BIOL10401 Online Practical 2: Algae for Biofuel PDF

23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science BIOL10401 Introduction to Laboratory Science Online resource 2: ALGAE FOR BIOFUELS > Online Practical 2: Algae for Biofuel Oil production by the green algae...

23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science BIOL10401 Introduction to Laboratory Science Online resource 2: ALGAE FOR BIOFUELS > Online Practical 2: Algae for Biofuel Oil production by the green algae Chlamydomonas reinhardtii in response to nitrogen limitation stress Staff: Prof Amanda Bamford and Dr Cecilia Medupin Welcome to Online Practical 2 If any of the videos do not work on this resource for you, please go the video portal for this unit to find another version! You simply need to visit the podcast video portal and login. There you will see a list of all of your courses which have podcasts available for them. see https://www.mypodcasts.manchester.ac.uk/student- faqs/#/ Aims This practical utilises the unicellular eukaryotic (click on these coloured words throughout this resource for more info) microalgae Chlamydomonas reinhardtii and looks at the physiological responses of this species to low nitrogen availability. The basis behind this experiment is the concept that organisms such as Chlamydomonas have evolved mechanisms to adapt and survive periods of nutrient limitation, including a reduction in growth and photosynthesis and alterations in metabolism, notably an increase in storage lipid biosynthesis. It is this accumulation of oils that make the algae interesting from a biofuel perspective as this accumulated oil is suitable to be converted into a biodiesel. During this online practical you will learn how to identify and quantify the responses of algae to nutrient limitation including changes in cell morphology, cellular physiology and lipid accumulation. In Lab session 2 you will handle Chlamydomonas and perform some of the activities described here. Intended Learning Outcomes At the end of this practical you will know how to: a) Transferable research skills Use a compound microscope to examine living organisms. Visualise cells under oil immersion Make cell extracts using ethanol solvent Separate small amounts of solid matter from liquid (bench-top centrifugation) Calibrate and use a spectrophotometer in both fixed wavelength and scanning mode. Measure and deliver small volumes of liquids accurately (automatic pipettes) b) Specific biological techniques Use microscopy techniques to examine the influence of nutrient limitation on cell biovolume Analyse and contrast the cellular structure of nutrient deficient and replete microalgae Extract and quantify chlorophyll pigments in an algal mixture Measure the visible light absorbance spectra of the chlorophyll extracts Calculate the unknown cell lipid concentration using the relationship between cell biovolume and lipid accumulation Useful Data-handling Learning Modules that apply to this practical 'Measurements and units' 'Moles and concentrations' 'Accuracy and precision' 'Functions and equations' 'Graphs and trendlines' https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 1/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Introduction Algae are a diverse group of organisms that possess chemical and metabolic characteristics that are of increasing interest for biotechnological exploitation. One such use of algae currently being investigated is as a sustainable source of biofuel is Chlamydomonas reinhardtii. Under certain growth conditions many species of algae can accumulate lipids that can be converted into a biofuel. Algae are autotrophs that can make sugars by photosynthesis, but they still need to obtain essential nutrients such as nitrogen and phosphorus. The availability of these nutrients are often limiting in the natural environment so algae have evolved mechanisms to survive periods of nutrient limitation. These include alterations to the cell metabolism, in particular changes to photosynthesis and lipid metabolism. Under situations of severe nutrient starvation, the cells enter a dormant phase with stored lipids as an energy source in order to prolong survival until the environmental conditions improve. It is this ability of algae to accumulate high concentrations of lipids that is of interest to researchers investigating the biofuel potential of algae. In this practical you will explore the responses of a common species of green algae, Chlamydomonas reinhardtii, to nitrogen limitation in 4 stages: Part 1: Observations of cell culture and cell morphology - light microscopy and oil immersion. Part 2: Measuring cell biovolume Part 3: Estimating lipid concentration in algae using cell biovolume data. Part 4: Examine and quantify changes in photosynthetic pigments using absorption spectroscopy. In this online practical we are going to investigate whether we can observe and measure these predicted changes after only 7 days of treatment with low N supply to the cells! Note that during this practical there will be activities for you to do to check your understanding Photos by Brian Piasecki (left) and