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Introduction to Laboratory Science > Algae for Biofuels > Flashcards

Algae for Biofuels Flashcards

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1
Q

What are the risks and safety procedures associated with this practical?

A
  • The species of microalgae is not hazardous to health although to avoid any potential skin sensitivity or allergic reaction care should be taken to avoid spilling any algae onto your skin
  • Ethanol is flammable and toxic solvent, so we have to avoid spillage and skin contact
  • Glass slides and cover slips are always disposed of in sharp bins (bright yellow). Care should be taken to avoid cuts if any glass is broken
  • Lab-coats are always worn in a laboratory
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2
Q

What do algae do under situations of severe nutrient starvation?

A

the cells enter a dormant phase with stored lipids as an energy source in order to prolong survival until the environmental conditions improve

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3
Q

What is the term ‘Algae’

A

a general non-scientific term for primitive eukaryotic organisms that possess the green pigment a to produce cellular energy by photosynthesis but lacks the features of more advanced photosynthetic organisms such as a vascular system of xylem and phloem.

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4
Q

Give features of the Chlamydomonas cell

A
  • filled by a single cup-shaped chloroplast which surrounds the nucleus and the other organelles
  • has two flagella for swimming
  • when viewed under a light microscope seen as a slightly oval cell of approximately 5-10 um i width and 10-15 um in length
  • will grow easily on a medium of inorganic salts using photosynthesis
  • can grow hetetrophically in darkness if acetate is provided as an alternate carbon source
  • It can grow mixotrophically when light and acetate is provided together allowing it to grow faster
  • Aeration by shaking or bubbling with air of 5% CO2 will also increase the growth rate
  • Optimal growth is usually at 20-25 degrees Celsius
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5
Q

When can photodamage to photosystems I and II take place?

A
  • when there is too much light

- when essential nutrients are in limited supply

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6
Q

What are photosystems?

A

chloroplast protein complexes that perform light absorption and energy transfer reactions of photosynthesis
- the PSI and PSII complexes contain chlorophyll a and b which capture light and transfer it to the reaction centres in each photostem

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7
Q

What happens during nutrient limitation?

A
  • 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 call 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 super-complex.
    • 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
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8
Q

Why is the abundance of total chlorophyll reduced in algae in response to nutrient limitation?

A
  • partly due to a reduction in PSII abundance

- due to impaired chlorophyll biosynthesis because nitrogen is required for chlorophyll a and b

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9
Q

A major response to nutrient limitations is modifications in cellular metabolism that allows the cell to enter a dormant phase that extends its ability to survive stress. What do these modifications in metabolism include?

A
  • the accumulation of the storage carbohydrate starch
  • accumulation of non-membrane glycerolipids, predominantly triacylglycerol
  • these accumulate in the cell in lipid droplets alongside starch granules
  • starch granules are found between thylakoid membrane stacks and surround a spherical structure located at the pole opposite the cilia known as the pyrenoid
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10
Q

What is the potential of drawing biofuels from algae?

A
  • Algae could become a source of biofuel due to their ability to produce lipids
  • Extracted triacylglycerol oils can be converted into biodiesel by a transesterification reaction
  • Because algae use light and CO2 the biofuel they produce will be carbon neutral and contribute to the reduction of greenhouse gas emissions
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11
Q

What are the drawbacks of drawing biofuels from algae?

A

It is hard and not cost effective to generate enough algal biomass

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12
Q

How is algae cultivated and what are the benefits and drawbacks of these methods?

A
  • Algae is cultivated in large volumes in either ponds or phot-bioreactors but it is expensive
  • Often ponds are cheaper 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
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13
Q

How should you view cells under a microscope (e.g. with algae)?

A
  • first place a drop of algae on a glass slide
  • place a cover slip gently on top
  • view the microscope under the slides first using the x10 objective then the x40 objective to find the cells and get them into focus
  • a x100 objective lens needs to be used to observe the cells in more detail. This has to be done under oil immersion (put a drop of oil on top of the coverslip)
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14
Q

How do you kill the algae and stop them swimming so you can view them in detail under a microscope?

