- Email: [email protected]
Cyanobacteria
CHAPTER FIVE
Cyanobacteria Steven L. Percival*, David W. Williams** *
Professor of Microbiology and Anti-infectives, Surface Science Research Centre and Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK Professor of Oral Microbiology, Tissue Engineering & Reparative Dentistry, School of Dentistry, Cardiff University, Heath Park, Cardiff, UK **
BASIC MICROBIOLOGY Cyanobacteria are Gram-negative prokaryotic microorganisms that were originally referred to as the ‘blue-green algae’. These microorganisms are very closely related to bacteria in terms of cellular structure, with no defined nucleus or membrane bound organelles present. Cyanobacteria have a cell wall containing peptidoglycan that is frequently surrounded by a mucilagenous sheath. Inside the cell wall is a typical cell membrane. Generally, cyanobacteria cells are larger than most other bacteria, ranging in size from 1 mm for unicellular types, to over 30 mm for multicellular species (Singh and Montgomery, 2011). Cyanobacteria are morphologically diverse and three basic morphological forms are described, and these are unicellular, filamentous forms without heterocysts, and filamentous forms with heterocysts (Singh and Montgomery, 2011). Heterocysts are differentiated and specialized cells that can fix nitrogen, and these are thought to promote survival under low nitrogen conditions (Kumar et al., 2010). Cyanobacteria are photoautotrophic, which means they use light energy to photosynthesize (mainly by chlorophyll-a) in order to generate their carbon cellular material, and in the process will produce oxygen. A number of species can also grow heterotrophically by using organic compounds as a source of carbon (Halm et al., 2011). Under phosphorus (P) and nitrogen (N)-rich conditions, cyanobacteria uptake and then intracellularly store these components; a property that has evolved over several billion years to allow them to exploit extreme environmental conditions (Shi et al., 2003; Lla´cer et al., 2008). Some cyanobacteria produce proteinaceous gas vacuoles that allow them to float in aquatic environments. The gas vacuoles are cylindrical in shape and sealed with conical end-caps (Hayes, 1988). Cyanobacteria can also exhibit gliding motility (Hoiczyk, 2000; Lyra et al., 2005). Importantly, cyanobacteria can grow prolifically under suitable conditions, thus generating so called ‘algal blooms’ in eutrophic freshwater lakes and reservoirs (Reichwaldt and Ghadouani, 2012). A number of secondary metabolites produced by cyanobacteria are potentially toxic, and during algal blooms can lead to harmful effects to both the local ecology and also the health of animals and humans. Microbiology of Waterborne Diseases ISBN 978-0-12-415846-7, http://dx.doi.org/10.1016/B978-0-12-415846-7.00005-6
Ó 2014 Elsevier Ltd. All rights reserved.
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NATURAL HISTORY Cyanobacteria are classified within the Kingdom Monera (Prokaryota), Division Eubacteria, class Cyanobacteria. However, there is still significant debate over classification at higher taxonomic levels regarding the composition of orders, families, genera and species. Many of these problems stem from historical failures to adequately recognize that cyanobacteria are prokaryotic and not eukaryotic. Since traditional identification and classification of cyanobacteria was through cellular morphology, these microorganisms were originally classified as blue-green algae (Cyanophyta) under botanical codes. It was not until the 1960s that the prokaryotic features of these microorganisms were established and the proposal made to include them within the bacteriological code. Traditional taxonomic principles have been complemented in recent years with molecular methods, such as sequencing of small-subunit (SSU) rRNA genes. Inconsistencies between the approaches are evident, although taxonomy of filamentous cyanobacteria with heterocysts as represented by Anabaena cylindrical, still hold true. There are an estimated 150 genera of cyanobacteria containing approximately 2000 species, of which around 46 have been reported as being toxicogenic (Hitzfeld et al., 2000; Ernst et al., 2006). Importantly, the genera and species which comprise problematic cyanobacteria are generally well recognized. Cyanobacteria are an ancient (occurring as long ago as 3500 million years; Schopf, 1993) and diverse group of microorganisms, and unsurprisingly, due to the extent of evolution, can exist in a wide range of habitats including fresh and marine water environments. Cyanobacterial habitats also include those that are considered extreme, such as frozen lakes, hot springs and salt works (Whitton, 1992).
METABOLISM AND PHYSIOLOGY Cyanobacteria are major components in the supply of global oxygen, sequestration of carbon dioxide (CO2) and nitrogen fixation, and as such play essential ecological roles. Currently, cyanobacteria are the only recognized prokaryotes that exhibit photosynthesis with the generation of oxygen. Protein complexes in thylakoid membranes of the cyanobacteria are responsible for both electron transport chains involved in photosynthesis and respiration. Cyanobacterial photosynthesis is essentially the same as seen in plants. Photosystem II exploits light energy to generate electrons from water, which in turn are transported via soluble electron carriers (plastocyanin or cytochrome c553) to the inner side of the thylakoid membrane. Photosystem I (chlorophyll) is then reduced leading to reduction of NADP. A proton gradient develops across the thylakoid membrane, which serves to drive ATP synthesis. Reduced NADPH from the process appears to be involved in carbon fixation. As a consequence, respiration in cyanobacteria is thought to involve succinate dehydrogenase activity as opposed to electron flow from NADPH.
