Reference Work Entry

The Prokaryotes

pp 758-777

The Genera Leptothrix and Sphaerotilus

  • Stefan Spring

Introduction

Representatives of the genera Leptothrix and Sphaerotilus were among the first microorganisms to be recognized in the environment and described in detail by scientists. The type species of the genus Leptothrix, L. ochracea, was already observed in the late eighteenth century and described by Roth (1797) under the synonym “Conferva ochracea.” Later, Kützing (1843) proposed to place this species within the genus Leptothrix. Ten years earlier, the same author had published a description of the species Sphaerotilus natans (Kützing, 1833), which is today still known under this name. These early publications were probably evoked by the observation of ocherous deposits (clearly visible to the naked eye) in ponds or slowly running water. A microscopic examination of these suspicious aggregates led then to the discovery of filamentous microorganisms, which were obviously responsible for the deposition of iron or ferromanganese oxides in a slimy matrix, resulting in the typical color of the formed amorphous flocs or surface films.

Further investigations of the Sphaerotilus-Leptothrix group were induced by their potential economic importance. The massive growth of these microorganisms at industrial sites can lead to technological problems like clogging of water distribution systems or the bulking of activated sludge (Dondero, 1975). More recently, the significance of Leptothrix species in the corrosion of steel could be demonstrated (Olesen et al., 2000; Rao et al., 2000). On the other hand, a potential application of these microorganisms in the biological clearance of heavy metals from contaminated water supplies is being discussed (Nelson et al., 1999; Solisio et al., 2000).

Both genera, Leptothrix and Sphaerotilus, have been always considered as closely related because of the conformity of some suspicious morphologic traits (Mulder and Van Veen, 1963). Later, their close relationship was confirmed by phylogenetic and phenotypic data, thereby justifying their treatment as a group. Characteristic traits, which distinguish members of this group from other phylogenetically related species, are the capability to form tubular sheaths and the precipitation of copious amounts of oxidized iron or manganese.

Phylogeny and Taxonomy

Phylogeny and Related Genera

Members of the genera Leptothrix and Sphaerotilus comprise a phylogenetically coherent cluster within the β1-subgroup of the Proteobacteria. The similarity values among 16S rRNA gene sequences representing strains of the Sphaerotilus-Leptothrix group are in the range between 96.3 and 99.8% (Pellegrin et al., 1999). It has to be noted, however, that to date no 16S rRNA sequence of L. ochracea (the type species of the genus Leptothrix) is available. In addition, the physiology of this species is largely unknown, because it could not be isolated in pure culture. Hence, it cannot be excluded that the type species of Leptothrix is phylogenetically only distantly related to the other species of this genus.

Representatives of the genera Aquabacterium, Ideonella, Rubrivivax, Roseateles and two misclassified species, [Alcaligenes] latus and [Pseudomonas] saccharophila, are phylogenetically closely related to the Sphaerotilus-Leptothrix group. Together they form the Rubrivivax line of descent, which is tightly associated with the Comamonadaceae (Wen et al., 1999). The relative branching order of most species within the Rubrivivax group is difficult to determine, mainly because of the restricted number of varying positions available for the estimation of phylogenetic distance values. Depending on the method and database used for tree reconstruction, it may happen that representatives from other genera get intermixed with species from the Sphaerotilus-Leptothrix group. Therefore, it is difficult to decide if the capability of sheath-formation within this group evolved from a common ancestor, i.e., if it is a monophyletic trait. Despite the close phylogenetic relationship of members of the Rubrivivax branch, a differentiation is easily possible based on phenotypic characteristics. It is noteworthy that the genera in this tight assemblage represent three basically different metabolic types, namely photoautotrophs (Rubrivivax), facultative chemolithoautotrophs ([Alcaligenes] latus and [Pseudomonas] saccharophila) and respiratory chemoorganotrophs (e.g., Ideonella and Sphaerotilus). In Figure 1, a representative phylogenetic tree based on 16S rRNA gene sequence data has been selected which adequately reflects the phenotypic classification within this group.

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Fig. 1.

Phylogenetic tree showing the position of members of the Sphaerotilus-Leptothrix group among representatives of the Rubrivivax-branch of the β-Proteobacteria. Sheath-forming species are labeled in red, photoautotrophic species in green, and facultatively chemolithoautotrophic species in blue. The GenBank/EMBL accession number for each sequence is shown in parentheses. With the exception of Sphaerotilus natans (DSM 565) and Leptothrix cholodnii (CCM 1827), sequences of type strains were used. This tree was reconstructed using the ARB program package (Ludwig and Strunk, 1997). It is derived from a distance matrix on a selection of 16S rRNA sequences using the neighbor-joining method of Saitou and Nei (1987). Phylogenetic distances were calculated as described by Felsenstein (1982). The sequence of Escherichia coli was used as an outgroup (not shown). The bar indicates 5% estimated sequence divergence.

Important phenotypic characteristics useful for distinguishing members of the genera Leptothrix and Sphaerotilus from other species belonging to the Rubrivivax-lineage are listed in Table 1.

Table 1.

Differential characteristics of genera and misclassified species belonging to the Rubrivivax group of the β-Proteobacteria.

Characteristic

[Alcaligenes] latus

[Pseudomonas] saccharophila

Roseateles

Rubrivivax

Ideonella

Aquabacterium

Leptothrix a

Sphaerotilus

Number of species

1

1

1

1

1

3

5

1

Flagellation

Peritrichous

One polar

Several polar

One polar

Several, polar or subpolar

One polar

One polar; subpolar tuft

Subpolar tuft

Formation of sheaths

+

+

Carotenoid pigments

+

+

Photoautotrophic growth

+

Autotrophic growth with H2

+

+

+

NR

NR

NR

NR

Anaerobic growth

+

+

+

Oxidation of Mn2+

NR

NR

NR

NR

NR

+

G + C content (mol%)

69–71

69

66

70–72

68

65–66

68–71

69

Isolation source

Soil

Mud

River water

Mud

Activated sludge

Drinking water

Freshwater, sediment

Freshwater, activated sludge

Symbols: +, present in all species; −, absent in all species; and NR, not reported.

aPhysiological data for Leptothrix lopholea and L. ochracea are not available.

From Kersters and De Ley (1984); Palleroni (1984); Mulder and Deinema (1992); Malmqvist et al. (1994); Spring et al. (1996); Kalmbach et al. (1999); and Suyama et al. (1999).

Phenotypic Characteristics

Common Traits

Some important phenotypic traits are shared by all members of the genera Leptothrix and Sphaerotilus: Cells are straight rods, Gram-negative and motile by flagella. Growth is filamentous under natural conditions, owing to the formation of tubular sheaths that surround single and linear chains of cells. Sheaths are produced by excretion of fibrillar polymeric substances that are crosslinked to form a mesh-like fabric closely fitting to the cells. In contrast to slimes or capsules, this matrix is not in intimate contact with the cells. In aquatic habitats rich in iron and manganese, sheaths get incrusted with ferric hydroxide (Sphaerotilus) or ferromanganese oxides (Leptothrix). Without these incrustations, they appear as very thin, almost transparent structures and are therefore not easily recognized by phase contrast microscopy. Occasionally, cells move out of the sheath or lyse and leave behind an empty space or gap within the filament, thereby facilitating the observation of the sheath. Treatment of slide preparations with ethanol may improve the visibility of sheaths under phase contrast.