from IGV Biotech (right) https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 2/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Background Information: Key concepts Background information explaining the key concepts and justification for this practical is summarised below 1. The green alga Clamydomonas rehardtii 'Algae' is a general non-scientific term for primitive eukaryotic organisms that possess the green pigment chlorophyll a to produce cellular energy by photosynthesis, but lack the features of more advanced photosynthetic organisms, such as a vascular system of xylem and phloem for internal transport of water and nutrients. Algae vary from small, single-celled forms (often called microalgae) to complex multi-cellular forms, including seaweeds (often called macroalgae). In this practical you are provided with cultures of Chlamydomonas reinhardtii, a single-celled eukaryotic alga of the class Chlorophyceae. It is a common freshwater alga but can also live on soil. Chlamydomonas is a 'model' algal species for laboratory studies, and has become widely used for studying photosynthesis, motility and stress responses, and general aspects of algal cell biology and biotechnology. The Chlamydomonas cell is filled by a single cup-shaped chloroplast which surrounds the nucleus and the other organelles (see Figure 1 below). It has two flagella for swimming. When viewed under a light microscope it is normally seen as a slightly oval cell of approximately 5-10 µm in width and 10-15 µm in length. Chlamydomonas will grow easily on a medium of inorganic salts, using photosynthesis to provide energy. It can also grow heterotrophically in darkness if acetate is provided as an alternative carbon source, or mixotrophically when light and acetate is provided together, allowing it to grow faster. Aeration by shaking or bubbling with air or 5% CO2 will also increase the growth rate. Optimal growth is usually at 20-25°C. Figure 1. Non- stressed cells. The morphology of Chlamydomonas reinhardtii. (a) Ultrastructure of the Chlamydomonas cell, showing the central nucleus (N) with nucleolus (Nu), surrounded by the cup-shaped chloroplast (C) containing thylakoid membranes (T), starch grains (S) and pyrenoid (P), within the stroma (St). An eye-spot (ES) is positioned against the inner envelope membrane of the chloroplast. Two flagella (F) project from the apical region of the cell, and vacuoles (V) might also be visible in the cytoplasm. (b) Chlamydomonas cells growing in liquid culture, viewed under 500× magnification. A single Chlamydomonas cell is usually about 10 μm in diameter. Images from https://doi.org/10.1016/S1360-1385(01)02018-0 The video below shows non-stressed Chlamydomonas reinhartii swimming - recorded using a light microscope under a range of magnifications. You can see them moving around using their flagella! See if you can work out if the flagella pushes them along or pulls them along? Note the magnifications shown at bottom right of the screen (apologies for the music!). Activity: Morphology The photo below is of a high N, non-stressed Chlamydomonas cell taken using x100 objective lens under oil immersion (Photo by Brian Piasecki; see later in resource to find more about oil immersion). Complete your first activity activity below to test your cell morphology knowledge. You might want to look at Figure 1 again. [Click on tick on top right of box when you are finished] https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 3/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science 2. Nutrient deficiency and photosynthesis Plants, algae and some bacteria use two photosystems in photosynthesis light capture. Photosystem I and Photosystem II (PSI and PSII) are chloroplast protein complexes that perform light absorption and energy transfer reactions of photosynthesis. Photosystem I is called I as it was the first one discovered but actually Photosystem II is the first one in the photosynthetic electron transport chain! When there is too much light, the photosystems can become photodamaged. Photodamage can also occur when essential nutrients are in limited supply because cell growth and metabolism has been slowed and absorbed sunlight energy cannot be fully used. During nutrient limitation, photosynthesis will reduce as a protective response to prevent photodamage. PSII is reduced under nutrient limitation and instead light is routed to a protein complex called light-harvesting complex II (LHCII), which is more efficient at converting excess light to heat. The PSII core is surrounded by variable numbers of these light-harvesting antenna complex II (LHCII), forming a PSII–LHCII supercomplex (see structure below). The LHCII and the remaining PSI are less sensitive to photodamage. The decline of PSII will also reduce the production of highly toxic and damaging reactive oxygen species (ROS), which will otherwise form when the excess light cannot be used by PSII. Figure 2. Photosynthetic protein changes that occur within the chloroplast to reduce cell damage during nutrient limitation. The abundance of PSII is reduced (unshaded, dashed line) to prevent photodamage