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Nitrogen fixation is the process of nitrogen gas reduction through the action of the nitrogenase enzyme complex. Nitrogen gas, whilst being highly abundant in the environment, is highly inert, and therefore its conversion to a nitrogen product that can be incorporated into cellular material and processes requires reduction. The process of nitrogen fixation is, however, inhibited by oxygen (Postgate, 1998) and the fact that cyanobacteria are both aerobic and also generate oxygen as a metabolic by-product of photosynthesis means that strategies have had to be developed by these microorganisms to enable nitrogen fixation. Indeed, and as mentioned previously, the extent of the problem is highlighted by the fact that cyanobacteria are the only oxygenic microorganisms that can also fix nitrogen. The way cyanobacteria have dealt with this is by effectively separating the processes, both spatially and temporally. In terms of temporal separation, cyanobacteria effectively fix carbon during the day, whilst fixing nitrogen at night. Spatial separation of the processes is also evident in the filamentous cyanobacteria, where specialist anaerobic heterocysts are responsible for the process of nitrogen fixation and this means nitrogen fixation can also occur during the day (Berman-Frank et al., 2007; Kumar et al., 2010). The cyanobacterial genera that are recognized as being able to fix nitrogen include Anabaena, Aphanizomenon and Gloeotrichia. The genera of Microcystis, Coelosphaerium and Oscilhtoria appear to be unable to fix nitrogen. A range of cyanotoxins can be produced by cyanobacteria and these can be designated as neurotoxins (e.g. anatoxin-a, saxitoxin and neosaxitoxin; Wiese et al., 2010), hepatotoxins (e.g. microcystins, nodularins and cylindrospermopsin; Labine and Minuk, 2009), tumourogenic toxins (e.g. microcystins; Zegura et al., 2011), and toxins that are irritants affecting the skin and gastrointestinal tract (e.g. lipopolysaccharides; Stewart et al., 2006). Cyanotoxins are normally produced intracellularly and then generally released into the environment when cells lyse. Cyanobacterial growth is promoted by the presence of specific trace metals within water and this is particularly evident with iron and molybdenum. The presence of iron is believed to enhance the process of both photosynthesis and nitrogen fixation, whilst molybdenum appears to increase the rate of carbon fixation. The presence of zinc has also been reported to enhance growth and toxin production by certain cyanobacteria.
CLINICAL FEATURES While high densities of cyanobacteria may be present in the faeces of infected animals, there is little evidence to support persistence of these microorganisms in the normal intestinal flora of healthy warm-blooded animals. Cyanobacteria are not infectious agents, although as mentioned earlier, some species produce toxins during algal blooms, which are triggered by nutrient enrichment from natural waters and industrial effluents. There are about 25 species of cyanobacteria associated with adverse health effects (Gold et al., 1989). The first recognition of cyanobacterial algal blooms appears to
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have been as early as the 12th century in Lake Llangors in Wales, UK. Since this time, toxic algal blooms have been reported in many parts of Europe, USA, Australia, Africa, Asia and New Zealand. One survey in the UK found that 75% of cyanobacterial blooms contain toxins (Baxter, 1991). Within algal blooms, perhaps the most frequently encountered toxins are the microcystins and these are the toxins most often linked with incidences of animal and human poisoning. Of the many different kinds of microcystins globally reported, microcystin-LR is the most frequently encountered. The problem of microcystins is compounded by their high degree of chemical stability within water at both a wide range of temperatures and pH. The two main categories of toxins produced by cyanobacteria are the neurotoxins and hepatotoxins. Observations of poisoning of domestic animals show that when mucous membranes are exposed to cyanobacterial neurotoxins, symptoms develop in 4–10 minutes and death within 30 minutes. The effects of hepatotoxins can cause symptoms in exposed animals within 30 minutes and death within 24 hours. Using cobra venom as a point of reference, the alkaloid neurotoxins produced by species of Anabaena flos-aquae and Nodularia spumigena can be equally potent, with 20 pg per kg body weight being the lethal dose (LD50 of 200 pg/kg). The hepatotoxins produced by Microcystis aeruginosa and some strains of Aphanizomenon flos-aquae are in fact over twice as potent as cobra venom with 9 pg/kg being the LD50. A number of these toxins have been found to be tumour promoters and oncogenic in laboratory animals. One of the toxins isolated from Anabaena is an alkaloid that can cause neuromuscular blocking. Another toxin, isolated from Microcystis aeruginosa, affects the cardiovascular system and produces lesions in the livers of a variety of laboratory animals when administered orally or by intraperitoneal injection. Fortunately, large doses of these toxins are necessary to produce the symptoms in animals and residual toxins that might pass through a drinking water supply after conventional treatment would be diluted by vast volumes of water. This safety factor places the concentration far below any known human toxicity level. Some cyanobacteria produce toxins that are irritants, and if these are ingested in sufficient concentration they may cause gastrointestinal upset. Body contact exposure through recreational water activity may also induce skin irritations that lead to a rash. Control of cyanobacteria is a problem, as research has shown that the toxins can remain potent for days, even after the microorganisms have been destroyed by chlorination and copper sulphate treatment (El Saadi et al., 1995). Whilst there is toxicity data from mouse models, further research is required on the acute and chronic toxicity of cyanobacterial toxins and suitable methods need to be developed for monitoring the types and concentrations of cyanobacterial toxins in natural as well as treated drinking water. The Engineering and Water Supply Department of South Australia have developed interim guidelines for acceptable numbers of cyanobacteria in water supplies (El Saadi, 1995).
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PATHOGENICITY AND VIRULENCE No species in the cyanobacteria group is classed as a true pathogen of humans and animals. Cyanobacteria do not invade the animal/human body, but as mentioned above can produce potent toxins. It has been hypothesized that these toxins are produced by cyanobacteria to kill fish, thereby releasing nutrients for growth. There are three main types of cyanobacterial toxins and these are lipopolysaccharide endotoxins, hepatotoxins and neutrotoxins. The presence of these toxins in waters is not usually a problem for humans, but farm animals can be poisoned by drinking water from ponds containing dense cyanobacterial blooms. When acute health effects occur in humans they tend to include gastroenteritis, liver damage, nervous system drainage, pneumonia, sore throat, earache and contact irritation of skin and eyes. The potential chronic health effects of long-term exposure to cyanobacterial toxins in drinking water are unknown. It has been suggested that high rates of liver cancer in parts of China may be linked to cyanobacterial hepatotoxins in drinking water (Carmichael, 1994). Neurotoxins tend to exhibit their effects rapidly and those produced by cyanobacteria are generally one of three types based on mode of action. These modes of action include neuronal depolarization, the inhibition of cholinesterase, or ion channel blockage. Hepatotoxins produced by cyanobacteria are cyclic peptides and induce damage to hepatocyte cell structure. Hepatotoxins exhibit slower effects compared with neurotoxins and have also been reported as having tumorigenic activity (Falconer, and Humpage, 2001). Allergies to cyanobacteria have also been reported (Torokne et al., 2001). Treatment and therapy for cyanobacterial poisoning is very limited and indeed largely unavailable for the neurotoxins.
