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Review

Actinomycetes in relation to taste and odour in drinking water: Myths, tenets and truths
Beryl Zaitlina,, Susan B. Watsonb,c
a

Department of Geology and Geophysics, University of Calgary, Calgary, Alberta, Canada T2L 1M3 Environment Canada, National Water Research Institute, Burlington, Ont., Canada L7R 4A6 c Department of Biological Sciences, Division of Ecology, University of Calgary, Calgary, Alberta, Canada T2N 1N4
b

art i cle info

Article history: Received 17 August 2005 Received in revised form 17 January 2006 Accepted 15 February 2006 Available online 4 April 2006 Keywords: Taste Odour Geosmin Methylisoborneol Actinobacteria Streptomyces

A B S T R A C T

Actinomycetes are a complex group of bacteria present in a wide variety of environments, either as dormant spores or actively growing. Some actinomycetes produce two potent terpenoids (geosmin and 2-methylisoborneol (MIB)) and pyrazines, common causes of drinking water off avours, and have been implicated in taste and odour episodes. However, isolation from a water source is not evidence that actinomycetes caused a taste and odour event. Dormant spores of actinomycetes may be isolated from aquatic environments in high concentrations, despite production in the terrestrial environment. Similarly, odourous compounds produced by actinomycetes may be produced terrestrially and washed into aquatic environments, with or without the actinomycetes that produced them. Actinomycetes may exist as actively growing mycelium in small, specialized habitats within an aquatic system, but their odourous compounds may inuence a wider area. This paper attempts to elucidate the types and activities of actinomycetes that may be found in, or interact with, drinking water supplies. & 2006 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. Introduction: actinomycetesan enigmatic and misjudged villain? . Ecological and socioeconomic signicance of actinomycetes . . . . . . Systematics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identication of actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Enumeration methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Isolation onto media . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Indications of activity . . . . . . . . . . . . . . . . . . . . . . . . . . Source tracking taste and odour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrates for growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1742 1742 1744 1745 1745 1745 1745 1746 1748 1748

5. 6. 7.

Corresponding author. Tel.: +1 403 289 1680.

E-mail address: zaitlin@ucalgary.ca (B. Zaitlin). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.024

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8. Chemical ecology 9. Benecial roles . . 10. Conclusions . . . . References . . . . . . . . .

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1. Introduction: actinomycetesan enigmatic and misjudged villain?

Musty/earthy odours in adequately treated water supplies cause concern among consumers, who may think water with these odours is unsafe to drink. Musty/earthy odours are the second most common cause of odour problems recorded by water utilities, behind chlorine (Suffett et al., 1996). Actinomycetes have long been associated with musty/earthy odours in water and sh (Adams, 1929; Thaysen, 1936; Silvey and Roach, 1953), but their actual contribution to odour in freshwater was unknown. In the late 1960s, the earthy/musty smelling secondary metabolites geosmin and 2-methylisoborneol (MIB) were identied from actinomycete cultures (Gerber and Lechevalier, 1965; Gerber, 1979, 1983). Since then actinomycetes have gained considerable notoriety throughout the water industry as major sources of drinking water taste and odour (T/O). Actinomycetes have been implicated in a number of documented T/O episodes (Raschke et al., 1975; Jensen et al., 1994; Sugiura and Nakano, 2000; Klausen et al., 2004a, b). In many cases, however, the evidence linking actinomycetes to individual T/O events is tenuous at best, in part because their taxonomic identity, distribution and activity in aquatic ecosystems remain ambiguous. This is important, because signicant effort and expense have often been expended in efforts to enumerate and control actinomycetes, sometimes without clear evidence of the actinomycetes role in a T/O event. This paper aims to elucidate current knowledge on actinomycete ecology and socioeconomic signicance, taxonomy, and odour production, and thereby evaluate actinomycetes as primary causes of source water odour.

2. Ecological and socioeconomic signicance of actinomycetes

Actinomycetes have major socioeconomic importance (Table 1). They include human pathogens such as Actinomyces israelii, a primary cause of tooth decay and other, often serious, infections in humans (Beaman, 1981; Berkow and Fletcher, 1992). Non-pathogenic strains play essential roles as decomposers in terrestrial systems. These actinomycetes are vital to nutrient recycling and are among a small number of organisms capable of breaking down complex organic materials such as chitin (Lacey, 1973). Actinomycetes in the genus Frankia are also extremely important to numerous plant genera, acting as root-nodulating, nitrogen-xing plant symbionts (Huss-Danell, 1997). Many genera, notably Streptomyces, produce commercially important antibiotics (Goodfellow and ODonnell, 1989) and an array of other secondary metabolites. These include aliphatic alcohols, lactones, biogenic sulphides, ketones, esters, thioesters, lactones, furanones and chymova et al., isoprenoids (Gerber, 1983; Wilkins, 1996; Ja 2002; Scho ller et al., 2002). Early recognition of the potential application of many of these compounds has resulted in extensive research into their use as antibiotics, enzymes, enzyme inhibitors and other pharmaceutically useful compounds (Goodfellow and ODonnell, 1989). Most relevant to this paper, some actinomycetes produce the two potent terpenoids geosmin and MIB and pyrazines, common causes of drinking water off avours (Kikuchi et al., 1973; Gerber, chymova et al., 2002), and have been 1979; Wilkins, 1996; Ja implicated in a number of documented T/O episodes (Raschke et al., 1975; Jensen et al., 1994; Sugiura and Nakano, 2000; Klausen et al., 2004a).

