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1 Year 2011-2012 Peter Bossier Aäron Plovie
Algae Culture Year Peter Bossier Aäron Plovie Algae Culture

2 -Algal Cultering Techniques from Robert Andersen
Theoretical courses: Mainly based on books -Algal Cultering Techniques from Robert Andersen -Live feeds in marine aquaculture from Josianne StØttrup and Lesley MvEvoy and various papers and reviews (see slides) Practical course: -microalgal growth curve, cell counting -chlorophyl analysis -dry weight determination Exam: Algae Culture

3 Phytoplankton Several hundred of billion tonnes of dry weight per year in the oceans, leading up to some 100 million tonnes of renewable resources per year; latest data say 50 billion ton in the oceans. Hence also important in aquaculture More than species in 15 major classes Taxonomy is ongoing Surface of a ball? 4 x pie x r x r Let them calculate the productivity per ha or square meter Max 500g/m2 or 5 ton /ha of 10 ton OM/ha Prod: 50x 10¨9 ton or 5 x 10^13 kg Earth surfac: 3,6 10^14 m2 So on average 100 g/m2

4 Ocean primary productivity

5 Chapter 1: What are algae
Chapter 1: What are algae? Phylogenetic relationships, endosymbiosis theory and general features. Algae Culture

6 Tree of life: Algae are spread all over the tree and are therefore a polyphyletic group. Algae Culture

7 All members of the group (D, E, G, H) have the same ancient ancestor (B). No other organism not in-cluded in this group (for example J) has that ancient ancestor (B). All members of the group (E, G) have different ancient ancestors (C, F). Other organisms not in-cluded in this group (D, H) share those ancient ancestors (C, F). Compare with real tree: One branch with all its leaves (monophyletic). Different leaves from different branches (polyphyletic). Algae Culture

8 2005 Sleeping sickness Uni-and multicellular Mostly multicellular
Malaria Uni-and multicellular Root of tree still unclear. Multicellularity also spread over tree. Malaria and sleeping sickness are placed on tree to show that algae are more related to totally different organisms than to other algae. Plastid loss 2005 Algae Culture

9 How come algal taxa are spread all over tree of life?
Endosymbiosis events and plastid losses cause huge algal diversity. Lepidodinium has obtaines a chloroplast from green algal lineage, Dinophysis had obtained a chloroplast from red algal lineage, while both are dinoflaggelates. Algae Culture

10 Primary endosymbiosis
Secundary endosymbiosis Lepidodinium has obtaines a chloroplast from green algal lineage, Dinophysis had obtained a chloroplast from red algal lineage, while both are dinoflaggelates. Serial secundary endosymbiosis Tertiary endosymbiosis Each endosymbiosis event involves gene transfer between host genome(s) and chloroplast genome(s)! Algae Culture

11 A closer look on endosymbiosis:
Algae Culture

12 According to endosymbiosis theory: all plastids in algae are
derived from one primary endosymbiosis. However,… (f) Paulinella is a genus of about nine species of freshwater amoeboids (Rhizaria, Cercozoa, Euglyphida). Its most famous member is the photosynthetic P. chromatophora which has recently (evolutionarily speaking) taken up a cyanobacterium as an endosymbiont. This is striking because the chloroplasts of all other known photosynthetic eukaryotes derive ultimately from a single cyanobacterium endosymbiont which was taken in probably over a billion years ago in plants (and subsequently adopted into other eukaryote groups, by further endosymbiosis events). The P. chromatophora symbiont was related to the Prochlorococcus and Synechococcus cyanobacteria.  Algae Culture

13 Algae species relevant to aquaculture
Algae Culture

14 Algae species relevant to aquaculture:
Microalgae Cyanobacteria Arthrospira platensis (spirulina) Algae Culture

15 Algae species relevant to aquaculture:
Microalgae Green algae (Chlorophyceae) Dunaliella salina Algae Culture

16 Algae species relevant to aquaculture:
Microalgae Green algae (Chlorophyceae) Chlorella virginica Algae Culture

17 Algae species relevant to aquaculture:
Microalgae Green algae (Prasinophyceae) Tetraselmis striata Algae Culture

18 Algae species relevant to aquaculture:
Microalgae Dinophyceae Crypthecodinium cohnii Algae Culture

19 Algae species relevant to aquaculture:
Microalgae Haptophyceae Isochrysis galbana Algae Culture

20 Algae species relevant to aquaculture:
Microalgae Haptophyceae Pavlova lutheri Algae Culture

21 Algae species relevant to aquaculture:
Microalgae Eustigmatophyceae Nannochloropsis gaditana Algae Culture

22 Algae species relevant to aquaculture:
Microalgae Bacillariophyceae (Diatoms) Skeletonema costatum Algae Culture

23 Algae species relevant to aquaculture:
Microalgae Bacillariophyceae (Diatoms) Chaetoceros calcitrans Algae Culture

24 Algae species relevant to aquaculture:
Microalgae Bacillariophyceae (Diatoms) Phaeodactylum tricornutum The pennate diatom Phaeodactylum tricornutum. (a) Light micrographs showing the three morphotypes of P. tricornutum: left, fusiform; top right, triradiate; bottom right, oval. (b) Light micrographs of a small cluster of cells of P. tricornutum. Each cell is approximately 15 μm in length. Images courtesy of Alessandra De Martino. Algae Culture