and the production of reactive oxygen species (ROS). Light is instead absorbed by LHCII which can dissipate excess light. PSII-LHCII form a supercomplex - for structure see https://doi.org/10.1073/pnas.1912462116 The PSI and PSII complexes contain chlorophyll a and chlorophyll b photosynthetic pigment molecules which capture light and transfer it to the reaction centre in each photosystem. The abundance of total chlorophyll has been found to reduce in algae in response to nutrient limitation. This is partly due to a reduction in PSII abundance. During nitrogen limitation stress, a reduced abundance of chlorophyll molecules is also due to impaired chlorophyll biosynthesis because nitrogen is required for chlorophyll and is a major component of the molecules of chlorophyll a and b (see Figure 3). Figure 3. Structure of chlorophylls. You can see that the Mg2+ co-factor is surrounded by four nitrogen atoms (image from Perez et al, 2006, Light management in ornamental crops). NB This background information explains why nutrient limitation decreases chlorophyll production. However, you will not be expected to know this theory for the assessment of the unit. 3. Nutrient-limitation induced accumulation of storage metabolites A major response to nutrient limitation is modifications in cellular metabolism that are essential for allowing the cell to enter a dormant phase that extends its ability to survive the stress. These include an accumulation of the storage carbohydrate starch and an accumulation of non-membrane glycerolipids , predominantly a lipid called triacylglycerol (TAG). These are synthesised from photosynthetic products (see Figure 4a below). Glycerolipids accumulate in the cell in lipid droplets alongside starch granules (see Figure 4b below). Starch granules are found between thylakoid membrane stacks and surrounding a spherical structure located at the pole opposite the cilia known as the pyrenoid. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 4/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Figure 4a. Proposed carbon flux in green algae. Green arrows represent the carbon (C) flux under nitrogen (N)‐replete (plenty of N) conditions. Yellow arrows represent redirected C flux under N starvation. Dotted arrow represents a minor contribution to total C flux under N‐replete conditions. TAG is triacylglycerol and Acy-CoA is a group of coenzymes that metabolise fatty acids. (diagram from https://doi.org/10.1111/pbi.12523) Figure 4b: An image of nutrient stressed Chlamydomonas cell photographed by a transmission electron microscope, showing the formation of starch granules (S) and lipid droplets (LD) and N = nucleus after 3 days of stress. Scale bar is 2 μm. (adapted from DOI: 10.1128/EC.00203-09). In Figure 5 below are some photos of Chlamydomonas cells from a paper by Dean et al (2010 https://doi.org/10.1016/j.biortech.2010.01.065) where they treated them for 21 days at high and low N. You can see the localisation of the lipids in the cells (yellow and green stains in left 2 panels) and chlorophyll (red stain in far left panel and green in far right panel). They stained for lipid and chlorophyll using Nile red and used Epi-fluorescence microscopy as well as bright field (light) microscopy. You can easily see that the low N and high N treatment cells are very different. Which treatment has the most lipids in the cells? Which has the most chlorophyll? What has happened to the shape of the cells? During this online practical we will be exploring whether we get similar changes after only 7 days of treatment. In lab session 2 you will investigate the effect of different nitrogen concentrations. Figure 5. Lipid and chlorophyll localisation in C. reinhardtii detected by Nile Red staining in response to N treatment. Top panel is under low N treatment and bottom panel is high N treatment. Left-hand panels show Nile Red fluorescence (yellow/green=lipid) with chlorophyll fluorescence (red), and middle panels show Nile Red fluorescence alone (false colour green). Right hand panels show cells under light microscope. (Scale bar = 10 μm. Adapted from https://doi.org/10.1016/j.biortech.2010.01.065) https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 5/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science So it seems that such cells under nutrient stress accumulate lipids in lipid bodies (see image above). The next video is really an amazing 3D Youtube video. It shows how Chlamydomonas rheinhardtii cells move and also how Chlamydomonas can be used to create fuel. It discusses the underlying processes involved by making use of stereoscopic 3D visualization. However, don't worry - you do not need to know about the details of the pathways! Chlamydomonas 3D - From Biological Cells to Biofuels. 