Chlorophyll is a pigment found in plant cells and some microorganisms, such as algae and cyanobacteria. It gives these organisms their characteristic green color by reflecting the green wavelengths found in sunlight and plays a central role in the process of photosynthesis.
There are several different chlorophyll molecule variants that exist. Chlorophyll-a is present in all photosynthetic organisms and is the primary pigment involved in photosynthesis. Some organisms have accessory chlorophyll pigments that augment light energy absorption at different ranges of the color spectrum. For example, chlorophyll-b is often found in green algae and more complex plants, while chlorophyll-c is typical in brown algae.
Chlorophyll and phycobiliprotein variants have their fluorescence peaks at different wavelengths.
The term blue-green algae is technically a misnomer because it actually refers to a group of bacteria called cyanobacteria. They are named such because, like true algae, cyanobacteria are photosynthetic and commonly found in aquatic or other highly moist environments as individual cells in filamentous colonies.
Their pigmentation, which comes from both chlorophyll and unique pigments called phycobiliproteins, produces their characteristic green or bluish-green color. Phycobiliproteins distinguish cyanobacteria as they are normally not found in true algae. Phycocyanin and phycoerythrin are the two primary phycobiliproteins used for monitoring blue-green algae.
Why Measure Chlorophyll and Blue-Green Algae?
Photosynthesis forms the basis for primary production in aquatic ecosystems by transforming light energy from the sun into chemical energy, primarily in the form of glucose (sugar), according to the following reaction:
Chlorophyll captures the light energy that drives the process of photosynthesis.
This process consumes carbon dioxide and creates two products essential to aquatic ecosystems. Firstly, it generates the oxygen necessary to sustain aquatic life forms like fish, aquatic invertebrates, zooplankton, aerobic bacteria, and plants. Secondly, it supports photosynthesizing primary producers like phytoplankton and aquatic plants (macrophytes) that form the base of the food web.
Chlorophyll is a critical component of the equation as it is the primary pigment responsible for capturing the light energy that drives the process of photosynthesis. Without chlorophyll, primary production would be greatly reduced or eliminated, and aquatic species at higher trophic levels could not thrive.
Measurement of chlorophyll can, thus, provide insight into the rate of primary production, the potential for higher life forms, and the overall health of an aquatic ecosystem.
While lack of chlorophyll can indicate poor production, very high chlorophyll levels can indicate eutrophication, the excess of nutrients. This can feed the rapid growth of algae and cyanobacteria (blue-green algae) in what is known as an algal bloom. When such events cause adverse effects to humans or aquatic ecosystems, they are classified as Harmful Algal Blooms (HABs).
In more severe cases, toxin-producing algal blooms can pose health risks to humans and animals that come in contact with or ingest the toxins. Certain species that thrive under eutrophic conditions, cyanobacteria (blue-green algae) in particular, are known to produce toxins in some situations. This can be especially problematic in waters used for recreation, drinking water supply, or seafood production.
When algae and cyanobacteria eventually exhaust their nutrient source, the resulting large-scale die-off leaves behind a mass of dead organic material that can continue to release toxins as it decays. Bacteria and other microorganisms begin to decompose the material, consuming oxygen in the process. This can lead to hypoxic conditions, potentially resulting in large fish kills and a general reduction of biodiversity.
In summary, chlorophyll is an important parameter because it provides information on the relative levels of bioproductivity in an aquatic ecosystem. Blue-green algae measurement helps to further categorize the species of phytoplankton (single-celled, photosynthesizing organisms) present and can provide additional valuable information to help predict and detect the outbreak of HABs.
A variety of species can contribute to chlorophyll presence in natural waters.
How Are Chlorophyll and Blue-Green Algae Measured?
Chlorophyll and blue-green algae are measured in field applications using fluorometers, which are instruments that measure the fluorescence of a substance following excitation by a light energy at a specific wavelength.
