A Guide to Cyanobacteria

Identification and Impact

Mark A. Nienaber, Miriam Steinitz-Kannan

• Publisher: The University Press of Kentucky
• Year: 2018
• ISBN:
  9780813175607
• Topic: Biological Sciences
• Subtopic: Microbiology
• Index: Biological Sciences

Blue-green algae (also known as cyanobacteria) and the toxins they can produce pose serious economic, environmental, and public health problems worldwide. Much of the scientific and public interest in these microorganisms arises from their tendency to undergo explosive population growth and form harmful blooms, which have inflicted damage in industries as diverse as health care, public utilities, agriculture, recreation, real estate, and commercial and sport fishing. Until now, water quality professionals and other individuals tasked with finding and eliminating cyanotoxins have lacked an accessible guide to these potentially deadly microorganisms.

Written for nonspecialists in a clear and straightforward style, this guide will help students, landowners, and citizen scientists identify different kinds of cyanobacteria and understand their impact on waterways, from neighborhood lakes and farm ponds to major river systems. The central feature of the book is a detailed key that systematically walks the reader through each step of the identification process. This key is linked to an extensive set of photographs and a companion smartphone app to assist readers in confirming their findings.

Authors Mark A. Nienaber and Miriam Steinitz-Kannan include an ample glossary to help newcomers to the subject get up to speed as well as an in-depth and current bibliography to aid advanced readers in further research. They also offer instructions on how to correctly collect and analyze cyanobacteria. Altogether, this accessible yet comprehensive resource makes important, complex material available to a wide range of professionals and laypeople engaged in combating harmful cyanotoxins.

1

Collecting Cyanobacteria

Methods for collecting algae differ based on the needs of the person doing the collecting. If the interest is only in the general types of algae present, then qualitative analysis is enough. If the interest is in the specific identities of the algae and their relative abundance, then a more complicated, quantitative analysis is needed. This is performed by environmental professionals to determine whether the type and amount of algae pose a potential threat to the public.

Qualitative Analysis

This is just algae identification. To identify which types of algae are by the shore, all you need is a turkey baster and a microscope. A drop of pond water under a microscope is fascinating. Because different types of algae live in different parts of a pond, you should collect from different parts of the pond to give you a better representation of the algae present—a representative sample. Some live by the shore; others live out in the middle. To collect the organisms floating out in the middle (the plankton) you need a plankton net. This is a cone-shaped net made of cloth that funnels down to a collection bottle or vial at the narrower end. The cloth is specially made with pores 63 micrometers (μm) in diameter that allow the water to pass through but direct the plankton into the collection bottle. There are coarser nets (> 80 μm pores) that are cheaper, but they allow small algae to pass through. Finer nets (< 25 μm pores) are good, but they easily clog. The net is pulled through the surface layer of the water to collect the photosynthetic organisms. This is very helpful if the algae are not growing in a conspicuously dense cluster or bloom.

Quantitative Analysis

This broad description is an overview of the techniques developed at the Water Research Lab of Northern Kentucky University for quantitative or total algal analysis. The techniques are modified from Standard Methods for the Examination of Water and Wastewater (Clesceri et al. 1998).

For a quantitative analysis of a body of water, the sampling methods must be much more precise to accurately count (quantify) the algae in a sample and compare it to other samples. These samples are called whole water samples and should ideally be taken from several depths and in dark (opaque) bottles. The sample volume required depends on the condition of the lake or stream. If the algae have formed visible mats or a dense surface scum, less will be needed. If the algae are not visible, a greater volume will be required. It is important to take enough different and representative samples to determine the overall condition of the body of water being analyzed. Representative sampling helps ensure that any conclusions reached about a set of samples can safely be extended to the body of water (population) as a whole. Samples are immediately preserved because the organisms collected will continue to degrade (eat) each other or may replicate in the bottle. Preservation is done with a chemical preservative (such as Lugol’s Iodine) or with temporary refrigeration (a cooler with ice packs). Standard wisdom is to add enough Lugol’s to make the sample the color of strong black tea. Refrigerated samples cannot be kept much more than a day and must be chemically preserved before proceeding.

The collected water sample is poured into a graduated, cylindrical container and allowed to settle for at least 24 hours. Excess water (that is, without organisms) is removed by aspiration, leaving 10 mL of sample. The sample is then poured and settled into a 10 mL well of a tissue culture plate that has a transparent, flat bottom. The algae are counted using an inverted microscope that looks up through the transparent bottom of the plate, usually using 20X magnification. Higher magnifications are used only to confirm identifications.