Table 1 Roles played by selected actinomycete taxa Human pathogens

Actinomyces Arachnia Dermatophilus Micromonospora Nocardia Oerskovia Rhodococcus Rothia Streptomyces Thermopolyspora

Root-nodulating
Frankia

Antibiotic producing
Actinomadura Actinoplanes Actinosynnema Dactylosporangium Kibdelosporangium Kitasatosporia Microbisporia Micromonospora Nocardia Saccharopolyspora Streptoalloteichus Streptomyces Streptosporangium Streptoverticillium

Geosmin/MIB producing
Microbispora Nocardia Streptomyces

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Table 2 Actinomycete groups, their morphological characteristics and genera that have been found in aquatic systems Aggregate group
Actinobacteria Actinoplanetes Nocardioform Streptomycetes

Characteristics
Aerobic, facultatively anaerobic, or anaerobic, rods, cocci or fragmenting branched laments, no aerial mycelium Aerobic, non-motile spores which may be enclosed within vesicles, no aerial mycelium Aerobic, occur as rods, cocci or branched laments, or form substrate and aerial mycelium that fragments Aerobic, spore forming, form extensively branched substrate and aerial mycelium, spores on aerial mycelium and occasionally on substrate mycelium Thermophilic (4560 1C), Produce single spores on aerial or substrate mycelium

Genera
Actinomyces Actinoplanes, Micromonospora Nocardia, Rhodococcus, Aeromicrobium Streptomyces, Kitasatosporia

Thermoactinomyces (not a true actinomycete)

Thermoactinomyces

Modied from Goodfellow and Williams (1983).

Despite a considerable body of research, the ecology of these bacteria is not well understood. Actinomycetes are common soil inhabitants, where their production of the earthy-smelling geosmin and MIB contributes signicantly to the characteristic odour of soil (Gerber and Lechevalier, 1965; Buttery and Garibaldi, 1976). It is perhaps less often recognized that other important soil organisms, notably cyanobacteria and fungi such as Penicillium spp. and Chaetomium spp., are also signicant producers of these metabolites (Kikuchi et al., 1983; Larsen and Frisvad, 1995). The cyanobacteria are frequently implicated in T/O events, and are found in both soil and water (Watson, 2003). The Penicillium and Chaetomium fungi are very common in soil, and may contribute signicant odour to soil, which may then enter water by uvial transport. Soil actinomycetes and fungi are thought to be aerobes, and unlikely to produce any compounds after settling out into the sediment. However, it is not known how long they may continue to be metabolically active while in the water column. Actinomycetes are also found in many aquatic environments (Table 2). They have been isolated from both marine (Okami and Okazaki, 1978; Weyland, 1981; Jensen et al., 1991; Bruns et al., 2003) and freshwater systems (Willoughby, 1969; Rowbotham and Cross, 1977; Niemi et al., 1982; Jiang and Xu, 1996; Terkina et al., 2002; Klausen et al., 2004b), and in salt marshes (Hunter et al., 1981). In freshwater systems, odourproducing actinomycetes have been found in association with cyanobacteria (Sugiura et al., 1994), with aquatic plants (Raschke et al., 1975; Zaitlin et al., 2003a), and with zebra mussels (Lange and Wittmeyer, 1997a; Zaitlin et al., 2003a). Actinomycetes have also been found in association with terrestrial plant litter that had fallen into streams (Raschke et al., 1975; Makkar and Cross, 1982), and with chitin exoskeletons in streams (Aumen, 1980). In articial environments, actinomycetes were found in association with cat-tail (Typha latifolia) and bulrush (Scirpus acutus) roots in a constructed wetland (Hatano et al., 1994), in drinking water pipeline deposits (Zacheus et al., 2001), and in sewage treatment scum (Lemmer, 1986; Jenkins et al., 1993). Many actinomycetes are capable of forming spores, which can survive adverse conditions (e.g. salinity; Weyland, 1969)

and play an important role in their widespread distribution by wind and water-borne sediment (Lloyd, 1969; Goodfellow and Williams, 1983). Molecular techniques are now widely applied to identify and trace pathogenic Escherischia coli and faecal indicator bacteria in source waters, where similar terrestrialaquatic interactions obscure the origins of these bacteria (Simpson et al., 2002). Recent work has similarly demonstrated molecular biology as a highly promising tool to source track actinomycetes, and provided important evidence that some of these taxa are both indigenous and highly active in aquatic environments. In a study by Bruns et al. (2003) DNA hybridization was used to trace the origins of the actinomycete Aeromicrobium marinum isolated from nearshore surface water of the German Wadden Sea. The authors estimated that this one taxon alone represented an astonishing 1 104 cells ml1, or $1%, of the total bacterial microora of these surface waters. Such high planktonic densities are unlikely to be sustained by shoreline runoff of terrestrial populations, and point instead to active growth in the marine environment. Bruns et al. (2003) found that cultures of the A. marinum isolate had an obligate salt requirement, also indicative of an autochthonous marine inhabitant. Other signicant work has similarly used molecular biology to identify indigenous taxa. Mincer et al. (2002) isolated a group of actinomycetes (MAR 1) from diverse shallow water (o30 m) tropical marine sediments. 16S rDNA phylogenetic sequence analysis showed these isolates as a distinct group within the Micromonosporaceae, which demonstrated an obligate requirement for high salinity in vitro. These taxa were isolated in high numbers, up to 104 colony-forming units (CFU)1 ml1 from nearshore Bahamian sediments, suggesting that this group is also an autochthonous and common marine inhabitant. A separate research group extracted Streptomyces rRNA from coastal salt marsh sediment at concentrations which represented 25% of the sediment community RNA. Dormant spores do not produce rRNA, and the presence of Streptomyces rRNA indicates active vegetative cells (Moran et al., 1995). There is interest in exploring these newly discovered marine actinomycete taxa for useful metabolites

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(Jensen and Fenical, 1996), but the ecological roles of these actinomycetes have not yet been explored.