25 Algal characteristics
Name feature Length x with (µm) Cell weight (pg) Arthrospira Cylindrical cells forming helicoidal trichomes x 10 Chaetoceros Free living brown , four long diagonal setae 5-16 11 Chlorella Free living, green, spherical, cell wall, 2-16 autospores 1,5-10 Crypthecodinium Free living, mobile, spherical or elliptic, incomplete cingulum, forming cyst 10-30 Isochrysis galbana Free living, mobile, yellow to golden brown, short subapical haptonema with two flagella, no cell wall 5-6 x 2-4 23-47 Algae Culture

26 Algal characteristics
Name feature Length x with (µm) Cell weight (pg) Nannochloropsis oculata Free living, or aggregated, ovoid, cell wall, no chlorofyl b 2-4 10 Pavlova lutheri Free living, mobile gold-brown, ovoid, short haptonema with 2 unequal flagella, no cell wall 7-9x 5-7 30-40 Phaeodactylum tricornutum Chained, oval, Y or spindle shaped 3-4 x 8 5 -10 organic weight Skeletonema costatum Chained, spherical to cylindrical cells attached together by a ring of external gutter-shaped processes 10 x5 52 Tetraselmis Free living, mobile , spindle-shaped cells, four polar flagella, cell wall , cyst form 9-11 x 7-8x 4-6 Algae Culture

27 Algae species relevant to aquaculture:
Macroalgae Rhodophyceae (Red algae) Porphyra spp. (Nori) Algae Culture

28 Algae species relevant to aquaculture:
Macroalgae Rhodophyceae (Red algae) Gracilaria spp. Algae Culture

29 Algae species relevant to aquaculture:
Macroalgae Phaeophyceae (Brown algae) Laminaria spp. Algae Culture

30 Chapter 2: Microalgal growth. Photosynthesis and its substrates.
Algae Culture

31 During the exponential phase: dC/dt = µC
Microbial growth: During the exponential phase: dC/dt = µC µ = specific growth rate dependent on temperature and light irradiance! Ct= C0 eµt µ = (lnCt – lnC0) / (t – t0) For heterotrophic bacteria mainly expressed in h-1, for algal autotrophs expressed in d-1! Algae Culture

32 Algae Culture

33 During the exponential phase:
Doubling time C(t+tD) = 2 C(t) C0 eµ(t+tD) = 2 C0 eµt C0 eµt eµtD = 2 C0 eµt EµtD = 2 µtD = ln(2) tD = ln(2) / µ Algae Culture

34 Algal growth rates Name Specific growth rate (day -1)
Culture conditions Arthrospira 2.47 1,68 maxima, 550, 12/12, 33°C, B platensis, 273, 24/0, 35, C Chaetoceros 0,87 1,37 Calcitrans, 60, 24/0, 18°C, B Gracilis, 165; 24/0, 18°C, B Chlorella 2,03 0,3 0,16 vulgaris, heterotrophy, B vulgaris, axenic, B vulgaris, open conditions, B Crypthecodinium 1,98 cohnii, heterotrophy, B Isochrysis galbana 1,4 Galbana, 36, 24/0, 25°C, B Algae Culture

35 Algal characteristics
Name Specific growth rate (day -1) Culture conditions Nannochloropsis oculata 0,28 Sp, 75, 12/12, 20°C, B Pavlova lutheri 0,92 Lutheri, 20°C, B Phaeodactylum tricornutum 1,5 Tricornutum, 23°C, B Skeletonema costatum 0,84 -2,88 Costatum , 135, 24/0, 20°C, B Tetraselmis 1,68 Sp, 25°C, B B: batch, C: continuous, energy in microEinstein per m-2s -1 Algae Culture

36 Example: Compare the doubling times of Pavlova lutheri grown in batch culture at 20°C and 60µEm-2s-1 and grown in continuous culture at 20°C in their exponential phases. tD = ln(2) / µ tD = / 0.25 = days tD = / 0.92 = days Calculate what cell concentration an axenic batch culture of Chlorella vulgaris will have after 25 days. At the start of the exponential phase, the cell concentration is 10,000 cells per ml. Ct= C0 eµt Ct = e0.3*25 Ct = 10,000 * 1808 Ct = 18, Algae Culture

37 Example: Compare the doubling times of Pavlova lutheri grown in batch culture at 20°C and 60µEm-2s-1 and grown in continuous culture at 20°C in their exponential phases. tD = ln(2) / µ tD = / 0.25 = days tD = / 0.92 = days Calculate what cell concentration an axenic batch culture of Chlorella vulgaris will have after 25 days. At the start of the exponential phase, the cell concentration is 10,000 cells per ml. Ct= C0 eµt Ct = 10,000 e0.3*25 Ct = 10,000 * 1808 Ct = 18,080,000 Algae Culture

38                                                                          Relationships

39 Production and productivity
P expresses production: increase in biomass per unit of time (g/day). We speak of productivity when P is related to the • surface of illumination: g/(m2 day) or • volume of the culture: g/(l day) Algae Culture

40 Productivity P= (C-C0)V/(t-t0) with V the volume of the reactor
In batch culture with no medium renewal: P= (C-C0)V/(t-t0) with V the volume of the reactor In continuous culture where the medium is renewed at an equivalent flow Q (for dilution or harvesting) The variation of concentration in the reactor is the difference between the growth of the biomass and the population reduction following dilution/harvesting or dC/dt= (µ - D)C where D is the dilution rate Q/V Algae Culture

41 dC/dt= (µ - D)C where D is the dilution rate Q/V
Productivity dC/dt= (µ - D)C where D is the dilution rate Q/V D>µ: growth can not compensate for dilution, concentration goes down D=µ: the culture stabilizes around a mean value of C D<µ: the culture has not reached its steady state and concentration is still increasing In a stabilized continuous culture (D=µ) the productivity of biomass (per unit of time) P is the product of the harvesting flow rate DV by the concentration P=DVC In theory, a continuous culture can last indefinitely, but there are practical problems such as deposit on light walls or contamination. Algae Culture