Further conspicuous morphological features of members of this group include the formation of slime, holdfasts, and false branching of filaments. These traits are frequently recognized in the genus Sphaerotilus, but are less common among representatives of the genus Leptothrix. The sheaths of S. natans and several Leptothrix species are surrounded by a slime layer that may be involved in the accumulation of iron hydroxide and in the oxidation of Mn(II). Holdfasts are sticky, disc-like evaginations or blebs formed at one cell pole opposite to the flagellum by which organisms attach to walls of containers, submerged plants, stones and other surfaces. Holdfast formation can be easily observed in pure cultures of Sphaerotilus natans and Leptothrix lopholea, but is absent or variable in other cultured Leptothrix species. False branching is characteristic for Sphaerotilus natans and several Leptothrix strains. It develops if a cell attaches to an existing filament and forms a new filament, thereby forming a branch.

Metabolism is strictly aerobic, respiratory and chemoorganoheterotrophic. The possibility of an autotrophic or mixotrophic growth of these bacteria with ferrous iron as electron acceptor has been discussed for many years, but up to now no definite evidence could be presented which would support this assumption. Representatives of both genera, Leptothrix and Sphaerotilus, require vitamin B12 as an essential growth factor in mineral media. A number of Leptothrix strains have been found to require in addition thiamine and biotin as growth factors (Rouf and Stokes, 1964). In broth culture, a flocculent growth is typical. Poly-β-hydroxybutyrate is stored as reserve material. The major quinone type detected is ubiquinone Q8 (>90% of total quinones). The four major components of the cellular fatty acids are cis-9 hexadecanoic acid (cis-9 16:1), hexadecanoic acid (16:0), cis-9,11 octadecanoic acid (cis-9,11 18:1) and decanoic acid (12:0). In all strains tested, the hydroxylated fatty acid 3-hydroxydecanoic acid (3-OH 10:0) can be detected, however in some cases, only in low amounts (Kämpfer, 1998). The G+C content of DNA is similar in both genera and ranges between 68 and 71 mol%. In general, chemotaxonomic features characteristic for the Sphaerotilus-Leptothrix group are very similar in other organisms belonging to the Rubrivivax group and Comamonadaceae, so that a clear differentiation of species or even genera within this phylogenetic group is hardly possible based solely on chemotaxonomic traits.

Distinguishing Traits

Albeit closely related, several phenotypic characteristics allow the differentiation of the genera Leptothrix and Sphaerotilus. The ability to oxidize soluble manganese (Mn2+) compounds to solid manganic (Mn4+) oxides is restricted to Leptothrix species. This trait has been originally used for differentiation because it can be easily observed in Mn2+-containing media or habitats. Further distinguishing traits, however, were only revealed by a detailed taxonomic study of cultured strains of both genera. They include the storage of polysaccharides as reserve material only by Sphaerotilus strains, size of cells, structure of the sheath surface (Leptothrix sheaths show a rough surface when viewed by electron microscopy, in contrast to the sheaths of Sphaerotilus strains which have a smooth surface), utilization of carbon sources, and the pronounced response of S. natans to an increase of organic nutrient concentration in contrast to a poor response of most Leptothrix species.

A summary of phenotypic characteristics useful for the classification of both genera is presented in Table 2.

Table 2.

Main morphological and physiological characteristics of the genera Leptothrix and Sphaerotilus.

Characteristic

Leptothrix a

Sphaerotilus

Cell dimensions

Width (µm)

0.6–1.5

1.2–2.5

Length (µm)

1.5–14

1–10

Flagella

Monotrichous, polar

+

Polytrichous, subpolar

+

+

Structure of sheath surfaceb

Rough

Smooth

Reserve material

Poly-β-hydroxybutyrate

+

+

Polysaccharide

+

Major fatty acidsc

cis-9 16:1, 16:0, cis-9,11 18:1

cis-9 16:1, 16:0, cis-9,11 18:1

Hydroxylated fatty acid

3-OH 10:0

3-OH 10:0

Major quinone type

Q-8

Q-8

Oxidation of Mn2+

+

Growth stimulation by increase of nutrient concentration

−/+d

+

Need for vitamin B12

+

+

Carbon sources used for growthe

l-Alanine

+

l-Asparagine

+

l-Aspartate

+

Butyrate

+

d-Fructose

D

+

d-Glucose

D

+

d-Gluconate

+

l-Ornithine

+

Symbols: +, 90% or more strains are positive; −, 90% or more strains are negative; D, 11–89% of the strains are positive.

aResults are based on strains available from culture collections.

bElectron microscopic observation of unstained preparations.

cNumber of carbon atoms:number of double bonds.

dMost freshly isolated strains show no pronounced response, with the exception of Leptothrix cholodnii strains.

eUtilization of carbon sources was tested in GMBN medium (Richard et al., 1985; Kämpfer, 1998).

From Van Veen et al. (1978); Spring et al. (1996); and Kämpfer (1998).

Problems in the Taxonomy of Members of the Sphaerotilus-Leptothrix Group

The Proposal of Pringsheim

Members belonging to the Sphaerotilus-Leptothrix group share some important phenotypic characteristics which are easily recognized. On the other hand, distinguishing traits are sometimes difficult to determine and require the investigation of defined pure cultures. Certain strains of this group show a considerable morphological variability, depending on the respective environmental conditions, leading to the effect that virtually identical or similar strains were described under different species names. These circumstances probably led Pringsheim (Pringsheim, 1949a; Pringsheim, 1949b) to believe that all strains of this group should be placed in the genus Sphaerotilus. He also concluded that L. ochracea would be a morphological variant of S. natans because both species lack visible deposits of manganese oxides on their sheaths. The investigations which led to these conclusions were however inadequate because they were based on only a few partly described strains or on the observation of uncultured bacteria in natural environments. Several studies in the following years clearly revealed the mistakes in Pringsheim’s nomenclature. Nevertheless, his assumptions produced a lot of confusion in the taxonomic literature on the Sphaerotilus-Leptothrix group between the 1950s and 70s. In that time, for instance, a variety of manganese-oxidizing strains, which probably belong to different Leptothrix species, were described under the name Sphaerotilus discophorus (e.g., Rouf and Stokes, 1964).

Instability of Phenotypic Characteristics

Several difficulties regarding the taxonomy of this group are due to the loss of important phenotypic traits upon cultivation (Van Veen et al., 1978). The reversible or nonreversible loss of physiological and morphological characteristics of strains in pure culture caused divergent descriptions of species, which were originally described on the basis of observations in the natural environment (e.g., L. discophora, see below). The ability to form sheaths has been irreversibly lost by several strains of Sphaerotilus natans and by most Leptothrix strains available from public culture collections, with the exception of L. cholodnii (formerly “L. discophora”) SP-6 (= LMG 8142). Gaudy and Wolfe (1961) reported that sheath formation in Spaerotilus natans is affected by the composition of the culture medium and inhibited at high concentrations of peptone. The colony morphology of freshly isolated strains corresponds to the ability of sheath formation and can be smooth (sheathless cells) or rough and filamentous (sheath-forming cells). Sometimes even various colony morphologies of the same strain can be observed on one agar plate. The manganese-oxidizing activity in Leptothrix strains is independent from the formation of sheaths and more stable in most strains, but loss of this trait has also been reported.