3.

Systematics

Actinomycetes have a wide range of morphologies, and many of the more widely studied taxa (notably odour-producing members of the genus Streptomyces) have a branching lamentous vegetative form supercially resembling fungal hyphae (Figs. 1A, B), although actinomycetes are not fungi. Filamentous streptomycetes represent one of many bacteria taxa belonging to the taxonomic order Actinomycetales which includes a diversity of morphotypes, including singlecelled rod-shaped cocci, fragmenting hyphae and branched mycelia (Goodfellow and Williams, 1983). Current systematics denes the order Actinomycetales as Gram-positive bacteria characterized by DNA rich in guanine and cytosine (Goodfellow, 1989). This classication groups a collection of organisms with dissimilar morphologies and ecological roles into a single order; many of the bacteria grouped in the Actinomycetales are entirely unrelated to source water odour. In fact, these bacteria are isolated from a wide range of terrestrial and aquatic systems. However, while viable isolates can be obtained from many habitats, these gram (+) bacteria are slow-growing and notoriously difcult to culture. Because of this, their distribution and ecology, and the degree to which they area able to colonize and adapt to different environments remain largely unresolved. This information is vital to our ability to trace the origins of T/O and distinguish between potential terrestrial or aquatic sources. The most common actinomycetes isolated from freshwater environments include Actinoplanes, Micromonospora, Rhodococcus, Streptomyces and Thermoactinomyces (Goodfellow and Williams, 1983) (Table 2). Less frequently, Actinomyces, Kitasatosporia and Nocardia (Cross, 1981; Jiang and Xu, 1996; Wohl and McArthur, 1998, 2001) are isolated from aquatic environments (Table 2). Thermoactinomyces, which are thermophilic and endospore-forming and Rhodococcus, which are generally

Fig. 1 Diagram and light microscope photograph of Streptomyces sp. showing features of an actinomycete. (a) Chains of arthrospores, which are made of segments of hyphae with thickened walls surrounded by a hydrophobic sheath; (b) aerial mycelium, growing above the substrate; and (c) substrate mycelium.

coprophilic (associated with manure), are thought to grow exclusively in the terrestrial environment but are washed frequently into aquatic environments (Al-Diwany and Cross, 1978; Cross, 1981; Goodfellow and Williams, 1983). These genera are not known to produce odourous metabolites. On the other hand, Actinoplanes is associated with allochthonous leaf litter in streams, and has motile zoospores, arguing for aquatic activity (Makkar and Cross, 1982). In fact, a phage specic to Actinoplanes has been isolated from stream and lake water, also indicating that active growth of Actinoplanes occurs in water (Willoughby et al., 1972). This genus also has never been shown to produce odour. With other taxa such as Micromonospora and Streptomycetes it is still unclear whether they are indigenous to both terrestrial and aquatic environments. Both Micromonospora and Streptomycetes are frequently isolated from the water column and lake sediments (Cross, 1981; Jiang and Xu, 1996; Terkina et al., 2002). Both genera are also common soil inhabitants, and it is unclear whether either is capable of active growth in aquatic environments. Micromonospora is often more abundant than Streptomyces in lake sediment based on CFU isolated (Cross, 1981; Jiang and Xu, 1996; Terkina et al., 2002), but this may be due to the long (4100 yr) survival times of Micromonospora spores in this milieu, as noted from sediment cores (Cross and Attwell, 1974). Micromonospora mycelium (i.e. the active growth form) has also been found in signicant amounts in lake sediments, indicating that species in this genus may be active in this environment (Johnston and Cross, 1976). In comparison, Streptomyces have been isolated as spores, suggesting a primarily dormant presence due to wash-in (Johnston and Cross, 1976). In the Chesapeake Bay, the highest diversity and counts of actinomycetes isolated from sediments occurred at the head of the bay, where the water is fresh and sediments are brought down by rivers. Further out in the bay, where there is less sediment and a greater marine inuence, there were fewer actinomycetes and they were less diverse, becoming predominantly Micromonospora spp. (Takizawa et al., 1993), indicating Micromonospora may be actively reproducing in the aquatic environment. However, several phages isolated from the surface of lake mud in England have not only been shown to be specic to species of Streptomyces but also appear different from phages isolated from terrestrial soil, indicating that some species of Streptomyces are active in freshwater lacustrine environments (Willoughby, 1974). Streptomyces is the primary actinomycete known to produce odourous metabolites. Among the last three genera, Actinomyces and Nocardia include human or animal pathogens (Beaman, 1981; Table 1). Nocardia species are also commonly observed in activated wastewater sludge, where they often cause problems with foaming (Jenkins et al., 1993). Two isolates of Nocardia have been shown to produce geosmin in the laboratory (Gerber, 1979), but there are no reported tests for odourous metabolite production from Nocardia spp. isolated from aquatic environments. Similarly, Actinomyces is not known to produce odourous metabolites. Kitasatosporia is a new genus, proposed in 1983, that culturally and morphologically resembles Streptomyces but can be distinguished on the basis of cell wall chemistry (Takahashi et al., 1983). Odour production by Kitasatosporia has not been documented.

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Isolates of other genera, including Microbispora and Actinomadura, have been shown to produce geosmin and/or MIB (Gerber, 1979), but these have not been isolated from aquatic environments. Overall, only members of the genus Streptomyces have been both isolated from aquatic environments and shown to produce odourous metabolites.

traditional plate counts are still widely used (Wohl et al., 1998; Sugiura and Nakano, 2000; Lanciotti et al., 2003; Klausen et al., 2004a).

