December 17, 2012
May 21, 2012
April 18, 2012
April 17, 2012
April 11, 2012
March 10, 2012
Protein Analysis
Protocol for Total Protein Determination -Lowry et
al., 1951 Last
updated 4/9/2012 Sitti
REAGENT C
2% Na2CO3 in 0.1N
NaOH - 50 ml
0.5% CuSO4.5H20 in 1%
Na Tartrate - 1 ml
REAGENT E
Diluted
1N Folin-Ciocalteu phenol reagent
PROTOCOL
1. Filter
10 ml aliquot/culture onto pre-combusted GF/F filter (fold and wrap in
aluminium foil - store at -80 oC
/LNG if not analyze) *make 3 replicates for each PBR tube.
2. Homogenize
filter in glass-teflon homogenizer in 3 mL 1N NaOH. Rinse 2-3 times homogenizer
with 1N NaOH and collect as maximum extract as possible in a falcon tube, bring
up the volume to 10 ml and cap tightly.
3. Keep
the extract at room temperature for 22-24 hours for complete extraction.
4. Vortex
for thorough mix and centrifuge the extract for 5 mins at 5000 rpm.
5. Take
0.5 mL of extract and mix with 5 mL of
freshly prepared reagent C.
6. Allow
to stand for 10 min. (in dark)
7. Add 0.5
mL freshly prepared reagent E, mixed well.
8. Allow
to stand for 30 minutes to develop maximum colour. (in dark)
9. Read
absorbance at 750 nm against a reagent blank.
10. Use Bovine
serum albumin prepared in 1N NaOH as standard for total protein contents
determination. To get the standard
curves or calibration curves, prepare standard solution at concentration 0 - 150
µg/µl
March 1, 2012
Chlorophyll analysis - spectrophoptometry
Chlorophyll Analysis
This guide is primarily for the lab researcher who will spectrophotometrically measure the chlorophyll content of laboratory cultures of phytoplankton. Those wishing to determine chlorophyll in natural seawater samples or in samples containing significant amounts of phaeophytin should consult fluorometric and other techniques. Some variation in this protocol maybe required for species that are particularly resistant to extraction. This may include overnight extractions in a freezer, boiling methanol extraction, and sonication.
- Sample preparation
- Filter a known volume of culture onto a 25 mm glass fiber filter.
- Whatman GF/F filters are better suited for small cells; Gelman A/E are adequate for cells greater than 4 um is size.
- Swirl your culture before sampling to ensure homogeneous sampling.
- Keep vacuum level low (< 10 in Hg) to prevent cell breakage and low filtration efficiency.
- Rinse funnel walls with small amount of filtered seawater.
- Remove the funnel. Fold the filter in quarters and place in a grinding vessel.
- Try not to touch the filter with your “oily, acidic” fingers. Use forceps and the tip of the grinding vessel mouth as extensions of your hands.
- The grinding vessel is pretty tough; you can tap the vessel bottom on the bench to get the filter to the bottom of the vessel.
- Place enough 90% acetone in the grinding vessel such that the filter is covered (about 1-2 mls).
- Keep in mind that you want to keep the total acetone extract volume for the entire analysis to less than 10 ml.
- For “tough” cells, use instead 80% Methanol instead of acetone and boil the sample for 5 minutes at 75-80 degrees C.
- Homogenize the filter using the motorized Teflon pestle.
- Use Safety glasses!
- Hold tightly to the vessel, insert the pestle into the vessel and turn on the motor (use REVERSE position and about setting 3 on the dial). Use an up & down motion while keeping the pestle immersed in the acetone. When filter is suitably macerated, turn off the motor while pulling the pestle from the narrow part of the vessel at the same time. This keeps the pestle from becoming stuck when it stops.
- Rinse the pestle into the vessel with a small amount of 90% acetone. The acetone volume should now be about ½ to ¾ of the narrow portion of the vessel.
- Place the vessel in a dark box and repeat the above steps for other samples.
- Clarify the sample preparations.
Samples can be clarified using filtration or centrifugation. The filtration method is best suited for situations in which you have many samples to analyze (see Kevin for help on this method). Centrifugation is more often used in our lab. It is more economical (but less time efficient).
- Remove the sample from the dark box and carefully pour sample into a 12 ml conical glass centrifuge tube.
- Gently vortex the sample prior to pouring into the centrifuge tube. This keeps the ground-up filter from “sticking” to the vessel when you try to pour it out.
- To make a vortex, hold the top of the vessel between your thumb and index finger. Flick the bottom of the vessel with the fingers of you other hand.
- Rinse the vessel 2 or 3 times with small amounts of 90% acetone. Pour the rinses into the centrifuge tube. Once again, try to keep your total acetone extract low.
- Vortex the centrifuge tube gently to ensure that the extract is homogeneous.
- Balance your sample against a blank tube or another sample and use the Dynac clinical centrifuge.
- Spin at top speed for 5 minutes.
- Remove the tubes from the centrifuge and note sample extract volumes.
- For best results, your extract should have some color to it, but not be too colored. Absorbencies beyond 1.5 result in peak flattening and inaccuracies. Experience will help you make good estimates of this using just your eyes.
- Spectrophotometric determination
In most circumstances, you will want to make your measurements using the Aminco DW-2000 or DW2 instruments. Please refer to the guides on using this instrument should you not be familiar with it. If needed, you can also use the HP 8451 Diode Array spectrophotometer. However, this is abit less desirable as it has a 2 nm bandpass and does not directly measure the absorbance at 647 nm required for chlorophyll b containing organisms.
- Make a wavelength scan of absorbance from 375 to 750 nm.
- Be sure to store the data on the computer
- From the UTILITIES menu, choose to transfer you data to a DOS text file.
- EXIT the DW2000 software and change directory to GWBASIC
- DOS command is: cd gwbasic
- Start the chlorophyll calculation program.
- Type: gwbasic dw2000
- Use the up/down arrow keys to select READ FROM DISK and press ENTER
- to see files, enter 2
- your file from step 2 should have a PRN extension
- enter the file name (without extension) that you want to read
- if successful, you should see the spectra displayed on the screen
- Select SETUP using the F2 function key
- then use the up/down arrow keys to choose DATA TYPE and enter 1 for CHLOROPHYLL
- use arrow keys to choose SPECIES TYPE and choose the appropriate chlorophyll content (chl b or c containing organisms) and solvent (acetone or methanol)
- use arrow keys to select FINISHED
- Enter volume filtered, volume extract, and cell count if known. If cell count is unknown, then enter a value of 1e6.
- Select RECALC. by pressing the F6 function key
- the chlorophyll calculations should be displayed and you should write down the appropriate numbers. The calculations are based upon the equations of Jeffrey and Humphrey, 1975.
- Use the ESC key to exit the program
- If you have a problem with the program, use CTRL + Break to halt the program and type SYSTEM to return to the DOS operating system. Start over from Step 4.
- Calculations and Reference
Multiply values by volume of acetone extract and divide by the volume filtered (all in milliliters). The results are in milligrams per liter. Note that each OD value is corrected for the absorbance at 750 nm (e.g. OD664 = Abs664 – Abs750).
For Chlorophyll c containing organisms in 90% Acetone:
Chl a = (11.47 * OD664) – (0.4 * OD630)
Chl c = (24.36 * OD630) – (3.73 * OD664)
And in 80% Methanol:
Chl a = (12.66 * OD665) – (0.5 * OD635)
Chl c = (31.25 * OD635) – (5.79 * OD665)
For Chlorophyll b containing organisms in 90% Acetone:
Chl a = (11.93 * OD664) – (1.93 * OD647)
Chl b = (20.36 * OD647) – (5.5 * OD664)
And in 80% Methanol:
Chl a = (16.5 * OD665) – (8.3 * OD650)
Chl b = (33.8 * OD650) – (12.5 * OD665)
There is a trichromatic calculation (90% Acetone) for determining chlorophylls in a mixed phytoplankton assemblage in which both chlorophyll b and c containing organisms are present.