42 Photosynthesis: Machinery
Algae Culture

43 There are three basic classes of pigments.
Chlorophylls are greenish pigments which contain a porphyrin ring. This is a stable ring-shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll "captures" the energy of sunlight. There are several kinds of chlorophyll, the most important being chlorophyll "a". All plants, algae, and cyanobacteria which photosynthesize contain chlorophyll "a". A second kind of chlorophyll is chlorophyll "b", which occurs only in green algae and in the plants. A third form of chlorophyll which is common is called chlorophyll "c", and is found only in the photosynthetic members of the Chromista as well as the dinoflagellates. Algae Culture

44 There are three basic classes of pigments.
Carotenoids are usually red, orange, or yellow pigments, and include the familiar compound carotene, which gives carrots their color. These compounds are composed of two small six-carbon rings connected by a chain of carbon atoms. As a result, they do not dissolve in water, and must be attached to membranes within the cell. Carotenoids cannot transfer sunlight energy directly to the photosynthetic pathway, but must pass their absorbed energy to chlorophyll. For this reason, they are called accessory pigments. Algae Culture

45 There are three basic classes of pigments.
Phycobilins are water-soluble pigments, and are therefore found in the cytoplasm, or in the stroma of the chloroplast. They occur only in Cyanobacteria and Rhodophyta. The bluish pigment phycocyanin is predominant in Cyanobacteria, which gives them their name. The reddish pigment phycoerythrin is predominant in Rhodophyta, which gives the red algae their common name. Algae Culture

46 Light visible by human eyes (wavelength of nm) is also mainly the spectrum that is relevant for photo-synthesis Algae Culture

47 Where are those pigments located in photosynthetisizing cell?
Chloroplast Where are those pigments located in photosynthetisizing cell? Algae Culture

48 Now we know where and how the light energy is captured
Now we know where and how the light energy is captured. What happens with the captured energy? Production of NADPH and ATP for production of fixed carbon (sugars) via Calvin cycle. Algae Culture

49 Captured energy brings electrons in electron transport chain (ETC) from water (2H2O -> 4e− + 4H+ + O2). Electrons are passed to NADP, forming NADPH. ETC creates also a proton (H+) gradient over thylakoid membrane for ATP production Algae Culture

50 Algae Culture

51 NADPH and ATP are used as energy source to produce
3-phosphoglycerate (building block of sugars) from C02. 3C5H10O5 + 3 CO2 gives 3 C6H12O6 + 3 O2 Write the equation of one Calvin cycle Rubisco can only use CO2 and not bicarbonate Algae Culture

52 Ribulose-1,5-bisphosphate carboxylase oxygenase, most commonly known by the shorter name RuBisCO, is an enzyme involved in the Calvin cycle that catalyzes the first major step of carbon fixation, a process by which the atoms of atmospheric carbon dioxide are made available to organisms in the form of energy-rich molecules such as glucose. RuBisCO is very important in terms of biological impact because it catalyzes the primary chemical reaction by which inorganic carbon permanently enters the biosphere. RuBisCO is also considered to be the most abundant protein on Earth. Algae Culture

53 -a simpler cellular structure
The photosynthetic mechanisms of algae is similar to that of land based plants, but due to -a simpler cellular structure -a submerged life style where they have efficient access to water, CO2 and other dissolved nutrients, they are generally more efficient in converting solar energy into biomass. Algae Culture

54 Substrates for photosynthesis: Light energy
Algae Culture

55 What is light? Wave–particle duality postulates that all matter exhibits both wave and particle properties. A central concept of quantum mechanics, this duality addresses the inability of classical concepts like "particle" and "wave" to fully describe the behavior of quantum-scale objects. The idea of duality originated in a debate over the nature of light and matter that dates back to the 17th century, when competing theories of light were proposed by Christiaan Huygens and Isaac Newton: light was thought either to consist of energy waves (Huygens) or of corpuscles particles (Newton). Through the work of Max Planck, Albert Einstein and many others, current scientific theory holds that all particles also have a wave nature (and vice versa). This phenomenon has been verified not only for elementary particles, but also for compound particles like atoms and even molecules. Algae Culture

56 What is light? From physics class, perhaps you learned that light is an electromagnetic wave. One of the properties of light is that it has a particular speed. This speed depends on what material the light is traveling through, but in a vacuum the speed of light is x 108 m/s. Since light is nothing more than exchange between an electric and magnetic field, it is a form of pure energy (no mass). Because light is an oscillating field, it has a frequency of oscillation. Because it is traveling through space at a constant speed, the light will cover a certain distance within one oscillatory cycle. This is called the wavelength of light. Finally, it turns out that the amount of energy in wave of light is proportional to its frequency – the higher the frequency of light, the higher the energy. High frequency also corresponds to a short wavelength, so wavelength and frequency are inversely related. Algae Culture

57 What is light? Single packets of light which are called photons.
Photons have only one property, frequency. This determines their energy (color). Thus, the fact that light comes in packets is intimately related to the fact that it has a distinct color. It is the energy per packet (the frequency of the oscillation in the packet) that determines the color. Now light can have almost any energy. Low energy light we know of as radio waves – electromagnetic radiation that travels through space between your radio and the radio station. The frequencies for this type of radiation are on the order of MHz (106 Hz or millions of times per second – Hz stands for hertz which is the number of times something repeats per second) to hundreds of megahertz. Up in the gigahertz range (billions of oscillations per second) is the microwave region. At somewhat higher frequencies we have infrared light (roughly a frequency of 1014 Hz), Visible red light, visible orange, yellow, green blue violet (on the order of 1015 Hz) and then the ultraviolet spectrum. At higher frequencies yet, there are X-rays (1017 Hz), gamma-rays and other very high energy photons. Algae Culture