A report on the disc- or holdfast-formation of an uncultured sheathed bacterium, which was tentatively identified as L. discophora (Carlile and Dudeney, 2001), illustrates the taxonomic problems within this group. Interestingly, the formation of basal discs or holdfasts at one end of the filament (discophora means disc-bearing) was mentioned in the original description of this species by Schwers (1912), which was based on photomicrographs of cells in natural environments. In later studies, however, disc formation was never again observed in pure cultures of this morphotype, so that it was concluded that the presence of this trait within the genus Leptothrix was confined to the species L. lopholea (Mulder, 1989a). If the authors of the abovementioned studies have in fact described the same species, the ability to form discs would not distinguish between L. discophora and L. lopholea, this trait being more stable in the latter. To avoid this kind of confusion, descriptions of new strains should be based on freshly isolated pure cultures that were studied, if possible, under conditions most similar to natural habitat conditions.

Lack of Reference Strains

A major problem in the taxonomy of the genus Leptothrix is the availability of reference strains. The type species of the genus Leptothrix, L. ochracea, is not cultured and the description is based only on morphological observations. Although the description of “L. pseudo-ochracea” was based on pure cultures, no type strain was designated. On the other hand, the type strains of L. lopholea, LVMW 124, and L. cholodnii, LVMW 99, are not available from public culture collections and have apparently been lost. The strain LMG 7171 can be used as reference strain for L. cholodnii until a neotype is designated, but for L. lopholea, no other strains are available, preventing detailed taxonomic studies on this species.

Ecology

Ecology of Sheath Formation

The production of a sheath takes place at the expense of energy and requires synthesis and excretion of a large amount of cellular material. Therefore, it can be assumed that sheath formation was developed by microorganisms under a selective pressure and offers several ecological and nutritional advantages in the environment. A linear arrangement of single cells within a tubular sheath enables bacteria to form filaments, without actual enlargement of cell size. It has been shown that filamentous growth is an effective strategy of bacteria to exceed the size limit of particles edible by protozoa, thereby allowing them to escape from grazing (Sommaruga and Psenner, 1995). In addition, sheaths provide cells with physical protection from infection by bacteriophages (Winston and Thompson, 1979), bacterial predators (Venosa, 1975), and bacterivorous metazoa. Obviously tough sheaths impregnated with ferric oxides cannot be consumed by metazoa and are not penetrated by bdellovibrios or phages.

Rigid sheaths in combination with extracellular slime represent an ideal matrix for the rapid build-up of biofilms in the form of floating aggregates or dense layers on solid surfaces. The ecological advantages of biofilm formation for bacteria are well known and include reduction of flow rates in running waters, adsorption of nutrients, and establishment of heterogenous microniches characterized by various chemical gradients. The reversible inhibition of sheath formation by high nutrient concentrations in some Sphaerotilus strains may be an indication for the important role played by the sheath surface in the accumulation of nutrients (Gaudy and Wolfe, 1961).

The sheath provides cells with an additional compartment close to their outer surface. Enzymes and proteins which are secreted by the cell can be incorporated in the sheath matrix, thereby allowing the extracellular production or degradation of compounds. It is possible that members of both genera use the sheath or the space between sheath and outer cell surface for the oxidation of iron and/or manganese to avoid toxic concentrations of metal compounds within the cell.

Methods for Studying Distribution and Abundance

Members of the Sphaerotilus-Leptothrix group are not only abundant in natural environments, but also may play a role in industrial processes leading to several technical problems. They were associated with bulking of activated sludge (Wagner et al., 1994), corrosion of stainless steel (Olesen et al., 2000), clogging of water distribution systems (Dondero, 1975), and slime formation in paper mill factories (Pellegrin et al., 1999). To prevent damage caused by the prolific growth of these organisms, it could be advantageous to monitor them in environmental samples without time-consuming enrichment and cultivation steps. Although in some samples the affiliation of bacteria to the Sphaerotilus-Leptothrix group may be assigned on the basis of phase-contrast microscopic appearance, distinction between genera or single species is hardly possible. Consequently, culture-independent methods for the identification and detection of these species were developed. Two approaches could be promising: whole cell hybridization with fluorescently labeled oligonucleotide probes and polymerase chain reaction (PCR) detection of Leptothrix species using primers targeting mofA, an enzyme gene involved in manganese oxidation.

Whole Cell Hybridization

Fluorescence in situ hybridization (FISH) using oligonucleotide probes is a well established technique for the identification and in situ detection of microorganisms based on signature regions of their 16S rRNA genes (Amann et al., 1995). Several probes were developed targeting 16S rRNA sequences of members of the Sphaerotilus-Leptothrix group. A check of published probe sequences against a current database of 16S rRNA gene sequences (Ludwig and Strunk, 1997) revealed, however, that some probes which were originally intended to be specific for a single species are in fact targeting a variety of different species, partly not even belonging to the Sphaerotilus-Leptothrix group. To minimize the risk of mistaken identification of uncultured bacteria by crossreactivity of probes, it is advisable to use simultaneously at least two different specific probes, distinguishable by fluorescence label. Suitable combinations of probes for the Sphaerotilus-Leptothrix group can be found in Table 3.

Table 3.

Specificity of oligonucleotide probes targeting 16S rRNA sequences of members of the Sphaerotilus-Leptothrix group.

Sequence of:

Hybridization with probea

Species

Strain

Accession no.

SNAb

PSP-6c

LDIb

PS-1c

Leptothrix cholodnii

CCM 1827

X97070

+

+

+

SP-6

L33974

+

+

+

L. discophora

SS-1T

L33975

+

+

L. mobilis

Feox-1T

X97071

+

+

Sphaerotilus sp.

IF4

AF072914

+

S. natans

6T

L33980

+

Target region of probe (E. coli positions)

656–673

138–155

649–666

66–82

Sequences with complementary target region, not affiliated to the Sphaerotilus-Leptothrix groupd

13

9

4

0

Symbols: +, hybridization of probe; −, no hybridization of probe under stringent conditions; and T, type strain.

aReactivity of the probe was either checked experimentally or determined by sequence comparison.

bData from Wagner et al. (1994).

cData from Siering (1997)Ghiorse (1997).

dOnly sequences of cultured strains available in public data bases were counted.

The identification of distinct species by FISH is still difficult, even by using a combination of specific probes, but the combined observation of morphological features (e.g., sheath formation) and hybridization signals should at least enable the identification of uncultured bacteria at the genus level. In Figure 2, the in situ detection of uncultured Leptothrix bacteria in an environmental wetland sample is shown.

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Fig. 2.

Laser scanned epifluorescence (a) and matching laser scanned phase contrast image (b) of previously unidentified filamentous bacteria labeled by the PS-1 oligonucleotide probe in a sample of particulate material from the Sapsucker Woods wetland, Ithaca, NY. From Ghiorse et al. (1996), with permission.

Low signal intensities of labeled cells and samples with low numbers of target cells represent frequently encountered problems of the FISH method. In habitats with low numbers of sheathed bacteria, e.g., water distribution systems or sediments, the application of microscope slides can be a promising approach to obtain suitable samples for hybridization experiments. Microscope slides were successfully used for the enrichment of sheathed bacteria by employing to advantage their ability to adhere efficiently to smooth surfaces (Spring et al., 1996; Carlile and Dudeney, 2001). After removal and cleaning, the overgrown side of these slides can be used for FISH experiments. To improve signal intensities of labeled cells, it may help to treat samples prior to hybridization with diluted hydrochloric or oxalic acid, to remove iron and manganese oxides from sheaths, which could otherwise prevent efficient penetration of oligonucleotide probes into cells.