Chl a = (11.85 * OD664) – (1.54 * OD647) – 0.08 * OD630)
Chl b = (-5.43 * OD664) + (21.03 * OD647) – (2.66 * OD630)
Chl c = (-1.67 * OD664) – (7.60 * OD647) + (24.52 * OD630)
*** Chl c calculation may lead to results which are 24% too low
Reference:
Jeffrey, S.W. and Humphrey, G. F. 1975. New Spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae, and natural phytoplankton. Biochem. Physiol. Pflanz. 167: 191-194.
Jeffrey, S.W. and Welschmeyer, N.A. Spectrophotometric and fluorometric equations in common use in oceanography. In Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods. Appendix F; 597-615
More recently, the following equations have been determined for 100% Methanol:
Chl a = (16.29 * OD665) – (8.54 * OD652)
Chl b = (30.66 * OD652) – (13.58 * OD665)
Reference:
Porra, R. J., Thompson, W.A., and Kriedemann, P.E. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta 975: 384-394.
For cyanobacteria extracted in boiling methanol, we have typically used:
Chl a = 13.42 * OD665
Reference:
Mackinney, G. 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140: 315-322.
However, more recent work has suggested that Mackinney’s extinction coefficients are too low. Hence, we should re-examine which equation to use for cyanobacteria.
Chlorophyll Fluorescence and Definitions
The following is a generalized description of the photosynthesis light reaction and the value of chlorophyll fluorescence in photosystem II. For more specific information please refer to papers cited in this discussion or contact Opti-Sciences for more information.
Light energy utilized in photosynthesis by higher plants and algae cells is collected first by an antenna pigment system and transferred to reaction centers where light quanta are converted to chemical energy by chlorophylls in a protein environment. Electron transfer takes place in the reaction center when a chlorophyll molecule transfers an electron to a neighboring pigment molecule. Pigments and protein involved in this primary electron transfer define the reaction center. This initial electron transfer is also called charge separation.
Competing models of energy capture and transfer exist. In the “puddle model” of Photosystem PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The “lake model” is considered more realistic for terrestrial plants.
Reaction centers are of two types, Photosystem II (PSII), and Photosystem I (PSI). both are located in the thylakoid membrane of a chloroplast of higher plants. In bacteria they are in a membrane surrounding the cytoplasm or in more intricate constructs. All plants the produce oxygen have both types of reaction centers.
It is in PSII that oxygen evolution and the splitting of water occurs. To reduce the PSII reaction center an electron is pulled from the water splitting complex.
After charge separation, electrons flow to other nearby plastoquinone molecules in the thylakoid membrane by oxidation reduction reactions. They act as energy transfer molecules in electron transport chain.
The next stop is a cytochrome b6f complex. Eventually this complex supplies an electron to reduce PSI. PSI then goes through a somewhat similar process to PSII, and eventually produces NADPH.
In addition, protons created from PSII functions, the splitting of water, and the reaction in the cytochrome b6f complex, enter the thylakoid lumen and are used by an ATP pump in the thylakoid membrane in the presence of ATP synthase, to create ATP from ADP. Both ATP and NADPH are used as energy sources to drive the Calvin Benson Cycle of carbon fixation.
PSI goes through a similar process to PSII, however, the low fluorescent signal of PSI does not vary with plant stress or with various photosynthetic functions as it does with PSII. Therefore PSII is of primary interest. Factors such as light levels, light quality, water availability, nutrient availability, heat, cold, herbicides, pesticides, pollution, heavy metals, disease, and genetic make up can all have an impact on CO2 assimilation, plant health and condition. They also are reflected in the fluorescence signal in PSII. Therefore, by using a chlorophyll fluorometer one can quantify the impact of these factors on plants to improve breeding and production programs, and to better understand plant functions.
The most prominent pigments that absorb this energy are chlorophyll-a and chlorophyll-b. Other accessory pigments may be involved such as carotenoids, or phycobilins in cyanobacteria, or bacteriochlorophyll in some bacteria.
Light energy absorbed initially by the antenna and transferred to the reaction centers is channeled by a number of different processes including photochemistry, photo-protective heat dissipation, other heat dissipation and about 3%-9% of the light energy absorbed by chlorophyll pigments is re-emitted as fluorescence. The emission peak is of a longer wavelength than the excitation energy. This effect was first observed more than 100 years ago, when N.J.C. Müller (1874) by visually using colored glass filters. He also noted that fluorescence changes that occur in green leaves are correlated with photosynthetic assimilation. Lack of appropriate technical equipment prevented a more detailed investigation of this phenomenon. The light energy drives photosynthetic electron transport through PSII and PSI leading to the oxidation of water, oxygen evolution, the reduction of NADP+ to NADPH, membrane proton transport and ATP synthesis.
The loss of light energy from the reaction center as fluorescence comes primarily from the PSII reaction. When the chloroplast or leaves have been dark-adapted, the pools of oxidation-reduction intermediates for the electron transport pathway return to an oxidized state. Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescent light emission from PSII followed by a decrease to steady state fluorescence as CO2 fixation starts to occur. Changes in the intensity of the fluorescence emission are a sensitive reflection of changes in the photosynthetic apparatus. Following many years of study, chlorophyll fluorescence has been shown to represent changes in the health and function of the photosynthetic process. This includes all the reactions from the oxidation of water, charge separation, electron transport, development of the electrochemical gradient, the photo-protective mechanisms of the xanthophyll cycle, and the change in ph of the thylakoid lumen. Even changes in the plant that affect stoma opening and gas exchange with the atmosphere are reflected by changes in the fluorescence characteristics of a leaf. Vast amounts of research have shown excellent correlation between carbon fixation and chlorophyll fluorescence data.
This is a typical Dark-Light kinetic pulse modulated trace.
Definitions are described below.
Figure 1-1 • Dark–Light Pulse modulated Chlorophyll Fluorescence Trace
Definitions:
Dark adapted
– This is a term that means that the area of the plant, or the entire plant, to be measured has been in the dark for an extended period of time before measurement. Dark adaption requirements may vary for dark adapted tests. Dark adaption times of ten minutes to 60 minutes are common, and some researchers use pre-dawn values. Dark adapted measurements include Fv/Fm, OJIP measurements, and most non-photochemical quenching parameters. Fv/Fm and most OJIP measurements take only a second to make a measurement. Non-photochemical quenching parameters take longer.
Light adapted
– Yield measurements are made when a sample is already exposed to existing light conditions. No dark adaption is required. When a sample is exposed to light it normally takes several second to a few minutes for photosynthesis to reach steady state. To obtain a reliable yield measurement photosynthesis must reach steady state. This is not a concern when using ambient sunlight or artificial greenhouse light however clouds and light fleks below a canopy level can cause problems. If one uses a built in fluorometer illuminator to measure yield make sure that steady state photosynthesis has been reached. This is normally a very fast test that take a second or two and it has the added advantage that it does not require dark adaption. Light adapted measurements include Yield or ΔF/Fm’.
PAR
– Photosynthetically Active Radiation between 400nm and 700nm. Measured in either µmls or µE The Term PAR means photosynthetically active radiation in the wave band between 400-700 nm. PAR can be measured in different dimensions such as Watts per meter or in micro- Einsteins or micro-moles. When using a PAR Clip, dimensions will always be in the equivalent terms, micro-Einsteins, or micro-moles
PPFD
- Photosynthetic Photon Flux Density is the photon flux density of PAR. Measured in either or. PPFD, or “photosynthetic photon flux density”, is the number of PAR photons incident on a surface in time and area dimensions. These terms are equivalent for PAR Clip leaf radiation measurements. Furthermore, both can be presented in either of the equivalent dimensions, micro-moles (µmls) or micro-Einsteins (µE).