58 Algae Culture

59 E = hf = hc/λ E = energy of one photon (Joule, J)
The energy carried by light is contained in the photons that travel as a wave: E = hf = hc/λ E = energy of one photon (Joule, J) 1 joule is equal to 6.24*1018 eV f = frequency (Hertz, Hz = s-1) c = speed of light (ms-1) λ (lambda) = wave length (m) h = Planck’s constant = 6.626*10-34 Js Algae Culture

60 How many photons does 1 Joule of light energy at 500 nm contain?
Example: What is the energy of a single photon of light at 500 nm? What is the frequency of that photon? E = hf = hc/λ E = 6.626*10-34 x 3.0*108/(500*10-9) E = *10-17 J f = E/h f = *10-17 / 6.626*10-34 f = 6*1014 s-1 How many photons does 1 Joule of light energy at 500 nm contain? N x E = 1 N = 1/E N = λ/hc N = 500*10-9 / (6.626*10-34 x 3.0*108) N = 25.15*1017 photons Algae Culture

61 How many photons does 1 Joule of light energy at 500 nm contain?
Example: What is the energy of a single photon of light at 500 nm? What is the frequency of that photon? E = hf = hc/λ E = 6.626*10-34 x 3.0*108/(500*10-9) E = *10-17 J f = E/h f = *10-17 / 6.626*10-34 f = 6*1014 s-1 How many photons does 1 Joule of light energy at 500 nm contain? N x E = 1 N = 1/E N = λ/hc N = 500*10-9 / (6.626*10-34 x 3.0*108) N = 25.15*1017 photons Algae Culture

62 So 25.15*1017 photons correspond to 0.000004177 moles.
N = 25.15*1017 photons To produce 1 Joule of energy by light at 500 nm, it requires a very large number of photons. To avoid having to deal with such large numbers, we can measure the number of photons in moles, where 1 mole of photons = 6.02*1023 photons (Avogadro’s number). So 25.15*1017 photons correspond to moles. And now micromoles (10-6 mole) is the easiest to use unit. 4.177 micromoles of photons are necessary for 1 Joule of light energy at 500 nm. Algae Culture

63 4.177 micromoles of photons are necessary for 1 Joule of light energy at 500 nm.
We know that Watt is the unit of power and 1 Watt is equal to 1 J/s light energy produced. We can conclude that micromoles photons per second are necessary to produce one Watt of light at 500 nm. Algae Culture

64 Relationship between wavelength of light and how many micromoles of photons are necessary to produce one Watt = 1 Joule energy per second Wavelength (nm) Micromoles/Watt Algae Culture

65 Light sources: What differs in the various light sources is the mechanism by which electrons are excited to emit photons and the composition of materials used to provide the electrons. -In an incandescent lamp, electrons in a tungsten (wolfram) filamental resistance are excited by heat. Those excited electrons emit photons. Approximately 90% of the power consumed by an incandescent light bulb is emitted as heat (infrared), rather than as visible light. -In a fluorescent lamp, free electrons are created between a cathode and anode. Those free electrons are used to energize mercury atoms, which emit photons in the UV range. These UV-photons excite the electrons of the lamp’s phosphor coating, which results in emitting photons of different wavelengths, depending on the mix of used phosphor atoms. Algae Culture

66 Light sources: -Metal halide lamps use a different approach in which atoms of metal halide gas are used along with mercury, and are energized by a plasmal arc between electrodes. -When a light-emitting diode (LED) is forward biased (switched on), electrons are able to recombine with electron holes within the device, releasing energy in the form of photons. This effect is called electroluminescence and the color of the light (corresponding to the energy of the photon) is determined by the energy gap of the semiconductor. early LEDs emitted low-intensity red light, but modern versions are available across the visible, UV and infrared wavelengths, with very high brightness. Algae Culture

67 When we characterize a light source, we are interested in determining how many photons it generates per unit of time. This is called the photon flux. We are also interested in how many photons land on a given area, usually 1 square meter (m2), and this is called the photon density. If we combine those two, we can measure the photon flux density (µmol photons m-2 s-1 also µEinstein m-2 s-1 or µE m-2 s-1 Finally, if we are only interested in the photons that are available for photosynthesis (photosynthetic photons), which are photons in the range of nm, we can measure the photosynthetic photon flux density (PPFD). This is the number of photons in the range of nm falling on 1 square meter per second. Algae Culture

68 Different light sources have different distributions of photons in the 400-700 nm range.
The light source can be characterized by determining this distribution of the photons, and this is done by using a spectroradiometer. This instrument has a sensor and associated hardware and software to determine the distribution of energy (measured as power density in Watts/m2) at different wavelengths of the electromagnetic spectrum. This is usually displayed as a graph with the wavelength on the X-axis and the power density on the Y-axis. This graph is call the spectral power distribution plot (SPD plot). Algae Culture

69 Spectral power distribution plots for various light sources:
Algae Culture

70 Note that for each wavelength the spectroradiometer measures the power density in watts/m2. This is termed the Spectral Irradiance. You may recall from that there is a direct relationship between power/energy at each wavelength and the number of photons. For example, as seen in the graph below, at 420nm the lamp produces 0.4 watts/m2 of power or 0.4 joules/m2/second of energy. Using the relationship between energy and wavelength, it can be determined how many photons/m2/sec at 420nm will be required to generate 0.4 joules of energy (1.46 micromoles). Thus, we can easily convert from watts/m2 to micromoles/m2/sec. If this is done for all wavelengths, we would get a plot that shows the distribution of the number of photons at each wavelength per meter squared per second. . Or 3,5 µM per square meter x 0.4= 1.46 (3,5: see graph 2 slides ahead) Algae Culture