Diagnostic PCR

An alternative approach for the detection of Leptothrix bacteria was developed by Siering and Ghiorse (1997b). They used specific PCR primers for the amplification of the gene mofA, encoding a putative multi-copper oxidase involved in manganese oxidation by Leptothrix species (see section “Oxidation of Fe2+ and Mn2+” in this Chapter).

The gene mofA was originally retrieved by cloning a DNA fragment of Leptothrix discophora SS-1, but homologous genes were identified also in several other Leptothrix species (Siering and Ghiorse, 1997b). The application of nondegenerate PCR primers enabled the in vitro amplification of a 706-bp portion of the gene mofA from a pure culture of L. discophora SS-1 and extracted nucleic acids from a water sample of a wetland iron seep, but not from nucleic acids extracted from a sediment sample of the same site. The obtained results were in agreement with previous observations which indicated that in this habitat, the growth of Leptothrix bacteria is restricted to ferromanganese surface films and the root zone of Lemna species in the water column. Potential advantages of this protocol are an increased specificity and sensitivity compared to the FISH method. However, in contrast to the latter method, no information on the morphology of the detected cells can be obtained.

The Genus Leptothrix

Habitats

Leptothrix species are widely distributed in the environment and can be easily found at sites which are characterized by a circumneutral pH, an oxygen gradient and a source of reduced iron and manganese minerals. Typical habitats include iron seeps of freshwater wetland areas, forest ponds, iron springs and the upper layers of sediments (Ghiorse and Ehrlich, 1992). At suitable sites, the massive growth of these organisms can be easily observed with the naked eye as ocherous masses emerging as surface films, solid mats or fluffy, dispersed material. Depending on the amount of oxidized manganese, the color can vary from yellowish-orange to dark brown. A wetland iron seep, shown in Fig. 3, is characterized by a pronounced ocherous color originating from a prolific growth of Leptothrix bacteria.

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Fig. 3.

Ellis Hollow iron seep (near Ithaca, NY). A wetland site with massive growth of Leptothrix species (mainly L. ochracea) resulting in typically colored surface films. From Ghiorse and Ehrlich (1992), with permission.

An iron-oxidizing microbial mat that developed at an iron seep where anoxic groundwater (rich in ferrous iron) flows over a stone wall was described in detail by Emerson and Revsbech (1994). They could demonstrate that Leptothrix species represent a significant fraction of the active biomass in the upper few millimeters of this dense microbial mat.

Further studies presented indirect evidence for the association of Leptothrix bacteria with lacustrine ferromanganous micronodules (Stein et al., 2001) or microbial mats in areas of possible deep-lake hydrothermal venting (Dymond et al., 1989).

Most Leptothrix species are restricted to natural, unpolluted environments with low concentrations of easily degradable organic nutrients. A prominent exception is L. cholodnii, which is frequently detected in sewage sludge (Kämpfer, 1997).

Isolation

Several methods for the enrichment of Leptothrix species from the environment are based on the tendency of these filamentous organisms to attach to surfaces. Mulder and Van Veen (1963) used continuous flow devices to imitate the natural growth conditions of these organisms. A schematic of such an apparatus is shown in Fig. 4.

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Fig. 4.

Apparatus for growing Leptothrix species in running artificial iron-containing ditch water. (1) Inlet for tap water. (2) Distributor for the incoming tap water. (3) Cylinder containing iron-stone soil. (4) Seitz filter. (5) Manifold for distributing the sterile iron-containing water. (6) Inlet for the gas mixture (1% O2, 5% CO2, and 94% N2). (7) Inoculation tube. (8) Erlenmeyer flask at a high level. (9) Erlenmeyer flask at a low level fitted with outlets (10). Reproduced with permission from Mulder and Deinema (1992).

For producing iron-containing ditch water, an iron-cylinder is filled with iron-stone soil supplemented with 1 to 2 g of ferric carbonate/kg of soil. After an incubation period of about 3 weeks, during which the soil is kept saturated with water, enough ferric iron is reduced to ferrous iron to start a continuous flow of tap water percolating through the soil. The soil extract running off the iron cylinder from the upper outlet is sterilized by filtration and supplied dropwise to the upper Erlenmeyer flasks, which can be inoculated with various samples containing Leptothrix bacteria. To avoid rapid nonbiological oxidation of ferrous iron, the upper Erlenmeyer flasks can be aerated with a gas mixture of low oxygen content. Comparable to the situation in unpolluted slowly running water, bacteria which are able to attach to solid surfaces have a selective advantage within this device because they obtain more nutrients than bacteria moving with the medium. The described apparatus can be also applied for the observation of pure cultures of Leptothrix bacteria under conditions similar to their natural environment.

A less laborious enrichment method was introduced by Rouf and Stokes (1964), who filled glass cylinders with water taken from the environment and added extracted alfalfa straw as nutrient source, MnCO3, and freshly precipitated Fe(OH)3. After several days, the flocculent growth of sheathed bacteria adhering to the walls of the cylinder indicated the enrichment of Leptothrix bacteria.

An alternative method, which is also based on the capability of Leptothrix species to attach to smooth surfaces, was applied by Spring et al. (1996). Microscope slides were put into a sample of freshwater sediment, and after several weeks, sheaths of filamentous bacteria encrusted with ferric oxides covered parts of the slides. The slides can be removed from the sediment and used for isolation after washing in sterile tap water to remove loosely attached sediment bacteria.

Isolation of pure cultures can be achieved by streaking material from enrichment cultures on previously dried agar plates containing low levels of nitrogen and carbon sources. Enrichment cultures may not be necessary if natural environments are studied in which Leptothrix bacteria can be detected by their flocculent growth. Flocculent cell material from these sites can be washed several times with sterile tap water and streaked directly on solid media.

The isolation medium used by Rouf and Stokes (1964) has the following composition (per liter of tap water): Peptone, 5.00 g; ferric ammonium citrate, 0.15 g; MgSO4 · 7 H2O, 0.20 g; CaCl2, 0.05 g; MnSO4 · H2O, 0.05 g; FeCl3 · 6 H2O, 0.01 g; and agar, 12.00 g. The plates are incubated at 25°C for 5–14 days in the dark. Colonies of Leptothrix strains can be easily distinguished from most contaminating bacteria on this medium by their dark-brown color.

Identification

So far six different species of Leptothrix can be distinguished on the basis of phenotypic characteristics. Reference strains for taxonomic studies are only available for three species, viz., L. cholodnii, L. discophora and L. mobilis. Descriptions of the remaining species are incomplete because they are based solely on microscopic observations (L. ochracea) or poorly characterized isolates (L. lopholea and “L. pseudo-ochracea”), hence preventing an accurate identification of newly isolated strains using culture-dependent methods. The name of “Leptothrix pseudo-ochracea” was not included in the Approved Lists of Bacterial Names (Skerman et al., 1980) and has therefore no formal taxonomic status. Differential characteristics of the known Leptothrix species are listed in Table 4. Detailed morphological descriptions follow below.

Table 4.

Differential characteristics of Leptothrix species.

