Lab Report

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BIO8201 Photosynthesis report

Module 5 in this course will be examined by a lab report which is worth 30% of your assessment.

Read through the following exercise by Dearnaley and McCabe (2013) and follow the instructions on how to write up the lab report on page 9. Use the marking scheme on page 10 as a guide as to how to structure the report and of what to include in each section. There are also guides to writing lab reports and how to use Harvard referencing on the Study Desk.

If you are uncertain about anything, contact the course examiner, John Dearnaley ([email protected]) for guidance.

Photosynthesis

By John Dearnaley and Bernadette McCabe (2013)

INTRODUCTION

Photosynthesis is unquestionably the most important series of chemical reactions that occurs on Earth. Life as we understand it would not occur without photosynthesis.

Photosynthesis is the process by which chlorophyll containing cells convert radiant energy from the sun into chemical energy (ATP and carbohydrates), releasing oxygen into the environment during the process. All the energy (food) requirements of organisms are directly or indirectly derived from green plants and therefore ultimately from the sun. In addition to providing food, photosynthesis maintains high oxygen levels in the atmosphere and in aquatic habitats. Fossil fuels (coal, gas, petroleum) were derived from the photosynthetic activity of plants and protistans that lived millions of years ago. A summary equation for photosynthesis is given below:

LIGHT

6 CO2 + 12H2O 6 (CH2O) + 6H2O + 6O2

Carbon water CHLOROPHYLL Carbohydrate Water Oxygen

Dioxide

Carbon dioxide provides the carbon and oxygen atoms used to synthesize CH2O (carbohydrate). The oxygen released as a by-product is derived from the spitting of water molecules.

There are two major phases to photosynthesis:

1. LIGHT REACTION (HILL REACTION OR PHOTOPHOSPHORYLATION)

When light energy strikes chlorophyll molecules, electrons are energized and passed from chlorophyll to a series of electron acceptor molecules, resulting in the synthesis of ATP and the reduction of an electron carrier molecule, called NADP. The electrons lost from chlorophyll are replaced by electrons derived from water.

2. CALVIN CYCLE (CARBOHYDRATE SYNTHESIS OR THE "SUGAR SHUFFLE".)

During the Calvin cycle CO2 is reduced by hydrogen derived from water, resulting in the formation of a carbohydrate (CH2O)n. The Calvin cycle does not require light. The energy required to synthesize carbohydrates is supplied by ATP and NADPH produced during the light reaction.

The first stable end product of photosynthesis in many plants is a 3 carbon compound, called phosphoglyceraldehyde (PGAL). Because PGAL is a 3 carbon compound, the Calvin cycle is often called C3 photosynthesis. PGAL is then used to synthesize glucose. In 1960 M.D. Hatch and C.R. Slack of Queensland discovered that certain tropical plants (sugar cane, corn and grasses) combine CO2 with a 3 carbon compound to form a 4 carbon compound. This metabolic pathway is called C4 photosynthesis.

Glucose can be used in several ways by the cell. It can be respired to yield energy in the form of ATP, converted into other sugars or transformed into starch which is stored.

OBJECTIVES

1. To study the photosynthetic pigments present in plant tissues.

2. To study the relationship between leaf anatomy and photosynthesis.

3. To study some of the adaptive modifications of leaves for photosynthesis in different environments.

PART A. SEPARATION OF PLANT PIGMENTS BY PAPER CHROMATOGRAPHY

There are a number of pigments (coloured molecules) involved in photosynthesis. The primary pigments are chlorophylls (a, b, c and d). The main photosynthetic pigment found in all green plants is chlorophyll a. It absorbs red (670-680 nm) and blue (440 nm) light more strongly than other wave lengths. Accessory pigments (eg. carotenes, xanthophylls, phycocyanins) absorb other wave lengths and pass the energy onto chlorophyll a.

PROCEDURE THAT WAS USED TO OBTAIN AND TEST PLANT PIGMENTS

1. 2-5 ml of solvent (acetone-petroleum ether 10:90) was poured into a chromatography jar. The lid was replaced and the jar was allowed to fill with solvent vapour.

2. 1-2g of Kikuyu grass (Pennisetum clandestinum) leaves were cut up and ground thoroughly with a mortar and pestle.

3. The leaf material was added to a test tube with 4 ml of acetone. The tube was stoppered and shaken vigorously It was let stand for 10 minutes.

4. 3 ml of water was added and the tube shaken again

5. 3 ml of petroleum ether was then added, the tube was shaken again and then let stand for several minutes or until the different pigments separated into layers.