71 Adding all the photons over the range of nm will provide the measure of the photosynthetically available radiation (PAR) measured in terms of PPFD. Technically, the photosynthetically available radiation would be the area under the curve. These computations are often performed by software that is available with the spectroradiometers. Since the power distribution and the photon distribution are mathematically interchangeable, either of them can be used as the basis for comparison of light output from different light sources Algae Culture

72 Example: Algae Culture

73 Example: Algae Culture

74 What is important to note is the following:
Because each photon's energy is different at different wavelengths, a different number of photons will be required to produce the same amount of energy at different wavelengths. To produce the same amount of total energy at 400nm would require 57% less photons than at 700nm, because the photons at 400nm have higher energy. 2) Because the PPFD is a summation of all photons in the nm range, two very different spectral distributions can have the same PPFD. What this means is that there is not a one-to-one relationship between PPFD and spectral distribution, so knowing a light source's PPFD does not tell us anything about how its photons are distributed. Different light sources with similar PPFD values can have very different spectral distributions. As seen in the figure below, the two lamps have very similar PPFD values, but their spectral distributions are very different. The independence of PPFD and spectral distribution is one reason that we must consider spectral distribution data as well as PPFD when comparing light sources. 3 Algae Culture

75 What is important to note is the following:
3) Also note that PPFD measures photons falling on a given area; the number of photons falling on this area changes as its distance from the light source increases. Hence, when comparing lamps‘ PPFDs it is very important to know the distance at which the measurements were taken, and only PPFD values at the same distance can be compared. Algae Culture

76 Algae Culture

77 As a reference, a bright sunny day is around 2000 µE/(m2 s)
The saturation light intensity Is is generally 300 to 600 µEinstein/(m2 sec). As a reference, a bright sunny day is around 2000 µE/(m2 s) Specific growth rate is 0.5 to 1.5 day-1. Algae Culture

78 Light as energy Photometric measurements are geared towards how the human eye perceives light. The sensitivity of human eyes is different for different wavelengths The human eye is more sensitive to light at 555 nm (green) and less sensitive to blues and reds. This characteristic of human vision established the standard observer response curve known as the luminous efficiency function to represent how the human eye responds to light at different wavelengths. Per this standard, detectors in the eye respond differently to different regions of the spectrum, and the response is scaled with respect to the peak values.

79 Light as energy The change in the eye's spectral response can be explained by the presence of two types of receptors, rods and cones, in the retina. Cones are active at high light levels and are most densely situated in the central part of the field of view. The cones' spectral response corresponds to the photopic sensitivity curve. The rods are responsible for human vision at low light levels and are prevalent in the peripheral field of view, away from our direct line of sight. As light levels are reduced, cones become less active and rods become active with established spectral sensitivity gradually switching toward the scotopic response curve. The peak spectral sensitivity for photopic vision is 555 nm, and 507 nm for scotopic vision. From this it is quite clear that the human eye finds light at 555 nm to be the brightest, with the blues and reds tending to be less bright. The luminous efficiency functions are shown in the next figure

80 Light as energy

81 Light as energy All photometric light measurements evaluate light in terms of this standard visual response described by the luminous efficiency function and, hence, all are weighted measures. Not all of the wavelengths are treated equally. The wavelength at 555 nm is assigned a weight of 1, and the others are scaled according to this function. According to this function, light at a wavelength of 450 nm is given a weight of This explains why a light source with large amounts of radiation in the "blue" region will have a low reading when using photometric units. Luminous Flux is the amount of radiation coming from a source per unit time, evaluated in terms of a standard visual response. Unit: lumen (lm). You will see most data from lighting companies refer to light output in terms of lumens. Think of this as the amount of light produced by the lamp as perceived by the human eye.

82 Light as energy Illuminance is the luminous flux per unit area. It is measured in lux (lumen/m2) or footcandles (lumen/ft2). The light emanating from a lamp is used to illuminate objects and the amount of light (measured in lumens) falling onto a specific area of the object, usually one square meter, is termed lux. When we measure this same area in square feet, the unit is footcandles. These units are often used in photography, where we are interested in how much light is falling onto the subject.

83 Light as energy Conversion from Radiometric Units to Photometric Units
The following method is used to convert between photometric units and radiometric units. As defined, 1 watt = 683 lumens at 555 nm (peak photopic response), and it is scaled for other wavelengths based on the Luminous Efficiency Function V (λ) shown in the previous figure . To determine a lamp's lux values, the spectral irradiance (in W/m2) at each wavelength (taken from the spectral power distribution) in the spectral range ( nm) is multiplied by the luminous efficiency function at the equivalent wavelengths. Then, all of these multiplied values are summed and multiplied by 683 to find the total lux output. As you can see, the conversion requires knowledge of the spectral power distribution and cannot be done without it.

84 Light as energy For the purpose of photosynthesis, light is termed Photosynthetically Available Radiation (PAR). This radiation's range is identical to what humans can see in the nm range, but each photon is treated uniformly in this measurement (unlike the photometric measurement, which weights the photons according to how the human eye sees them). The reason for expressing PAR as a number of photons instead of energy units is that the photosynthetic reaction takes place when a plant absorbs the photon, regardless of the photon's wavelength (provided it lies in the range between 400 and 700 nm). That is, if a plant absorbs a given number of blue photons, the amount of photosynthesis that takes place is exactly the same as when the same number of red photons is absorbed. Note, however, that the plant or coral may have an absorption response that preferentially absorbs more photons of certain wavelengths PAR is measured as PPFD, which are Einstein/m2/s or µmoles/m2/s. One Einstein = 1 mole of photons = 6.022×1023 photons, hence, 1 µEinstein = 6.022×1017 photons.