4.1.1.

Isolation onto media

4.

Identication of actinomycetes

Identication of isolated actinomycetes may follow one of several methods of classication. Major works on the identication of actinomycetes have been published in a review by Goodfellow and ODonnell (1989) and in Holt et al. (1994). Identication to genus can usually be accomplished by using a combination of morphological and chemical properties (LeChevalier and LeChevalier, 1980; Goodfellow 1989), but characterization to species is often more difcult. 16S rRNA sequencing has also been used to differentiate between genera of actinomycetes, which generally agree with morphological and chemical taxonomy, although there are some differences (Embley and Stackebrandt, 1994). In terms of odour production, identication of an actinomycete to genus or species may give limited information: Although Streptomycetes are the major known odour producers (Kikuchi et al., 1973; Sugiura et al., 2000; Zaitlin et al., 2003a), not all Streptomycetes produce odours (Zaitlin et al., 2003a), and those that do, do not produce them under all circumstances (Sivonen, 1982).

4.1.

Enumeration methods

Actinomycetes in water are difcult to quantify. When water is sampled by traditional plating methods, it is not possible to distinguish between actively growing inhabitants of the aquatic environment and dormant spores. Moreover, taxa that do not grow on the nutrient media used will be missed. Nevertheless, traditional plate counts give an idea of the potential actinomycete population, and allow laboratory isolation of taxa present in an area. For these reasons,

Isolation onto growth media captures both actively growing forms and dormant spores. The dormant spores rapidly become active on the growth medium and are indistinguishable from the forms captured in an active state. All methods of isolation are selective to some degree, and the common practice of diluting a substrate, then isolating from the diluted material, selects those forms and species which are most prevalent in the substrate, whether they are actively growing or not. Nevertheless, isolation from a substrate does give an indication of the potentially active community. Common isolation media include colloidal chitin (Lingappa and Lockwood, 1962; Hsu and Lockwood, 1975), starch casein (Ku ster and Williams, 1964), M3 (Rowbotham and Cross, 1977) or diluted standard microbiological media such as nutrient or potato-dextrose (Difco Manual, 1969) (Table 3). Antifungal agents such as nystatin or actididione are generally added (Williams and Davies, 1965). Air-drying or heating samples before plating is often done to favour isolation of sporeforming strains (Goodfellow and ODonnell, 1989; Jayashree et al., 1991), and water samples may be membrane ltered or centrifuged before plating (Goodfellow and ODonnell, 1989). Nalidixic acid may also be added to plates to suppress Gramnegative bacterial growth (Takizawa et al., 1993). It should be emphasized, however, that isolation of actinomycetes from a given environment does not give an accurate assessment of their activity there, but simply reects viability and adaptability to the culture conditions applied (Goodfellow and ODonnell, 1989).

4.1.2.

Indications of activity

As the actinomycetes of interest in T/O research are all lamentous, one technique is to compare the numbers of laments and spores from samples treated with different periods of homogenization. Filaments break into smaller

Table 3 Common media used for isolation of actinomycetes from water Media
Actinomycete isolation agar Chitin agar Starch-casein Czapeks agar M3 agar

Ingredients (per l d/d water)

Difcos 4 g ground colloidal chitin, 0.3 g KH2PO4, 0.7 g K2HPO4, 0.5 g MgSO4 7H2O, 0.01 g FeSO4 7H2O, 0.001 g ZnSO4 7H2O, 0.001 g MnCl 4H2O, 20 g agar, adjust pH to 6.87.0 after autoclaving 10.0 g starch, 2.0 g KNO3, 0.3 g casein, 2.0 g NaCl, 2.0 g K2HPO4, 0.05 g MgSO4 7H20, 0.01 g FeSO4 7H2O, 18 g agar, adjust pH to 6.87.0 after autoclaving 30 g sucrose, 3 g NaNO3, 1.0 g K2HPO4, 0.15 g MgSO4 7H20, 0.01 g FeSO4 7H2O, 15 g agar, autoclave 0.5 g K2HPO4, 0.75 g Na2HPO4, 0.1 g KNO3, 0.3 g NaCl, 0.1 g MgSO4 7H20, 0.02 g CaCO3, 0.2 g sodium propionate, 0.002 g FeSO4 7H2O, 0.0018 g ZnSO4 7H2O, 0.0002 g MnSO4 4H2O, 18 g agar, add sterile-ltered 0.04 g thiamine HCl after autoclaving 0.0001 g yeast extract, 15 g agar, autoclave

Water agar

100 mg ml1 cycloheximide or 50 mg ml1 nystatin and/or 20 mg ml1 polymixin B sulphate may be added by sterile ltration after the medium has been autoclaved and slightly cooled. Nalidixic acid may also be added at 10 mg ml1.

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segments, so plate counts will increase with increasing homogenization until a lethal effect is reached; conversely, spores are largely unaffected by homogenization and plate counts derived from spores remain stable with increasing periods of homogenization (Skinner, 1951; Johnston and Cross, 1976). Digital image analysis offers a potential supplemental or alternative approach. In this analysis, a digital photograph is taken of a microscopic eld containing laments, and then test bars are digitally superimposed across the image. The number of laments that intercept the test bars can be used to calculate the cumulative length of the laments. This approach has been used to quantify cyanobacteria (Nedoma et al., 2001), but to our knowledge, has not yet been applied to actinomycetes. A third technique to determine the numbers of active actinomycetes in an aquatic environment is to measure the number of lytic phage for a particular actinomycete species in that environment. For this method, enrichment broth is inoculated with water or sediment from the aquatic environment in question. After several days of enrichment, the broth is ltered through a 0.22 mm pore size lter to remove all bacteria. The resulting ltrate is then spread on a lawn of an actinomycete isolated from that environment. As phage are generally very specic, the number of plaques that form will give an indication of the activity of phage in that environment, which in turn indicates the activity of the actinomycete in that environment (Willoughby et al., 1972). An advantage of this technique is that it can be done in any reasonably equipped biology laboratory, and requires no specialized equipment. RNA probes have also been used to determine actinomycete activity. 16S ribosomal RNA (rRNA) contains highly variable regions which can be used to identify a specic group of bacteria. Short (approximately 20 nucleotide) probes are made and tagged with uorescent material. These probes may then be applied to a batch of bacteria ltered out of a natural source and prepared for hybridization. The probes