SURVIVAL IN THE ENVIRONMENT Since cyanobacteria are not dependent on a fixed source of carbon they are widely distributed throughout aquatic environments. Habitats include freshwater and marine environments and some soils. In fact, cyanobacteria are found in the early stages of soil formation, being associated with converting bare rock or decomposing debris. Stagnant water, sediments and soil appear to be the significant reservoirs for these organisms. In the environment, cyanobacteria exhibit a selective advantage over eukaryotes, due to the ability to fix nitrogen under adverse conditions.
Survival in Water and Epidemiology Cyanobacteria naturally occur in stream sediments, slow-moving streams, receiving waters for waste discharges and treatment effluents, rural storm runoff, drainage canals and marine waters. Prolific growth of these bacteria often occurs during summer months in surface waters. Densities of 500 cells or more per ml have been recorded at these times
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of the year. Polluted surface waters that become stagnant due to slow flows under summer drought conditions often support persisting populations of cyanobacteria. The growth of cyanobacteria is stimulated by high water temperature and high concentrations of inorganic nitrogen (N) and phosphorus (P). Indeed, to this end, cyanobacteria are able to exploit a wide range of dissolved organic forms of both phosphorus and nitrogen compounds through extracellular phosphatases and aminopeptidases. Their ability to be a source of biological nitrogen fixation in soils and water is also a significant contributor to their long-term survival and plays an important role in the ecological succession of microorganisms in the environment. In 1976, an outbreak of intestinal illness in Pennsylvania was associated with a cyanobacterial bloom in a municipal water supply and affected 62% of the population (Carmichael et al., 1985). Recreational water, particularly in the USA, has been implicated in causing adverse health effects following exposure to cyanobacterial toxins (Carmichael et al., 1985). There have been a number of cyanobacterial poisoning incidents through surface water contact (El Saadi et al., 1993; Soong et al., 1992). The most frequent mode of transmission of cyanobacterial toxins is by water ingestion, although in areas of massive cyanobacteria blooms, contact with the body through water sports is also of serious concern. There have been numerous reports of poisonings of livestock, pets and wildlife with waters containing cyanobacteria blooms. Outbreaks of human gastroenteritis from ingestion of toxic cyanobacteria in the public water supplies occurred in Charleston, West Virginia, USA, and the area served by the Anacostia Reservoir near Washington, DC, USA, during the drought years of 1930 and 1931. In 1981, an outbreak of human poisoning occurred in Northeastern Pennsylvania where 12 children and one adult were affected by an Anabaena species bloom. In another case study, in 1990, a localized outbreak of diarrhoea occurred among residents of a Chicago apartment building. This incident was traced to cyanobacteria toxins in an open water supply storage tank. Apparently, the cover to the tank had been inadvertently left open so that light, airborne cyanobacteria in dust particles gained access, grew and, in time, released toxic byproducts throughout the plumbing system. For utilities using surface water supplies, cyanobacteria are well known for their association with taste and odour problems, often regarded as a matter of aesthetics. In light of recent information on cyanobacteria, granular activated carbon (GAC) may be very important in toxin removal. Furthermore, for those water systems using disinfection as the only surface water treatment, there is always the threat of a seasonal passage of cyanobacteria and deposition of dead cells in the distribution pipe network. Such an occurrence provides a source of assimilable organic carbon (AOC), which is a potential nutrient for bacterial regrowth. The US Environmental Protection Agency (USEPA) or European governments regulate neither cyanobacteria nor their metabolites, except under guidelines, stating
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that drinking water must be potable. As defined, potable water must not only be safe but also clear and free from any objectionable tastes and odours, regardless of origin.
EVIDENCE FOR GROWTH IN A BIOFILM Cyanobacteria have the ability to grow as biofilms (Gaylarde et al., 2004; Rossi et al., 2012). In particular, cyanobacterial populations have been detected in their filamentous forms on buildings, with coccoid morphotypes also being shown to grow on artificial media as biofilms (Gaylarde et al., 2004; de los Rı´os et al., 2007).
METHODS OF DETECTION Monitoring water supplies for cyanobacterial presence is an important aspect to reducing harmful effects on human and animal health. Quantitative analysis of the microorganism and cyanotoxin levels are key measurements. The simplest approach to detect cyanobacterial algal blooms is visual inspection of water systems. Unfortunately, algal blooms may be present below the surface of the water and therefore may not readily be seen until a significant level of cyanobacteria is present. Analytical analysis of water samples for both the microorganism as well as toxins is therefore the most appropriate approach. To detect cyanobacteria, water samples are first blended with glass beads or treated by ultrasound to break filamentous forms prior to streaking on agar plates (AWWA, 1999). There are a variety of selected mineral media available (D-medium, ASM-1, BG-11 and WC) and these are incubated at 25 C under cool white fluorescent light. Cyanobacteria may grow extremely slowly, and may require several months of culture to generate visible colonies (Castenholz, 1988). Some recalcitrant cyanobacteria may not be freed easily of contaminants, thus, physical and chemical separation schemes may be necessary. Success in achieving pure cultures depends on persistence and patience. Microscopic examination using simple wet-mount staining techniques with India ink and methylene blue are used to identify morphological characteristics.
RISK ASSESSMENT Health Effects: Occurrence of Illness, Degree of Morbidity and Mortality, Probability of Illness Based on Infection • There are about 25 species of cyanobacteria that have been associated with adverse health effects. • Cyanobacterial toxins act primarily as hepatotoxins and neurotoxins, but can also cause skin irritation. They are extremely potent toxins and can potentially be fatal to
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humans. However, acute oral or dermal exposures have not resulted in any known human deaths. Reported illnesses in humans exposed to cyanobacterial toxins range from dermatitis and gastroenteritis to hepatitis and allergic reactions. Illness is selflimited. • Cyanobacterial toxins have been shown to be tumour promoters in animal studies and epidemiological evidence in humans suggests that chronic exposure to microcystins in drinking water is associated with an increase in hepatocellular cancer.