Characteristic

L. ochracea

“L. pseudo-ochracea”

L. lopholea

L. cholodnii

L. discophora

L. mobilis

Cell dimensions

Width (µm)

1.0

0.8–1.3

1.0–1.4

0.7–1.5

0.6–0.8

0.6–0.8

Length (µm)

2–4

5–12

3–7

2.5–15

2.5–12

1.5–12

Flagella

Monotrichous polar

+

+

+

+

+

Polytrichous subpolar

+

Holdfasts

+

V

False branching

+

+

Deposition of MnO2 on sheath surface

+

+

+

+

ND

Growth at

35°C

ND

ND

ND

+

+

pH 8.5

ND

ND

ND

+

+

Growth ona

d-Fucose

ND

ND

ND

+

Fumarate

ND

ND

ND

+

dl-Lactate

ND

ND

ND

+

G+C content (mol%)

ND

ND

ND

68–70

71

68

Symbols: +, present in all strains; −, absent in all strains; ND, not determined; and V, variable (depending on growth conditions).

aGrowth was determined in GMBN medium supplemented with the respective carbon source (Kämpfer et al., 1995).

L. ochracea

Leptothrix ochracea is the most common iron-precipitating ensheathed bacterium that probably occurs all over the world in slowly running ferrous iron-containing waters, poor in readily decomposable organic matter. Low oxygen tensions seem to be required for prolific growth of this species (Emerson and Revsbech, 1994). The pronounced development and activity of L. ochracea in iron- and manganese-containing waters give rise to the accumulation and deposition of large masses of ferric oxide and, probably, manganese dioxide (MnO2), which are thought to be responsible for the formation of bog ore (see for instance, Ghiorse and Chapnick, 1983).

Descriptions of this species are based on observations in the natural environment or in the laboratory on enrichment cultures of slowly running soil extract. The most typical characteristic of L. ochracea is the formation of large numbers of almost empty sheaths within a relatively short time. The mechanism of this procedure can be followed in a slide culture of the organism, in an iron-containing soil extract medium, under a phase-contrast microscope. In this way, the behavior of L. ochracea in crude culture can be observed continuously. It will be seen that chains of cells leave their sheath at the rate of 1–2 µm/min, continuously producing a new smooth and hyaline sheath connected with the old envelope (Fig. 5a). Impregnation and covering of the sheaths with iron probably take place after the cells have left the envelopes. Aged golden-brown and highly refractile sheaths are brittle, so that they are easily broken into relatively short fragments. Viewed under phase contrast, such fragments are very characteristic and appear similar to broken glass capillaries (Fig. 5b, c). In Mn(II)-containing environments or enrichment cultures, sheaths of this species can be easily distinguished from sheaths of the other Leptothrix species which are characterized by irregular encrustations of granular MnO2. In contrast, the sheaths of L. ochracea are only impregnated with ferric oxides and lack MnO2 deposits, leading to a smooth and slender appearance of aged sheaths. The lack of MnO2 encrustations in sheaths of L. ochracea can be detected either by using an electron microscope equipped with an X-ray energy dispersive microanalysis system (Ghiorse and Ehrlich, 1992) or by treatment with oxalic acid (Carlile and Dudeney, 2001). A diluted solution (1%) of oxalic acid, a solvent for hydrated ferric oxide, can make the sheaths of L. ochracea transparent and almost disappear, whereas sheaths of other Leptothrix species are unaffected owing to the impregnation with manganese oxides.

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Fig. 5.

Morphological characteristics of L. ochracea. Bar = 10 µm. (a) Phase contrast micrograph of cells moving out of sheaths and subsequently forming new sheaths. From Van Veen et al. (1978), with permission. (b) Broken old sheaths covered and impregnated with ferric hydroxide in slowly running iron(II)-containing soil extract. From Mulder and Van Veen, 1963, with permission. (c, d) Acridine orange staining of cells partly covered by a hyaline sheath. Same field viewed by differential interference contrast (c) and epifluorescence microscopy (d). Intact bacterial cells stained with acridine orange fluoresce bright green under violet illumination. Note that the chain of single cells appears as continuous filament when viewed by interference contrast. Courtesy of W. C. Ghiorse.

Investigators who have studied and described L. ochracea under natural conditions were unable to obtain pure cultures (Cholodny, 1926; Charlet and Schwartz, 1954). Others who thought they had isolated L. ochracea had, in fact, described one of the other species of this genus (Winogradsky, 1888; Winogradsky, 1922; Molisch, 1910; Lieske, 1919; Cataldi, 1939; Präve, 1957). In some instances, an organism resembling L. ochracea has been isolated, viz., “L. pseudo-ochracea.” A potential lithoautotrophic metabolism of L. ochracea with Fe(II) as electron donor could, therefore, never be proved with pure cultures and is uncertain, since the organism normally grows at a pH value of 6–7, at which Fe(II) is readily oxidized nonbiologically. In addition, its Mn (II)-oxidizing capacity, which probably occurs under natural conditions, has never been confirmed. It is, however, possible that in this species, the Mn(II) oxidizing factors are secreted into the surrounding environment leading to the precipitation of MnO2 granules away from the sheath surface.

L. pseudo-ochracea

Cells are more slender than those of the other Leptothrix species (Table 4), and are very motile by one thin polar flagellum. Even chains of 6–10 cells may show an undulatory locomotion after leaving their sheath. This characteristic may account for the relatively large number of empty sheaths in culture, compared with the number found in cultures of most other Leptothrix species; however, L. ochracea possesses even more empty sheaths. In slowly flowing ferrous iron-containing soil extract, the sheaths become impregnated with ferric oxide and appear yellow-brown. In this respect, the organism resembles L. ochracea. However, in media with added manganese compounds, the sheaths are covered with small granules of MnO2, enabling an easy distinction from the sheaths of L. ochracea which are not impregnated with manganese oxides.

On Mn(II)-containing agar, the black-brown colonies are very filamentous and may exceed a width of 10 mm. On basal agar media containing 0.1% peptone and 0.1% glucose, the organism may grow in concentric rings.

The normal habitat of “L. pseudo-ochracea” is the slowly running, unpolluted, iron- and manganese-containing freshwater of ditches and brooklets. This species may also be found in slightly polluted water.

L. lopholea

Leptothrix lopholea resembles S. natans to a greater extent than do the other Leptothrix species. It produces polytrichous subpolar flagellation and forms holdfasts and false branches. Strains may grow also in rich media. Cells usually develop short-sheathed filaments radiating from a cluster of holdfasts, giving rise to many tiny flocs when the cells are grown in liquid media (Fig. 6).

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Fig. 6.

Leptothrix lopholea. (a) Bacterial flocs with black-brown MnO2 deposits. Reproduced with permission from Mulder and Deinema (1992). (b) Many trichomes radiating from common holdfasts. Bar = 10 µm. From Van Veen et al. (1978), with permission.

Deposition of iron and manganese oxides is more pronounced on holdfasts than on filaments. On Mn(II)-containing agar media, encrustation of sheaths with MnO2 is retarded, so that colonies at first are white and later become black-brown. Cell growth responds poorly to an increased supply of organic nutrients. Strains that do show a good response oxidize manganese more slowly.

This species may be isolated from slowly flowing, unpolluted or polluted freshwater and from activated sludge.