A

Add 20ul of Lugol’s iodine solution to 1 ml sample of algae in a microfuge tube and mix

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15
Q

What do you observe with high nitrogen cells under a microscope?

A
  • fast swimming, oval cells with two flagella
  • very green
  • cupped shape chloroplast and possibly some other organelles visible
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16
Q

What do you observe with low nitrogen cells under a microscope?

A
  • usually do not swim or swim much slower (no flagella)
  • much less green in colour
  • small granular bodies
  • now creating storage products and saving energy so they do not swim and photosynthesis is inhibited so there is less chlorophyll
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17
Q

How does bright field microscopy work?

A

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 places 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.

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18
Q

What is the eyepiece?

A

also known as the ocular. this is the part used to look through the microscope. Its found at the top of the microscope. Its standard magnification is 10x with an optional eyepiece having magnifications from 5X – 30X.

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19
Q

What is the eyepiece tube?

A

it’s the eyepiece holder. It carries the eyepiece just above the objective lens. In some microscopes such as the binoculars, the eyepiece tube is flexible and can be rotated for maximum visualization, for variance in distance. For monocular microscopes, they are not flexible

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20
Q

What are the objective lenses?

A

These are the major lenses used for specimen visualization. They have a magnification power of 40x-100X. There are about 1- 4 objective lenses placed on one microscope, in that some are rare facing and others face forward. Each lens has its own magnification power.

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21
Q

What is the nose piece?

A

also known as the revolving turret. It holds the objective lenses. It is movable hence it can revolve the objective lenses depending on the magnification power of the lens.

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22
Q

What are the adjustment knobs?

A

These are knobs that are used to focus the microscope. There are two types of adjustment knobs i.e fine adjustment knobs and the coarse adjustment knobs

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23
Q

What is the stage of a microscope?

A

This is the section on which the specimen is placed for viewing. They have stage clips hold the specimen slides in place. The most common stage is a mechanical stage, which allows the control of the slides by moving the slides using the mechanical knobs on the stage instead of moving it manually.

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24
Q

What is the aperture?

A

This is a hole on the microscope stage, through which the transmitted light from the source reaches the stage

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25
Q

What is the microscopic illuminator?

A

This is the microscopes light source, located at the base. It is used instead of a mirror. it captures light from an external source of a low voltage of about 100v.

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26
Q

What are the condensers?

A

These are lenses that are used to collect and focus light from the illuminator into the specimen. They are found under the stage next to the diaphragm of the microscope. They play a major role in ensuring clear sharp images are produced with a high magnification of 40oX and above. The higher the magnification of the condenser, the more the image clarity. More sophisticated microscopes come with an Abbe condenser that has a high magnification of about 1000X.

27
Q

What is the diaphragm?

A

also known as the iris. It’s found under the stage of the microscope and its primary role is to control the amount of light that reaches the specimen. It’s an adjustable apparatus, hence controlling the light intensity and the size of the beam of light that gets to the specimen. For high-quality microscopes, the diaphragm comes attached with an Abbe condenser and combined they are able to control the light focus and light intensity that reaches the specimen.

28
Q

What’s the condenser focus knob?

A

this is a knob that moves the condenser up or down thus controlling the focus of light on the specimen.

29
Q

What is the abbe condenser?

A

this is a condenser specially designed on high-quality microscopes, which makes the condenser to be movable and allows very high magnification of above 400X. The high-quality microscopes normally have a higher numerical aperture than that of the objective lenses.

30
Q

What’s the rack stop?

A

It controls how far the stages should go preventing the objective lens from getting too close to the specimen slide which may damage the specimen. It is responsible for preventing the specimen slide from coming too far up and hit the objective lens.

31
Q

How should you set up a light microscope?