µmls
- is a micro mol (also abbreviated µmol. This a dimension that involves both time and area. It is the equivalent to the micro Einstein. Both terms have been used extensively in biology and radiation measurements.
µE
– is a micro Einstein This a dimension that involves both time and area. It is the equivalent to the micro mol. Both terms have been used extensively in biology and radiation measurements.
1 µE = 1 µmol m-2 s-1 = 6.022 x1023 photons m-2 s-1
Actinic Light source
– This is any light source that drives photosynthesis. It may be the Sun, or an artificial light. Higher end fluorometers contain one or more built-in artificial actinic light sources for experimentation with specific repeatable radiation (or light) levels.
Saturation pulse
– is short pulse of very intense light designed to fully reduce a leaf's PSII system. It is created by an artificial light source in all modulated fluorometers.
Far red light
– is a light source that provides light above 730 nm to allow the transfer of electrons to PSI and allow the rapid re-oxidation of PSII.
Modulated light Source
– This is the measuring light source in a modulated fluorometer. It is a precision light diode that is usually either red or blue. Most fluorometers usually have one or the other. The OS5p has both. They use a very low intensity, usually between 0.2 µmls and 1µml, to allow measurement without driving photosynthesis.
Fo
– is the dark adapted initial minimum fluorescence.
Fm
– is maximal fluorescence measured during the first saturation pulse after dark adaption.
Fs
– also known as F’ is the fluorescence level related to the actinic light and is a reflection of level of photosynthetic activity.
Fms
– also known as Fm’ is the saturation pulse value that is not dark adapted. They are lowered values due to NPQ or non-photochemical quenching. When this parameter has reached steady state, it is used to calculate photosynthetic Yield along with Fs. Fms at steady state is also used to calculate qN, NPQ, qP, qL, Y(NPQ), Y(NO), qE, qT, and qI.
Fod
– also known as Fo’ is the minimal value after the far red light is turned on for several seconds after the actinic light has been turned off. It represents Fo with non-photochemical quenching.
Ft
– is the current instantaneous fluorescent signal.
Fv/Fm
= (Fm – Fo) / Fm This is a dark adapted test used to determine Maximum quantum yield. This ratio is an estimate of the maximum portion of absorbed quanta used in PSII reaction centers. It is important to properly dark adapt samples for this test. Fo will be raise and Fm will be lowered if dark adaption is inadequate. Since dark adaption requirement can vary with species, varieties, mutants, and sun vs. shade leaves testing should be done to ensure proper dark adaption. (Kitajima and Butler, 1975)
Y
= (Fms – Fs) / Fms This test is also known as ΔF/Fm’. Yield of PSII is a light adapted test normally taken at steady state photosynthesis levels. It provides a measure of actual or effective quantum yield. This ratio is an estimate of the effective portion of absorbed quanta used in PSII reaction centers. (Genty, 1989)
Quenching Parameters
Quenching parameters allow the quantification of both the effective photochemical state of the PSII regarding the fraction of PSII centers that remain open or oxidized at any time, and the non-photochemical photosynthetic mechanisms involved in photo-protection, state 1 and state 2 transition quenching, photo-inhibitor and photo-damage.
Puddle Model and Lake Model of Antennae
In the “puddle model” of Photo-system PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The lake model has proven to be the more realistic model for terrestrial plants. Research has shown that some marine species have a distinct antenna architecture. With this in mind, some of the quenching parameters of the “puddle model” are incompatible with those of the “lake model”. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
qP = (Fm' - F) / (Fm' - Fo')
NPQ = (Fm - Fm') / Fm'
NPQ = qE + Qt + qI
qN = (Fm - Fm') / (Fm - Fo)
qL = qP(Fo'/F')
Y(NO) = 1/NPQ + 1 + qL((Fm/Fo)-1)
Y(NPQ) = 1 - Y - Y(NO)
Understanding the graphs above:
In each case the sample is first dark adapted. The test is started and Fo, or minimal fluorescence, is measured without actinic light. Then a saturation pulse occurs and completely closes all receptors in PSII by completely reducing PSII. Fm, maximal fluorescence, is the result. After the saturation pulse, an actinic light is turned on and the fluorescent signal declines slowly with the onset of CO2 fixation until it reaches steady state. Photochemical quenching a measure of open PSII centers, photo-protective non-photochemical quenching and other heat dissipation mechanisms occur. Saturation pulses during steady state photosynthesis provide Fm', maximal fluorescence in this situation after NPQ has reached equilibrium with photochemistry. qP, or qL in the lake model, now represents the fraction of PSII receptors that remain open or oxidized. F (or Fs) represents fluorescence related to current steady state photochemical levels. At the point where the actinic light is turned off, far red illumination is turned on to allow the transfer of electrons quickly to reduce PSI, and allow the re-oxidation of PSII. Fo' represents this value with un-relaxed non-photochemical quenching. The rising values of the saturation pulses after the actinic light has been turned off represent the relaxation of NPQ over time. A portion of NPQ, qE (or Y(NPQ) in the lake model), represents ph and the xanthophyll cycle.photo-protection mechanisms of thylakoid lumen The remainder of NPQ represents qT, and qI, (or Y(NO) in the lake model). qT is quenching due to state 1 and state 2 transitions and is negligible in higher plants. qI represent photo-inhibition and photo-damage.
Definitions for Quenching Parameters:
NPQ
is non-photochemical quenching and is a measure of heat dissipation and a combined total for the combination of photo-protective mechanisms, state 1 and state 2 transition quenching, and photo-inhibition and photo-damage. NPQ = qE + qT + qI. NPQ is an alternate expression of non-photochemical quenching. It provides an estimate of quenching without knowledge of Fo. The advantage of NPQ over qN depends on the specific application. NPQ is more heavily affected by non-photochemical quenching that reflects heat-dissipation of excitation energy in the antenna system. So it may be thought of as an indicator of 'excess excitation energy'. Alternatively, NPQ is relatively insensitive to the part of non-photochemical quenching associated with qN values lower than 0.6 This range of qN is affected by ΔPH of the thylakoid lumen which is an important aspect of photosynthetic regulation. (Bilger & Björkman, 1990)
qN
is similar to NPQ but requires Fod or Fo’ in the calculation. qN is defined as the coefficient of non-photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN value. By using the Far-Red source after actinic illumination, the PSII acceptors re-oxidized and PSI is reduced. A new Fod value is measured and used for corrections to the quenching coefficients. (puddle model) (Van Kooten & Snel, 1990)
qE
is the quenching parameter that represents the photo-protective mechanisms in the leaf that allow rapid compensation for changes in light levels due to cloud cover and increased light intensity. It is directly related to Δph of the thylakoid lumen and the xanthophyll cycle. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
qT
is the quenching parameter that represents state 1 and state 2 transitions. This value is negligible in higher plants. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
qI
is the quenching parameter that represents photo-inhibition and photo-damage. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
Y(NPQ)
is a lake model quenching parameter that represents heat dissipation related to all photo-protective mechanisms. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
Y(NO)
is a lake model quenching parameter that represents all other components of non-photochemical quenching that are not photo-protective. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
qP
is the quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. qP is defined as the coefficients of photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN and qP values. By using the Far-Red source for post illumination, the PSII acceptors may be re-oxidized through the illuminations affect on PSI. A new Fod value may be measured and used for corrections to the quenching coefficients. This assumes the PSI acceptors are properly activated, which may not be the case in a dark adapted sample. therefore the Fod determination should be done after induction of photosynthesis has been done for several minutes. (puddle model) (Van Kooten & Snel, 1990)
qL
is the lake model quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
Equations for quenching parameters:
qP
= (Fm’- F) / (Fm’-Fo’)
NPQ
= (Fm - Fm’) / (Fm’)
NPQ
= qE + qT + qI
qE
= Fm’ after rapid relaxation is complete with the actinic light turned off usually one to ten minutes - Fm’ during steady state fluorescence with actinic light on/Fm’ at steady state.