85 Light as energy: practically
PPF (μmol m-2 s-1) to Lux Lux to PPF (μmol m-2 s-1) Sunlight 54 0.0185 Cool White Fluourescent Lamps 74 0.0135 High Pressure Sodium Lamps 82 0.0122 High Pressure Metal Halide Lamps 71 0.0141 Multiply the PPF by the conversion factor to get Lux. For example, full sunlight is 2000 μmol m-2 s-1 or 108,000 Lux (2000 ∗ 54). Multiply the Lux by the conversion factor to get PPF. For example, full sunlight is 108,000 Lux or 2000 μmol m-2 s-1 (108,000 ∗ ).

86 Light as energy: practically
PPF (μmol m-2 s-1) Lux (Sunlight) Lux (HPS) Lux (Metal Halide) Lux (Fluorescent) 10 540 820 710 740 100 5,400 8,200 7,100 7,400 200 10,800 16,400 14,200 14,800 300 16,200 24,600 21,300 22,200 600 32,400 49,200 42,600 44,400 1000 54,000 82,000 71,000 74,000 2,000 108,000 164,000 142,000 148,000

87 Light as energy: summary of units

88 Algae Culture

89 Photoinhibition is light-induced reduction in the photosynthetic capacity of a plant, alga or a cyanobacterium. Photosystem II (PSII) is more sensitive to light than the rest of the photosynthetic machinery, and most researchers define the term as light-induced damage to PSII. The chloroplast of plants is a remarkable system that converts solar energy into chemical energy with a high efficiency. However, reactive forms of oxygen can be produced during illumination of chloroplasts, especially when the absorption of light energy exceeds the capacity of photosynthesis. Algae Culture

90 Indeed, at high photon flux densities (PFDs), the accumulation of excitation energy in the light-harvesting chlorophyll antennae (LHC) of the photosystems favors the production of triplet excited chlorophyll molecules that can interact with O2 to generate reactive singlet oxygen (1O2). Overreduction of the photosynthetic electron carrier chain would also favor the direct reduction of O2 by photosystem I (PSI) and the subsequent production of damaging reactive oxygen species, such as superoxide (O2-), hydrogen peroxide (H2O2), and the hydroxyl radical (.OH). Algae Culture

91 Protection against photoinhibition: Xanthophyll cycle
The xanthophyll cycle involves the enzymatic removal of epoxy groups from xanthophylls (e.g. violaxanthin, diadinoxanthin) to create so-called de-epoxidised xanthophylls (e.g. zeaxanthin, diatoxanthin, dinoxanthin) These enzymatic cycles were found to play a key role in stimulating energy dissipation within light harvesting antenna proteins by non-photochemical quenching- a mechanism to reduce the amount of energy that reaches the photosynthetic reaction centers. Non-photochemical quenching is one of the main ways of protecting against photoinhibition.  Algae Culture

92 Protection against photoinhibition: Xanthophyll cycle
During light stress violaxanthin is converted to zeaxanthin via the intermediate antheraxanthin, which plays a direct photoprotective role acting as a lipid-protective anti-oxidant and by stimulating non-photochemical quenching within light harvesting proteins. This conversion of violaxanthin to zeaxanthin is done by the enzyme violaxanthin de-epoxidase (VDE), while the reverse reaction is performed by zeaxanthin epoxidase (ZE). In diatoms and dinoflagellates the xanthophyll cycle consists of the pigment diadinoxanthin, which is transformed into diatoxanthin (diatoms) or dinoxanthin(dinoflagellates), at high light.  Algae Culture

93 Dha:Dehydroxyascorbinezuur (Engels:DHA)
Violaxanthin de-epoxidase (EC  , VDE) is an enzyme with system name violaxanthin:ascorbate oxidoreductase.[1][2][3][4][5][6][7] This enzyme catalyses the following chemical reaction violaxanthin + 2 L-ascorbate  zeaxanthin + 2 L-dehydroascorbate + 2 H2O (overall reaction)(1a) violaxanthin + L-ascorbate  antheraxanthin + L-dehydroascorbate + H2O(1b) antheraxanthin + L-ascorbate  zeaxanthin + L-dehydroascorbate + H2OViolaxanthin de-epoxidase is a part of the xanthophyll (or violaxanthin) cycle for controlling the concentration of zeaxanthin inchloroplasts. Algae Culture

94 The violaxanthin (Vx) cycle is found in vascular plants and the green (Chlorophyta) and brown (Phaeophyceae) algae. The Vx cycle is also present in the algal groups, which contain the diadinoxanthin (Ddx) cycle. However, in these algae, the Ddx cycle is the main xanthophyll cycle and the pigments of the Vx cycle serve mainly as intermediate products in the biosynthesis of the Ddx cycle pigments. De-epoxidation is reversed under low light intensities or in darkness. Algae Culture

95 Protection against photoinhibition: UV-absorbing compounds
The major pigments absorbing ultraviolet light (UV) in algae are thought to be mycosporine-like amino acids (MAAs) with absorbance maxima from 310 to 360 nm. These pigments are found in all classes of algae. Some other compounds may participate in protection from UV such as phenolic compounds and alginates. Mycosporine-like amino acids (MAAs) are small secondary metabolites produced by organisms that live in environments with high volumes of sunlight, usually marine environments. So far there are up to 20 known MAAs identified. They are commonly described as “microbial sunscreen”.  When MAAs absorb UV light the energy is dissipated as heat. Do students know what secondary metabolites are? No function in primary metabolism but in stress situations. Algae Culture