Exposure Assessment: Routes of Exposure and Transmission, Occurrence in Source Water, Environmental Fate • World-wide, the number of humans acutely affected by cyanobacterial toxins is low compared with other waterborne contaminants. However, because of decreasing water quality, the potential for an increase in incidents is high. • Routes of exposure are primarily ingestion through drinking water or recreational water contact, and also dermal exposure or possible aerosolization. • Most acute exposures occur from recreational water use and low levels in drinking water are associated with an increase in hepatocellular cancer in certain exposed populations. • Cyanobacteria are found in all types of water: lakes, rivers, marine environment, and drinking water reservoirs. Surface waters that receive waste effluents are at special risk for contamination. High water temperatures and concentrations of inorganic nitrogen and phosphorus stimulate growth. • Surface water systems exposed to a disinfectant may have deposition of dead cells in the distribution system with potential for regrowth. • Cyanobacterial toxins are ubiquitous, though their occurrence is dependent on conditions that contribute to algal bloom formation. Concentrations vary widely depending on the species of bloom and the stage of its formation and deterioration. Toxin concentrations range from 0.2 mg/l to 8.5 mg/l. • The toxic dose is unknown for human. The no observed adverse effect level (NOAEL) for mice dosed orally with microcystin-LR has been reported to be 40 mg/kg/day for 13 weeks. The NOAEL for mice dosed orally with anatoxin-a has been reported to be 0.1 mg/kg/day. Intraperitoneal and intranasal exposure is more potent than oral ingestion for both toxins.
Risk Mitigation: Drinking Water Treatment, Medical Treatment • There is some question as to the efficacy of standard drinking water treatment (e.g. coagulation, sedimentation, disinfection and filtration) for removing all but large concentrations of cyanobacterial toxins, though current methods are effective enough to prevent any acute effects.
Cyanobacteria
• Evidence on the efficacy of chlorine on degrading microcystins is equivocal; chlorine is effective on anatoxin-a. Activated carbon treatment appears to be the best removal method for treated water. • Preventing formation of blooms in the source water is the best way to assure cyanobacteria-free drinking water. • Membrane filtration technology has the potential to remove virtually any cyanobacteria or their associated toxins from drinking water. • Efficacious medical treatment is unknown in acute exposure; however, antihistamines, and steroids may be helpful for allergic reactions. If given in a timely manner, activated charcoal or an emetic could have a positive effect on the toxic response.
REFERENCES AWWA, 1999. Manual of water supply practices: waterborne pathogens. American water works association, Denver. Baxter, P.J., 1991. Toxic marine and freshwater algae: an occupational hazard? Br. J. Ind. Med. 49, 505–506. Berman-Frank, I., Quigg, A., Finkel, Z.V., et al., 2007. Nitrogen-fixation strategies and Fe requirements in cyanobacteria. Limnol. Oceanogr. 52 (5), 2260–2269. Castenholz, R.W., 1988. Culturing methods for cyanobacteria. Methods Enzymol. 167, 68–93. Carmichael, W.W., Jones, C.L.A., Mahmood, N.A., Thesis, W.C., 1985. Algal toxins and water based diseases. Crit. Rev. Environ. Control. 15, 275–313. Carmichael, W.W., 1994. The toxins of cyanobacteria. Sci. Am. 270, 78–86. de los Rı´os, A., Grube, M., Sancho, L.G., Ascaso, C., 2007. Ultrastructural and genetic characteristics of endolithic cyanobacterial biofilms colonizing Antarctic granite rocks. FEMS Microbiol. Ecol. 59 (2), 386–395. El Saadi, O., Camerson, A.S., 1993. Illness associated with blue-green alga. Med. J. Aust. 158, 792–793. El Saadi, O., Easterman, A.J., Camerson, S., Roder, D.M., 1995. Murray River water, raised cyanobacterial cell counts and gastrointestinal and dermatological symptoms. Med. J. Aust. 162, 122–125. Ernst, B., Neser, S., O’Brien, E., Hoeger, S.J., Dietrich, D.R., 2006. Determination of the filamentous cyanobacteria Planktothrix rubescens in environmental water samples using an image processing system. Harmful. Algae 3, 281–289. Falconer, I.R., Humpage, A.R., 2001. Preliminary evidence for in vivo tumor initiation by oral administration of extracts of the blue-green alga Cylindrospermopsis raciborskii containing the toxin cylindrospermopsin. Environ. Toxicol. 16, 192–195. Gaylarde, C., Gaylarde, P., Copp, J., Neilan, B., 2004. Polyphasic detection of cyanobacteria in terrestrial biofilms. Biofouling 20 (2), 71–79. Gold, G.A., Bell, S.G., Brooks, W.P., 1989. Cyanobacterial toxins in water. Water Sci. Technol. 21, 1–13. Hayes, P.K., 1988. Gas vesicles: chemical and physical properties. Methods Enzymol. 167, 213–222. Halm, H., Lam, P., Ferdelman, T.G., et al., 2011. Heterotrophic organisms dominate nitrogen fixation in the South Pacific Gyre. ISME J. 15. Hoiczyk, E., 2000. Gliding motility in cyanobacterial: observations and possible explanations. Arch. Microbiol. 174 (1–2), 11–17. Hitzfeld, B.C., Ho¨ger, S.J., Dietrich, D.R., 2000. Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment. Environ. Health Perspect. 108, 113–122. Kumar, K., Mella-Herrera, R.A., Golden, J.W., 2010. Cyanobacterial heterocysts. Cold. Spring. Harb. Perspect. Biol. 2 (4), a000315. Lyra, C., Laamanen, M., Lehtima¨ki, J.M., et al., 2005. Benthic cyanobacteria of the genus Nodularia are non-toxic, without gas vacuoles, able to glide and genetically more diverse than planktonic Nodularia. Int. J. Syst. Evol. Microbiol. 55 (Pt 2), 555–568.
