Development of In Situ Sensors for Chlorophyll Concentration Measurement
ton absorption coefficient, the phytoplankton photosynthetic . best biomass proxy by providing simple relationships for both Pmax and α. . chlorophyll a fluorescence, which is due to the absorption by all photosynthetic pig-. Running title: The size-scaling of phytoplankton chlorophyll content. 8 Phytoplankton cells adjust the Chl-a to carbon biomass ratio imbalance between the rate of light absorption and the energy demands for photosynthesis . relationship between the FlowCAM measured fluorescence per cell and the. field estimates of phytoplankton biomass that can be directly correlated to Introduction to Phytoplankton & Photosynthetic Pigments . Illustration of how the in vivo chlorophyll a fluorescence sensor works on Hydrolab sondes taking of occasional grab samples used for quantitative laboratory analysis, a relationship.
They proposed a conceptually straightforward theoretical model to optimize the factors affecting the fluorescence-capture capability of the capillary-based probe. By measuring the fluorescence spectra of Cy5. Meanwhile, the intensity of collected LSE signal was doubled by reflection of interference filters F1 and F2. A commercial version named Aquatic Laser Fluorescence Analyzer [ 42 ] has been used in the field, but only in flow-through measurement. Very recently, a lensless miniature portable fluorometer [ 43 ] was designed to measure chlorophyll and CDOM concentration in aquatic environments.
In order to obtain better spatial resolution and light collection efficiency as well as smaller size, power, and cost, contact fluorescent imaging method was used. This method utilized LEDs for fluorescence excitation and a single contact fluorescent imaging pixel array for fluorescence collection.
Considering the low concentration of chlorophyll and overlap of excitation and emission light, researchers used absorption filters with ultrathin glass strips to attenuate excitation light.
Although the limit of detection was 0. It has been concluded that theoretical models and numerical simulations are necessary to improve the design of fiber probes. However, because of the small numerical aperture, variability, and low quantum yield, detection of fluorescence is challenging for a fiber-optic probe. Detecting Angle The angle between excitation and emission light is important for collection efficiency and minimizing interference.
As is known, the beams of excitation and emission are orthogonal in most benchtop and in situ fluorometers. However, for optimizing the structure and improving SNR of in situ instruments, other angles have also been reported as improving SNR. They used a blue diode laser for chlorophyll excitation peak nm. The positioning of both the excitation and the receiver diode on a single flat surface further reduced the possibility of recirculation within the sample volume due to mixing caused by irregularities in the probe shape.
A numerical simulation was developed to approximate the optical geometry of the dual-fiber-optic sensor. This permitted a visual representation of the fluorescence distribution within the sensor sampling volume. A Monte Carlo simulation was used to evaluate sampling variability associated with the number and distribution of particles within the sampling volume. It was proved that laboratory observations and previously published results were generally consistent with model predictions.
The exciting and measuring channels were of separate design, and the light beams were not orthometric. In this case, the fluorescence zone grew in size; meanwhile, it reduced the influence of laser radiation propagating inside the glass and penetrating into the receiving fiber.
In order to protect the photosensitive receiving part from direct and scattered sunlight, a metal construction was designed. The design as a whole improved reliability and reduced background illumination. An opposite structure was used in a polydimethylsiloxane-based microfluidic chip, which integrated filters, source, detector, and electronically controlled valves [ 48 ].
A very compact low-cost LED with a peak emission wavelength of nm served as the excitation source. A silicon detector, located on the side of the microchannel opposite the source, was placed as close as possible to the channel to maximize the captured fluorescence signal. To achieve the highest possible detection sensitivity from the opposite structure, two filters were used for detector shielding from the excitation source signal.
One filter was the excitation source filter, which passed the low wavelength signal nm while attenuating any signal the source produced at the fluorescence signal wavelengths nm. Detecting angle is related to optical structure and is crucial to receiving maximum fluorescence. By combining an appropriate detecting angle with a highly selective filter, interference signals can be minimized. As a result, the signal-to-noise ratio SNR can be maximized to improve the accuracy and stability.