96 Algae Culture

97 α = extinction coefficient C = algal concentration p = path length
Self-shading is the phenomenon that describes the decrease of light irradiance while the light is penetrating dense algal cultures or blooms. Law of Lambert-Beer I = I0 e-αCp I = light intensity α = extinction coefficient C = algal concentration p = path length Algae Culture

98 Example: Calculate the light intensity in the middle of a cylindrical culture vessel with a diameter of 30 cm. The culture vessel contains a Tetraselmis striata culture with a concentration of 0.5 g/L. The extinction coefficient of this alga is 0.05 L/g mm and the initial light intensity is 800 µmolphoton/m2 s. Law of Lambert-Beer I = I0 eαCp I = 800 e0.05 * 0.5 * 150 I = 800 e0.0235 I = µmolphoton/m2 g Algae Culture

99 Example: Calculate the light intensity in the middle of a cylindrical culture vessel with a diameter of 30 cm. The culture vessel contains a Tetraselmis striata culture with a concentration of 0.5 g/L. The extinction coefficient of this alga is 0.05 L/g mm and the initial light intensity is 800 µmolphoton/m2 g. Law of Lambert-Beer I = I0 eαCp I = 800 e0.05 * 0.5 * 150 I = 800 e0.0235 I = µmolphoton/m2 g Algae Culture

100 Substrates for photosynthesis: Inorganic carbon
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101 Problem: Availability of CO2 is extremely low in sea water.
Algae Culture

102 Solution: Carbonic anhydrases
The carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons or (vice-versa), a reversible reaction that occurs rather slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zinc ion; they are therefore classified as metalloenzymes. In the oceans, where zinc is nearly depleted, diatoms use cadmium as a catalytic metal atom in cadmium carbonic anhydrase (CDCA). The reaction catalyzed by carbonic anhydrase is(under high CO2) CO2 + H2O --- HCO3 + H+ The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. Typical catalytic rates of the different forms of this enzyme ranging between 104 and 106 reactions per second. An anhydrase is defined as an enzyme that catalyzes the removal of a water molecule from a compound, and so it is this "reverse" reaction that gives carbonic anhydrase its name, because it removes a water molecule from carbonic acid. (in lungs and nephrons of the kidney - low CO2 concentration, in plant cells) CDCA1 consists of three tandem CA repeats (R1–R3), which share 85% identity in their primary sequences. Algae Culture

103 CA families (f) There are at least five distinct CA families (α, β, γ, δ and ε). These families have no significant amino acid sequence similarity and in most cases are thought to be an example of convergent evolution. The α-CAs are found in humans. α-CA The CA enzymes found in mammals are divided into four broad subgroups, which, in turn consist of several isoforms. β-CA Most prokaryotic and plant chloroplast CAs belong to the beta family. γ-CA The gamma class of CAs come from methane-producing bacteria that grow in hot springs. δ-CA The delta class of CAs has been described in diatoms. The distinction of this class of CA has recently come into question, however. ε-CA The epsilon class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes. Recent 3-dimensional analyses suggest that ε-CA bears some structural resemblance to β-CA, particularly near the metal ion site. Thus, the two forms may be distantly related, even though the underlying amino acid sequence has since diverged considerably. Algae Culture

104 Algae Culture

105 Substrates for photosynthesis: Other inorganic substrates and Redfield ratio
Algae Culture

106 Algae Culture

107 early field studies in marine ecosystems, and
It is generally believed that the rate of nitrate uptake by phytoplankton is severely reduced by the presence of ammonium. This effect is reffered to either as ‘inhibition of nitrate uptake by ammonium’ or ‘preference for ammonium’, and in its most extreme form it is believed to result in no nitrate uptake above a treshold ammonium concentration of ca 1 µM. Evidence for the negative effect of ammonium on nitrate utilization arizes from 3 sources: early laboratory studies of nitrate utilization in freshwater green algae, early field studies in marine ecosystems, and theoretical considerations of the relative energy requirements for the utilization of nitrate and ammonium, due to the number of electrons required to reduce nitrate to ammonium. In many of these early studies it was assumed that nitrate uptake (transport into cell) and reduction were so tightly coupled that uptake of nitrate must be inhibited by ammonium because the enzyme nitrate reductase is strongly inhibited. It is now known that nitrate uptake and reduction are frequently uncoupled during transient conditions in marine phytoplankton and the nitrogen uptake and assimilation are so complex that it is difficult to explain the interaction between nitrate and ammonium uptake by one mechanism. Algae Culture

108 an indirect interaction, which will be termed ‘preference’,
A thorough review of the literature, however, indicates that ‘inhibition’ or ‘preference’ is neither as universal nor as severe a phenomenon as is generally believed. (i.e. Ammonium does not always ‘inhibit’ nitrate uptake and even when it does, nitrate uptake rarely ceases intirely). In addition, it has also been reported that nitrate can sometimes inhibit ammonium uptake and that small amounts of ammonium may actually stimulate nitrate uptake. The interaction between ammonium and nitrate uptake can be simplified by dividing it into 2 distict processes: an indirect interaction, which will be termed ‘preference’, and a direct interaction, which will be called ‘inhibition’. These 2 interactions are not mutually exclusive; one or both can occur in phytoplankton. They are, however, influenced differently by environmental conditions, and vary in importance from species to species. Preference means that ammonium is more readily utilized than nitrate and is independent of the ammonium concentration. It can only be assessed by measuring nitrate uptake in the absence of ammonium and ammonium uptake in the absence of nitrate. Inhibition results when the presence of one nitrogen source prevents or reduces the uptake of the other. It can only be quantified by comparing the uptake rate in the absence of the inhibiting nitrogen source with uptake rates in the presence of increasing concentrations of the inhibitor. Algae Culture