The detection process begins with a light source in the sensor, such as a high-intensity LED or laser, that directs light through an emission filter. This narrows the light to a small band of wavelengths known to excite photon-emitting molecules called fluorophores in the substance of interest.
Upon excitation, electrons in the fluorophores move to a higher energy state. As the electrons return to ground state, they release excess energy as fluorescence, which a photodetector in the sensor measures.
Fluorometers emit light at one wavelength and measure returned fluorescence at another wavelength.
The wavelength of the excitation light source will be higher energy (shorter wavelength) than the lower energy (longer wavelength) of the emitted fluorescence. Many different substances can be measured with fluorometers, each with its particular excitation and emission wavelengths that form a “signature” for the substance.
In the case of chlorophyll, most fluorometers are tuned to detect chlorophyll-a since it is the most common form of chlorophyll found in all phytoplankton. For chlorophyll-a, excitation is centered around the 470 nm wavelength in the blue range of the visible spectrum, with emission generally occurring somewhere in the 650-700 nm red range.
The wavelengths used by fluorometers for chlorophyll detection fall within the visible range of the light spectrum.
Blue-green algae sensors are tuned to focus on specialized light-harvesting structures called phycobilisomes contained within cyanobacteria. Phycobilisomes organize phycobiliproteins together with chlorophyll molecules to broaden the spectrum of light absorption for photosynthesis.
Phycobilisomes are light-harvesting antennae found in cyanobacteria that stack phycocyanin and phycoerythrin on chlorophyll molecules.
Blue-green algae sensors are normally selected based on whether they will be used in a freshwater or saltwater environment. Cyanobacteria in freshwater environments tend to produce significant amounts of the fluorescing phycobiliprotein pigment phycocyanin. Saltwater cyanobacteria typically produce phycocyanin as well, but they are distinguishable by an additional pigment called phycoerythrin that has different fluorescence characteristics.
Since a function of cyanobacteria’s phycobiliproteins is to transfer light energy to chlorophyll to augment photosynthesis, it is normal for some of the energy used to excite phycocyanin or phycoerythrin to cause a lesser degree of excitation in chlorophyll molecules. Thus, some chlorophyll fluorescence may be detected along with blue-green algae. This effect should be considered for any potential interferences between sensors or influence on the interpretation of data.
How to Select a Chlorophyll or Blue-Green Algae Sensor?
On the topic of data interpretation, this is one of the key factors to consider when selecting a fluorometer, along with anti-fouling features and maintenance needs.
The units of measurement for chlorophyll and blue-green algae fluorometers can be somewhat complex. While units such as cell counts per milliliter (cells/mL) and pigment concentration in micrograms per liter (μg/L) or parts per billion (ppb) seem to give the most absolute and directly comparable measurements, it is important to understand how the sensor arrives at these units and the potential limitations.
For example, a sensor that gives a direct μg/L output has most likely based this value on a laboratory standard created from pigment extracts or other surrogates. This may or may not correlate well with field measurements, depending on the exact species of algae and/or cyanobacteria present at the field site. Pigments in extractions (in vitro) may additionally have different absorption and emission characteristics than pigments contained within cell structures (in vivo).
Therefore, such units should be used only in conjunction with supplementary lab analysis of grab samples to confirm correlation. An improved correlation can be developed over time for a specific site, but this again depends on the consistency of photosynthetic species present.
Given the caveats associated with absolute measurement units, many sensors provide fluorescence values in relative fluorescence units (RFUs) in addition to or instead of concentration.
RFUs are arbitrary units that represent the fluorescence intensity detected by the photodetector of a given sensor. In a long-term monitoring application using the same sensor, measurement in RFUs gives good comparability of the scale of algal bloom events. A baseline condition is established over time, and grab samples can be taken to build a correlation with other measurement units.
Regardless of preferred measurement units, obtaining high-quality data depends on the fluorometer’s emission light source and photodetector window remaining unobstructed.