Excitation Source The excitation source can greatly affect the quality of fluorescence signal because of its importance as a prerequisite of light-induced fluorescence measurement. Various light sources can be used to stimulate fluorescence of Chlsuch as lamps, lasers, and LED. Different light sources have their own advantages and disadvantages.
Thus, a well-selected light source and good excitation protocol can improve accuracy and reduce interference, size, and cost. Following are the most relevant achievements related to the scope of this paper.
Various Light Sources 3. Lamps Broadband lamps, which are bulky and have high power requirements, were used in initial benchtop fluorometers.
In order to minimize power and optimize the excitation of fluorescence, a flashing xenon lamp was chosen as the radiation source [ 4950 ]. However, broadband radiation has the potential to cause overlap with fluorescence.
Consequently, one or more blue optical filters were typically used to reduce interference from other spectra in the region. With the forward-scatter fluorescence geometry and Spectralon as the sample cell material, the sensitivity was increased.
The data show that as little as 0. Broadband lamps are also used in multiparameter instruments. This allowed the yellow substances, suspended particles, and phytoplankton to be measured simultaneously through absorption, scattering, and fluorescence.
Otherwise, the broadband excitation results in respective broadening of the Raman spectral band, an effect that significantly complicates its discrimination from the constituent fluorescence. Another reason for using broadband lamps is to calibrate the interference of other components in water.
As is known, single wavelength instruments meet the demands of low exposure of samples and fast response time.
On the other hand, single wavelength measurement does not allow measurement of interfering signals, where deconvolution using several spectral points is needed to extract contributions from individual components. Because of their size and power requirements, broadband lamps are seldom used for in situ measurement at present.
However, some special applications such as single-cell fluorescence excitation spectroscopy systems [ 54 ] and multivariate optical computing instruments [ 55 ] require super high power Xe arc lamps 75 W. Lasers Lasers emit coherent light beams of high intensity and directionality, which can improve selectivity and efficiency of excitation and reduce the spectral overlap between the water Raman scattering and fluorescence bands of aquatic constituents.
They used an air-cooled argon laser with a wavelength of nm as the excitation and used a He-Ne laser in the alignment procedure. A series of measurements indicated that ambient Chl concentrations could be detected in situ and could be calibrated using Raman scattering signal. Though lasers have many advantages, the earlier generation lasers have significant disadvantages, which are relatively large size, cost and power consumption, and a limited number of excitation wavelengths.
Laser diodes also provide high energy output, monochromaticity, and broad wavelength availability from nm to infrared, though some are cost prohibitive. The wavelengths of nm [ 57 ] and nm [ 58 ] were commonly used as excitation in the s. Recently, blue and green narrow-band laser excitation has been used to selectively stimulate the constituent fluorescence and simplify the overlapped spectral patterns [ 59 ]. It was capable of reasonably comprehensive characterization of aquatic fluorescence constituents, including ChlPBP pigments, variable fluorescencea measure of the potential quantum yield, here,where Fo was the minimal fluorescence and Fm was the maximal fluorescenceand CDOM in estuarine and fresh waters.
In order to measure phytoplankton photophysiological assessments and spectral discrimination between oil and CDOM fluorescence, additional UV nm and blue nm light were used together with a nm laser diode.
The three-laser ALF provided additional variable fluorescence with nm excitation for improved measurement of Chl. YLF laser diode with nm was off-the-shelf, Bensky et al. The resulting fluorescence at nm was instantaneously recorded during each laser pulse using a streak camera. Because of the nanosecond time scale, the short laser pulse did not reveal any pulse-to-pulse hysteresis such as that seen with pump sources with flashes lasting milliseconds.