qT
= Fm’ after rapid relaxation is complete usually with the actinic light turned off usually one hour - Fm’ at qE /Fm’ at steady state.
qI
= Fm-Fm’ at qT/ Fm’ at steady state.
qN
= Fm - Fm’/ Fm-Fo
qL
= qP(Fo’/F’)
Y(NO)
= 1/NPQ +1 + qL((Fm/Fo)-1)
Y(NPQ)
= 1 - Y - Y(NO)
1
= qL + Y(NPQ) + Y(NO)
Relative Electron Transport Rate
Relative Electron Transport Rate, written ETR
µmls = (Y) (PAR) (.84) (.5) = (quantum yield of PSII) (measured photosynthetically active radiation measured in uMols quanta m-2 s-1.)( leaf absorption coefficient)(fraction of absorbed light by PSII antennae). ETR is valuable for many types of plant stress investigations. This relative form of ETR provides a great deal of information as yield varies with PAR radiation (light intensity). For more information see the stress guide listed on this web site.
Absolute electron transport rate is measured by CO2 gas exchange measurements.
Relative ETR does not correlate exactly because while most of radiation is absorbed in the upper layers and provide fluorescent information, some radiation does enter lower layers and the information is not captured in fluorometry. CO2 gas exchange ETR includes information from deeper layers. (U. Schreiber 2004).
Nitrogen deficiency Test (FRFex360/FRFex440)
This Test is available with the purchase of the Universal Par clip.
Nitrogen Stress in plants can be determined by the ratio of UV excited and blue excited far red fluorescence, (FRFex360/FRFex440). Unlike leaf absorption techniques used for nitrogen testing, nitrogen stress can be distinguished from sulfur stress with this measurement (Sampson 2000). FRFex360/FRFex440 measures the concentration of UV absorbing compounds in the leaf epidermis. Based on the Carbon/nutrient balance hypothesis, excess fixed carbon relative to nutrient uptake stimulates the Shikimate acid pathway. This creates production of phenolic and other carbon based compounds that reside primarily in the epidermis (Cerovic 1999), (Price 1989), (Waterman and Mole 1994). A decrease of this ratio indicates higher concentrations of phenolic compounds due to nitrogen stress. These compounds absorb UV light and cause a decrease in excitation of chlorophyll molecules in the mesophyll. The blue light fluorescence acts as a measuring standard because it passes through the epidermis unaffected by these compounds. Therefore a ratio of unabsorbed UV light far red fluorescence to unabsorbed blue light far red fluorescence provides a sensitive test for nitrogen deficiency. It was found by Sampson (Sampson 2000) that sulfur deficiency did not affect the ratio.
The nitrogen test is a fast test taking less than two second. One thousand measurements are made and the average value is reported. The test can be done with steady state fluorescence.
With this test field plants will measure lower ratios than green house plants and older leaves will measure lower ratios than newer leaves due to the creation of some flavenoids by solar UV exposure. Controlling these variables is important. One should choose the same leaf and leaf age on plants to be measured. A plant that is without nitrogen stress can be used as a standard to set the ratio at one. Take care to use a field plant for field tests, and a green house plant for green house tests.
OJIP stress testing
OJIP is a fast, dark adapted test protocol that uses a high signal capture rate for analysis of fluorescence changes with emphasis on the initial fluorescence rapid rise kinetics using strong actinic light. In the initial fluorescence kinetic rise it has been found that the resulting curve displays intermediate peak values before reaching Fm, or P, maximum Fluorescence. These intermediate peaks or steps are designated J, I, and P with O being the initial measured fluorescence signal value after 20 µsec (Strasser 2004).
Besides the JIP steps an additional step called the K step appears during specific types of stress. (R. J. Strasser 2004).
Stress identification example: The K step = F270µs (a fluorescence step that appears 270 microseconds after the actinic light is turned on) in the OKJIP test. The K peak is activated when the OEC, or oxygen evolution cycle, is affected. Research has shown that heat stress, nitrogen stress, and water stress can activate the K peak (R. J. Strasser 2004). The performance index parameter captures this information.
Performance index: PI
Probably the most used parameter in the OJIP test is performance index: PI. Stresses that do not directly affect PSII do not cause a decrease of the Fv/Fm-value. PI was developed in an effort to solve that problem (G. Schansker 2008).
Performance index, PI, is an indicator of three main attributes, which determine potential photosynthetic activity; reaction centers density, probability that an absorbed photon is used for a charge separation, and forward electron transfer. The idea is that if a stress will affect any of these components, the effect will show up in the performance index (G. Schansker http://come.to/bionrj). In other words, on a less mechanistic level, besides capturing Fm, and Fo, information as found in Fv/Fm, PI also incorporates O and J information and the initial slope of O-K.
Measured and calulated OJIP Values with direct readout
1. Ft
= Fluorescence at the onset of actinic illumination
2. F20µs
= F20µs; fluorescence intensity at 20 µs =
O location
3. F70µs
= fluorescence at 70µs
4. F270µs
= fluorescence at
270µs =step K
5. Fj
= fluorescence intensity at
J-step
(at 2 ms)
6. Fi
= fluorescence intensity at
I-step
(at 60 ms)
7. FP
= maximal fluorescence intensity = Fm =
P-step
8. tFm
= time to reach maximum fluorescence intensity
9. Area
= area above the fluorescence curve to Fm (or P). If area decreases compared to a non stressed plant, the stress is linked to donor side related stress. If Area increases, the stess factor is an acceptor side linked stress.
10. PIABS
= Perfomance index on an absorption basis.
11. Fv/Fm
= Optimal quantum yield.
Derived Values in Measuring File
1. Fv
= Fm - Fo (maximal variable fluorescence)
2. VJ
= (Fj - Fo) / (Fm - Fo) (variable fluorescence for J step) represents the fraction of closed reaction centers at the J step.
3. Fm / Fo
= Fm / Fo
4. Fv / Fo
= Fm - Fo / Fo
5. Fv / Fm
= Tro /ABS or Fm - Fo / Fm. Optimal fluorescence yield.
6. Mo
= 4(F270us – F20us) / Fm-F20us) Approx initial slope in ms-1.
7. Sm
= area / Fm – F20us Multiple turn-over, or reductions of QA. Normalized Area used to compare different samples.
8. Ss
= Vj/Mo The smallest Sm turn-over (single turn-over or reduction of QA) representing the area corresponding to the O to J phase.
9. N
= Sm (Mo) (I / Vj) Turn-over number: How many timese QA has been reduced from 0 to tFM.
10. Vav
= 1 - (Sm/tFm) Average relative variable fluorecense from t=0 to tFm.
(source: http://www.optisci.com/cf.htm)
The following is a generalized description of the photosynthesis light reaction and the value of chlorophyll fluorescence in photosystem II. For more specific information please refer to papers cited in this discussion or contact Opti-Sciences for more information.
Light energy utilized in photosynthesis by higher plants and algae cells is collected first by an antenna pigment system and transferred to reaction centers where light quanta are converted to chemical energy by chlorophylls in a protein environment. Electron transfer takes place in the reaction center when a chlorophyll molecule transfers an electron to a neighboring pigment molecule. Pigments and protein involved in this primary electron transfer define the reaction center. This initial electron transfer is also called charge separation.