Prior statistical analyses have tested the ability of this method to accurately determine the relative abundance of the organisms in a body of water, so that observers can evaluate whether the abundance is a problem and can make meaningful comparisons to other bodies of water. The analyses showed that to obtain a large enough sample size to achieve the required rigor and accuracy, a minimum of 300 algal units had to be counted, or the algal units in a minimum of 300 fields had to be counted (Clesceri et al. 1998). An algal unit is determined by the type of algae being counted. For single-cell types of algae, one individual cell equals one algal unit. For colonial algae types, algal units are defined as the area covered by the colony using a calibrated square. A field is a square region seen in the microscope view area that is in place to mark out a specific area, usually measured in square microns. This is superimposed on the microscope’s view area by using a Whipple Disc. A Whipple Disc is a circular piece of glass that fits inside one of the ocular lenses (eyepieces) and has a square etched on it. This is used to determine the area of the square in the view area, and to calculate the number of algal units per square micron. This method allows for quantitative comparisons among water samples and bodies of water.

As previously stated, cyanobacteria rarely make toxins when not blooming. If blooms are being sampled to measure toxins, remember that some toxins bind irreversibly to plastics, which throws off the measurement. It is best to collect the samples directly into glass containers. If this is not possible, transfer samples to glass as soon as possible, certainly before freezing the sample (Oehrle and Westrick 2003; Oehrle et al. 2010). Also, please wear gloves to protect yourself.

2

Description

Cyanobacteria are a very diverse group of microorganisms that, without close inspection, look and act like other common algae. They are found in similar places and conditions. They are, as a rule, both green. However, we will demonstrate that there are basic differences that set cyanobacteria apart from the other algae.

Internal Anatomy

Cyanobacteria are classified as prokaryotes, like other bacteria. In Greek, pro- means “before” and karyon means “nut” or “kernel,” in contrast to eukaryotes, in which eu- means “well” or “true” kernel. The prokaryotes are thought of as more primitive organisms and eukaryotes are thought of as more complex organisms. This is reflected in their internal structures, as well. Prokaryotes have fewer and simpler internal structures (sometimes called bacterial microcompartments), which are surrounded (bound) by layers of protein (Komárek 2013). Eukaryotes have a wide array of internal structures, called organelles, which are surrounded by phospholipid membranes. Examples of organelles are the nucleus and the chloroplast.

Though cyanobacteria do not have membrane-bound organelles, there are observable structures inside the cell. In some cyanobacterial cells there is a hollow structure called an aerotope (or gas vacuole). The protein wall surrounding the aerotope will allow gases to pass through air, but not water (Oliver and Ganf 2000). As the aerotope collects gas, it makes the cell it is inside more buoyant in water so it can float to the surface. This moves the cell (or group of cells) into the light so it can photosynthesize. As the cell accumulates denser carbohydrate through photosynthesis, it become less buoyant and sinks. As the cell consumes carbohydrate and produces carbon dioxide, the cell becomes more buoyant and the cycle continues. Cyanobacteria are the only photosynthetic plankton with aerotopes, which make possible the regulation of buoyancy and the resulting vertical migration through water. There are many advantages to this vertical migration. There are often higher concentrations of chemical nutrients (such as nitrogen or phosphorus) in the lower water levels and less competition for them. There are also dangers closer to the surface: predators are more numerous, and high light intensity can damage pigments and cell structures. (Please also see pages 19–20, Vertical mobility.)

Though almost all types of cyanobacteria can produce aerotopes, generally those that form surface blooms produce the greatest numbers of aerotopes. These genera are Microcystis, Dolichospermum, and Aphanizomenon. These usually form surface films or scums when the water is calm and stagnant and the cells have lost their ability to regulate their buoyancy (Walsby 1994). There are other bloom-forming cyanobacteria that have aerotopes (Cylindrospermopsis and Planktothrix) but do not form surface scums.

Aerotopes can also collect the carbon dioxide produced by carbohydrate breakdown (metabolism) for reuse in photosynthesis (Rae et al. 2013). When aerotopes are present in great numbers in a cell, they refract the light going through the cell, which makes them appear dark brown or even black under a microscope.

Some of the other internal structures in cyanobacteria have direct roles in photosynthesis. One is another type of protein-walled bacterial micro-compartment, called a carboxysome (Rae et al. 2013). This structure acts to increase the efficiency of photosynthesis in cyanobacteria by increasing the concentration of carbon dioxide (CO₂) around a crucial enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase, abbreviated RuBisCO. This enzyme is involved in the first major step of photosynthesis: the process by which atmospheric CO₂ is incorporated into the series of chemical reactions that will eventually store energy in the form of carbohydrate. This key initial step is also referred to as carbon fixation: mobile, atmospheric inorganic carbon (read CO₂) is pulled (fixed) into a more stable, structural form in living things. It is suggested that cyanobacteria could not attain the environmental dominance they sometimes achieve without this increased efficiency in photosynthesis (Rae et al. 2013).