The lack of pulse-to-pulse hysteresis proved useful for direct phytoplankton mapping as a function of concentration since the fluorescent emission from the plankton is linear with pump energy. Having a somewhat complicated drive and high cost, laser diodes have not been widely used in in situ fluorometers.
The development of laser diodes having higher performance and lower cost will allow for their greater applicability in fluorometers. The advantages of LED commonly include the following: Commercially available LEDs ranging from ultraviolet to near-infrared wavelengths and having different output powers and encapsulations can be selected.
For example, in order to measure in situ Chl concentrations related to individual algal groups, an exact LED wavelength is selected in order to excite individual algae. Moreover, multiple wavelengths can be used to distinguish and correct different phytoplankton group compositions.
Recently, LEDs have also been used as actinic light, which is applied to drive photosynthesis. Following are the most relevant achievements using LED excitation. Narrow band LED is an alternative to traditional lamp or laser light. The fluorometer had an extremely high sensitivity for investigating the growth of adhering phototrophic microorganisms. The fluorometer was used to determine Chl and Chl concentrations in a variety of organisms containing different ratios of chlorophylls.
Multiwavelength LEDs are used to quantify total Chl and estimate the phytoplankton group compositions. Firstly, the norm spectra of four spectral algal groups were obtained in advance.
Using these norm spectra and actual five-point excitation spectrum of a water sample, a separate estimate of the respective Chl concentration was rapidly obtained for each algal group. An integrated multiwavelength fluorescence sensors prototype was fabricated by Starikov et al. Those researchers used eight LEDs with different colors from UV to red, which were arranged in a circular design and mounted in light tight housing.
The sensors could be operated in the absorption scattering and fluorescence mode, which exhibited a lower detection limit and a larger dynamic range. Moreover, a multiexcitation fluorometer with nine wavelength excitation LEDs was developed and evaluated by Yoshida et al. After measuring the nine excitation spectra, using a mathematical process, and solving the optimization problem, the total phytoplankton biomass was quantified, and the phytoplankton group compositions were estimated.
In order to gather more data, multiexcitation and mathematical processes are both necessary and effective. This application is due to the development of white LEDs with superior performance in terms of both power efficiency and emission spectrum.
Multicolor PAM in particular is a very versatile instrument that provides six colors of pulse-modulated measuring light,and nm and five colors of actinic light, andin addition to having white — nm and far-red nm light sources. This allows adjustment to suit very different phytoplankton groups.
We can conclude that LEDs are very suitable to in situ fluorometers. However, directionality is a serious shortcoming, especially as compared to laser diodes.
This problem could be addressed to a great extent by a collimator lens and optical fibers. Excitation Method Unlike sun-induced chlorophyll fluorescence, fluorometers always use various active light sources. Variable fluorescence, introduced from plant physiology, is used to study and monitor phytoplankton physiology. Huot and Babin described the theory, basic concepts, and practice of fluorescence protocols [ 67 ]. Here, we will describe the excitation method from the viewpoint of how they have improved measurement accuracy.
To improve the accuracy of fluorescence concentration measurements, it is desirable to minimize a potential variability in the fluorescence efficiency associated with environmental factors or constituent functional state. This can be achieved by appropriate selection of the measurement protocol [ 68 ]. On the other hand, the variability of in vivo Chl fluorescence can be stimulated using various active fluorescence techniques to retrieve valuable information about phytoplankton photophysiology and photochemical efficiency [ 41 ].
The principle of PAM is to selectively monitor the fluorescence yield of a weak measuring beam; it is not affected by even extremely high intensities of actinic light. By repetitive application of short light pulses of saturating intensity, the fluorescence yield at complete suppression of photochemical quenching is repetitively recorded, allowing the determination of continuous plots of photochemical quenching and nonphotochemical quenching. Very recently, a new type of multicolor PAM chlorophyll fluorometer [ 66 ] demonstrated high accuracy and reliability for measurements of photodamage [ 72 ].