4. The potential of biofuels from algae There are many biotechnological applications of algae. One of great interest is that algae could become a source of biofuel due to their ability to produce lipids. Extracted triacylglycerol oils can be converted into biodiesel by a chemical reaction (transesterification). Because algae can use light and CO2 with few other inputs needed, they have the potential to generate a low-cost biofuel that is also carbon-neutral and will therefore contribute towards the reduction of greenhouse gas emission. However, one of the hurdles to overcome is to be able to generate enough algal biomass. Algae can be cultivated in very large volumes, either in ponds or in photo-bioreactors (see Figure 6), but the feasibility of the process still needs to be demonstrated on a large scale and a big challenge is to make the process cost-effective. Open ponds are cheaper to build and maintain but the culture is harder to manage and contamination could occur. Photo-bioreactors provide algae containment and controlled growth conditions but are more expensive to build and run. Figure 6. Large scale cultivation methods for the commercial growth of algae can include enclosed photo-bioreactors (top) or open ponds (right). The photo on left shows a tubular glass photobioreactor for the cultivation of microalgae. It has an operational volume of 4000 litres and the principle was developed in the late 1990s. This type of photobioreactor is suitable for the scaled production of microalgae-based high-value products (photo by IGV Biotech). The other photo (right) shows a June 2010 satellite photo of raceway open ponds in southern California (Photo by Pacific Northwest National Laboratory). If you want to read more about the potential and process of converting algae to biofuels click on this link to an article in the journal of Frontiers in Bioengineering and Biotechnology https://doi.org/10.3389/fbioe.2014.00090 You can also find out more from Alaswad, A. & Dassisti, Michele & Prescott, T. & Olabi, Abdul Ghani. (2015). Technologies and developments of third generation biofuel production. Renewable and Sustainable Energy Reviews. 51. 1446- 1460. link to paper So to summarise: this practical utilises Chlamydomonas reinhardtii and looks at the responses of this species to 7 days of low nitrogen availability. The basis behind this experiment is the concept that organisms such as Chlamydomonas have evolved mechanisms to adapt and survive periods of nutrient limitation, including a reduction in growth and photosynthesis and alterations in metabolism, notably an https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 6/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science increase in storage lipid biosynthesis. It is this accumulation of oils that make the algae interesting to scientists! The rest of this resource will now go through the practical. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 7/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 1: Making observations of cell movement and morphology using a light microscope A culture of Chlamydomonas reinhardtii was grown for 7 days either in non-stressed conditions with a high concentration of nitrogen, in the form of ammonium (7 mM NH4Cl) or in nitrogen limitation conditions (0.7 mM NH4Cl). These cultures have been grown in an acetate-containing medium (TAP medium) in the presence of light at 22°C. There are two algae cultures from each growth treatment (High N and Low N, see Figure 7). We are going to make observations and measurements of the morphology and physiology of these cells. First we will need to look at them under a light microscope. [detailed instructions on how to set up and use a light microscope are on the next page.] Figure 7. Flasks of cell cultures of Chlamydomonas under High N (left) and Low N (right) conditions. Notice the difference in colour! You will see these in Lab session 2 (Adapted from doi:10.1371/journal.pone.0122600.g005) Below is your first instruction video recorded in our labs. Watch the video and read the text below that outlines the steps. Firstly a drop of algae is placed onto a glass slide and a cover slip gently placed on top of the liquid. Under the the microscope the slides are then viewed using the x10 objective then the x40 objective to find the cells and get them into focus. However, to observe the cells in more detail a high x100 objective lens has to be used and then the cells should be viewed under oil immersion. [If we really want to see the cells in detail, but they were swimming too fast, we could add 20 µl of Lugol's iodine solution to 1 ml algal sample in a microfuge tube and mix – this will kill the algae and stop them swimming!] Below are some videos taken with a digital light microscope in our lab. Can you see any differences between the 2 treatments? How fast are the cells moving? Are they different sizes? Can you see any flagella? High N cells under x10 and then under x40 objective lenses ( if the video fails try this link https://youtu.be/mTg__eF8U1I). Low N cells under x10 and then under x40 objective lenses ( if the video fails try this link https://youtu.be/mx8S6xRlrZA). Activity: Morphology and movement sorting This is your next opportunity to test your knowledge. Watch the videos of high and low N cells moving on this page and earlier ones on previous pages and the photos. Then sort the characteristics on the cards into the High N and low N treatments in the activity. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 8/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science PART 1: THINKING POINT on morphology and movement What were the main differences between the cells in low nitrogen conditions compared to high nitrogen conditions? What does this tell you? What is happening inside the cells at low N that may affect their mobility? https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 9/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 1: How to view cells under a light microscope To see Chlamydomonas cells clearly we must employ a microscope. In bright field microscopy visible light is focused by a condenser lens through a specimen mounted on the stage. The image is then magnified by two further lenses placed at both ends of a light-tight tube. The degree of detail viewed depends on the limit of resolution, illumination and contrast. Adjustments to the condenser can affect resolution and contrast. Study the picture of the light microscope below and learn more about the parts of a microscope by clicking on this link https://microbenotes.com/parts- of-a-microscope/ photo by CNX OpenStax Figure 8. Labelled bright field light microscope. You will use the microscope in in Lab sessions 2-4. here is a video that you may find useful as preparation: Activity: Test yourself and see if you can remember the names of the parts of the microscope Try this simple test (photo by pngimg.com) https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 10/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 2: Measuring cell biovolume To complement the observations made in Part 1, we will need to measure the biovolume of the High N and Low N cells to examine whether accumulation of lipid bodies and starch granules alters the size and thus volume of the cells. We measure biovolume by observing cells under the microscope (x 40 objective lens) and measuring the length and width of a few cells using an eyepiece reticle (scalebar) then calibrating the reticle with a stage micrometer. this gives us the dimensions in µm. You will be introduced to the reticle and the calibration process in Online Practical 4 and will use the reticle in Lab session 4, so here we will just give you the measured dimensions. RESULTS Table 1. Cell dimension measurements (µm)- to one decimal place. Activity: Converting cell width and length dimensions (µm) to cell biovolume (µm3). STEP 3: Now use the formula below for a spheroid shape to calculate the final cell biovolume (µm3) from the width and length measurements for Replicate 1 in Table 1. Cell biovolume (μm3) =(π /6) x (width2 ) x length [Note: π (pi) = 3.14159] ​ If you can't get to the correct answer to these multiple choice questions then ask in Lab session 2. You would normally repeat this process for many cells (in this practical we used 3 replicates per treatment) and work out the mean value for low and high N cells. The means for the 3 cells measured in this practical are shown in Table 2 below. RESULTS Table 2. Biovolumes of high N cells and low N cells (mean values of 3 replicates). https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 11/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 2: THINKING POINT on cell biovolume How did the size of the cells change in low nitrogen conditions compared to high nitrogen conditions? What does this tell you? What is happening biologically to make them different sizes? https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 12/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 3: Lipid estimation Previous measurements by Dean et al. (2010) have been made that show a positive correlation between Chlamydomonas cell biovolume and cell glycerolipid content. This correlation data is shown in Figure 18 below. We can use the line data equation (y = 0.0204 x) below and the cell biovolume values determined in PART 2 of this practical to calculate lipid content per cell (Table 3). Figure 18. Correlation between cell biovolume (μm3 ) and lipid content per cell (pg cell-1) determined for Chlamydomonas in response to varying N limitation. RESULTS Table 3. Lipid content of high and low N cells. Given that the biovolumes are 270 μm3 (high N) and 545 μm3 (low N) ACTIVITY: Calculate lipid content from biovolume. Have a go at using the equation from Figure 18 to calculate lipid content from a biovolume value https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 13/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 3: THINKING POINT on lipid estimation Was there a predicted change in storage lipid content between the cell treatments? If so, does this change explain any of the morphological differences seen in Part 1. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 14/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 4: Photosynthetic pigment analysis using spectroscopy In this part of the practical we will use spectroscopy, as introduced in Online practical 1. This is a very valuable tool for analysing the molecular composition and concentration of solutions. You can use UV-visible spectrophotometers to identify chemicals in mixtures by looking at where the peaks are and comparing to known values in the literature. Studies on water quality monitoring have used UV-visible absorption spectra to monitor for water pollution events https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855095/ In this practical you will learn how to apply this technique to an investigation of the photosynthetic pigments in our Chlamydomonas samples. You are expected to be familiar with some the background information on spectrophotometry from Online Practical 1. Background Information on pigments Pigments are chemical compounds that reflect only certain wavelengths of visible light. This makes them appear "colourful". Leaves and flowers contain different pigments that give them their colour. More important than their reflection of light is the ability of pigments to absorb certain wavelengths for photosynthesis. However, the light-absorbing photosynthetic pigments do not absorb all wavelengths of light equally. The absorption of different wavelengths, or absorption spectrum, by the photosynthetic pigments can be demonstrated using a spectrophotometer. A spectrophotometer can measure how much light is absorbed by a substance when it is irradiated with light of a specific wavelength. Photosynthetic pigments all have characteristic absorption spectra (see Figure 8 below for typical spectra for purified chlorophyll a and b and a range of marine algal pigments). The wavelength(s) at which the maximum (best) absorbance occurs is known as absorbance maxima λ max ). The absorbance is greatest at λ max and this wavelength is selected to perform quantitative measurements. The absorbance maxima for chlorophyll a and b is typically 430 and 665 nm (for Chl a) and 460 and 649 nm (for Chl b). Note: maxima values vary depending on the solvent used to extract the pigment. An absorption spectrum is a graph of light absorption (dependent variable) as a function of wavelength (independent variable). It is important to remember that in fact there is a whole range of photosynthetic pigments, some of which are only found in certain types of algae e.g phycocyanin! You can see from the spectra in Figures 19 & 20 below that chlorophylls absorbs maximally in red and blue regions of the spectrum. So how do you think how well plants would grow if just grown under green lights? https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 15/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Figures 19 and 20. Absorption spectra of marine algal pigments (top image from Yarish et al 2012) and purified chlorophylls a and b (bottom image by Daniele Pugliesi) Why are plants green? click on this link for the answer blog from John Innes Centre https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 16/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Step 1: Spinning down the algal cells and extracting chlorophyll from the cells Here are some videos of the procedure - you will do this in lab Session 2. 1A: First we first need to spin down the cells from the culture solution (making sure the tubes are balanced in the centrifuge): 1B: After this first spin, we now need to extract the chlorophyll from the algal cells using ethanol: This ''introduction to the centrifuge'' video below is very detailed however you do not have to watch all of it! You can use the times in the chapters to skip parts. Word document transcript for '' introduction to the centrifuge'' https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 17/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Step 2: Recording the absorbance spectra of chlorophyll The absorbance spectra for one high N and one low N sample can then measured using a spectrophotometer set to read between 400nm-700nm using scanning mode as you did in Lab session 1. RESULTS Figure 21. Chlorophyll absorption spectrum from High N cells. Figure 22. Chlorophyll absorption spectrum from Low N cells. Note the different scales on the side of the spectra! At what region of wavelengths are the peaks? Can you pick out the chlorophyll a and b peaks by comparing these spectra to Figures 19 and 20 on earlier page? Remember that our ethanol cell extracts are a mixture and not a purified extract of different chlorophylls, as used in Figures 19 and 20, so some of the peaks will be 'hidden' underneath bigger ones due to some chlorophylls being present at much higher concentrations than others. Also other pigments such as carotenoids can also be detected. Have a go at identifying the peaks then check your answers in the photo below! https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 18/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science.... https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 19/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science ​Step 3: Measuring Chlorophyll concentration Next we are going to see the steps to measure the absorbance of chlorophyll a and b (chl a and chl b) using the absorption maxima values of 3 samples from high N and 3 samples from Low N that were prepared in step 1 above. From these readings we can then calculate the concentration of chlorophyll a and b and also total chlorophyll