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Labine, M.A., Minuk, G.Y., 2009. Cyanobacterial toxins and liver disease. Can. J. Physiol. Pharmacol. 87 (10), 773–788. Lla´cer, J.L., Fita, I., Rubio, V., 2008. Arginine and nitrogen storage. Curr. Opin. Struct. Biol. 18 (6), 673–681. Postgate, J.R., 1998. Nitrogen fixation, third ed. Cambridge Univ Press, Cambridge. Reichwaldt, E.S., Ghadouani, A., 2012. Effects of rainfall patterns on toxic cyanobacterial blooms in a changing climate: between simplistic scenarios and complex dynamics. Water Res. 46 (5), 1372–1393. Rossi, F., Micheletti, E., Bruno, L., et al., 2012. Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments. Biofouling 28 (2), 215–224. Schopf, J.W., 1993. Microfossils of the Early Archean Apex Chert – new evidence of the antiquity of life. Science 260, 640–646. Shi, X., Yang, L., Niu, X., et al., 2003. Intracellular phosphorus metabolism of Microcystis aeruginosa under various redox potential in darkness. Microbiol. Res. 158 (4), 345–352. Singh, S.P., Montgomery, B.L., 2011. Determining cell shape: adaptive regulation of cyanobacterial cellular differentiation and morphology. Trends. Microbiol. 19 (6), 278–285. Soong, F.S.E., Maynard, E., Kirke, K., Luke, C., 1992. Illness associated with blue-green algae. Med. J. Aust. 156, 67. Stewart, I., Schluter, P.J., Shaw, G.R., 2006. Cyanobacterial lipopolysaccharides and human health – a review. Environ. Health 24 (5), 7. Torokne, A., Palovics, A., Bankine, M., 2001. Allergenic (sensitization, skin and eye irritation) effects of freshwater cyanobacteria – experimental evidence. Environ. Toxicol. 16 (6), 512–516. Wiese, M., D’Agostino, P.M., Mihali, T.K., et al., 2010. Neurotoxic alkaloids: saxitoxin and its analogs. Mar. Drugs 8 (7), 2185–2211. Whitton, B., 1992. Diversity, ecology and taxonomy of the cyanobacteria. In: Mann, H., Carr, N. (Eds.), Photosynthetic prokaryotes. Plenum Press, New York, pp. 1–51. Zegura, B., Straser, A., Filipic, M., 2011. Genotoxicity and potential carcinogenicity of cyanobacterial toxins – a review. Mutat. Res. 727 (1-2), 16–41.
Cyanobacteria Steven L. Percival*, David W. Williams** *
Professor of Microbiology and Anti-infectives, Surface Science Research Centre and Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK Professor of Oral Microbiology, Tissue Engineering & Reparative Dentistry, School of Dentistry, Cardiff University, Heath Park, Cardiff, UK **
BASIC MICROBIOLOGY Cyanobacteria are Gram-negative prokaryotic microorganisms that were originally referred to as the ‘blue-green algae’. These microorganisms are very closely related to bacteria in terms of cellular structure, with no defined nucleus or membrane bound organelles present. Cyanobacteria have a cell wall containing peptidoglycan that is frequently surrounded by a mucilagenous sheath. Inside the cell wall is a typical cell membrane. Generally, cyanobacteria cells are larger than most other bacteria, ranging in size from 1 mm for unicellular types, to over 30 mm for multicellular species (Singh and Montgomery, 2011). Cyanobacteria are morphologically diverse and three basic morphological forms are described, and these are unicellular, filamentous forms without heterocysts, and filamentous forms with heterocysts (Singh and Montgomery, 2011). Heterocysts are differentiated and specialized cells that can fix nitrogen, and these are thought to promote survival under low nitrogen conditions (Kumar et al., 2010). Cyanobacteria are photoautotrophic, which means they use light energy to photosynthesize (mainly by chlorophyll-a) in order to generate their carbon cellular material, and in the process will produce oxygen. A number of species can also grow heterotrophically by using organic compounds as a source of carbon (Halm et al., 2011). Under phosphorus (P) and nitrogen (N)-rich conditions, cyanobacteria uptake and then intracellularly store these components; a property that has evolved over several billion years to allow them to exploit extreme environmental conditions (Shi et al., 2003; Lla´cer et al., 2008). Some cyanobacteria produce proteinaceous gas vacuoles that allow them to float in aquatic environments. The gas vacuoles are cylindrical in shape and sealed with conical end-caps (Hayes, 1988). Cyanobacteria can also exhibit gliding motility (Hoiczyk, 2000; Lyra et al., 2005). Importantly, cyanobacteria can grow prolifically under suitable conditions, thus generating so called ‘algal blooms’ in eutrophic freshwater lakes and reservoirs (Reichwaldt and Ghadouani, 2012). A number of secondary metabolites produced by cyanobacteria are potentially toxic, and during algal blooms can lead to harmful effects to both the local ecology and also the health of animals and humans. Microbiology of Waterborne Diseases ISBN 978-0-12-415846-7, http://dx.doi.org/10.1016/B978-0-12-415846-7.00005-6
Ó 2014 Elsevier Ltd. All rights reserved.
79 j
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Steven L. Percival and David W. Williams
NATURAL HISTORY Cyanobacteria are classified within the Kingdom Monera (Prokaryota), Division Eubacteria, class Cyanobacteria. However, there is still significant debate over classification at higher taxonomic levels regarding the composition of orders, families, genera and species. Many of these problems stem from historical failures to adequately recognize that cyanobacteria are prokaryotic and not eukaryotic. Since traditional identification and classification of cyanobacteria was through cellular morphology, these microorganisms were originally classified as blue-green algae (Cyanophyta) under botanical codes. It was not until the 1960s that the prokaryotic features of these microorganisms were established and the proposal made to include them within the bacteriological code. Traditional taxonomic principles have been complemented in recent years with molecular methods, such as sequencing of small-subunit (SSU) rRNA genes. Inconsistencies between the approaches are evident, although taxonomy of filamentous cyanobacteria with heterocysts as represented by Anabaena cylindrical, still hold true. There are an estimated 150 genera of cyanobacteria containing approximately 2000 species, of which around 46 have been reported as being toxicogenic (Hitzfeld et al., 2000; Ernst et al., 2006). Importantly, the genera and species which comprise problematic cyanobacteria are generally well recognized. Cyanobacteria are an ancient (occurring as long ago as 3500 million years; Schopf, 1993) and diverse group of microorganisms, and unsurprisingly, due to the extent of evolution, can exist in a wide range of habitats including fresh and marine water environments. Cyanobacterial habitats also include those that are considered extreme, such as frozen lakes, hot springs and salt works (Whitton, 1992).