Numerous applications have used various fluorometers based on PAMs to estimate photosynthetic activity, biomass productivity, and related factors [ 73 ].
However, Beer and Axelsson reported that the PAM fluorometry was limited when measuring photosynthetic rates of macroalgae at high irradiances [ 74 ]. A detailed description and comparison with PAM fluorometry are given in various sources [ 7576 ]. Recently, Kocsis et al. A single laser diode was used for both pumping and probing. The apparatus offered high sensitivity and excellent performance and could become a versatile device for a range of demanding applications.
The basic theory and applications in aquatics can be seen in Suggett et al. In conclusion, the three variable-fluorescence methods can determine photosynthetic parameters of natural phytoplankton.
For determining the concentration of Chl, simple pulse modulation is commonly used. Meanwhile, there are some special excitation methods that could provide unique perspectives on fluorescence measurement. Although fluorescence can be measured precisely by PAM technology, the energy of pulse-modulated fluorescence cannot be used effectively at the same time due to sidelobes in the frequency domain.
Therefore, Zhang et al. A laboratory-based SAM Chl fluorometer was presented for phytoplankton classified measure. In contrast to a PAM fluorometer, which is excited by sequentially switching each light source, the SAM fluorometer used three high power LEDs, and nm simultaneously excited by three different modulation frequencies, and Hz.
Detection time was shortened to 1 s. The SAM fluorometer achieved a better detection limit, as low as 0. A novel phase fluorometer was designed and demonstrated based on fluorescence lifetime and time-correlated single-photon counting TCSPC [ 85 ]. The fluorometer used a blue LED driven by an oscillator with a sinusoidal signal instead of a pulse of light. The fluorescence signal was detected by a high-speed, low-capacitance, and wide bandwidth 1 GHz silicon photodiode, after which the phase difference between the fluorescence and reference was measured.
Unfortunately, LOD with 3. By combining with simulations for steeper cutoff and higher quality optical filters, the detection limit of chlorophyll decreased to 0. However, the phase fluorometer approach could be considered to design in situ instruments. A pseudorandom sequence modulation was introduced into the amplitude measurement of the fluorescence by Hu and colleagues [ 86 ]. Experiments show that the sensor had a minimum detectable level of 0.
The pseudorandom sequence method remarkably improved sensitivity and interference suppression ability of the chlorophyll sensor and could be further applied in other ultraweak signal amplitude measurements. Detectors A detector is the receiver of the fluorescence. Because chlorophyll fluorescence is very weak, especially in vivo and in situ, selecting a high-performance detector with low noise can improve both the detection limit and the measurement accuracy.
Moreover, in order to improve selectivity, chlorophyll fluorescence detection is most often performed using a PMT in combination with an appropriate emission filter.
Because of the large volume and high voltage needed, the original in vivo fluorometer with PMT detector used shipboard operation [ 22 ]. Beutler and colleagues developed several fluorometers for phytoplankton analysis in situ. Every generation of fluorometer has its own characteristics. Subsequently, in order to study red cyanobacteria and cryptophyta, four PMTs were used [ 88 ]. The fluorescence detector was a PMT behind a nm band-pass filter.
The fluorometer used six LEDs for measuring light and four laser diodes for maximal fluorescence Fm. The laser diodes were chopped by 8 kHz, which is double the frequency of the LEDs. In order to prevent overload of the PMT, the detector was switched sensitive for It also took advantage of a bit waveform digitizer PS, PicoScope with increased input sensitivity to improve measurements of laser-stimulated emission LSE fluorescence induction.
Thus, ultralow Chl concentration could be detected, even below 0.
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In conclusion, PMT has a better performance for weak Chl fluorescence. However, the application of PMT on in situ fluorometers is limited by size and power because traditional PMT always requires high voltage, which is difficult to supply in the field. There exists a possibility of developing fluorometers based on PMT with higher accuracy for in situ measurement.