6. Using a Pasteur pipette, the upper dark green solvent layer was removed and placed in a small beaker.

7. Using a capillary tube, six small drops of pigment extract were added to the chromatography paper strip. The drop was placed about 1 cm from the end of the paper and the pigment allowed to dry thoroughly before adding another drop to the same spot.

8. The paper strip was carefully added into the chromatography jar until the tip of the paper just touched the solvent. The paper strip was secured, making sure the paper did not touch the sides of the jar.

9. The jar was left on the bench and observed at frequent intervals.

10. The paper was removed from the jar before the solvent reached the top of the paper.

11. The paper was let dry for a few minutes.

12. 4 different colours were observed:

(a) chrome yellow-carotene,

(b) greenish yellow (dirty yellow) - xanthophyll,

(c) bluish green - chlorophyll a,

(d) yellowish green - chlorophyll b.

(The pigments are listed in order of separation from top to bottom of the paper strip).

13. Elution of Pigments for Spectral Analysis:

(a) Each pigment band from the paper strip was cut out and placed into separate beakers containing small amounts of acetone.

(b) After the pigments have been removed from the paper (ie. eluted), each pigment-acetone solution was poured into a separate cuvette.

(c) A spectrophotometer was used to measure the absorbance of each pigment solution at 10 nm intervals over the range 380 - 720 nm, reading against acetone as the blank. The spectrophotometer was zeroed at each wavelength setting before testing the samples.

14. Prepare absorption spectra for each pigment on the same set of axes, by graphing the relative absorbance (y-axis) against wavelength (x-axis) (the information in the Tables). Note, the experiment was conducted five times for each pigment, so you first need to determine the mean value of each pigment at each wavelength. Plot your graph with means and standard error bars at each wavelength.

13

Table 1. Absorbance of light by plant pigments - carotene.

Wavelength (nm)

380

390

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

Experiment 1

0.020

0.020

0.080

0.082

0.100

0.103

0.116

0.143

0.134

0.121

0.112

0.103

0.080

0.044

0.030

0.029

0.025

0.020

Experiment 2

0.019

0.024

0.081

0.082

0.105

0.107

0.119

0.145

0.128

0.123

0.109

0.103

0.081

0.042

0.029

0.030

0.023

0.018

Experiment 3

0.023

0.022

0.076

0.084

0.103

0.106

0.112

0.134

0.130

0.120

0.114

0.107

0.076

0.040

0.031

0.027

0.025

0.021

Experiment 4

0.024

0.020

0.084

0.083

0.101

0.103

0.115

0.140

0.130

0.117

0.112

0.108

0.078

0.041

0.033

0.028

0.026

0.019

Experiment 5

0.017

0.021

0.080

0.088

0.100

0.105

0.115

0.139

0.132

0.120

0.111

0.101

0.076

0.047

0.030

0.026

0.022

0.020

Wavelength (nm)

560

570

580

590

600

610

620

630

640

650

660

670

680

690

700

710

720

Experiment 1

0.021

0.022

0.019

0.018

0.015

0.016

0.015

0.014

0.013

0.013

0.013

0.013

0.012

0.014

0.004

0.003

0.003

Experiment 2

0.019

0.021

0.016

0.017

0.013

0.013

0.015

0.012

0.012

0.013

0.012

0.012

0.010

0.011

0.009

0.006

0.003

Experiment 3

0.017

0.015

0.015

0.016

0.014

0.015

0.014

0.011

0.011

0.010

0.012

0.010

0.011

0.010

0.009

0.007

0.001

Experiment 4

0.017

0.020

0.018

0.014

0.015

0.012

0.011

0.014

0.011

0.010

0.011

0.011

0.011

0.010

0.006

0.005

0.005

Experiment 5

0.016

0.019

0.019

0.015

0.014

0.014

0.013

0.015

0.010

0.011

0.011

0.011

0.010

0.012

0.008

0.005

0.001

Table 2. Absorbance of light by plant pigments - xanthophyll.

Wavelength (nm)

380

390

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

Experiment 1

0.018

0.020

0.023

0.027

0.059

0.074

0.134

0.126

0.093

0.056

0.079

0.121

0.090

0.059

0.046

0.027

0.018

0.016

Experiment 2

0.016

0.022

0.021

0.025

0.047

0.068

0.135

0.130

0.091

0.053

0.076

0.124

0.089

0.057

0.044

0.028

0.015

0.013

Experiment 3

0.011

0.020

0.022

0.023

0.049

0.066

0.128

0.128

0.095

0.055

0.081

0.120

0.091

0.056

0.042

0.027

0.017

0.019

Experiment 4

0.015

0.021

0.020

0.024

0.055

0.072

0.129

0.125

0.092

0.056

0.078

0.119

0.091

0.061

0.045

0.031

0.016

0.015

Experiment 5

0.017

0.019

0.022

0.027

0.052

0.071

0.130

0.121

0.093

0.057

0.079

0.122

0.089

0.060

0.047

0.026

0.018

0.015

Wavelength (nm)