will attach only to bacteria to which they are complementary, and these bacteria will be visible by uorescence microscopy (Klausen et al., 2004b). This technique allows enumeration of bacteria which are difcult to culture, and allows the proportion of tagged bacteria to total bacterial population to be estimated.

5.

Source tracking taste and odour

As noted above, no standard and denitive method has been developed to trace the origins and in situ activities of actinomycetes isolated from aquatic systems and thereby evaluate their supposed link to T/O events. There is still considerable debate whether these cells and associated volatile organic compounds (VOCs) are produced in terrestrial environments, and are introduced into water, or if actinomycetes actively grow and produce VOCs in aquatic environments. A second major question is whether they inhabit aquatic environments, and, if so, what ecological niches they occupy. After chlorine, earthy/musty odours are the second most common type of reported T/O (Suffett, 1996), and most often caused by the metabolites geosmin and MIB (Wnorowski, 1992 and many others). As observed above, some actinomycetes (notably Streptomyces) produce one or both of these terpenoids, as well as other musty/earthy smelling metabolites (Table 4). Actinomycetes have been associated with musty/earthy odours in water and sh since the early 1900s (Adams, 1929; Thaysen, 1936). However, such off-avours may not necessarily be derived from these bacteria. Many cyanobacteria (blue-green algae) are major producers of geosmin and/or MIB (e.g. Anabaena, Geitlerenema, Hyella, Jaaginema, Leiblenia, Oscillatoria, Phormidium, Planktothrix, Porphyrosiphon, Pseudoanabaena, Schizothrix, Symploca and Tychonema (Watson, 2003; Izaguirre and Taylor, 2004), and odour source-tracking must certainly consider both groups as potential sources. In some documented cases where actinomycetes and cyanobacteria are both candidate causes, cyanobacteria

Table 4 Major odorous secondary metabolites produced by actinomycetes Secondary metabolite Actinomycete taxa reported to produce
Streptomyces Streptomyces Actinomadura Streptomyces Streptomyces Streptomyces Pseudonocardia Saccharomonospora Thermoactinomyces Thermomonospora Thermoactinomyces

Odour description
Earthy Musty

References

Trans-1,10-dimethyl-trans-9-decalol (Geosmin) 1,2,7,7-tetramethyl-2-norbornanol (2-methylisoborneol) 6-ethyl-3-isobutyl-2-pyrone (mucidone) 2-isobutyl-3-methoxypyrazine or 2isopropyl-3-methoxypyrazine Dimethyl disulde or Dimethyl trisulde

Gerber (1979), Scho ller et al. (2002), chymova et al. (2002) Ja Gerber (1979), Scho ller et al. (2002), chymova et al. (2002) Ja Gerber (1979) Gerber (1979) Wilkins (1996) Scho ller et al. (2002)

Musty Potatolike

6-methyl-5-hepten-2-one

Wilkins (1996)

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abundance has been found to correlate closely with odour concentration while actinomycete numbers did not (Leventer and Eren, 1970; Persson, 1980, 1981). In other studies, neither actinomycetes nor cyanobacterial numbers were correlated with muddy odour or geosmin levels (GAP, 2000). One study performed fungal and actinomycete counts in source water, but found no relationships between either actinomycete or fungal counts with odour intensity (Popovska, 1983). Other studies have found a signicant relationship between actinomycete levels, particularly during low river ows and high (417 1C) temperatures (Raschke et al., 1975). Actinomycete counts in source waters have been found to correlate with increased turbidity and increased odour levels, implicating terrestrial runoff as the primary source (Jensen et al., 1994). In some cases, both actinomycete and cyanobacteria abundances were found to correlate with increased geosmin and odour levels (Sugiura and Nakano, 2000). In a study in the River Arno (Italy), cyanobacterial counts were correlated with high odour periods in the spring and summer, while actinomycete counts were correlated with periods of high odour production in the autumn and winter (Lanciotti et al., 2003). There are several possible reasons for this ambiguity. Actinomycetes and cyanobacteria are not the only organisms that produce geosmin, MIB and other musty-earthy compounds. These compounds are produced by gliding bacteria (Lysobacter; Emde et al., 1992), an amoeba species; or bacterial symbionts within (Hayes et al., 1991); and several genera of myxobacteria (Trowitzsch et al., 1981; Yamamoto et al., 1994; Schulz et al., 2004). Many of these organisms are poorly studied as components of aquatic microora and some authors suggest that these taxa may contribute in situ to geosmin in fresh water (Hayes et al., 1991; Emde et al., 1992). In addition to the actinomycetes, common soil fungi in the genera Penicillium (Bo rjesson et al., 1993; Larsen and Frisvad, 1995), Aspergillus (Bo rjesson et al., 1993), and Chaetomium (Kikuchi et al., 1983; Bjurman and Kristensson, 1992) are also known to produce geosmin/MIB. Actinomycetes and fungi are generally predominant in terrestrial soil environments (Stahl and Parkin, 1994), although cyanobacteria are often also present (Baldwin and Whitton, 1992). In terrestrial environments, it has been estimated that up to 8.7 mg geosmin and 12.2 mg MIB kg1 of soil may be produced, and these compounds may be purged from the soil by stirring the soil in water (Stahl and Parkin, 1996). In water, geosmin is detectable as an off-avour at an average threshold of 0.016 mg l1 and MIB at 0.018 mg l1 (Young et al., 1996). This implies that less than 1.5 g of soil in a litre of water could produce detectable odours. Geosmin and other earthymusty compounds produced in the terrestrial environment may be transported into water by runoff. Elevated geosmin/odour levels have been associated with periods of increased runoff (Raschke et al., 1975; Hrudey et al., 1992; Jensen et al., 1994), and increased actinomycete levels are associated with increased ow rate or turbidity kova et al., 2002a; Lanciotti et al., (Jensen et al., 1994; Uhna 2003). Actinomycetes have also been isolated from sediment near or in water (Niemi et al., 1982; Wood et al., 1983; Takizawa, 1993; Zaitlin et al., 2003a). It is important to note