Photodiode Silicon photodiodes are suitable for in situ fluorometers because of their good response time, small size, and low cost, though photodiodes have higher noise and interference immunity than PMT.
The development of PD technique has promoted and accelerated the performance of in situ fluorometers [ 64 ]. Additionally, there exist some techniques to improve the SNR. Because of the weak current signal detected from PD, the transimpedance amplifier has been used to translate the current to voltage first [ 85 ].
In order to simplify and improve the design, a monolithic PD with on-chip transimpedance amplifier was used by Lamb et al. In order to offset the high noise of PD, Aiken [ 50 ] used two low noise operational amplifiers producing a nonlinear response.
Results are shown in Figure 2 whereas the chemical composition of BBM medium and the lecheate are indicated in Table 3. Culture samples were taken once each day 3rd, 7th and 9th in order to determine absorption by using the absorptance technique as explained previously, i.
This can be explained due to the absorption of light by dissolved substances present in the lecheate, what affects the turbidity of the medium.
After three days, cell number was higher in lecheate treatments than in BBM. EPAR diminished after 7 days culture in lecheate because of the increase in cell density Fig.
This can be related to the low cell density and turbidity associated to BBM treatment. The increase of the cell number throughout the time was higher under lecheate treatment than that in BBM Fig.
ETR presented similar pattern as cell number in the 7th and 9th days of culture Figs. ETR increased from the 3rd to the 7th day as well as the number of cells. However, ETR decreased in spite of the increase in cell number in day 9. A possible uncoupling between growth and photosynthesis, i.
In order to evaluate the uncoupling between growth and photosynthetic rate, the expression of growth as biomass, i. However, in this case it was not possible to determine g DW per volume as the sampling volume used in the experiment was too small.
The positive effect of the lecheate of urban sewage on the growth of Chlorella fusca could be explained by the efficient use of the ammonium, at high concentration in lecheate medium Table 2. In order to assess the quality of the data obtained with the Pocket PAM fluorometer, in vivo chlorophyll fluorescence was also measured by another and more commonly used fluorometer, Water PAM emitter-detector unit of fiber version, Water EDF.
Thus, possible artifacts by scattering or reabsorption of fluorescence seem not to be significant. Modulated fluorescence excitation is produced by blue LED maximum wavelength emission: Actinic light is provided by the same power LED as for modulated light.
The light guide is made of plastic fiber and it has 40 cm x 1. The height of the layer in the thin compartment 2 can be changed by locating a fiberglass rectangular piece at the end of the plate before the cascade. Cells were pumped from the tank compartment 3 through PVC tubes back again to compartment 1 Fig. The TLC cultivator has three different parts: Compartment-1 with a surface of 0.
The production is expressed per unit area m2 instead of using volume units m3 as the TLC is provided with a fine culture film flowing on a plate that provides a high exposure surface. Effective quantum yield was measured every two minutes by introducing the light guide of Junior PAM into the compartment 1 Figs.
To integrate the bio-optical properties of the algae in the culture media, the specific attenuation coefficient Kc, m2 mg-1 Chl-a has been considered since it takes into account all variables affecting light absorption as cell size, chlorophyll concentration and algal density Figueroa et al.
The fiber of the Water PAM red light version was located in the compartment 1 of the TLC the same as the fiber of Junior PAM and simultaneously measurements of effective quantum yield were conducted at six different periods in the day from the morning to the afternoon 10 min measurements in each period.
On 18 Februaryphoton fluence rates decreased, showing higher variations than that observed on 16 February as a consequence of intermittent clouds Fig. In the first period, cells were grown in 2 cm layer culture on the exposure area until it reached the stationary phase.
Measurements were carried out in this phase during two days. After this time, the culture was diluted to the density necessary to be in exponential phase again. After that, a layer height of 1 cm was applied to the culture for another two days Fig. The whole experiment was conducted from 1 to 15 July Irradiance and effective quantum yield were measured online. Results are shown in Figure 4 and they were described above. In addition, culture samples were taken throughout the day in order to determine absorption by using the absorptance technique as it was explained previously.
