)Procedure (cont’d) We used a micropipette to We used a micropipette to

transfer HgCltransfer HgCl22 and FeCl and FeCl33

Then we observed the Then we observed the AnacystisAnacystis nidulansnidulans every 3 or 4 days by every 3 or 4 days by two methods.two methods.

First, we used a First, we used a spectrophotometer to measure spectrophotometer to measure absorbance levels at 750nm.absorbance levels at 750nm.

Second, we used hand tally Second, we used hand tally counters to count the cells counters to count the cells under a microscope, on a under a microscope, on a special slide called a special slide called a hemacytometer, or a bacterial hemacytometer, or a bacterial counting chamber.counting chamber.

Results: ControlResults: Control

0.1

1

10

100

Cel

l # (

^107

)

0

0.5

1

1.5

2

O.D

. 750

0 5 10 15 20 25 30

Day

Standard Growth Curve of Cyanobacteria Anacystis nidulans

Mean Cell

Mean O.D.

Results: Flask 2Results: Flask 2

0.1

1

10

100

Cel

l# (

^107

)

0

0.5

1

1.5

2

O.D

. 750

0 5 10 15 20 25 30

Day

A.nidulans with 50mg/L FeCl3 and .5mg/L HgCl2

Mean Cell

Mean O.D.

Results: Flask 3Results: Flask 3

0.1

1

10

100

0

0.5

1

1.5

2

O.D

. 750

0 5 10 15 20 25 30

Day

A.nidulans with 50mg/L FeCl3 and 1mg/L HgCl2

Mean Cell

Mean O.D.

0.1

1

10

100

Cell#

(^

10

7 )

0

0.5

1

1.5

2

O.D

.

75

0

0 5 10 15 20 25 30

A.nidulans with 50mg/L FeCl 3 and 5mg/L HgCl 2

Results: Flask 4Results: Flask 4

0.1

1

10

100

Cel

l# (

^107

)

0

0.5

1

1.5

2

O.D

. 750

0 5 10 15 20 25 30

Day

A.nidulans with 50mg/L FeCl 3 and 5mg/L HgCl 2

Mean Cell

Mean O.D.

pH ResultspH Results

DayDay Flask Flask #1#1

Flask Flask #2#2

Flask Flask #3#3

Flask Flask #4#4

Day 1Day 1 7.957.95 7.957.95 7.957.95 7.957.95

Day Day 2727 99 9.49.4 9.159.15 7.417.41

ConclusionsConclusions

After extensive growth of After extensive growth of AnacystisAnacystis

nidulansnidulans in flasks 2 and 3, a resistance in flasks 2 and 3, a resistance is built to the mercuric chloride and is built to the mercuric chloride and ferric chloride. The cyanobacteria ferric chloride. The cyanobacteria thrives after growing a resistance and thrives after growing a resistance and grows at a rate faster then that of the grows at a rate faster then that of the control. However, after a stationary control. However, after a stationary point is reached at a high enough point is reached at a high enough concentration, the cyanobacteria begin concentration, the cyanobacteria begin to die. to die.

Conclusions Conclusions (cont’d)(cont’d)

In flask 4, the In flask 4, the concentrations of concentrations of the heavy metals the heavy metals were too high for were too high for the cyanobacteria to the cyanobacteria to be able to resist it. be able to resist it. The cyanobacteria The cyanobacteria had no chance to had no chance to grow, and began to grow, and began to die very rapidly, die very rapidly, from the beginning from the beginning of the experiment.of the experiment.

Thank You!Thank You!