METABOLISM AND PHYSIOLOGY Cyanobacteria are major components in the supply of global oxygen, sequestration of carbon dioxide (CO2) and nitrogen fixation, and as such play essential ecological roles. Currently, cyanobacteria are the only recognized prokaryotes that exhibit photosynthesis with the generation of oxygen. Protein complexes in thylakoid membranes of the cyanobacteria are responsible for both electron transport chains involved in photosynthesis and respiration. Cyanobacterial photosynthesis is essentially the same as seen in plants. Photosystem II exploits light energy to generate electrons from water, which in turn are transported via soluble electron carriers (plastocyanin or cytochrome c553) to the inner side of the thylakoid membrane. Photosystem I (chlorophyll) is then reduced leading to reduction of NADP. A proton gradient develops across the thylakoid membrane, which serves to drive ATP synthesis. Reduced NADPH from the process appears to be involved in carbon fixation. As a consequence, respiration in cyanobacteria is thought to involve succinate dehydrogenase activity as opposed to electron flow from NADPH.
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Nitrogen fixation is the process of nitrogen gas reduction through the action of the nitrogenase enzyme complex. Nitrogen gas, whilst being highly abundant in the environment, is highly inert, and therefore its conversion to a nitrogen product that can be incorporated into cellular material and processes requires reduction. The process of nitrogen fixation is, however, inhibited by oxygen (Postgate, 1998) and the fact that cyanobacteria are both aerobic and also generate oxygen as a metabolic by-product of photosynthesis means that strategies have had to be developed by these microorganisms to enable nitrogen fixation. Indeed, and as mentioned previously, the extent of the problem is highlighted by the fact that cyanobacteria are the only oxygenic microorganisms that can also fix nitrogen. The way cyanobacteria have dealt with this is by effectively separating the processes, both spatially and temporally. In terms of temporal separation, cyanobacteria effectively fix carbon during the day, whilst fixing nitrogen at night. Spatial separation of the processes is also evident in the filamentous cyanobacteria, where specialist anaerobic heterocysts are responsible for the process of nitrogen fixation and this means nitrogen fixation can also occur during the day (Berman-Frank et al., 2007; Kumar et al., 2010). The cyanobacterial genera that are recognized as being able to fix nitrogen include Anabaena, Aphanizomenon and Gloeotrichia. The genera of Microcystis, Coelosphaerium and Oscilhtoria appear to be unable to fix nitrogen. A range of cyanotoxins can be produced by cyanobacteria and these can be designated as neurotoxins (e.g. anatoxin-a, saxitoxin and neosaxitoxin; Wiese et al., 2010), hepatotoxins (e.g. microcystins, nodularins and cylindrospermopsin; Labine and Minuk, 2009), tumourogenic toxins (e.g. microcystins; Zegura et al., 2011), and toxins that are irritants affecting the skin and gastrointestinal tract (e.g. lipopolysaccharides; Stewart et al., 2006). Cyanotoxins are normally produced intracellularly and then generally released into the environment when cells lyse. Cyanobacterial growth is promoted by the presence of specific trace metals within water and this is particularly evident with iron and molybdenum. The presence of iron is believed to enhance the process of both photosynthesis and nitrogen fixation, whilst molybdenum appears to increase the rate of carbon fixation. The presence of zinc has also been reported to enhance growth and toxin production by certain cyanobacteria.
CLINICAL FEATURES While high densities of cyanobacteria may be present in the faeces of infected animals, there is little evidence to support persistence of these microorganisms in the normal intestinal flora of healthy warm-blooded animals. Cyanobacteria are not infectious agents, although as mentioned earlier, some species produce toxins during algal blooms, which are triggered by nutrient enrichment from natural waters and industrial effluents. There are about 25 species of cyanobacteria associated with adverse health effects (Gold et al., 1989). The first recognition of cyanobacterial algal blooms appears to
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have been as early as the 12th century in Lake Llangors in Wales, UK. Since this time, toxic algal blooms have been reported in many parts of Europe, USA, Australia, Africa, Asia and New Zealand. One survey in the UK found that 75% of cyanobacterial blooms contain toxins (Baxter, 1991). Within algal blooms, perhaps the most frequently encountered toxins are the microcystins and these are the toxins most often linked with incidences of animal and human poisoning. Of the many different kinds of microcystins globally reported, microcystin-LR is the most frequently encountered. The problem of microcystins is compounded by their high degree of chemical stability within water at both a wide range of temperatures and pH. The two main categories of toxins produced by cyanobacteria are the neurotoxins and hepatotoxins. Observations of poisoning of domestic animals show that when mucous membranes are exposed to cyanobacterial neurotoxins, symptoms develop in 4–10 minutes and death within 30 minutes. The effects of hepatotoxins can cause symptoms in exposed animals within 30 minutes and death within 24 hours. Using cobra venom as a point of reference, the alkaloid neurotoxins produced by species of Anabaena flos-aquae and Nodularia spumigena can be equally potent, with 20 pg per kg body weight being the lethal dose (LD50 of 200 pg/kg). The hepatotoxins produced by Microcystis aeruginosa and some strains of Aphanizomenon flos-aquae are in fact over twice as potent as cobra venom with 9 pg/kg being the LD50. A number of these toxins have been found to be tumour promoters and oncogenic in laboratory animals. One of the toxins isolated from Anabaena is an alkaloid that can cause neuromuscular blocking. Another toxin, isolated from Microcystis aeruginosa, affects the cardiovascular system and produces lesions in the livers of a variety of laboratory animals when administered orally or by intraperitoneal injection. Fortunately, large doses of these toxins are necessary to produce the symptoms in animals and residual toxins that might pass through a drinking water supply after conventional treatment would be diluted by vast volumes of water. This safety factor places the concentration far below any known human toxicity level. Some cyanobacteria produce toxins that are irritants, and if these are ingested in sufficient concentration they may cause gastrointestinal upset. Body contact exposure through recreational water activity may also induce skin irritations that lead to a rash. Control of cyanobacteria is a problem, as research has shown that the toxins can remain potent for days, even after the microorganisms have been destroyed by chlorination and copper sulphate treatment (El Saadi et al., 1995). Whilst there is toxicity data from mouse models, further research is required on the acute and chronic toxicity of cyanobacterial toxins and suitable methods need to be developed for monitoring the types and concentrations of cyanobacterial toxins in natural as well as treated drinking water. The Engineering and Water Supply Department of South Australia have developed interim guidelines for acceptable numbers of cyanobacteria in water supplies (El Saadi, 1995).