These differences cannot be explained by different cell densities among the cultures since the average cell density in 2 cm culture was similar to that in 1 cm layer culture, i. However, in 1 cm layer culture, cell weight per number of cells or per culture volume was higher than that in 2 cm culture layer Table 5. This can be related to the higher level of pigments and proteins per cell found in 1 cm layer culture than that found in 2 cm one data not shown. The decrease of daily integrated irradiance in the culture during the 8 and 9 days of experiment is related to the bio-optical characteristics in 1 cm culture, i.
In this study, Kc and cell weight per cell and volume were negatively related to photosynthetic production ETR. This result suggests photaccli-mation of the cell culture. Increase in photoinhibition decrease of ETRmax with increasing Kc was previously reported by Figueroa et al.
Bio-optical and biological variables of cell cultures of Chlorella fusca in a thin-layer cultivator of 4 m2 of exposure surface and surface: The initial cell density was 1. Effective quantum yield and photon fluence rate of PAR were measured for 2 days in 2 cm water layer when the stationary phase was reached, the culture was followed for another 2 days but the layer height of the culture in the planar plate of the cultivator was 1 cm.
Thus, the increase in light absorption in 1 cm culture and increase of Kc are related to the acclimation and reduction of algal productivity, i.
The maximum value of According to Tredicimaximal efficiency of photosynthesis in microalgae is This maximal value can be reached in short-term experiment under low irradiance but it can be maintained during long-term period under other stress condition as increased temperature Tredici, In our study the efficiency was lower than 5.
The measured biomass productivity in the 2 cm layer culture C. The huge differences among the physical variables measured in both sites could explain this result. A more productive strain, in the case of this study, under high irradiance and temperature Table 3 compared to the Chlorella sp.
Thus, C assimilation can be estimated from ETR as follows: The PQ value used was 1. PQ should be 1. The average internal content of C in C. The estimated biomass productivity eBP was higher in 2 cm layer culture than that under 1 cm Table 5 as occurred with the measured biomass productivity mBP. Thus, in 1 cm layer culture, the difference between measured and estimated values was much higher than that in 2 cm layer culture.
The reason of this discrepancy cannot be explained but it could be related to the bio-optical variation in 1 cm layer culture, i.
Interestingly, Kc is higher in 1 cm layer culture and it has been previously shown that an increase of Kc in phytoplankton different species, chlorophyll content and cells sizes was related to an increase in photoinhibition Figueroa et al. However, Kromkamp et al. In this study with C. Linear relation between estimated and measured biomass productivity has been determined in Chlorella sp.
No good correlation was found in the culture in which 1 cm layer was applied cells with higher cell weight per number of cells compared to 2 cm layer culture.
This indicate that the use of ETR as estimator of biomass yield cannot be generalized since it does not take into account the variability of alternative electron cycling and the variability of the reduction degree of the biomass. The higher cell weight per number of cells or culture volume in 1 cm compared to 2 cm layer culture Table 5 could be related with the accumulation of lipids or proteins and it is not adequate to assume a constant photosynthetic quotient.
As final conclusion, chlorophyll fluorescence can be considered as a good tool to control microalgal cultivation by using online monitoring of photochemical activity to optimize the cultivation regime, i. More investigations are necessary for the estimation of biomass yield by using ETR data taking into account the contribution of cyclic and non cyclic electron transport and the bio-optical and biochemical characteristics of different species under different culture and environmental conditions.
Light-harvesting among photosynthetic organisms. Determination of phytoplankton absorption coefficient in natural seawater samples: The water-water cycle in chloroplasts: Real-time coastal observing systems for marine ecosystem dynamics and harmful algal blooms. Chlorophyll fluorescence as a probe of photosynthetic productivity.