L. cholodnii

Cells of freshly isolated strains are usually found in long chains inside the sheaths. Single motile cells may be seen outside the sheaths. In the presence of Mn(II), the sheaths become covered with granular MnO2 (Fig. 7a). At some sheath locations, the MnO2 deposits may even exceed 10 µm. Leptothrix cholodnii, in contrast to other Leptothrix species, responds to an increased supply of organic nutrients (Table 2). This results in relatively large colonies (up to 5 mm in diameter) on nutrient-rich agar media. On Mn(II)-containing agar, black-brown hairy colonies are formed (Fig. 7b), particularly when the organism is seeded densely.

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Fig. 7.

Leptothrix cholodnii. (a) Sheaths encrusted with MnO2. Bar = 10 µm. Reproduced with permission from Mulder and Deinema (1992). (b, c) Colony morphologies. (b) Filamentous colony on agar with MnCO3 (medium 1, Mulder and Van Veen, 1963). Bar = 100 µm. Reproduced with permission from Mulder and Deinema (1992). (c) Smooth colony on the agar medium of Rouf and Stokes (1964). Bar = 500 µm.

Most strains display a strong tendency to dissociate spontaneously and to produce smooth rather than the typical rough colonies (Fig. 7c). Such mutant strains are largely sheathless and oxidize manganese slightly or not at all (Mulder and Van Veen, 1963; Rouf and Stokes, 1964; Stokes and Powers, 1965).

In agreement with its nutritional requirements, L. cholodnii is found in slowly running iron- and manganese-containing unpolluted waters or in polluted waters, particularly in activated sludge.

L. discophora

Cells are relatively small compared to those of the other Leptothrix species described (Table 4). They may occur in narrow sheaths or be free-swimming; free cells are motile by a thin polar flagellum at one or both poles. The manganese-oxidizing and ferric oxide-storing capacities of this organism are very pronounced. In the presence of Mn(II), the sheaths are heavily but irregularly encrusted with MnO2, giving rise to sheaths of sometimes 10 µm thickness. Under natural conditions, holdfasts may be formed and sheaths are covered with a slime capsule which tapers toward the growing tip and is impregnated with hydrated ferric oxides (Carlile and Dudeney, 2001). In enrichment media with both manganese (II) and iron (II), as in slowly flowing soil extract, the sheaths become covered with a thick, dark brown, fluffy layer of ferric oxide and MnO2 which may increase the diameter of the trichomes up to about 20–25 µm (Fig. 8a).

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Fig. 8.

Leptothrix discophora. (a) Sheaths covered with ferric hydroxide and manganese dioxide in running iron(II)- and manganese(II)-containing soil extract. Bar = 10 µm. From Mulder and Van Veen (1963), with permission. (b) Cells of a sheathless strain showing false branching. Bar = 10 µm. From Spring et al. (1996), with permission. (c) Smooth colony on MnSO4-containing agar. MnO2 is present within the colony and in a halo containing no bacteria. Bar = 100 µm. From Mulder and Van Veen (1963), with permission.

Following isolation, the ability to form sheaths is easily lost in this species (Adams and Ghiorse, 1986). False branching is regularly observed, even with sheathless strains (Fig. 8b). In older cultures, coccoid bodies and cell evaginations are formed. Colonies on the solid medium of Rouf and Stokes (1964) are about 1 mm in diameter, more or less circular in shape, flat, and dark-brown (Fig. 8c). Under certain conditions, filamentous colonies may be formed. Increasing the supply of nutrients such as glucose, peptone, methionine, purine bases, vitamin B12, biotin and thiamine only increases growth slightly. Visible aggregates are formed when grown in liquid media.

The normal habitat is slowly running, unpolluted, iron- and manganese-containing water of ditches, rivers or ponds.

L. mobilis

Cells are similar in width to L. discophora cells (Fig. 9a), but are usually shorter in length. False branching, which is typical for L. discophora, is absent in cultures of L. mobilis. Cells are highly motile by a single polar flagellum. Sheaths are not formed under laboratory conditions. Colonies on the agar medium of Rouf and Stokes (1964) are about 1 mm in diameter, circular sometimes with frayed edges, flat, smooth and dark-brown (Fig. 9b). Visible aggregates are formed when grown in liquid media.

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Fig. 9.

Leptothrix mobilis. (a) Dimensions of single cells grown in the medium of Rouf and Stokes (1964). Bar = 10 µm. From Spring et al. (1996), with permission. (b) Colony morphology on agar medium of the same composition. Dark-brown, granular deposits of MnO2 are visible in the frayed edge of the colony. Bar = 500 µm.

The species description is based on only one strain isolated from the sediment of a freshwater lake (Spring et al., 1996).

Preservation

Stock cultures can be stored on agar slants of the medium of Rouf and Stokes (1964) for about two months at 4°C. Rouf and Stokes (1964) reported a better survival of their strains if stored at room temperature instead of refrigerated. Most Leptothrix strains do not survive lyophilization. For the long-term preservation of these strains, freezing in liquid nitrogen is recommended using suspensions of cells in freshly prepared medium supplemented with 5% dimethyl sulfoxide (DMSO) as cryoprotectant.

Physiology

The genus Leptothrix is characterized by two remarkable metabolic activities: Formation of sheaths and oxidation of iron and manganese.

Oxidation of Fe2+ and Mn2+

It could be demonstrated in several studies that the deposition of metal-oxides in Leptothrix species is biologically controlled. Oxidation of manganese and iron is catalyzed by various metal-oxidizing factors actively secreted by the cell (De Vrind-de Jong et al., 1990).

At circumneutral pH, biological oxidation of iron is difficult to distinguish from the chemical oxidation by oxygen. Only with the identification of an iron-oxidizing protein with a molecular weight of 150 kDa in spent culture medium of the sheathless strain Leptothrix discophora SS-1, the capability of biological iron-oxidation in Leptothrix species could be clearly demonstrated (Corstjens et al., 1992). The function of iron-oxidation is still unknown, but it seems unlikely to play a role in the generation of energy. It has to be noted, however, that Gallionella ferruginea, which is also neutrophilic and microaerophilic, thrives in the same habitats as L. ochracea and is able to gain energy from chemolithoautotrophic iron-oxidation (Hallbeck et al., 1993).

Several manganese-oxidizing factors could be identified in culture supernatants of Leptothrix species. One component with an estimated molecular weight of 110 kDa could be purified and partly characterized. It is called “manganese-oxidizing factor” (MOF) and consists of protein and probably polysaccharide (Emerson and Ghiorse, 1992). It is assumed that this component is part of a larger complex that originates from membranous blebs and is associated with the sheath structure (Brouwers et al., 2000). Antibodies raised against the MOF protein of L. discophora SS-1 allowed retrieval of the gene mofA, encoding a putative multi-copper oxidase. A strong indication for an active role of this copper-dependent enzyme in manganese-oxidation is the observation that addition of small amounts of copper to actively growing cultures enhances the rates of manganese oxidation considerably (Brouwers et al., 2000). In addition to multi-copper oxidases, c-type hemes could be involved in metal-oxidation by Leptothrix species. Genes encoding proteins with potential heme-binding sites were identified as part of the operon encoding mofA.

So far, the beneficial effects of manganese oxidation are largely unknown, but it is unlikely that the deposition of manganese oxides has been developed without conferring an important advantage to these bacteria. The benefits of manganese oxidation are probably so difficult to determine because they are only effective under natural conditions difficult to imitate in the laboratory. Traditionally, it was assumed that the positive effects of Mn(II)-oxidizing enzymes and manganese oxides are based on the detoxification of harmful oxygen species (superoxide and peroxide) or on the adsorption of toxic compounds (e.g., heavy metals). Recently, an interesting new perspective was introduced by Sunda and Kieber (1994). They found that manganese oxides are strong chemical oxidants able to attack complex organic compounds (e.g., humic substances), thereby leading to the release of small organic molecules which can be easily assimilated by bacteria.