560

570

580

590

600

610

620

630

640

650

660

670

680

690

700

710

720

Experiment 1

0.015

0.012

0.006

0.006

0.006

0.003

0.003

0.002

0.003

0.005

0.002

0.001

0.002

0.004

0.001

0.002

0.002

Experiment 2

0.014

0.011

0.005

0.007

0.006

0.002

0.001

0.001

0.002

0.004

0.001

0.003

0.002

0.002

0.001

0.003

0.001

Experiment 3

0.015

0.011

0.006

0.005

0.004

0.001

0.002

0.002

0.004

0.001

0.001

0.002

0.003

0.004

0.002

0.001

0.001

Experiment 4

0.013

0.010

0.004

0.006

0.003

0.002

0.001

0.003

0.003

0.002

0.003

0.002

0.001

0.001

0.002

0.001

0.002

Experiment 5

0.016

0.012

0.005

0.006

0.004

0.003

0.002

0.001

0.001

0.001

0.002

0.001

0.002

0.001

0.001

0.002

0.002

Table 3. Absorbance of light by plant pigments – chlorophyll a.

Wavelength (nm)

380

390

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

Experiment 1

0.180

0.211

0.280

0.320

0.511

0.675

0.486

0.163

0.108

0.102

0.058

0.031

0.024

0.022

0.028

0.030

0.030

0.030

Experiment 2

0.176

0.214

0.277

0.328

0.525

0.684

0.487

0.170

0.107

0.105

0.050

0.033

0.026

0.020

0.025

0.025

0.028

0.030

Experiment 3

0.184

0.198

0.282

0.326

0.530

0.665

0.480

0.169

0.105

0.102

0.056

0.030

0.023

0.019

0.026

0.024

0.025

0.031

Experiment 4

0.181

0.206

0.281

0.318

0.527

0.667

0.478

0.166

0.104

0.104

0.051

0.032

0.022

0.023

0.027

0.026

0.027

0.030

Experiment 5

0.182

0.202

0.279

0.322

0.519

0.672

0.483

0.164

0.106

0.103

0.055

0.031

0.021

0.023

0.026

0.029

0.024

0.032

Wavelength (nm)

560

570

580

590

600

610

620

630

640

650

660

670

680

690

700

710

720

Experiment 1

0.040

0.059

0.069

0.069

0.074

0.088

0.106

0.111

0.246

0.538

0.341

0.069

0.015

0.008

0.005

0.004

0.004

Experiment 2

0.043

0.057

0.067

0.066

0.073

0.086

0.105

0.112

0.248

0.535

0.343

0.071

0.011

0.009

0.004

0.002

0.003

Experiment 3

0.041

0.057

0.068

0.066

0.074

0.089

0.101

0.110

0.242

0.530

0.345

0.072

0.014

0.007

0.004

0.001

0.002

Experiment 4

0.039

0.055

0.066

0.068

0.072

0.087

0.104

0.119

0.246

0.532

0.342

0.080

0.012

0.009

0.005

0.002

0.001

Experiment 5

0.042

0.056

0.065

0.069

0.071

0.086

0.103

0.111

0.250

0.536

0.340

0.070

0.011

0.007

0.003

0.004

0.001

Table 4. Absorbance of light by plant pigments – chlorophyll b.

Wavelength (nm)

380

390

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

Experiment 1

0.080

0.112

0.129

0.174

0.229

0.281

0.440

0.490

0.320

0.066

0.031

0.022

0.010

0.010

0.009

0.009

0.007

0.006

Experiment 2

0.081

0.115

0.127

0.178

0.232

0.290

0.437

0.491

0.322

0.080

0.030

0.024

0.009

0.007

0.009

0.008

0.006

0.005

Experiment 3

0.079

0.113

0.128

0.185

0.240

0.285

0.442

0.488

0.320

0.076

0.035

0.020

0.010

0.009

0.007

0.009

0.005

0.004

Experiment 4

0.082

0.114

0.130

0.182

0.228

0.283

0.445

0.485

0.321

0.069

0.032

0.023

0.012

0.008

0.007

0.006

0.006

0.005

Experiment 5

0.083

0.111

0.133

0.175

0.239

0.287

0.448

0.486

0.325

0.075

0.029

0.022

0.008

0.007

0.006

0.007

0.007

0.004

Wavelength (nm)