concentration in μg ml-1. We now need to measure the absorbance of the ethanol extract at two different wavelengths: 649 nm and 665 nm. This time the spectrophotometer is set to single wavelength mode. We then use the following equations to calculate the concentration of total chlorophyll (total Chl = Chl a + Chl b) in the extracts. Chlorophyll a (µg ml-1) = (13.95 x A665nm) - (6.88 x A649nm) x dilution factor Chlorophyll b (µg ml-1) = (24.96 x A649nm) - (7.32 x A665nm) x dilution factor (Reference: Lichtenthaler & Wellburn 1983 Biochemical Society Transactions 11: 591-592) Remember we only use a dilution factor if the solution was too dark (i.e. the chlorophyll content was very high so the peaks were off the scale and we had to dilute the sample). When we measured the absorbance readings with our spectrophotometer we obtained the values below in Table 4 and we didn't have to dilute any of our samples. RESULTS Table 4. Absorbance measurements. Using the formulas above we then calculated chlorophyll a and b and total chlorophyll for each replicate (Table 5). Finally we calculated the mean values for the 2 treatments (Table 6). Before looking at the answers, can you calculate them yourself using the equations?..... Answers: Table 5. Chlorophyll concentrations calculated from equations. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 20/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science. Table 6. Final mean total chlorophyll concentrations. Activity: Calculate total chlorophyll concentration in cell cultures. To test yourself on the using equations for chlorophyll concentration by calculating the total chlorophyll concentration for the example in the activity below. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 21/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Part 4: THINKING POINT on Photosynthetic pigments Were there any differences in the concentration of chlorophyll between the two nitrogen treatments? How did they change and what does this indicate? https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 22/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Conclusion of Practical Below is a summary of questions for the THINKING POINTS for this practical. It is not enough to just measure things, we have to try to think of WHY they are different, what is the data telling us about what is going on in our treatments and how Chlamydomonas responds to nitrogen levels. Can you remember the answers and explanations? If not look over the videos under each THINKING POINT again. Part 1: Morphology and culture characteristics What were the main differences between the cells in low nitrogen conditions compared to high nitrogen conditions? What does this tell you? Part 2: Measuring cell biovolume How did the size of the cells change in low nitrogen conditions compared to high nitrogen conditions? What does this tell you? Part 3: Lipid estimation Was there a predicted change in storage lipid content between the cell treatments? If so, does this change explain any of the morphological differences that were observed? Part 4: Photosynthetic pigment analysis Did you see any difference in the concentration of chlorophyll between the nitrogen treatments? How did they change and what does this indicate? https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 23/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science Self-assessment Check you have achieved the following specific biological learning outcomes: Learning outcomes Know how to view cells under bright field (light) microscope. Observed and described Chlamydomonas reinhardtii cellular structure and morphology. Able to calculate cell biovolumes. Know how to extract and quantify photosynthetic pigments Chlorophyll a and b and determine absorption spectra of an extract. Know how to calculate lipid content based on biovolume measurements. https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 24/25 23/10/2023, 12:15 BIOL10401 Introduction to Laboratory Science End of Online Practical 2 Well done! You can go over the material in this online practical at any time. In order to register your completion of this resource, please take the quiz in Blackboard entitled 'Test your Understanding' Quiz of Online Practical 2 for which you need to obtain at least 60%. The quiz can be taken as often as needed to reach this total. This is accessed on Bb - Lefthand menu 'Test Your Understanding' or in the Content folder for Online Practical 2. The ILOs of this online practical are examinable in the BIOL10401 MCQ examination in January. If you struggle with any of the content, you can ask in the associated Lab session 1 that follows this OP1 or you can post a question on the Bb discussion site You can also discuss these activities in the PASS workshops (BIOL10401& DHS study group) Don't forget to complete any outstanding Data-Handling Skills Learning Modules and to attend the Data-Handling clinics if you have problems (see timetable). You may also want to look through the Lab Session 2 link on Bb before your session https://www.softchalkcloud.com/lesson/files/OgUunY75wx0tz8/prac2localv32_print.html 25/25