here that in fact, taste and odour episodes may not in fact involve either geosmin or MIB. Hrudey et al. (1992) found that odorous aldehydes were a major component of water odour in the North Saskatchewan River. Butyric acid was found to be a major odour-causing compound produced by actinomycetes isolated from lake mud (Weete et al., 1979). These compounds often go undetected because of low odour threshold levels and presence usually at subnanogram levels, requiring advanced and highly sensitive analysis. This could provide one very important explanation for the apparent discrepancies between actinomycete numbers, odour and geosmin or MIB levels. Although it is still debatable as to whether actinomycetes are capable of active growth in open water, evidence suggests that they are active on submerged substrates. It has been suggested that actinomycetes may live on zebra mussel (Dreissena sp.) interstices and faecal material (Lange and Wittmeyer, 1997a, b). One isolate of Streptomyces, capable of high levels of geosmin production was found in association with zebra mussels, although it was not determined whether this bacterium was associated with a specic tissue, or faecal/ biolm material (Zaitlin et al., 2003a). Streptomyces were found in the mantle, coelomic uid and stomach of the marine mussel Crenomytilus grayanus, suggesting that actinomycetes may be part of an autochthonous microora associated with this mussel (Ivanova and Mikhailov, 1992). In constructed wetlands used for wastewater treatment, streptomycetes formed the majority of bacterial isolates found on submerged cattail (Typha latifola) and bulrush (Scirpus acutus) stems, along with isolates from the actinomycete genera Micromonospora, Actinoplanes and Streptosporangium (Hatano et al., 1994). Streptomycetes were also the dominant genera isolated from aquatic plants in the Savannah River, South Carolina, and at least eight other genera of actinomycetes were also isolated from these plants (Wohl and McArthur, 1998). Actinomycetes have also been found in soft deposits in water distribution pipes, at counts up to 1.5 103 CFU l1 (Zacheus et al., 2001). Filamentous organisms, assumed to be bacteria, were found on loose deposits and corroded particles from a Delaware water hydrant (Herson et al., 1991). The ability of these actinomycetes to produce T/O compounds was not tested; however, actinomycetes in distribution systems are potentially an overlooked source of taste and odour problems. Simple counts of actinomycete propagules may not correlate with geosmin or MIB levels. In vitro studies have found considerable variation among strains in the presence/absence of production and in the per capita yield of one or both of these terpenoids under laboratory conditions (Sivonen 1982; Schrader and Blevins, 1993; Zaitlin et al., 2003a, b). For those strains that do produce geosmin and MIB, culture conditions inuence the amount of geosmin/MIB/earthy odour produced signicantly (Sivonen, 1982; Dionigi et al., 1992; Dionigi and Ingram, 1994; Blevins et al., 1995; Schrader and Blevins, 2001). The actinomycetes tested in these reports showed increased geosmin production at temperatures that are unlikely to be seen in most aquatic systems, (3540 1C) with far less production at more representative temperatures of 15 1C (Aoyama et al., 1993; Dionigi and Ingram, 1994; Blevins et al., 1995). However, many of the actinomycetes tested were isolated from sediments (Blevins et al., 1995; Schrader and

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Blevins, 1999; Schrader and Blevins, 2001), implying the actinomycetes in question may have had a terrestrial origin, where soil temperatures may regularly reach 15 1C or more. Other factors, such as pH and carbon sources also have been found to inuence production, and again this has been found to vary among different isolates. For example, an isolate of S. halstedii from sediment showed optimal growth, as measured by biomass, at pH 67, but optimal geosmin production at pH 9, and was able to produce geosmin over the extensive range of pH 611 (Blevins et al., 1995).

6.

Substrates for growth

Studies on the inuence of different carbon (energy) sources have shown intriguing results with important implications for activity and growth in the environment. In a study by Schrader and Blevins (2001), the effect of carbon source, phosphorous and other nutrients was evaluated. More readily assimilated carbon sources such as glucose were found to increase biomass but not geosmin production of S. halstedii, while more recaltricant sources such as mannitol increased per capita yield. However, growth of S. halstedii on a particular carbon source was not a good predictor of geosmin production on that carbon source. Increasing phosphorous from 0 up to 36 mM increased geosmin per capita and total biomass production while zinc, copper and iron inhibited geosmin production. Potassium appeared to have little effect. The range of these compounds was at levels unlikely to be seen in most surface waters, but likely found in soils, biolms and sediments. Conversely, Dionigi et al. (1996) found that copper sulphate addition increased biomass and per capita yield of geosmin in S. tendae, while manganese, magnesium, iron, cobalt, nickel and zinc had limited effects on both growth and per capita geosmin production. Increased atmospheric carbon dioxide levels and whole cells or lysed cells of the cyanobacterium Oscillatoria tenuis also increased geosmin production, though not biomass production (Schrader and Blevins, 1999). Geosmin production may also be related to growth stage of the actinomycete. Mutants of Streptomyces sp. that lost the ability to produce spores or aerial mycelium also stopped producing geosmin (Redshaw et al., 1979; Bentley and Meganathan, 1981). Normal isolates grown on medium that was not conducive to sporulation reduced their geosmin biosynthesis compared to those grown on medium that promoted sporulation (Dionigi et al., 1992).