560

570

580

590

600

610

620

630

640

650

660

670

680

690

700

710

720

Experiment 1

0.008

0.008

0.009

0.009

0.021

0.040

0.140

0.310

0.250

0.095

0.090

0.080

0.040

0.030

0.014

0.008

0.007

Experiment 2

0.007

0.008

0.010

0.012

0.019

0.041

0.147

0.319

0.246

0.094

0.090

0.082

0.043

0.032

0.017

0.006

0.005

Experiment 3

0.006

0.009

0.009

0.011

0.020

0.046

0.141

0.315

0.252

0.090

0.089

0.081

0.041

0.027

0.012

0.007

0.006

Experiment 4

0.005

0.006

0.008

0.013

0.018

0.042

0.145

0.312

0.261

0.092

0.092

0.085

0.038

0.033

0.015

0.009

0.004

Experiment 5

0.006

0.008

0.007

0.009

0.017

0.045

0.144

0.320

0.254

0.091

0.089

0.085

0.039

0.031

0.013

0.008

0.003

PART B. LEAF STRUCTURE AND FUNCTION

With few exceptions, most photosynthesis occurs in the leaves of terrestrial plants.

In this exercise leaves of two plants from different environments were chemically fixed (killed), embedded in resin and thinly sectioned with a machine known as a microtome. Thin sections were stained with toluidine blue and mounted on microscope slides.

1. LEAF ANATOMY - PrIVET LEAF

(a) Examine the image of a transverse section (T.S.) of a privet (Ligustrum sp.) leaf below. This image was taken on a compound microscope using the X10 objective.

(b) If present label, the following structure or tissues

cuticle - a waxy, waterproof layer of non-cellular material covering the upper epidermis (a single layer of cells).

palisade mesophyll - consists of cells rich in chloroplasts elongated perpendicularly to the leaf surface. Leaves may have one or more rows of palisade cells.

spongy mesophyll - loosely arranged cells of various shapes, frequently with branches extending from one cell to the other. Note the large volume of air space within the spongy mesophyll.

lower epidermis with stoma. Each stoma (singular) consists of 2 guard cells and a pore which leads into the air spaces within the spongy mesophyll.

(c) If each palisade mesophyll cell is approximately 30µm wide, add a scale bar to the image.

Q1. Which layer of the leaf has the majority of chloroplasts and why?

2. LEAF ANATOMY - EUCALYPTUS LEAF

(a) Examine the image of a transverse section (T.S.) of a Eucalyptus leaf below. This image was taken on a compound microscope using the X10 objective.

(b) Label the structure and tissues in this diagram (see above).

(c) If each palisade mesophyll cell is approximately 20µm wide, add a scale bar to the image.

(d) Compare and contrast the structure of this leaf with that of the privet leaf.

Figure 1. Transverse section of privet leaf.

Figure 2. Transverse section of Eucalyptus leaf.

REPORT WRITE UP

Make sure you include all relevant data in your report ie. Tables 1-4, the absorbance spectrum graph, labelled Figure 1 and labelled Figure 2.

In your discussion re-state firstly the main results you obtained. Next use the scientific literature to explain these results eg. the practical manual, textbooks, journal articles. If the results were not in agreement with expected theory, discuss why this might be, outlining any possible errors in the procedure and improvements that could be used in future.

Here are the areas we expect you to detail in your discussion.

-What were the major absorption peaks of the four pigments and why are they different?

-What region of the leaf had the most chloroplasts and why?

-How did the Eucalyptus leaf differ in anatomy from the typical dicot leaf and what is the reason for this?

BIO8201 - BIOLOGY FOUNDATIONS 2020

PHOTOSYNTHESIS

This report will be marked out of 100 and is worth 30% of your total assessment for this unit. It should be written as a brief report (max 5 typed (preferably) pages plus tables, figures etc). Proper report format for scientific writing must be used. Consult the sample lab report on the Study Desk if unsure. This report is to be submitted via the course Study Desk by 11:59pm on Friday September 11th. A late penalty of 5% per day will apply.

Attach this form to your report

Criteria

Weighting

Introduction: Provide a brief background to photosynthesis.

10

Objectives: Provide a clear statement (in your own words) of the objectives of the exercise.

4

Methods: Briefly outline the experimental approach in Part A and B.

8

Results:

Part A - Present the results as absorption spectra for each pigment, drawn on one set of axes and labelled correctly. Briefly outline the main trends or patterns in the absorption spectra.

Part B - Present the labelled figures including a scale bar. Briefly describe the results.

21

13

Discussion:

Main results obtained.

Theory behind main results.

Possible sources of error.

Improvement(s).

10

18

2

2

General conclusion

2

General presentation: correct format, spelling, grammar, expression, correct use of references.

10

Total

100