A
  1. Wipe the objectives and eyepiece lenses with lens tissue
  2. Locate the condenser lens and raise it to its upper limit, just below the stage
  3. Push the power switch on and turn the light intensity control (on left of base) until adequate light is obtained (not too high)
  4. Fully open the condenser diaphragm using the lever just below the stage so that maximum light is seen
  5. Swing the canning objective (x4 or x10) into the optical path. Make sure the objective clicks properly into position
  6. Put the slide on the stage using the secure clips, and put the specimen into the ring of light by using the stage control knobs
  7. Adjust the distance between the two eyepieces by pulling them apart or pushing them closer together so that the two fields of vision overlap. Use both eyepieces when focusing on an image
  8. Turn the coarse adjustment knob away from you as far as it will go, so the slide/stage is as near to the objective lens as it will go (the x10 should not hit the slide, be careful with higher magnification lenses: you could break the slide or wet the lens)
  9. Look through the eyepiece; turn the course adjustment knob towards you until the specimen comes into focus. You may need to move the stage so you can see a moving image when adjusting the focus
  10. When you get a glimpse of your specimen, come back to that point and slowly use the fine focus knob to get a sharp image
    (If you do not know what the specimens look like start by focusing something known in the same plane of focus)
    (If the focus is not great, re-adapt the binoculars to you inter-pupillary distance and set up the eye pieces according to your eyesight. You may also want to play with the contrasting features of the microscope (by varying diaphragm aperture and light intensity)
  11. Once you are in the right plane of focus scan the slide using the stage control knobs in search of the best specimen.
  12. Once your specimen of interest is in focus and in the centre of the field, move the next objective lens into the optical path, double checking that the lens will not crush the slide. If you use oil you need to ensure that only oil immersible lens touch the oil
  13. If you have not moved the stage up or down you should get a sharp focus on your specimen just by using the fine adjustment
    (for a normal preparation there is no need to move the stage as the microscope is built so you can switch the lenses and not touch the slide. Only lower the stage slightly if you have a high mount preparation)
  14. When you have finished your observation under oil, lower the stage to remove your slide
    Glass slides are disposed of in sharp bins or disinfectant containers if they contain bacteria. If you have used oil, you need to make sure that you clean the lens well with lens tissue.
32
Q

What should you do if your microscope is not set up correctly and not centred?

A
  • Using the x10 objective check that the specimen is in sharp focus
  • Close the field diaphragm to its minimum. The edge of the diaphragm should be in focus. If it is not then carefully move the condenser lens away from the stage until it is
  • Bring the field diaphragm to the centre of the field of view by manipulating the centring screws just above the field diaphragm ring
  • Open the field diaphragm so that the image of the diaphragm is about the same size as the field of view
  • The field diaphragm controls the diameter of the light beam coming up through the condenser
  • So when the diaphragm is nearly closed the light comes straight up through the condenser lens and contrast is high
33
Q

What oil is normally used for oil-immersion lenses and why?

A
  • Cedar - wood oil
  • it has the same refractive index as the glass of the cover slip, so that the object is effectively immersed in it
  • the presence of the oil increases the effective aperture of the object, thus increasing resolution
34
Q

How would you know whether to use oil when viewing something under a microscope?

A

Check the writing on the side of the lens for ‘oil’

35
Q

What is the procedure to follow for a x100 lens?

A
  1. Once you have a clear focus on the specimen at x40, make sure that it is in the centre of the light field and do not move the stage
  2. Move the x40 lens out of the optical path so that no lens is directly above your sample. Place a dot of immersion oil onto the slide at the point where you can see the circle of light on the specimen. If there is no cover slip, be careful the tip of the oil bottle does not touch your specimen
  3. Move the x100 objective into the optical path making sure the lens is well immersed in the oil and focus using the fine focussing knob. To obtain a crisp picture you may need to increase the amount of light and decrease the aperture to improve brightness and contract
  4. Do not move other lenses onto the oil. If you have lost focus of your specimen, find your sample again using x3, x10 or x20 lenses which are short and won’t touch the oil
  5. When you are finished viewing under oil immersion, immediately wipe the lens with lens tissue to remove any oil before it dries on the lens and blocks the view
36
Q

Why is the final magnification x400 when using the x40 objective lens?

A

Because the eyepiece lens has a magnification of x10 and you need to multiply the objective lens by the eyepiece lens to get the final magnification

37
Q

What is an eyepiece reticle?

A

a glass insert in an eyepiece lens with a scale inscribed on it which allows the investigator to make measurements of specimens. It can also be called a graticule and reticule

38
Q

Why is the eyepiece reticule only an intermediate tool to measure the size of your specimen?