pathways, but produce geosmin precursors (e.g. farnesols) via the MEP pathway (Seto et al., 1996; Spiteller et al., 2002). In comparison, some studies indicate that cyanobacteria use the MVA route to synthesize geosmin and MIB (Naes et al., 1989; Utkilen and Frshaug, 1992; Zimba et al., 1999), as also found for geosmin production by liverworts (Spiteller et al., 2002). The ability to produce geosmin/MIB may be lost through repetitive subculturing (Izaguirre, Zaitlin, unpublished data), which implies that production of these secondary metabolites is inducible and selected for by certain factors; once in culture and under idealized conditions, there is no selection to produce these compounds. A gene necessary for geosmin production has been localized to the cyc 2 gene in Streptomyces coelicolor, which is also responsible for a step in the mevalonic acid pathway (Gust et al., 2003). However, the synthesis of odourous compounds may not be entirely genomically regulated. Ishibashi (1992) demonstrated the loss of odorous compounds (presumably geosmin and/or MIB) from several strains of Streptomyces spp. following plasmid curing, suggesting that production of these compounds may be plasmid mediated. In plasmid curing, protoplast cells are grown under conditions that permit replication of genomic DNA but inhibit replication of extragenomic DNA such as plasmids. The cured strains may then be regenerated to form normal, but plasmidfree cells. However, in other cases, plasmid-containing isolates of S. halstedii and S. violaceusniger were cured of plasmids, resulting in reduction of odour by more than 50%, but production was not entirely eliminated. This suggests that although production of these compounds may be plasmid mediated, actual coding for these compounds may be chromosomal (Saadoun et al., 1998).

8.

Chemical ecology

7.

Biosynthesis

The biosynthesis of these geosmin and MIB compounds by different organisms is known to occur via two common pathways, the mevalonic acid (MVA) and the 1-deoxy-Dxylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) routes (Wanke et al., 2001). Some taxa use either or both routes depending on growth stage (W. Boland, pers. comm.). Bentley and Meganathan (1981) suggested that geosmin and MIB may be produced by actinomycetes from mevalonate. Later studies of geosmin production by Streptomyces indicate that these taxa maintain both biosynthetic

The purpose, if any, of MIB and geosmin production by actinomycetes is unclear. For some time these metabolites have been considered simply as metabolic byproducts, particularly in respect to cyanobacteria where geosmin was thought to be a side product of carotenoid synthesis (e.g. Utkilen and Frshaug, 1992). More recently, these and many other secondary metabolites have been re-examined for their potential bioactivity, in an attempt to understand the triggers, mode and dynamics of production (Watson, 2003; Watson and Cruz Rivera, 2003). The increased per capita production under certain circumstances suggests that rather than an overow waste-disposal mechanism, geosmin may function as a quorum sensing signal molecule. Quorum sensing is a mechanism that has been shown to exist widely in Gram-negative bacteria, where a chemical signal is released that enables a group of Gramnegative bacteria to collectively control gene expression and synchronize group behaviour (Lazdunski et al., 2004). There is evidence that they may act as allelogens in some environments. Thus Dionigi et al. (1993) found that concentrations of MIB and geosmin above 45.2 and 18.1 mg l1 respectively were inhibitory to Salmonella typhimurium suggesting a possible antagonistic role against competing microbial ora. These levels are far above what would normally be seen in the plankton, and at the high end of what might be

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seen in the soil, but are conceivably possible at a microscale level in sediments or biolms (Watson and Ridal, 2004). et al. (1999) observed In regard to higher organisms, Gagne that high concentrations of MIB reduced cell viability in trout hepatocytes, although geosmin did not. DNA damage in trout hepatocytes occurred at 0.45 mg l1 geosmin and 10 mg l1 MIB. Such levels might conceivably be experienced by organisms in close vicinity to producers in littoral or benthic areas of the aquatic environment. Thus inhibition of sea urchin embryos was found to occur above 40 mg l1 geosmin or 100 mg l1 MIB (Nakajima et al., 1996), although the choice of sea urchins as test organisms is perhaps not ideal, as geosmin and MIB are not known to be produced in marine systems. The long-term effects of chronic exposure have not been studied. Other work indicates that actinomycetes engage in chemical interactions where the compounds involved are yet to be identied. There is some evidence to suggest that the presence of actinomycetes may reduce the activity of other aquatic bacteria and some fungi. Aumen (1980) observed that chitinous exoskeletons of craysh introduced into a small stream were heavily colonized by actinomycetes after initial colonization by other bacteria and fungi. A single Streptomyces sp. became the dominant organism from approximately 18 days onward until the experiment was terminated at 46 days. In a study by Wohl and McArthur (2001), Kitasatosporia griseola, K. azaticus and two Streptomyces sp. Were found to inhibit the growth of two aquatic hyphomycetous fungi (Anguillospora liformis and Saprolegnia sp.) and to slow decomposition of detached leaves of the aquatic macrophyte Sparganium americanum. Similarly, a study of unidentied bacteria isolated from aquatic plant litter was shown to inhibit fungal growth on submerged plant material in the laboratory (MilleLindblom and Tranvik, 2003). However, in a study of organic matter decomposition in sediment in shallow Australian lakes, actinomycete population densities did not correlate with organic matter decomposition rates (Boon, 1989). Overall, the evidence suggests that actinomycete roles in the aquatic environment are similar to those they play in terrestrial environments: Decomposition of recaltricant substances such as chitin and inhibition of growth of fungi (Lockwood, 1959; Williams, 1978; Goodfellow and Williams 1983; Zaitlin et al., 2004).