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PATHOGENICITY AND VIRULENCE No species in the cyanobacteria group is classed as a true pathogen of humans and animals. Cyanobacteria do not invade the animal/human body, but as mentioned above can produce potent toxins. It has been hypothesized that these toxins are produced by cyanobacteria to kill fish, thereby releasing nutrients for growth. There are three main types of cyanobacterial toxins and these are lipopolysaccharide endotoxins, hepatotoxins and neutrotoxins. The presence of these toxins in waters is not usually a problem for humans, but farm animals can be poisoned by drinking water from ponds containing dense cyanobacterial blooms. When acute health effects occur in humans they tend to include gastroenteritis, liver damage, nervous system drainage, pneumonia, sore throat, earache and contact irritation of skin and eyes. The potential chronic health effects of long-term exposure to cyanobacterial toxins in drinking water are unknown. It has been suggested that high rates of liver cancer in parts of China may be linked to cyanobacterial hepatotoxins in drinking water (Carmichael, 1994). Neurotoxins tend to exhibit their effects rapidly and those produced by cyanobacteria are generally one of three types based on mode of action. These modes of action include neuronal depolarization, the inhibition of cholinesterase, or ion channel blockage. Hepatotoxins produced by cyanobacteria are cyclic peptides and induce damage to hepatocyte cell structure. Hepatotoxins exhibit slower effects compared with neurotoxins and have also been reported as having tumorigenic activity (Falconer, and Humpage, 2001). Allergies to cyanobacteria have also been reported (Torokne et al., 2001). Treatment and therapy for cyanobacterial poisoning is very limited and indeed largely unavailable for the neurotoxins.
SURVIVAL IN THE ENVIRONMENT Since cyanobacteria are not dependent on a fixed source of carbon they are widely distributed throughout aquatic environments. Habitats include freshwater and marine environments and some soils. In fact, cyanobacteria are found in the early stages of soil formation, being associated with converting bare rock or decomposing debris. Stagnant water, sediments and soil appear to be the significant reservoirs for these organisms. In the environment, cyanobacteria exhibit a selective advantage over eukaryotes, due to the ability to fix nitrogen under adverse conditions.
Survival in Water and Epidemiology Cyanobacteria naturally occur in stream sediments, slow-moving streams, receiving waters for waste discharges and treatment effluents, rural storm runoff, drainage canals and marine waters. Prolific growth of these bacteria often occurs during summer months in surface waters. Densities of 500 cells or more per ml have been recorded at these times
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of the year. Polluted surface waters that become stagnant due to slow flows under summer drought conditions often support persisting populations of cyanobacteria. The growth of cyanobacteria is stimulated by high water temperature and high concentrations of inorganic nitrogen (N) and phosphorus (P). Indeed, to this end, cyanobacteria are able to exploit a wide range of dissolved organic forms of both phosphorus and nitrogen compounds through extracellular phosphatases and aminopeptidases. Their ability to be a source of biological nitrogen fixation in soils and water is also a significant contributor to their long-term survival and plays an important role in the ecological succession of microorganisms in the environment. In 1976, an outbreak of intestinal illness in Pennsylvania was associated with a cyanobacterial bloom in a municipal water supply and affected 62% of the population (Carmichael et al., 1985). Recreational water, particularly in the USA, has been implicated in causing adverse health effects following exposure to cyanobacterial toxins (Carmichael et al., 1985). There have been a number of cyanobacterial poisoning incidents through surface water contact (El Saadi et al., 1993; Soong et al., 1992). The most frequent mode of transmission of cyanobacterial toxins is by water ingestion, although in areas of massive cyanobacteria blooms, contact with the body through water sports is also of serious concern. There have been numerous reports of poisonings of livestock, pets and wildlife with waters containing cyanobacteria blooms. Outbreaks of human gastroenteritis from ingestion of toxic cyanobacteria in the public water supplies occurred in Charleston, West Virginia, USA, and the area served by the Anacostia Reservoir near Washington, DC, USA, during the drought years of 1930 and 1931. In 1981, an outbreak of human poisoning occurred in Northeastern Pennsylvania where 12 children and one adult were affected by an Anabaena species bloom. In another case study, in 1990, a localized outbreak of diarrhoea occurred among residents of a Chicago apartment building. This incident was traced to cyanobacteria toxins in an open water supply storage tank. Apparently, the cover to the tank had been inadvertently left open so that light, airborne cyanobacteria in dust particles gained access, grew and, in time, released toxic byproducts throughout the plumbing system. For utilities using surface water supplies, cyanobacteria are well known for their association with taste and odour problems, often regarded as a matter of aesthetics. In light of recent information on cyanobacteria, granular activated carbon (GAC) may be very important in toxin removal. Furthermore, for those water systems using disinfection as the only surface water treatment, there is always the threat of a seasonal passage of cyanobacteria and deposition of dead cells in the distribution pipe network. Such an occurrence provides a source of assimilable organic carbon (AOC), which is a potential nutrient for bacterial regrowth. The US Environmental Protection Agency (USEPA) or European governments regulate neither cyanobacteria nor their metabolites, except under guidelines, stating
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that drinking water must be potable. As defined, potable water must not only be safe but also clear and free from any objectionable tastes and odours, regardless of origin.
EVIDENCE FOR GROWTH IN A BIOFILM Cyanobacteria have the ability to grow as biofilms (Gaylarde et al., 2004; Rossi et al., 2012). In particular, cyanobacterial populations have been detected in their filamentous forms on buildings, with coccoid morphotypes also being shown to grow on artificial media as biofilms (Gaylarde et al., 2004; de los Rı´os et al., 2007).
METHODS OF DETECTION Monitoring water supplies for cyanobacterial presence is an important aspect to reducing harmful effects on human and animal health. Quantitative analysis of the microorganism and cyanotoxin levels are key measurements. The simplest approach to detect cyanobacterial algal blooms is visual inspection of water systems. Unfortunately, algal blooms may be present below the surface of the water and therefore may not readily be seen until a significant level of cyanobacteria is present. Analytical analysis of water samples for both the microorganism as well as toxins is therefore the most appropriate approach. To detect cyanobacteria, water samples are first blended with glass beads or treated by ultrasound to break filamentous forms prior to streaking on agar plates (AWWA, 1999). There are a variety of selected mineral media available (D-medium, ASM-1, BG-11 and WC) and these are incubated at 25 C under cool white fluorescent light. Cyanobacteria may grow extremely slowly, and may require several months of culture to generate visible colonies (Castenholz, 1988). Some recalcitrant cyanobacteria may not be freed easily of contaminants, thus, physical and chemical separation schemes may be necessary. Success in achieving pure cultures depends on persistence and patience. Microscopic examination using simple wet-mount staining techniques with India ink and methylene blue are used to identify morphological characteristics.