Sensor fouling is a common source of interference. For long-term monitoring, particularly in waters with a high degree of biofouling, the sensor or multi-parameter platform should incorporate anti-fouling features such as a mechanical wiper or copper guard.
In addition to built-in anti-fouling features, periodic cleaning and recalibration may be necessary to ensure reliable measurement, especially in extended deployments.
What to Consider When Preparing a Chlorophyll or Blue-Green Algae Sensor?
Calibration of chlorophyll and blue-green algae fluorometers is somewhat flexible, but there are several important points to understand in order to obtain good calibration and comparable data across sites and from different sensors.
Begin calibration by first selecting a standard. While a pure chlorophyll, phycocyanin, or phycoerythrin pigment extract may seem to be the ideal calibration standard, such extracts can be expensive, unstable (subject to light and oxygen degradation), and difficult to source.
Instead, a stable secondary standard such as a Rhodamine WT dye dilution is common practice. Solid secondary standards that provide a stable fluorescence signal may also be used for correlation and periodic verification of fluorometer sensitivity as the sensor ages.
A general rule is to perform a two-point calibration whenever possible. A one-point calibration may be used under certain circumstances, but a two-point calibration using a prepared standard and a zero point solution will yield the best results and allow for good comparison between sensors.
When calibrating with the prepared standard, check product documentation for information about the effect of temperature on the standard value. Fluorescence and temperature have an inverse relation, so the temperature of the standard should be accounted for to improve accuracy.
For the zero point, the ideal standard would be source water that does not contain any of the analyte to eliminate the effect of any background fluorescence, but deionized (DI) water can also be used if it is more practical.
Exercise caution if using any absolute units like ppb, μg/L, or cell counts rather than units expressing relative sensor response (e.g., RFUs). The value reported by the sensor placed in the calibration standard may correlate well with field measurements, but this should be verified with supporting laboratory analysis. Remember that chlorophyll and blue-green algae measurements encompass a wide range of algae and cyanobacteria species that may produce variable fluorescence responses.
How to Deploy a Chlorophyll/Blue-Green Algae Monitoring System?
Chlorophyll and blue-green algae monitoring systems can be established either from floating buoy platforms or existing structures. For example, mounting at a water intake structure may be appropriate for a drinking water supply reservoir with algal bloom concerns. In an inland lake, port, or other coastal area with recreational activity, a dock can often provide a convenient structure for placing the fluorometer in a spot that will produce representative measurements.
Fluorometers can be mounted on docks or other structures in many chlorophyll applications.
A buoy-based deployment is useful for placing sensors at any location that lacks a natural mounting structure. If near-surface measurement is sufficient, sensors can be securely placed in a perforated deployment pipe that allows flow through to the fluorometer while offering easy access for maintenance without having to remove the buoy from the water.
For monitoring in open waters, fluorometers may also be mounted on floating buoy platforms.
If deeper measurement is desired, perhaps because the known species of algae and cyanobacteria in a body of water tend to be present at a certain depth, buoys can help to facilitate this as they also allow for sensor strings to be suspended below the buoy hull.
A data logger mounted on the buoy solar tower (or pole-mounted in the case of a dock) contains multiple sensor points for interfacing with multi-sensor systems. Wireless communications provide data in near real-time, and a solar-charged battery ensures continuous operation.
Conclusion
The increased occurrence of algal blooms in water bodies around the world has elevated the interest in establishing real-time algae monitoring systems. Chlorophyll is the primary pigment that drives the process of photosynthesis in phytoplankton, the single-celled primary producers in an aquatic ecosystem that includes both true algae and blue-green algae, which are actually cyanobacteria.
Monitoring chlorophyll levels with fluorometers can offer valuable insights into the overall biological activity within a body of water. Blue-green algae fluorometers can complement chlorophyll monitoring by detecting the presence of cyanobacteria, which are a significant contributor to the release of toxins associated with harmful algal blooms (HABs).
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