Also inside the cyanobacterial cell are extensions of the external membrane that fold back on themselves, increasing the surface area. These protrude into the protoplasm in a somewhat organized manner. It is on these surfaces, called thylakoids, that the photosynthetic pigment complexes are displayed to capture solar energy (Olive et al. 1997). The pigments in these complexes are phycocyanin (blue green), allophycocyanin (blue), chlorophyll-a (green), and phycoerythrin (red) (Colyer et al. 2005). The phycocyanins are coupled with proteins to form visible structures called phycobilisomes. These are the primary light-collecting structures for photosynthesis in cyanobacteria (Grossman et al. 1993). This is particularly true at low light levels. These structures also give the blue-green algae their characteristic color.

The external surface of a cyanobacterial cell consists of peptidoglycan (a polymer composed of polysaccharide and protein chains) and an outer membrane composed of lipopolysaccharide, or LPS (a large molecule composed of lipids and sugars joined by chemical bonds). This is very similar to that of the common gram-negative bacteria. This is important for several reasons. One is that these molecules contribute to the structural integrity of the bacteria and protect it from some chemicals. Second, the LPS layer can induce immunological (allergic) responses in humans and other mammals and is considered an endotoxin (Sivonen and Jones 1999). LPS exposure also induces fever, nausea, and other problems. All these problems have been observed from exposure to cyanobacteria (Sivonen and Jones 1999).

External Anatomy

In addition to these internal variations, cyanobacteria also exhibit external differences in appearance. Their cells usually have relatively thick, gelatinous cell walls (Kangatharalingam et al. 1992). The level of organization of these cells ranges from simple, uniform, single cells to fairly complex groups or colonies of multiple cells with a limited degree of specialization.

Cyanobacteria colonies have two primary shapes or forms: filamentous and coccoid. In the filamentous colony form, the cells are in linear chains called filaments. The cells in filaments usually look square to rectangular. Many are disk shaped and look like stacked hockey pucks. When filament cells divide, the new cell wall is produced at a 90° angle between the two new cells. It is also perpendicular to the length, or long axis, of the filament. This extends the chain (like stacking blocks or pucks) and maintains the connections between the neighboring cells. On the other hand, the cells in the coccoid-form colonies are usually round to oval. The term coccus is derived from the Greek word for a round berry. When coccoid cells divide, the new cell wall is not produced in the same way as it is in filamentous cells, but in seemingly random, multiple planes of division. Furthermore, the new cells will often separate from one another, so they do not form linear chains. This occurs generation after generation, so coccoid colonies have a wide variety of shapes. Some coccoid colonies are orderly spheres, others are two-dimensional sheets, and still others are amorphous blobs. Coccoid colonies take on a wide variety of forms.

Sheath

Many cyanobacterial cells and colonies can surround themselves in a jellylike coating called a sheath. This sheath can envelop an individual cell, an entire colony, or both. The sheath is made up of extracellular polymers that vary among genera and include high molecular weight polysaccharides. The sheath acts to protect the cyanobacterial cell or colony from predation, desiccation, and ultraviolet radiation from the sun. It also provides structural integrity to the colony. Some sheaths are thin and watery, others are slimy, and still others are stiff, thick gels. Sheaths range from clear and colorless to densely dark yellow and brown—some are almost opaque. This is why cyanobacteria may or may not look blue green when we see them in the field (Garcia-Pichel and Castenholz 1991). The appearance depends on the organism and the environment in which it grows.

Cellular Anatomy

Cyanobacterial cells vary in size, shape, and complexity. Most cells are thin-walled vegetative cells. They are the primary structural unit of both filamentous and coccoid colonies. They can be smaller than 1 micron in diameter (one-millionth of a meter, or µm). It is difficult to see detail at this size with a light microscope. These cells can be as large as 400 µm (the size of a human hair) and are visible to the unaided human eye (Komárek et al. 1999).

Cyanobacteria can have a wide variety of cell shapes. Filamentous cells generally appear square or rectangular, but there are also many other shapes. Cells in coccoid colonies tend to be spherical or oval, but again, there are many other cell shapes. Note that cell shapes are generally consistent for a specific organism in its environment. The cell shape of species X in colony 1 will be very similar to the cell shape of species X in colony 2. But biological organisms always vary in terms of cell size, shape, and complexity.

Changes in complexity in cyanobacterial cells, such as seasonal dormancy and replication, often occur in response to unfavorable environmental conditions, such as nutrient deficiency. Organisms and their cells can and must change in response to their circumstances. For cyanobacteria, it is usually the vegetative cells that divide and change into other types of cells. Depending on the cyanobacteria and their conditions, the vegetative cells can generally form one of three types of complex or specialized cells: akinetes, apical cells,...