RISK ASSESSMENT Health Effects: Occurrence of Illness, Degree of Morbidity and Mortality, Probability of Illness Based on Infection • There are about 25 species of cyanobacteria that have been associated with adverse health effects. • Cyanobacterial toxins act primarily as hepatotoxins and neurotoxins, but can also cause skin irritation. They are extremely potent toxins and can potentially be fatal to
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humans. However, acute oral or dermal exposures have not resulted in any known human deaths. Reported illnesses in humans exposed to cyanobacterial toxins range from dermatitis and gastroenteritis to hepatitis and allergic reactions. Illness is selflimited. • Cyanobacterial toxins have been shown to be tumour promoters in animal studies and epidemiological evidence in humans suggests that chronic exposure to microcystins in drinking water is associated with an increase in hepatocellular cancer.
Exposure Assessment: Routes of Exposure and Transmission, Occurrence in Source Water, Environmental Fate • World-wide, the number of humans acutely affected by cyanobacterial toxins is low compared with other waterborne contaminants. However, because of decreasing water quality, the potential for an increase in incidents is high. • Routes of exposure are primarily ingestion through drinking water or recreational water contact, and also dermal exposure or possible aerosolization. • Most acute exposures occur from recreational water use and low levels in drinking water are associated with an increase in hepatocellular cancer in certain exposed populations. • Cyanobacteria are found in all types of water: lakes, rivers, marine environment, and drinking water reservoirs. Surface waters that receive waste effluents are at special risk for contamination. High water temperatures and concentrations of inorganic nitrogen and phosphorus stimulate growth. • Surface water systems exposed to a disinfectant may have deposition of dead cells in the distribution system with potential for regrowth. • Cyanobacterial toxins are ubiquitous, though their occurrence is dependent on conditions that contribute to algal bloom formation. Concentrations vary widely depending on the species of bloom and the stage of its formation and deterioration. Toxin concentrations range from 0.2 mg/l to 8.5 mg/l. • The toxic dose is unknown for human. The no observed adverse effect level (NOAEL) for mice dosed orally with microcystin-LR has been reported to be 40 mg/kg/day for 13 weeks. The NOAEL for mice dosed orally with anatoxin-a has been reported to be 0.1 mg/kg/day. Intraperitoneal and intranasal exposure is more potent than oral ingestion for both toxins.
Risk Mitigation: Drinking Water Treatment, Medical Treatment • There is some question as to the efficacy of standard drinking water treatment (e.g. coagulation, sedimentation, disinfection and filtration) for removing all but large concentrations of cyanobacterial toxins, though current methods are effective enough to prevent any acute effects.
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• Evidence on the efficacy of chlorine on degrading microcystins is equivocal; chlorine is effective on anatoxin-a. Activated carbon treatment appears to be the best removal method for treated water. • Preventing formation of blooms in the source water is the best way to assure cyanobacteria-free drinking water. • Membrane filtration technology has the potential to remove virtually any cyanobacteria or their associated toxins from drinking water. • Efficacious medical treatment is unknown in acute exposure; however, antihistamines, and steroids may be helpful for allergic reactions. If given in a timely manner, activated charcoal or an emetic could have a positive effect on the toxic response.
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Labine, M.A., Minuk, G.Y., 2009. Cyanobacterial toxins and liver disease. Can. J. Physiol. Pharmacol. 87 (10), 773–788. Lla´cer, J.L., Fita, I., Rubio, V., 2008. Arginine and nitrogen storage. Curr. Opin. Struct. Biol. 18 (6), 673–681. Postgate, J.R., 1998. Nitrogen fixation, third ed. Cambridge Univ Press, Cambridge. Reichwaldt, E.S., Ghadouani, A., 2012. Effects of rainfall patterns on toxic cyanobacterial blooms in a changing climate: between simplistic scenarios and complex dynamics. Water Res. 46 (5), 1372–1393. Rossi, F., Micheletti, E., Bruno, L., et al., 2012. Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments. Biofouling 28 (2), 215–224. Schopf, J.W., 1993. Microfossils of the Early Archean Apex Chert – new evidence of the antiquity of life. Science 260, 640–646. Shi, X., Yang, L., Niu, X., et al., 2003. Intracellular phosphorus metabolism of Microcystis aeruginosa under various redox potential in darkness. Microbiol. Res. 158 (4), 345–352. Singh, S.P., Montgomery, B.L., 2011. Determining cell shape: adaptive regulation of cyanobacterial cellular differentiation and morphology. Trends. Microbiol. 19 (6), 278–285. Soong, F.S.E., Maynard, E., Kirke, K., Luke, C., 1992. Illness associated with blue-green algae. Med. J. Aust. 156, 67. Stewart, I., Schluter, P.J., Shaw, G.R., 2006. Cyanobacterial lipopolysaccharides and human health – a review. Environ. Health 24 (5), 7. Torokne, A., Palovics, A., Bankine, M., 2001. Allergenic (sensitization, skin and eye irritation) effects of freshwater cyanobacteria – experimental evidence. Environ. Toxicol. 16 (6), 512–516. Wiese, M., D’Agostino, P.M., Mihali, T.K., et al., 2010. Neurotoxic alkaloids: saxitoxin and its analogs. Mar. Drugs 8 (7), 2185–2211. Whitton, B., 1992. Diversity, ecology and taxonomy of the cyanobacteria. In: Mann, H., Carr, N. (Eds.), Photosynthetic prokaryotes. Plenum Press, New York, pp. 1–51. Zegura, B., Straser, A., Filipic, M., 2011. Genotoxicity and potential carcinogenicity of cyanobacterial toxins – a review. Mutat. Res. 727 (1-2), 16–41.