Competing models of energy capture and transfer exist. In the “puddle model” of Photosystem PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The “lake model” is considered more realistic for terrestrial plants.
Reaction centers are of two types, Photosystem II (PSII), and Photosystem I (PSI). both are located in the thylakoid membrane of a chloroplast of higher plants. In bacteria they are in a membrane surrounding the cytoplasm or in more intricate constructs. All plants the produce oxygen have both types of reaction centers.
It is in PSII that oxygen evolution and the splitting of water occurs. To reduce the PSII reaction center an electron is pulled from the water splitting complex.
After charge separation, electrons flow to other nearby plastoquinone molecules in the thylakoid membrane by oxidation reduction reactions. They act as energy transfer molecules in electron transport chain.
The next stop is a cytochrome b6f complex. Eventually this complex supplies an electron to reduce PSI. PSI then goes through a somewhat similar process to PSII, and eventually produces NADPH.
In addition, protons created from PSII functions, the splitting of water, and the reaction in the cytochrome b6f complex, enter the thylakoid lumen and are used by an ATP pump in the thylakoid membrane in the presence of ATP synthase, to create ATP from ADP. Both ATP and NADPH are used as energy sources to drive the Calvin Benson Cycle of carbon fixation.
PSI goes through a similar process to PSII, however, the low fluorescent signal of PSI does not vary with plant stress or with various photosynthetic functions as it does with PSII. Therefore PSII is of primary interest. Factors such as light levels, light quality, water availability, nutrient availability, heat, cold, herbicides, pesticides, pollution, heavy metals, disease, and genetic make up can all have an impact on CO2 assimilation, plant health and condition. They also are reflected in the fluorescence signal in PSII. Therefore, by using a chlorophyll fluorometer one can quantify the impact of these factors on plants to improve breeding and production programs, and to better understand plant functions.
The most prominent pigments that absorb this energy are chlorophyll-a and chlorophyll-b. Other accessory pigments may be involved such as carotenoids, or phycobilins in cyanobacteria, or bacteriochlorophyll in some bacteria.
Light energy absorbed initially by the antenna and transferred to the reaction centers is channeled by a number of different processes including photochemistry, photo-protective heat dissipation, other heat dissipation and about 3%-9% of the light energy absorbed by chlorophyll pigments is re-emitted as fluorescence. The emission peak is of a longer wavelength than the excitation energy. This effect was first observed more than 100 years ago, when N.J.C. Müller (1874) by visually using colored glass filters. He also noted that fluorescence changes that occur in green leaves are correlated with photosynthetic assimilation. Lack of appropriate technical equipment prevented a more detailed investigation of this phenomenon. The light energy drives photosynthetic electron transport through PSII and PSI leading to the oxidation of water, oxygen evolution, the reduction of NADP+ to NADPH, membrane proton transport and ATP synthesis.
The loss of light energy from the reaction center as fluorescence comes primarily from the PSII reaction. When the chloroplast or leaves have been dark-adapted, the pools of oxidation-reduction intermediates for the electron transport pathway return to an oxidized state. Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescent light emission from PSII followed by a decrease to steady state fluorescence as CO2 fixation starts to occur. Changes in the intensity of the fluorescence emission are a sensitive reflection of changes in the photosynthetic apparatus. Following many years of study, chlorophyll fluorescence has been shown to represent changes in the health and function of the photosynthetic process. This includes all the reactions from the oxidation of water, charge separation, electron transport, development of the electrochemical gradient, the photo-protective mechanisms of the xanthophyll cycle, and the change in ph of the thylakoid lumen. Even changes in the plant that affect stoma opening and gas exchange with the atmosphere are reflected by changes in the fluorescence characteristics of a leaf. Vast amounts of research have shown excellent correlation between carbon fixation and chlorophyll fluorescence data.
This is a typical Dark-Light kinetic pulse modulated trace.
Definitions are described below.
Figure 1-1 • Dark–Light Pulse modulated Chlorophyll Fluorescence Trace
Definitions:
Dark adapted
– This is a term that means that the area of the plant, or the entire plant, to be measured has been in the dark for an extended period of time before measurement. Dark adaption requirements may vary for dark adapted tests. Dark adaption times of ten minutes to 60 minutes are common, and some researchers use pre-dawn values. Dark adapted measurements include Fv/Fm, OJIP measurements, and most non-photochemical quenching parameters. Fv/Fm and most OJIP measurements take only a second to make a measurement. Non-photochemical quenching parameters take longer.
Light adapted
– Yield measurements are made when a sample is already exposed to existing light conditions. No dark adaption is required. When a sample is exposed to light it normally takes several second to a few minutes for photosynthesis to reach steady state. To obtain a reliable yield measurement photosynthesis must reach steady state. This is not a concern when using ambient sunlight or artificial greenhouse light however clouds and light fleks below a canopy level can cause problems. If one uses a built in fluorometer illuminator to measure yield make sure that steady state photosynthesis has been reached. This is normally a very fast test that take a second or two and it has the added advantage that it does not require dark adaption. Light adapted measurements include Yield or ΔF/Fm’.
PAR
– Photosynthetically Active Radiation between 400nm and 700nm. Measured in either µmls or µE The Term PAR means photosynthetically active radiation in the wave band between 400-700 nm. PAR can be measured in different dimensions such as Watts per meter or in micro- Einsteins or micro-moles. When using a PAR Clip, dimensions will always be in the equivalent terms, micro-Einsteins, or micro-moles
PPFD
- Photosynthetic Photon Flux Density is the photon flux density of PAR. Measured in either or. PPFD, or “photosynthetic photon flux density”, is the number of PAR photons incident on a surface in time and area dimensions. These terms are equivalent for PAR Clip leaf radiation measurements. Furthermore, both can be presented in either of the equivalent dimensions, micro-moles (µmls) or micro-Einsteins (µE).
µmls
- is a micro mol (also abbreviated µmol. This a dimension that involves both time and area. It is the equivalent to the micro Einstein. Both terms have been used extensively in biology and radiation measurements.
µE
– is a micro Einstein This a dimension that involves both time and area. It is the equivalent to the micro mol. Both terms have been used extensively in biology and radiation measurements.
1 µE = 1 µmol m-2 s-1 = 6.022 x1023 photons m-2 s-1
Actinic Light source
– This is any light source that drives photosynthesis. It may be the Sun, or an artificial light. Higher end fluorometers contain one or more built-in artificial actinic light sources for experimentation with specific repeatable radiation (or light) levels.
Saturation pulse
– is short pulse of very intense light designed to fully reduce a leaf's PSII system. It is created by an artificial light source in all modulated fluorometers.
Far red light
– is a light source that provides light above 730 nm to allow the transfer of electrons to PSI and allow the rapid re-oxidation of PSII.
Modulated light Source
– This is the measuring light source in a modulated fluorometer. It is a precision light diode that is usually either red or blue. Most fluorometers usually have one or the other. The OS5p has both. They use a very low intensity, usually between 0.2 µmls and 1µml, to allow measurement without driving photosynthesis.
Fo
– is the dark adapted initial minimum fluorescence.
Fm
– is maximal fluorescence measured during the first saturation pulse after dark adaption.
Fs
– also known as F’ is the fluorescence level related to the actinic light and is a reflection of level of photosynthetic activity.
Fms
– also known as Fm’ is the saturation pulse value that is not dark adapted. They are lowered values due to NPQ or non-photochemical quenching. When this parameter has reached steady state, it is used to calculate photosynthetic Yield along with Fs. Fms at steady state is also used to calculate qN, NPQ, qP, qL, Y(NPQ), Y(NO), qE, qT, and qI.
