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Algae Culture Year 2011-2012 Peter Bossier Aäron Plovie Algae Culture 2011-20121.

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Presentation on theme: "Algae Culture Year 2011-2012 Peter Bossier Aäron Plovie Algae Culture 2011-20121."— Presentation transcript:

1 Algae Culture Year Peter Bossier Aäron Plovie Algae Culture

2 2 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:

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

4 Ocean primary productivity

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

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

7 Algae Culture 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).

8 Algae Culture Plastid loss Malaria Sleeping sickness Uni-and multicellular Mostly multicellular 2005

9 Algae Culture How come algal taxa are spread all over tree of life? Endosymbiosis events and plastid losses cause huge algal diversity.

10 Algae Culture Primary endosymbiosis Secundary endosymbiosis Tertiary endosymbiosis Serial secundary endosymbiosis Each endosymbiosis event involves gene transfer between host genome(s) and chloroplast genome(s)!

11 Algae Culture A closer look on endosymbiosis:

12 Algae Culture 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.

13 Algae Culture Algae species relevant to aquaculture

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

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

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

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

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

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

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

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

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

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

24 Algae Culture Algae species relevant to aquaculture: Microalgae Bacillariophyceae (Diatoms) Phaeodactylum tricornutum

25 Algal characteristics NamefeatureLength x with (µm) Cell weight (pg) ArthrospiraCylindrical cells forming helicoidal trichomes x 10 ChaetocerosFree living brown, four long diagonal setae ChlorellaFree living, green, spherical, cell wall, 2-16 autospores 1,5-10 CrypthecodiniumFree living, mobile, spherical or elliptic, incomplete cingulum, forming cyst Isochrysis galbanaFree living, mobile, yellow to golden brown, short subapical haptonema with two flagella, no cell wall 5-6 x Algae Culture

26 Algal characteristics NamefeatureLength x with (µm) Cell weight (pg) Nannochloropsis oculata Free living, or aggregated, ovoid, cell wall, no chlorofyl b Pavlova lutheriFree living, mobile gold- brown, ovoid, short haptonema with 2 unequal flagella, no cell wall 7-9x Phaeodactylum tricornutum Chained, oval, Y or spindle shaped 3-4 x organic weight Skeletonema costatum Chained, spherical to cylindrical cells attached together by a ring of external gutter-shaped processes 10 x552 TetraselmisFree living, mobile, spindle- shaped cells, four polar flagella, cell wall, cyst form 9-11 x 7-8x Algae Culture

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

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

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

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

31 Algae Culture Microbial growth: During the exponential phase: dC/dt = µC µ = specific growth rate dependent on temperature and light irradiance! C t = C 0 e µt µ = (lnC t – lnC 0 ) / (t – t 0 ) For heterotrophic bacteria mainly expressed in h -1, for algal autotrophs expressed in d -1 !

32 Algae Culture

33 Algae Culture During the exponential phase: Doubling time C(t+t D ) = 2 C(t) C 0 e µ(t+t D ) = 2 C 0 e µt C 0 e µt e µt D = 2 C 0 e µt E µt D = 2 µt D = ln(2) t D = ln(2) / µ

34 Algal growth rates NameSpecific growth rate (day -1 ) Culture conditions Arthrospira2.47 1,68 maxima, 550, 12/12, 33°C, B platensis, 273, 24/0, 35, C Chaetoceros0,87 1,37 Calcitrans, 60, 24/0, 18°C, B Gracilis, 165; 24/0, 18°C, B Chlorella2,03 0,3 0,16 vulgaris, heterotrophy, B vulgaris, axenic, B vulgaris, open conditions, B Crypthecodinium1,98cohnii, heterotrophy, B Isochrysis galbana1,4Galbana, 36, 24/0, 25°C, B Algae Culture

35 Algal characteristics NameSpecific growth rate (day -1 ) Culture conditions Nannochloropsis oculata0,28Sp, 75, 12/12, 20°C, B Pavlova lutheri0,92Lutheri, 20°C, B Phaeodactylum tricornutum 1,5Tricornutum, 23°C, B Skeletonema costatum0,84 -2,88Costatum, 135, 24/0, 20°C, B Tetraselmis1,68Sp, 25°C, B B: batch, C: continuous,energy in microEinstein per m -2 s -1 Algae Culture