A

Because when you look into your eyepiece reticule, the markings of the scale are always the same whatever the magnification, but the size of the image superimposed under them gets larger with more magnification

39
Q

What is a stage micrometer?

A

A microscope slide with a scale etched on the surface. A typical micrometer scale is 1mm long and etched with 100 divisions of 0.01mm (10um) each)

40
Q

How do you measure a specimen with an eyepiece reticule?

A
  1. Focus on your specimen at your desired magnification (x40 in this case).
  2. Replace one of your eyepieces with the eyepiece reticule, align the scale with your specimen and read off the scale the size of your specimen in eyepiece units (epu, sometimes called ocular units)
  3. Remove your specimen slide and replace with a slide micrometer to calibrate your eyepiece reticule at the same magnification
  4. Find the scale on the slide micrometer. Use low power to first locate the circle surrounding the scale, and then the scale itself. The ring encircling the micrometer scale is visible with the naked eye and should be used to position the stage micrometer in the centre of the microscope optical path (stage aperture).
  5. Rotate the eyepiece scale into position and ensure that both scales (the stage micrometer and the eyepiece reticule) are visible in the field of view, simultaneously in focus and are parallel to each other.
  6. Position the eyepiece reticule directly over the slide micrometer and align the left-hand rule in the eyepiece with one of the longer, numbered (100 micrometer) division lines on the slide micrometer. It will depend upon the objective magnification factor how much of the slide scale you will be able to see.
  7. Determine two points at which the eyepiece reticule and micrometer scales exactly match or align the zeros on the two scales if possible. For the most accurate measurements, utilize the largest possible range of divisions on both scales. [Only occasionally do reticlue and stage micrometer graduations coincide over the entire length visible in the eyepieces!]. Finally, determine the apparent length of the eyepiece scale in reference to the divisions on the stage micrometer. The micrometer value for the objective in use can be then calculated by dividing the known length of the selected region of stage micrometer by the corresponding number of divisions of the eyepiece scale.
41
Q

How do you calculate the micrometer value for the objective lens in use?

A

divide the known length of the selected region of the stage micrometer by the corresponding number of divisions of they eyepiece scale

42
Q

How does the size of the cells change in low nitrogen conditions compared to high nitrogen conditions and what does this tell you?

A
  • the low nitrogen cells increased in volume
  • this happened because of the synthesis of storage products that accumulate in oil bodies and starch granules in the chloroplast
  • accumulation of these granules causes the cell to swell in size
43
Q

What is the equation for cell biovolume?

A

Cell biovolume = (pi/6) x (width^2) x length

44
Q

What is the equation to work out lipid content?

A 
y = 0.0204x
y = lipid content 
x = biovolume
45
Q

How do you calculate lipid content per cell?

A

Use the line data equation for lipid content and the cell biovolume values

46
Q

How does the lipid content with low nitrogen cells change and why?

A
  • low nitrogen cells had increased lipid content
  • due to accumulation of oil bodies
  • explains the increase in cell size
47
Q

What happens to the number of cells in low nitrogen treatment?

A

Decreased number of cells as they find it harder to divide and grow because they don’t have enough nitrogen

48
Q

What is some background information on pigments?

A
  • Chemical compounds that can only reflect certain wavelengths of visible light making them seem colourful
  • Pigments absorb certain wavelengths for photosynthesis
  • The pigments do not absorb all wavelengths of light equally
  • Photosynthetic pigments all have characteristic absorption spectra. The wavelengths at which the maximum absorbance occurs is known as absorbance maxima. The absorbance is greatest at (lamba max) and this wavelength is selected to perform quantitative measurements
49
Q

How is the absorption spectrum of photosynthetic pigments found?

A

By using spectrophotometry

50
Q

What is the absorbance maxima for chlorophyll a and b?

A

typically 430 and 665 nm

51
Q

What do maxima values vary depending on?

A

The solvent used to extract the pigment

52
Q

What is the process of obtaining an absorbance spectra for chlorophyll from algal cells?