Fod
– also known as Fo’ is the minimal value after the far red light is turned on for several seconds after the actinic light has been turned off. It represents Fo with non-photochemical quenching.
Ft
– is the current instantaneous fluorescent signal.
Fv/Fm
= (Fm – Fo) / Fm This is a dark adapted test used to determine Maximum quantum yield. This ratio is an estimate of the maximum portion of absorbed quanta used in PSII reaction centers. It is important to properly dark adapt samples for this test. Fo will be raise and Fm will be lowered if dark adaption is inadequate. Since dark adaption requirement can vary with species, varieties, mutants, and sun vs. shade leaves testing should be done to ensure proper dark adaption. (Kitajima and Butler, 1975)
Y
= (Fms – Fs) / Fms This test is also known as ΔF/Fm’. Yield of PSII is a light adapted test normally taken at steady state photosynthesis levels. It provides a measure of actual or effective quantum yield. This ratio is an estimate of the effective portion of absorbed quanta used in PSII reaction centers. (Genty, 1989)
Quenching Parameters
Quenching parameters allow the quantification of both the effective photochemical state of the PSII regarding the fraction of PSII centers that remain open or oxidized at any time, and the non-photochemical photosynthetic mechanisms involved in photo-protection, state 1 and state 2 transition quenching, photo-inhibitor and photo-damage.
Puddle Model and Lake Model of Antennae
In the “puddle model” of Photo-system PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The lake model has proven to be the more realistic model for terrestrial plants. Research has shown that some marine species have a distinct antenna architecture. With this in mind, some of the quenching parameters of the “puddle model” are incompatible with those of the “lake model”. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
qP = (Fm' - F) / (Fm' - Fo')
NPQ = (Fm - Fm') / Fm'
NPQ = qE + Qt + qI
qN = (Fm - Fm') / (Fm - Fo)
qL = qP(Fo'/F')
Y(NO) = 1/NPQ + 1 + qL((Fm/Fo)-1)
Y(NPQ) = 1 - Y - Y(NO)
Understanding the graphs above:
In each case the sample is first dark adapted. The test is started and Fo, or minimal fluorescence, is measured without actinic light. Then a saturation pulse occurs and completely closes all receptors in PSII by completely reducing PSII. Fm, maximal fluorescence, is the result. After the saturation pulse, an actinic light is turned on and the fluorescent signal declines slowly with the onset of CO2 fixation until it reaches steady state. Photochemical quenching a measure of open PSII centers, photo-protective non-photochemical quenching and other heat dissipation mechanisms occur. Saturation pulses during steady state photosynthesis provide Fm', maximal fluorescence in this situation after NPQ has reached equilibrium with photochemistry. qP, or qL in the lake model, now represents the fraction of PSII receptors that remain open or oxidized. F (or Fs) represents fluorescence related to current steady state photochemical levels. At the point where the actinic light is turned off, far red illumination is turned on to allow the transfer of electrons quickly to reduce PSI, and allow the re-oxidation of PSII. Fo' represents this value with un-relaxed non-photochemical quenching. The rising values of the saturation pulses after the actinic light has been turned off represent the relaxation of NPQ over time. A portion of NPQ, qE (or Y(NPQ) in the lake model), represents ph and the xanthophyll cycle.photo-protection mechanisms of thylakoid lumen The remainder of NPQ represents qT, and qI, (or Y(NO) in the lake model). qT is quenching due to state 1 and state 2 transitions and is negligible in higher plants. qI represent photo-inhibition and photo-damage.
Definitions for Quenching Parameters:
NPQ
is non-photochemical quenching and is a measure of heat dissipation and a combined total for the combination of photo-protective mechanisms, state 1 and state 2 transition quenching, and photo-inhibition and photo-damage. NPQ = qE + qT + qI. NPQ is an alternate expression of non-photochemical quenching. It provides an estimate of quenching without knowledge of Fo. The advantage of NPQ over qN depends on the specific application. NPQ is more heavily affected by non-photochemical quenching that reflects heat-dissipation of excitation energy in the antenna system. So it may be thought of as an indicator of 'excess excitation energy'. Alternatively, NPQ is relatively insensitive to the part of non-photochemical quenching associated with qN values lower than 0.6 This range of qN is affected by ΔPH of the thylakoid lumen which is an important aspect of photosynthetic regulation. (Bilger & Björkman, 1990)
qN
is similar to NPQ but requires Fod or Fo’ in the calculation. qN is defined as the coefficient of non-photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN value. By using the Far-Red source after actinic illumination, the PSII acceptors re-oxidized and PSI is reduced. A new Fod value is measured and used for corrections to the quenching coefficients. (puddle model) (Van Kooten & Snel, 1990)
qE
is the quenching parameter that represents the photo-protective mechanisms in the leaf that allow rapid compensation for changes in light levels due to cloud cover and increased light intensity. It is directly related to Δph of the thylakoid lumen and the xanthophyll cycle. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
qT
is the quenching parameter that represents state 1 and state 2 transitions. This value is negligible in higher plants. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
qI
is the quenching parameter that represents photo-inhibition and photo-damage. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)
Y(NPQ)
is a lake model quenching parameter that represents heat dissipation related to all photo-protective mechanisms. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
Y(NO)
is a lake model quenching parameter that represents all other components of non-photochemical quenching that are not photo-protective. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
qP
is the quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. qP is defined as the coefficients of photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN and qP values. By using the Far-Red source for post illumination, the PSII acceptors may be re-oxidized through the illuminations affect on PSI. A new Fod value may be measured and used for corrections to the quenching coefficients. This assumes the PSI acceptors are properly activated, which may not be the case in a dark adapted sample. therefore the Fod determination should be done after induction of photosynthesis has been done for several minutes. (puddle model) (Van Kooten & Snel, 1990)
qL
is the lake model quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)
Equations for quenching parameters:
qP
= (Fm’- F) / (Fm’-Fo’)
NPQ
= (Fm - Fm’) / (Fm’)
NPQ
= qE + qT + qI
qE
= Fm’ after rapid relaxation is complete with the actinic light turned off usually one to ten minutes - Fm’ during steady state fluorescence with actinic light on/Fm’ at steady state.
qT
= Fm’ after rapid relaxation is complete usually with the actinic light turned off usually one hour - Fm’ at qE /Fm’ at steady state.
qI
= Fm-Fm’ at qT/ Fm’ at steady state.
qN
= Fm - Fm’/ Fm-Fo
qL
= qP(Fo’/F’)
Y(NO)
= 1/NPQ +1 + qL((Fm/Fo)-1)
Y(NPQ)
= 1 - Y - Y(NO)
1
= qL + Y(NPQ) + Y(NO)
Relative Electron Transport Rate
Relative Electron Transport Rate, written ETR
µmls = (Y) (PAR) (.84) (.5) = (quantum yield of PSII) (measured photosynthetically active radiation measured in uMols quanta m-2 s-1.)( leaf absorption coefficient)(fraction of absorbed light by PSII antennae). ETR is valuable for many types of plant stress investigations. This relative form of ETR provides a great deal of information as yield varies with PAR radiation (light intensity). For more information see the stress guide listed on this web site.
Absolute electron transport rate is measured by CO2 gas exchange measurements.
Relative ETR does not correlate exactly because while most of radiation is absorbed in the upper layers and provide fluorescent information, some radiation does enter lower layers and the information is not captured in fluorometry. CO2 gas exchange ETR includes information from deeper layers. (U. Schreiber 2004).
Nitrogen deficiency Test (FRFex360/FRFex440)
This Test is available with the purchase of the Universal Par clip.