36 Algae Culture Example: Compare the doubling times of Pavlova lutheri grown in batch culture at 20°C and 60µEm -2 s -1 and grown in continuous culture at 20°C in their exponential phases. t D = ln(2) / µ t D = / 0.25 = days t D = / 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. C t = C 0 e µt C t = e 0.3*25 C t = 10,000 * 1808 C t = 18,

37 Algae Culture Example: Compare the doubling times of Pavlova lutheri grown in batch culture at 20°C and 60µEm -2 s -1 and grown in continuous culture at 20°C in their exponential phases. t D = ln(2) / µ t D = / 0.25 = days t D = / 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. C t = C 0 e µt C t = 10,000 e 0.3*25 C t = 10,000 * 1808 C t = 18,080,000

38 Relationships

39 Algae Culture 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/(m 2 day) or volume of the culture: g/(l day)

40 Algae Culture Productivity In batch culture with no medium renewal: P= (C-C 0 )V/(t-t 0 ) 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

41 Algae Culture 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.

42 Algae Culture Photosynthesis: Machinery

43 Algae Culture 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.

44 Algae Culture 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.

45 Algae Culture 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.

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

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

48 Algae Culture 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.

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

50 Algae Culture

51 Algae Culture NADPH and ATP are used as energy source to produce 3-phosphoglycerate (building block of sugars) from C0 2. Rubisco can only use CO2 and not bicarbonate Write the equation of one Calvin cycle

52 Algae Culture 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.

53 Algae Culture 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, CO 2 and other dissolved nutrients, they are generally more efficient in converting solar energy into biomass.

54 Algae Culture Substrates for photosynthesis: Light energy

55 Algae Culture 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.

56 Algae Culture 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 10 8 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.

57 Algae Culture 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 (10 6 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 Hz), Visible red light, visible orange, yellow, green blue violet (on the order of Hz) and then the ultraviolet spectrum. At higher frequencies yet, there are X-rays (10 17 Hz), gamma-rays and other very high energy photons.

58 Algae Culture

59 Algae Culture E = energy of one photon (Joule, J) 1 joule is equal to 6.24*10 18 eV f = frequency (Hertz, Hz = s -1 ) c = speed of light (ms -1 ) λ (lambda) = wave length (m) h = Plancks constant = 6.626* Js The energy carried by light is contained in the photons that travel as a wave: E = hf = hc/λ

60 Algae Culture 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* x 3.0*10 8 /(500*10 -9 ) E = * J f = E/h f = * / 6.626* f = 6*10 14 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* x 3.0*10 8 ) N = 25.15*10 17 photons

61 Algae Culture 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* x 3.0*10 8 /(500*10 -9 ) E = * J f = E/h f = * / 6.626* f = 6*10 14 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* x 3.0*10 8 ) N = 25.15*10 17 photons

62 Algae Culture N = 25.15*10 17 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*10 23 photons (Avogadros number). So 25.15*10 17 photons correspond to moles. And now micromoles (10 -6 mole) is the easiest to use unit micromoles of photons are necessary for 1 Joule of light energy at 500 nm.

63 Algae Culture 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.

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

65 Algae Culture 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 lamps phosphor coating, which results in emitting photons of different wavelengths, depending on the mix of used phosphor atoms.

66 Algae Culture 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.

67 Algae Culture 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 (m 2 ), 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.

68 Algae Culture Different light sources have different distributions of photons in the 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/m 2 ) 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).

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

70 Algae Culture Note that for each wavelength the spectroradiometer measures the power density in watts/m 2. 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/m 2 of power or 0.4 joules/m 2 /second of energy. Using the relationship between energy and wavelength, it can be determined how many photons/m 2 /sec at 420nm will be required to generate 0.4 joules of energy (1.46 micromoles). Thus, we can easily convert from watts/m 2 to micromoles/m 2 /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..