Structure and Composition of the Sheath

The overall structure and chemical composition of sheaths formed by Leptothrix species resemble those of Sphaerotilus natans, although several distinguishing traits were found. One characteristic which can be used for the differentiation between both genera is the structure of the sheath surface. In electron micrographs of unstained preparations, the sheath surface appears rough in Leptothrix species and smooth in strains of Sphaerotilus (Figs. 10 and 12b).

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Fig. 10.

Electron micrograph of a Leptothrix sheath showing a rough surface. Bar = 1 µm. Reproduced with permission from Mulder and Deinema (1992).

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Fig. 12.

Sphaerotilus natans. (a) Cells with internal granules of poly-β-hydroxybutyrate (white arrowhead) and filaments showing holdfasts (black arrowheads). Bar = 10 µm. (b) Electron micrograph of a single cell with a subpolar tuft of flagella and sheath with smooth surface. Bar = 1 µm. (c) Cells with and without a sheath and empty sheaths. Bar = 10 µm. All figures reproduced with permission from Mulder and Deinema (1992).

Emerson and Ghiorse (Emerson and Ghiorse, 1993a; Emerson and Ghiorse, 1993b) performed a detailed investigation of the ultrastructure and chemical composition of the Leptothrix sheath with the sheath-forming strain L. cholodnii (formerly “L. discophora”) SP-6. They reported that it consists of a condensed fabric of 6.5-nm-diameter fibrils underlying a more diffuse outer capsular layer. The inner sheath layer has a thickness of 30–100 nm and seems to be associated with the outer layer of the Gram-negative cell wall by membrane evaginations. The purified sheath substance contains approximately 34% polysaccharide, 24% protein, 8% lipid, and 4% inorganic material. As major components of the polysaccharide moiety, uronic acids and amino sugars, which are probably in the N-acetylated form, were identified, whereas neutral sugars could not be detected. The sheath proteins are rich in cysteine residues (6 mol%), which confer numerous disulfide- and sulfhydryl-groups to the sheath. The heteropolysaccharide and protein moieties appear to be tightly associated or connected. On the basis of these findings, it was concluded that the principal structural elements of the sheath are proteoglycan fibrils that are covalently linked to each other by interfibril disulfide bonds resulting in a stable fabric. It is possible that the difference between the condensed inner layer and diffuse capsular layer is caused by an increased amount of free sulfhydryl groups in the exterior sheath-layer. A further characteristic of the sheath is its negative charge due to free carboxyl groups, originating mainly from the uronic acids of the sheath-proteoglycans. Both chemical groups, the free sulfhydryl and the free carboxyl groups, provide the sheath with numerous sites for binding of metal cations, especially Mn2+ and Fe2+. According to the working model presented in Fig. 11, the anionic sheath-fibrils capture and concentrate soluble Mn(II) ions within the sheath, where they can be efficiently oxidized by excreted manganese-oxidizing factors associated with the sheath (Emerson and Ghiorse, 1993a). Manganese- and iron-oxidizing factors are probably excreted from the cell and transported to the sheath as membranous blebs containing protein complexes consisting of multi-copper oxidases and cytochromes (Emerson and Ghiorse, 1992).

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Fig. 11.

Schematic model of the architecture and potential function of the sheath in L. cholodnii SP-6. PHA, polyhydroxyalkanoate. From Emerson and Ghiorse (1993a), with permission.

The Genus Sphaerotilus

Habitat

Members of the genus Sphaerotilus are part of the natural microbial community in slowly running freshwater streams, ditches and ponds (Stokes, 1954). In contrast to Leptothrix sp., massive growth is typical in freshwater streams that receive high levels of organic pollution from sewage or industrial wastes, especially from paper, potato, dairy or other agricultural industries. When Sphaerotilus becomes established in a polluted river, it frequently proliferates so extensively that it lines the riverbed for long distances with a wooly, filamentous carpet (Dondero, 1975).

In poor settling activated sludge (so-called “bulking sludge”), Sphaerotilus is frequently found among other filamentous microorganisms (Eikelboom, 1975). Bulking or foaming of activated sludge is a common problem in water purification plants and prevents a ready separation of bacterial sludge flocs from the treated water. It appears that conditions which stimulate the growth of Sphaerotilus natans and/or other filamentous bacteria favor also the bulking of activated sludge. The correlation of conditions which favor the prolific growth of a distinct bacterial species with sludge bulking is however hardly possible owing to the high phenotypic diversity of filamentous bacteria which may be involved in this phenomenon (Kämpfer, 1997).

The uncontrolled growth of Sphaerotilus in artificial environments bears several risks of economic importance. It can contribute pyrogenic material to purified water for medical injection (Dondero, 1975) and cause damage to technical equipment or machines (Pellegrin et al., 1999). On the other hand, beneficial effects of the growth of S. natans are also discussed, e.g., the biosorption of heavy metals in wastewater at low pH values (Solisio et al., 2000; Esposito et al., 2001).

Isolation

Enrichment Procedures

In many environments sheathed bacteria occur only in low numbers, for instance in activated sludge or nonpolluted water samples. The use of of enrichment cultures may facilitate the successful isolation of Sphaerotilus sp. from such habitats. For that purpose, variations of Winogradsky’s hay infusion technique have been used (Mulder and Deinema, 1992). Extracted alfalfa straw (Stokes, 1954) or extracted pea straw (Mulder and Van Veen, 1963) serve as the nutrient material. Most of the soluble organic matter should be removed to prevent proliferation of undesirable organisms. This can be achieved by boiling and extracting the straw after it has been cut into small pieces. Suspensions of the extracted straw in tap water (1–8% [w/v]) are distributed in Erlenmeyer flasks and used as enrichment medium. After inoculation with 5–20% of a water sample or activated sludge and incubation for about one week at 22–25°C, filaments of Sphaerotilus may be seen in the medium upon microscopic observation. Often tufts of filaments can be found attached to the pieces of straw and also to the side of the flasks at or near the surface of the medium, thereby enabling easy removal using glass capillaries.

Pure cultures can be obtained from homogenized portions of the enriched material as described in the following paragraph.

Direct Isolation

Often slimy masses of Sphaerotilus are found attached to submerged surfaces in polluted, slowly running water. Material from such sites can be successfully used for the direct isolation of S. natans or related strains by streaking on agar plates. To remove contaminating cells, the collected material should be washed with sterile tap water several times. Homogenization of the washed flocs by blending for a very short time may be useful. However, activated sludge flocs containing many filaments of Sphaerotilus should be streaked directly on the agar plates without homogenization step to avoid release of numerous, nonfilamentous cells by destroying the floc structure. The agar plates used should be dry and contain only low levels of nitrogen and carbon to limit the size of undesirable bacterial colonies, leaving large areas for the filamentous organisms. A further reason for the use of nutritionally poor media is the observation that Sphaerotilus often forms smooth colonies on nutrient-rich agar, instead of the typical rough colonies, which can be easily recognized. To inhibit the growth of contaminating fungi, the agar medium can be supplemented with cycloheximide (0.005 g · liter–1; Pellegrin et al., 1999).