Nitrogen Stress in plants can be determined by the ratio of UV excited and blue excited far red fluorescence, (FRFex360/FRFex440). Unlike leaf absorption techniques used for nitrogen testing, nitrogen stress can be distinguished from sulfur stress with this measurement (Sampson 2000). FRFex360/FRFex440 measures the concentration of UV absorbing compounds in the leaf epidermis. Based on the Carbon/nutrient balance hypothesis, excess fixed carbon relative to nutrient uptake stimulates the Shikimate acid pathway. This creates production of phenolic and other carbon based compounds that reside primarily in the epidermis (Cerovic 1999), (Price 1989), (Waterman and Mole 1994). A decrease of this ratio indicates higher concentrations of phenolic compounds due to nitrogen stress. These compounds absorb UV light and cause a decrease in excitation of chlorophyll molecules in the mesophyll. The blue light fluorescence acts as a measuring standard because it passes through the epidermis unaffected by these compounds. Therefore a ratio of unabsorbed UV light far red fluorescence to unabsorbed blue light far red fluorescence provides a sensitive test for nitrogen deficiency. It was found by Sampson (Sampson 2000) that sulfur deficiency did not affect the ratio.
The nitrogen test is a fast test taking less than two second. One thousand measurements are made and the average value is reported. The test can be done with steady state fluorescence.
With this test field plants will measure lower ratios than green house plants and older leaves will measure lower ratios than newer leaves due to the creation of some flavenoids by solar UV exposure. Controlling these variables is important. One should choose the same leaf and leaf age on plants to be measured. A plant that is without nitrogen stress can be used as a standard to set the ratio at one. Take care to use a field plant for field tests, and a green house plant for green house tests.
OJIP stress testing
OJIP is a fast, dark adapted test protocol that uses a high signal capture rate for analysis of fluorescence changes with emphasis on the initial fluorescence rapid rise kinetics using strong actinic light. In the initial fluorescence kinetic rise it has been found that the resulting curve displays intermediate peak values before reaching Fm, or P, maximum Fluorescence. These intermediate peaks or steps are designated J, I, and P with O being the initial measured fluorescence signal value after 20 µsec (Strasser 2004).
Besides the JIP steps an additional step called the K step appears during specific types of stress. (R. J. Strasser 2004).
Stress identification example: The K step = F270µs (a fluorescence step that appears 270 microseconds after the actinic light is turned on) in the OKJIP test. The K peak is activated when the OEC, or oxygen evolution cycle, is affected. Research has shown that heat stress, nitrogen stress, and water stress can activate the K peak (R. J. Strasser 2004). The performance index parameter captures this information.
Performance index: PI
Probably the most used parameter in the OJIP test is performance index: PI. Stresses that do not directly affect PSII do not cause a decrease of the Fv/Fm-value. PI was developed in an effort to solve that problem (G. Schansker 2008).
Performance index, PI, is an indicator of three main attributes, which determine potential photosynthetic activity; reaction centers density, probability that an absorbed photon is used for a charge separation, and forward electron transfer. The idea is that if a stress will affect any of these components, the effect will show up in the performance index (G. Schansker http://come.to/bionrj). In other words, on a less mechanistic level, besides capturing Fm, and Fo, information as found in Fv/Fm, PI also incorporates O and J information and the initial slope of O-K.
Measured and calulated OJIP Values with direct readout
1. Ft
= Fluorescence at the onset of actinic illumination
2. F20µs
= F20µs; fluorescence intensity at 20 µs =
O location
3. F70µs
= fluorescence at 70µs
4. F270µs
= fluorescence at
270µs =step K
5. Fj
= fluorescence intensity at
J-step
(at 2 ms)
6. Fi
= fluorescence intensity at
I-step
(at 60 ms)
7. FP
= maximal fluorescence intensity = Fm =
P-step
8. tFm
= time to reach maximum fluorescence intensity
9. Area
= area above the fluorescence curve to Fm (or P). If area decreases compared to a non stressed plant, the stress is linked to donor side related stress. If Area increases, the stess factor is an acceptor side linked stress.
10. PIABS
= Perfomance index on an absorption basis.
11. Fv/Fm
= Optimal quantum yield.
Derived Values in Measuring File
1. Fv
= Fm - Fo (maximal variable fluorescence)
2. VJ
= (Fj - Fo) / (Fm - Fo) (variable fluorescence for J step) represents the fraction of closed reaction centers at the J step.
3. Fm / Fo
= Fm / Fo
4. Fv / Fo
= Fm - Fo / Fo
5. Fv / Fm
= Tro /ABS or Fm - Fo / Fm. Optimal fluorescence yield.
6. Mo
= 4(F270us – F20us) / Fm-F20us) Approx initial slope in ms-1.
7. Sm
= area / Fm – F20us Multiple turn-over, or reductions of QA. Normalized Area used to compare different samples.
8. Ss
= Vj/Mo The smallest Sm turn-over (single turn-over or reduction of QA) representing the area corresponding to the O to J phase.
9. N
= Sm (Mo) (I / Vj) Turn-over number: How many timese QA has been reduced from 0 to tFM.
10. Vav
= 1 - (Sm/tFm) Average relative variable fluorecense from t=0 to tFm.
(source: http://www.optisci.com/cf.htm)
February 21, 2012
Aurantiochytrium sp
Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101.
Hong WK, Rairakhwada D, Seo PS, Park SY, Hur BK, Kim CH, Seo JW.
Source
Microbe-based Fusion Technology Research Center, Jeonbuk Branch Institute, KRIBB, Jeongeup, Jeonbuk 580-185, South Korea.
Abstract
In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.
February 16, 2012
Reproduction
Algae are able to reproduce sexually and asexually. Sexual reproduction in algae is very rare because of the complexity of gamete transportation and because of this most algae may never get to reproduce sexually. There are two types of asexual reproduction in which algae take part in, they are daughter colony formation and sporulation. In daughter colony formation a group of algae will begin to create small duplicates of itself inside of the parent algae. After a while the parent will burst and let loose the daughter algae. Aside from daughter colony formation, algae also use sporulation as a means to reproduce. Sporulation is used by algae more than daughter colony formation. It consists of the parent algae creating reproductive cells within the cell walls. The zoospores(reproductive cells) escape from the parent cell and attach themselves to surface where they turn into filaments. Algae also reproduce sexually and the combination of gametes identifies isogamy and heterogamy,which are two classes of sexual reproduction. Isogametes are gametes that are alike in size and shape. In isogamy, the gametes that are being combined do not have any physical peculiarity amid male and female gametes. The isogametes side with each other's flagellar extremities touching and after a short time they form one, immobile diploid zygote. Unlike isogamy, heterogamy is the uniting of a male gamete and a female gamete. The algae produces sperm in large amounts and the egg cell that is produced is bigger than the sperm and unlike the sperm cell, it is immobile.
February 14, 2012
February 12, 2012
January 28, 2012
Microalgae - Growth
There are 5 reasonably well defined phases of algal growth in batch cultures (Fogg and Thake, 1987)
1 lag; 2 exponential; 3 declining growth rate; 4 stationary; 5 death. Each of the phases is described below and in Fig 1 (goto fig). Alternatively Jump straight to growth rate equation
Lag phase
The condition of the innoculum has a strong bearing on the duration of the lag phase (Spencer, 1954). An innoculum taken from a healthy exponentially growing culture is unlikely to have any lag phase when transferred to fresh medium under similar growth conditions of light, temperature and salinity. In general the length of the lag phase will be proportional to the length of time the innoculum has been in phases 3-5. A lag phase may also occur if the innoculum is transferred from one set of growth conditions to another.