from the Danube River were able to degrade phthalates, benzene and benzoate derivatives and aliphatic alcohols kova et al., 2002b). Nocardia corallina was shown in vitro (Uhna to degrade diphenylmethane dyes, including crystal violet 3, a common commercial dye which is generally recaltricant to decomposition (Yatome et al., 1993). Actinomycetes may play a role in decomposition of bluegreen algae and other bacteria, although it is unclear whether the actinomycetes are active antagonists, or if they are decomposing organisms that have senesced (Silvey, 1964). In one study, an actinomycete identied as Streptomyces phaeofaciens strain S-9 was shown to lyse cells of the cyanobacterium Microcystis aeruginosa, suggesting that this actinomycete might be useful as a biocontrol agent for cyanobacteria, or that it may already be doing so naturally (Yamamoto et al., 1998). Finally, actinomycetes may be useful in archaeological studies: Actinomycetes in the genus Thermoactinomyces are closely associated with agricultural activity, as they are active in decomposing hay and manure (Cross, 1981). These actinomycetes produce spores that can be long-lived and isolated from aquatic sediments years after they entered them (Johnston and Cross, 1976). In a study in Sweden, viable Thermoactinomyces spores were isolated from 9000 year old sediment cores in lakes, and the concentrations of these spores correlated with pollen data indicating agricultural activity in the vicinity of these lakes (Nilsson and Renberg, 1990).

10.

Conclusions

9.

Benecial roles

In addition to the production of commercially valuable metabolites and activity in soils and recycling processes discussed above, actinomycetes have the potential for other benecial activities. The production of useful compounds by actinomycetes from aquatic environments is just beginning to be studied, but this area looks extremely promising (Jensen and Fenical, 1996). Furthermore, some actinomycetes appear to facilitate the degradation of anthropogenic compounds in water. An isolate of Micromonospora purpurea, isolated from bottom sediments of Lake Baikal, was found to be able to degrade the otherwise-recaltricant phthalate bis-(2-ethylhexyl) phthalate in cold (4 1C) aqueous suspension in the laboratory (Azarova et al., 2003). Isolates of Streptomyces sp.

Actinomycetes are widely found in nearly all aquatic environments, from freshwater to saltwater. Isolation of actinomycetes from these environments does not necessarily mean they are actively growing there, as spores may wash in from terrestrial environments and persist in the aquatic environment for extended periods of time. However, Rhodococcus marinonascens, Aeromicrobium marinum and species of Streptomyces, Actinoplanetes and Micromonospora appear to be autochthonous marine inhabitants. In freshwater, the genus Actinoplanes appears to unquestionably inhabit aquatic environments, but the evidence is less clear for other genera. There appear to be species in the genera Streptomyces, Kitasatosporia and Micromonospora which are at least facultatively aquatic. The location of actinomycetes in freshwater systems also warrants further investigation. Isolation from open water and sediment may select for actinomycetes that are from terrestrial sources, however, habitats such as mussels, pipes and periphyton have only had limited examination for actinomycetes. Even if actinomycetes are present in low numbers across a basin, their impact in causing taste and odour episodes may be greatly magnied if they are colonising water intake pipes and similar structures. Furthermore, there may be actinomycetes specialized for survival in these environments, and it is these actinomycetes that should be examined for their potential odour production when problems arise with off-avours in drinking water supplies. The abundances of all actinomycetes in a freshwater system may have little relation to T/O in that system,

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and the current state of research does not support measuring overall abundance of actinomycetes to determine the source of T/O in a freshwater system. The roles actinomycetes play in aquatic environments is, if anything, less well studied than the prevalence in aquatic systems. In freshwater habitats, actinomycetes appear to play similar roles to their terrestrial counterparts, breaking down complex polymers and recaltricant compounds. Actinomycetes may also inhibit the growth of aquatic fungi on submerged substrates. Utilization of actinomycetes to decompose pollutants in aquatic systems is an area that shows much promise, but efforts will have to be made to determine the most effective species, and what can be done to enrich polluted water bodies in the species most useful for pollutant degradation. Production of odourous metabolites by actinomycete species from aquatic environments should also be investigated. Other genera than the Streptomycetes have been shown to produce T/O, but there are no reports on investigations as to whether isolates of the clearly aquatic actinomycete genus, Actinoplanetes, are capable of producing T/O. Nocardia is common in wastewater, and isolates in this genus have been shown to produce geosmin, but there is no further discussion in the literature regarding the capacity of Nocardia spp. to contribute to T/O events. The survival of terrestrial actinomycetes in aquatic environments may also be of interest. If terrestrial actinomycetes are major producers of geosmin, it is not known if their production of this compound ceases when they enter an aquatic environment, or if they continue to produce geosmin for some time after wash-in, then slowly cease as they become dormant. It is likely that terrestrial actinomycetes survive for extended periods on well-oxygenated surface muds, then become dormant as oxygen levels decrease over the summer. When lakes overturn in fall, it is possible that the increased oxygen may reactivate some of the actinomycetes in the sediment. To date, none of these questions have been examined.
R E F E R E N C E S

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