109 Algae Culture

110 Example of ‘inhibition’:
Microalgae actively take up NO3− and NH4+ from the environment and assimilate inorganic N into organic molecules (proteins) through the coordinated activities of assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase. Nitrate reductase is cytosolic and is regulated at the transcriptional and translational levels in many photosynthetic eukaryotes, including diatoms. In Chlorella and other algae, NR is not induced in the presence of NH4+. Nitrite (NO2−), once reduced from NO3−, induces oxidative stress in the cytosol and is actively transported into the chloroplast where it is further reduced to NH4+ by nitrite reductase. Algae Culture

111 Special case: Nitrogen fixation by cyanobacteria
Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria are able to utilize a variety of inorganic and organic sources of combined nitrogen, like nitrate, nitrite, ammonium, urea, or some amino acids.   2-Oxoglutarate has turned out to be the central signaling molecule reflecting the carbon/nitrogen balance of cyanobacteria. Central players of nitrogen control are the global transcriptional factor NtcA, which controls the expression of many genes involved in nitrogen metabolism, as well as the PII signaling protein, which fine-tunes cellular activities in response to changing C/N conditions. These two proteins are sensors of the cellular 2-oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to nitrogen starvation involves heterogeneous responses in different strains. Nitrogen fixation by cyanobacteria in coral reefs can fix twice the amount of nitrogen than on land–around 1.8 kg of nitrogen is fixed per hectare per day. Algae Culture

112 Algae Culture

113 Pi is taken up by algae using phosphate transporters
Pi is taken up by algae using phosphate transporters. These transporters are membrane-spanning proteins that are highly regulated, both temporally and spatially depending on the prevailing phosphorus conditions in the cell and its environment. Unlike nitrate, the PO4−3 ion does not undergo reduction prior to assimilation. It enters metabolic pathways through adenosine, wherein a phosphate ester bond is formed between Pi and ADP by the enzyme ATPase to produce ATP; or it bonds with hydroxyl groups of carbon chains to form simple phosphate esters (e.g., sugar phosphates). The primary process of Pi incorporation, through adenosine, occurs in mitochondria during respiration via oxidative phosphorylation, and in chloroplasts during the light reactions of photosynthesis via photophosphorylation. Algae Culture

114 Two ways in which phosphates play a crucial role in metabolism:
(f) Two ways in which phosphates play a crucial role in metabolism: Structural role as component of DNA and RNA backbone. Role as energy supplier in numerous biosynthesis reactions. (f) Algae Culture

115 RO-PO3H2 + H20 -> ROH + H3PO4
Because Pi is critically important in algae metabolism, adequate supplies of Pi are necessary to ensure optimum metabolic performance. Under low Pi conditions and/or increased metabolic Pi demand, algae generally increase phosphatase activity (that is, the activities of acid- and alkaline-phosphatases or PAs). Increased PA activities enhance the ability of plants to recycle internal Pi, and to utilize Pi from environmental sources. These enzymes tend to be substrate-nonspecific, thereby catalyzing the release of Pi from a broad range of P-containing compounds. The phosphatases are enzymes important to life because of their ability to hydrolyze phosphate esters or to transfer phosphate from one organic group to another. Phosphomonoesterases hydrolyze monoesters of phosphoric acid, RO-PO3H2 + H20 -> ROH + H3PO4 And are classified as alkaline- or acid-phosphatases depending on the pH at which maximum activity occurs. Other phosphatases hydrolyze diesters of phosphoric acid, pyrophosphates or metaphosphates. Algae Culture

116 Phosphatases in algae have been reported by several workers
Phosphatases in algae have been reported by several workers. An alkaline phosphatase of Chlorella vulgaris was reported to be localized at the cell surface. Many species of algae are capable of obtaining phosphorus from esters in order to sustain growth in the absence of orthophosphate. Algae Culture

117 The Redfield ratio is the molecular ratio of carbon, nitrogen and phosphorus in plankton.
The term is named after the American oceanographer Alfred C. Redfield, who first described the ratio in his article in 1934 (Redfield 1934). As a physiologist, Redfield participated in several voyages on board Atlantis. Alfred Redfield analyzed thousands of samples of marine biomass from all ocean regions. He found that globally the elemental composition of marine organic matter (dead and living) was remarkably constant. The ratios of carbon to nitrogen to phosphorus remained the same from coastal to open ocean regions. Algae Culture

118 Redfield explained the remarkable congruence between the chemistry of the deep ocean and the chemistry of living things in the surface ocean. When nutrients are not limiting, the molar element ratio C:N:P in most phytoplankton is 106:16:1. Redfield thought it wasn't purely coincidental that the vast oceans would have a chemistry perfectly suited to the requirements of living organisms. Although the Redfield ratio is remarkably stable in the deep ocean, phytoplankton may have large variations in the C:N:P composition, and their life strategy play a role in the C:N:P ratio, which has made some researchers speculate that the Redfield ratio perhaps is a general average rather than specific requirement for phytoplankton growth as no theoretical justification for Redfield ratio has ever been found. Diatoms need, in addition to other nutrients, silicic acid to create biogenic silica for their frustules (cell walls), and the proposed Redfield-Brzezinski nutrient ratio for diatoms is C:Si:N:P = 106:15:16:1. Algae Culture

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