Exponential phase and calculating growth rates
The growth rate of a microalgal population is a measure of the increase in biomass over time and it is determined from the exponential phase. Growth rate is one important way of expressing the relative ecological success of a species or strain in adapting to its natural environment or the experimental environment imposed upon it. The duration of exponential phase in cultures depends upon the size of the innoculum, the growth rate and the capacity of the medium and culturing conditions to support algal growth. Biomass estimates need to be plotted over time, and logistical constraints determine their frequency but once every one to two days is generally acceptable. Cell count and dry weight are common units of biomass determination. In-vivo fluorescence and turbidity can be used as surrogate measures which enable higher temporal resolution due to the logistical ease of measurement (correlations between fluorescence or turbidity and cell count can be established but they will become less accurate as experimental conditions are varied. For example cell fluorescence may vary with temperature so an experiment with several test temperatures may need correlations to be determined for each temperature. Correlations also become innacurate as cultures move into stationary phase so fluorescence can not be used as a substitute for cell counts where an estimate of final cell yield is needed). Once the growth phase has been plotted (time on x-axis and biomass on logarthmic y-axis) careful determination of the exponential (straightline) phase of growth is needed. Two points, N1 and N2, at the extremes of this linear phase (see fig below) are taken and substituted into the equation
Where N1 and N2 = biomass at time1 (t1) and time2 (t2) respectively; Levasseur et al (1993).
Divisions per day and the generation or doubling time can also be calculated once the specific growth rate is known.
Divisions per day ; Div.day-1 = K' / Ln2
Generation time ; Gen' t = 1 / Div.day-1
For healthy cells of a robust species, small innoculums equal to 0.5 % of the volume of the new culture will normally generate new healthy cultures. If the species is delicate or the culture less healthy then a larger innoculum of ~ 10% may be needed to support a new culture. (Many of the stock cultures in CMARC are transferred with a 0.5 to 1 mL innoculum into 40 mL fresh medium representing a 1.25 % to 2.5% innoculum).
Declining growth
Declining growth normally occurs in cultures when either a specific requirement for cell division is limiting or something else is inhibiting reproduction. In this phase of growth biomass is often very high and exhaustion of a nutrient salt, limiting carbon dioxide or light limitation become the primary causes of declining growth. When biomass is increasing exponentially a constant supply of air (or air plus CO2) will only be in balance with growth at one point during exponential phase. At low cell densities too much CO2 may lower the pH and depress growth. CO2 limitation at high cell densities causes any further biomass increase to be linear rather than exponential (with respect to time) and proportional to the input of CO2.
Light limitation at high biomass occurs when the cells absorb most of the incoming irradiation and individual cells shade each other (hence the often quoted term “self-shading”). Growth in most phytoplankton is saturated at relatively low irradiances of 50-200 μmol. photons m-2 s –1 (cf noontime irradiance at the water surface in the tropics of 2000 μmol. photons m-2 s –1 ). Microalgae are therefore generally well adapted to surviving conditions of low incident light and may survive for extended periods under these conditions.
Stationary phase
Cultures enter stationary phase when net growth is zero, and within a matter of hours cells may undergo dramatic biochemical changes. The nature of the changes depends upon the growth limiting factor. Nitrogen limitation may result in the reduction in protein content and relative or absolute changes in lipid and carbohydrate content. Light limitation will result in increasing pigment content of most species and shifts in fatty acid composition. Light intensities that were adequate or optimal for growth in the first 3 phases can now become stressful and lead to a conditon known as photoinhibition. It is important that while the measured light intensity within the culture will decrease with increasing biomass if the incident illumination is maintained relatively high then a large proportion of cells may become stressed, photoinhibit and the culture can be pushed into the death phase. This is especially the case if the culture is also nutrient stressed. It is preferable for many species to halve or further reduce the incident light intensity when cultures enter stationary phase to avoid photoinhibition. Some green algae and cyanobacteria may survive in the vegetative state (ie not as cysts) for over 6 – 12 months under very low illumination. For many species lower temperature combined with lower irradiance can further reduce stress. Survival is inversely proportional to temperature but only in darkness. Some algal species may form long lived cysts or temporary resting cysts with greatly reduced metabolism under different conditions of stress. The shut down of many biochemical pathways as stationary phase proceeds means that the longer the cells are held in this condition the longer the lag phase will be when cells are returned to good growth conditions.
Death phase
When vegetative cell metabolism can no longer be maintained the death phase of a culture is generally very rapid, hence the term “culture crash” is often used. The steepness of the decline is often more marked than that represented in the accompanying growth figure. Cultures of some species will lose their pigmentation and appear washed out or cloudy, whereas cells of other species may lyse (no recognizable cells) but the culture colour will be maintained. The latter is an important consideration and one reason why colour should not be relied upon to guage culture health. Bacteria which may have been kept in check during exponential and early stationary phase may “explode” as cell membrane integrity become progressivley compromised or leaky and a rich carbon source for bacterial growth is released. Free pigment and bacterial growth are further reasons why measures of turbidity or fluorescence should not be used beyond early stationary phase as surrogate biomass indicators, or especially as indicators of culture health. Occassionally cell growth of some species can reoccur after a culture has apparently died. In this instance most vegetative cells will have died, and possibly most of the bacteria, releasing nutrients back into the media. Then either the very few remaining vegetative cells or more likely germination of cysts or temporary cysts will be able to fund this secondary growth.
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Fig 1 General pattern of microalgal growth in batch cultures
source:http://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm
January 20, 2012
January 16, 2012
January 15, 2012
ALGAE BIODIESEL CHARACTERISTIC
Characteristics of algae biodiesel that differ from petro diesel:
1. Algae biodiesel has virtually no sulfur content.
2. Biodiesel has superior lubricating properties, reducing fuel system wear, and increases the life of fuel injection equipment.
3. Algae biodiesel has more aggressive solvent properties than petro diesel and will dissolve leftover varnish residue. Fuel filters should be changed shortly after introducing biodiesel into systems formerly running on petrodiesel to avoid clogging
4. Biodiesel has about 5-8 percent less energy density than petrodiesel, but with its higher combustion efficiency and better lubricity to partially compensate, its overall fuel efficiency decrease is only about 2 percent.
5. The cloud point, or temperature at which pure (B100) biodiesel starts to gel, is about 32 0F. A blend of B20 (20% biodiesel, 80% petrodiesel) generally does not gel in cold weather. Various additives will lower the gel point of B100.
6. Biodiesel's flash point (lowest temperature at which it can vaporize to form an ignitable mixture in air) is 2660F, significantly higher than petrodiesel's 1470F, or gasoline's 520F.
7. Biodiesel reduces particulate matter by about 47 percent as compared to petroleum diesel. Biodiesel has less dangerous particulate matter because it reduces the solid carbon fraction on the particulate matter while increasing the amount of oxygen.
Advantages of biodiesel produced from algae:
1. Higher yield and hence – hopefully – lower cost
2. The most significant benefit is however in the yield of algal oil, and hence biodiesel. According to some estimates, the yield (per Acre say) of oil from algae is over 200 times the yield from the best-performing plant/vegetable oils. While soybean typically produces less than 50 gallon of oil per acre and rapeseed generates less than 130 gallon per acre, algae can yield up to 10,000 gallons per acre.
3. Algae can grow practically in every place where there is enough sunshine
4. The biodiesel production from algae also has the beneficial by-product of reducing carbon and NOx Emissions from power plants, if the algae are grown using exhausts from the power plants.
5. Algae produce a lot of polyunsaturates, which may present a stability problem since higher levels of polyunsaturated fatty acids tend to decrease the stability of biodiesel. But polyunsaturates also have much lower melting points than monounsaturates or saturates, thus algal biodiesel should have much better cold weather properties than many other bio-feedstock. Since one of the disadvantages of biodiesel is their relatively poor performance in cold temperatures, it appears that algal biodiesel might score well on this point.
http://www.oilgae.com/algae/oil/biod/char/char.html Algal Biodiesel Characteristics & Properties
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