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 Algae Culture Example:

73 Algae Culture Example:

74 Algae Culture What is important to note is the following: 1)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

75 Algae Culture 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.

76 Algae Culture

77 Algae Culture The saturation light intensity I s is generally 300 to 600 µEinstein/(m 2 sec). As a reference, a bright sunny day is around 2000 µE/(m 2 s) Specific growth rate is 0.5 to 1.5 day -1.

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 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/m 2 ) or footcandles (lumen/ft 2 ). –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/m 2 /s or µmoles/m 2 /s. One Einstein = 1 mole of photons = 6.022×10 23 photons, hence, 1 µEinstein = 6.022×10 17 photons.

85 Light as energy: practically PPF (μmol m -2 s -1 ) to LuxLux to PPF (μmol m -2 s -1 ) Sunlight54Sunlight Cool White Fluourescent Lamps 74 Cool White Fluourescent Lamps High Pressure Sodium Lamps 82 High Pressure Sodium Lamps High Pressure Metal Halide Lamps 71 High Pressure Metal Halide Lamps 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 ( ). 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, ).

86 Light as energy: practically PPF (μmol m - 2 s -1 ) Lux (Sunlight ) Lux (HPS) Lux (Metal Halide) Lux (Fluores cent) ,4008,2007,1007, ,80016,40014,20014, ,20024,60021,30022, ,40049,20042,60044, ,00082,00071,00074,000 2,000108,000164,000142,000148,000

87 Light as energy: summary of units

88 Algae Culture

89 Algae Culture 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.

90 Algae Culture 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 O 2 to generate reactive singlet oxygen ( 1 O 2 ). Overreduction of the photosynthetic electron carrier chain would also favor the direct reduction of O 2 by photosystem I (PSI) and the subsequent production of damaging reactive oxygen species, such as superoxide (O 2 - ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (. OH).

91 Algae Culture 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.

92 Algae Culture 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.

93 Algae Culture

94 Algae Culture 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.

95 Algae Culture 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.

96 Algae Culture

97 Algae Culture 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 = I 0 e- αCp I = light intensity α = extinction coefficient C = algal concentration p = path length

98 Algae Culture 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/m 2 s. Law of Lambert-Beer I = I 0 e αCp I = 800 e0.05 * 0.5 * 150 I = 800 e I = µmolphoton/m 2 g

99 Algae Culture 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/m 2 g. Law of Lambert-Beer I = I 0 e αCp I = 800 e0.05 * 0.5 * 150 I = 800 e I = µmolphoton/m 2 g

100 Algae Culture Substrates for photosynthesis: Inorganic carbon

101 Algae Culture Problem: Availability of CO 2 is extremely low in sea water.

102 Algae Culture 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 10 4 and 10 6 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)

103 Algae Culture 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.

104 Algae Culture

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

106 Algae Culture

107 Algae Culture 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: (1)early laboratory studies of nitrate utilization in freshwater green algae, (2)early field studies in marine ecosystems, and (3)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.

108 Algae Culture 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.

109 Algae Culture

110 Algae Culture Example of inhibition: Microalgae actively take up NO 3 and NH 4 + 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 NH 4 +. Nitrite (NO 2 ), once reduced from NO 3, induces oxidative stress in the cytosol and is actively transported into the chloroplast where it is further reduced to NH 4 + by nitrite reductase.

111 Algae Culture 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 P II 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.

112 Algae Culture

113 Algae Culture P i 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 PO 4 3 ion does not undergo reduction prior to assimilation. It enters metabolic pathways through adenosine, wherein a phosphate ester bond is formed between P i 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 P i incorporation, through adenosine, occurs in mitochondria during respiration via oxidative phosphorylation, and in chloroplasts during the light reactions of photosynthesis via photophosphorylation.

114 Algae Culture 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) (f)

115 Algae Culture Because P i is critically important in algae metabolism, adequate supplies of P i are necessary to ensure optimum metabolic performance. Under low P i conditions and/or increased metabolic P i 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 P i, and to utilize P i from environmental sources. These enzymes tend to be substrate-nonspecific, thereby catalyzing the release of P i 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-PO 3 H 2 + H 2 0 -> ROH + H 3 PO 4 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.

116 Algae Culture 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.

117 Algae Culture 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.

118 Algae Culture 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.


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