A
  1. take 5ml algal samples from each treatment and transfer each sample into a centrifuge falcon tube
  2. the pair of tubes are put into a bench top centrifuge with the tubes opposite each-other. The samples are then centrifuged at maximum speed. This extracts the cells from the culture solution
  3. the tubes are removed from the centrifuge and then the supernatant (liquid on top) is removed and discarded without disrupting the pellet.
  4. We resuspend the pellet in 1ml of 96% ethanol by adding 1 ml to each pellet and mixing using a pipette to suck it in and out until there are no large lumps
  5. then an additional 4 ml of ethanol is added to each sample to make it up to 5 ml volume in total.
  6. the sample is then centrifuged again for 5 minutes
  7. carefully pipette each supernatant into a cuvette without disturbing the pellet at the bottom
  8. Use a cuvette of ethanol as a blank
  9. the absorbance spectra for one high nitrogen and one low nitrogen sample can be measured using a spectrophotometer set to read between 400-700nm using scanning mode
53
Q

Why when centrifuging do the tubes need to be opposite eachother?

A

The centrifuge must be balanced

54
Q

Why is ethanol mixed with the cell pellet?

A

Because chlorophyll is soluble in ethanol so this will separate it out

55
Q

Why do we use the spectrophotometer in scanning mode here?

A

Because we can record the extract absorbance at a range of wavelengths

56
Q

What are some things to note about the proper use of cuvettes?

A
  • Do not touch the back of a cuvette or the sides through which light is directed
  • Do not allow samples to sit in a cuvette for a long period of time.
  • Make sure the cuvette is properly aligned in the spectrophotometer.
  • Be certain to only use clean cuvettes.
  • Gently tap the cuvette to remove any bubbles.
57
Q

How do you use a spectrophotometer in single wavelength mode?

A
  1. put in a cuvette containing ‘blank’
  2. on the home screen touch ‘single wavelength’
  3. on the next screen touch ‘Simple (ABS %T)’
  4. enter desired wavelength. No need to name sample
  5. touch ‘blank’. After a few seconds the reading should reveal 0 for absorbance
  6. keep the cuvette and solution to use as a blank another time
  7. transfer sample to new cuvette
  8. touch ‘Read’ and record the absorbance on the screen
58
Q

Why would we want to measure the absorbance of chlorophyll a and b using the absorption maxima values from the high nitrogen and low nitrogen samples?

A

Because we can then calculate the concentration of chlorophyll a and b and also the total chlorophyll concentration in ug ml^-1

59
Q

How do you measure the absorbance of chlorophyll a and b using the absorption maxima values from the high nitrogen and low nitrogen samples?

A
  1. Fill a 1.5ml cuvette with 1 ml 96% ethanol (as a blank). Fill a 2nd cuvette with the extract supernatant (without disturbing the cell pellet) from your samples.
  2. We now need to measure the absorbance of the ethanol extract at two different wavelengths: 649 nm and 665 nm. This time you need to set the spectrophotometer to single wavelength mode.
  3. Placing the blank cuvette in the measuring position, set the spectrophotometer to the first wavelength and zero it.
  4. Place each sample in the measuring position and record the absorbance. Repeat this procedure for the second wavelength. It is important to re-zero the spectrophotometer with the blank for each wavelength
  5. If the absorbance is greater than 1.5 at any of the wavelengths, dilute your sample with a known amount of clean ethanol and re-measure.
  6. record the absorbance values and any dilution factor
  7. repeat with each sample, using a rinsed cuvette
60
Q

How do we work out the total concentration of total chlorophyll in the extracts?

A

concentration of chlorophyll a + concentration of chlorophyll b

61
Q

How do we work out the concentration of chlorophyll a?

A

Chlorophyll a (ug ml ^-1) = (13.95 x A665nm) – (6.88 x A649 nm) x dilution factor

62
Q

How do we work out the concentration of chlorophyll b?

A

Chlorophyll b(ug ml^-1 ) = (24.96 x A649) – (7.32 x A665nm) x dilution factor

63
Q

What’s the difference in chlorophyll concentration between the two nitrogen treatments?

A
  • the low nitrogen cells had significantly lower chlorophyll concentrations
  • these are for the reasons stated earlier

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