A suitable isolation medium was proposed by Mulder (1989b), having the following basal composition per liter of distilled water: KH2PO4, 27 mg; K2HPO4, 40 mg; Na2HPO4 · 2 H2O, 40 mg; CaCl2, 50 mg; MgSO4 · 7 H2O, 75 mg; FeCl3 · 6 H2O, 10 mg; MnSO4 · H2O, 5 mg; ZnSO4 · 7 H2O, 0.1 mg; CuSO4 · 5 H2O, 0.1 mg; Na2MoO4 · 2 H2O, 0.05 mg; cyanocobalmin, 0.005 mg; peptone, 1 g; glucose, 1 g; and agar, 7.5 g.

Upon inoculation and incubation of these plates at 20–30°C, colonies of Sphaerotilus may be seen and tentatively identified within a few days by their characteristically flat, dull, cottonlike appearance. Confirmation of the identification may be achieved by microscopic observation.

Sphaerotilus may also be isolated by spread plate techniques with or without a previous centrifugation or homogenization step on a variety of agar media (Eikelboom, 1975; Williams and Unz, 1985; Ziegler et al., 1990), including the commercially available R2A agar (Seviour et al., 1994).

Identification

The genera Sphaerotilus and Leptothrix are closely related and share many phenotypic characteristics. Nevertheless, a clear differentiation of both genera is possible on the basis of phenotypic traits summarized in Table 2. The only recognized species of Sphaerotilus is currently S. natans. Typical morphological characteristics of this species are false branching of filaments, extensive slime production, attachment of filaments to solid surfaces by holdfast formation (Fig. 12a), deposition of hydrated ferric oxides on sheaths, and motility of cells by means of a bundle of subpolar flagella (Fig. 12b). In contrast to those of Leptothrix, the sheaths of Sphaerotilus occurring in natural habitats are usually thin and hyaline without encrustations by ferric or manganese oxides (Fig. 12c). However, the capability of Fe(II) oxidation can be easily demonstrated in this species by cultivation in media containing soluble iron compounds and low nutrient concentrations, e.g., soil extract enriched with ferrous iron. Under these growth conditions, the sheaths of S. natans turn yellow-brown and resemble in appearance those of Leptothrix ochracea. Most of the pronounced morphological characteristics of S. natans are largely dependent on the strain and cultivation conditions. Sheath formation can be inhibited by high levels of nutrients in the medium—peptones being more effective than carbohydrates—resulting in the formation of smooth instead of rough, filamentous colonies (Mulder and Van Veen, 1963). This loss of sheath-forming capability is reversible and can occur also spontaneously.

Growth of pure cultures in liquid media is usually flocculent, but sometimes pellicular or homogenous (Pellegrin et al., 1999). Upon prolonged incubation, large, circular bodies resembling protoplasts may appear in broth cultures. Their formation is probably due to the production of enzymes involved in the decomposition of cell walls during the death phase (Phaup, 1968).

The nutritional versatility of S. natans and related strains is remarkable. They can utilize a variety of carbon and nitrogen sources, tolerate a wide range of nutrient concentrations and can grow under low partial pressures of oxygen. Utilization of fructose, glucose, maltose, sucrose, lactate, pyruvate, and succinate as sole sources of carbon was reported by Stokes (1954), Mulder and Van Veen (1963), Kämpfer (1998), and Pellegrin et al. (1999). Numerous other carbon sources can be assimilated by strains of S. natans, but the substrate utilization patterns of strains within this species differ widely. In contrast to most Leptothrix strains, S. natans is able to assimilate relatively high concentrations of substrates from which it synthesizes considerable amounts of cellular material. Cells may contain large amounts of poly-β-hydroxybutyrate either as numerous small globules or as a few large globules (Fig. 12a). Polysaccharides may also accumulate. The synthesis of both reserve compounds is stimulated by a high carbon/nitrogen ratio in the medium or by oxygen deficiency (Mulder and Van Veen, 1963).

Preservation

Stock cultures of S. natans on agar slants of the previously described media can be stored for about 3 months at 4°C. Addition of 2–3 ml of sterile tap water to the agar slants may prolong the viability for another 3 months. Preservation for longer periods is accomplished by common lyophilization techniques; however, it must be stressed that some Sphaerotilus strains do not survive lyophilization. For the long-term preservation of these strains, freezing in liquid nitrogen can be applied.

Physiology

Metabolism

Sphaerotilus is rarely found associated with deposits of metal oxides in natural environments and typically thrives in habitats with normal concentrations of ferrous iron. So far, the mechanism of iron oxidation in this microorganism is poorly understood and it could be that in S. natans, iron oxidation is only a side effect of a biological reaction with a different metabolic function.

Sphaerotilus natans is obligately aerobic and respiratory, but it can grow well with low concentrations of oxygen. It can readily adapt to various nutrient concentrations, growth temperatures (10–40°C) and pH values (pH 5.4–9.0), but seems to be sensitive to an increase in NaCl concentration (upper limit between 0.3 and 0.7%; Dondero, 1975). Glucose is dissimilated via the phosphogluconoate pathway and the tricarboxylic cycle. In the presence of glucose, the oxidation of other sugars, amino acids, and compounds of the tricarboxylic acid cycle seems to be repressed (Dondero, 1975).

Structure and Composition of the Sheath

The sheath of S. natans is, like the sheath of Leptothrix, resistant to proteases and composed of a complex of polysaccharide, protein and lipid. Romano and Peloquin (1963) detected in purified sheath material 36% carbohydrate, 28% protein and 5.2% lipid, whereas Takeda et al. (1998) obtained values of 54.1, 12.2 and 1–3%, respectively. These discrepancies may be due to the investigation of different strains of S. natans or the application of various cultivation conditions or sheath purification methods. The surface of the S. natans sheath is smooth and covered with an acidic exopolysaccharide composed of fucose, galactose, glucose and glucuronic acid (Gaudy and Wolfe, 1962). Slime production by exopolysaccharide secretion is much more pronounced in this species than in members of the genus Leptothrix. In contrast to the exopolysaccharide of the slime capsule, the sheath carbohydrate is free of acidic sugars and contains only glucose and galactosamine which is probably in its N-acetylated form. A heteropolysaccharide composed of glucose and galactosamine (in a molar ratio of 1:4) can be released from the sheath by hydrazine treatment which completely degrades the sheath structure (Takeda et al., 1998). The sheath of S. natans can be also degraded enzymatically by a kind of eliminase (produced by a Paenibacillus sp.) which attacks the polysaccharide moiety of the sheath (Takeda et al., 2000).

Although the sheath structure is resistant to protease treatment, part of the sheath protein appears to be sensitive to protease attack and can be removed from the sheath by treatment with agents that reduce disulfide bonds. However, in contrast to the Leptothrix sheath, disulfide bonds apparently do not play a role in maintaining the sheath structure of S. natans. In sheaths treated with protease, the most abundant amino acids were glycine and cysteine. Only three or four other major amino acids were detected, and it was concluded that the sheath protein may consist of repeating subunits of a small peptide which is crosslinked with a polysaccharide backbone (Takeda et al., 1998).

The revealed differences in the fine structure and composition of sheaths from Sphaerotilus and Leptothrix may be an indication for different functions of these structures for the respective microorganisms.

Acknowledgments

I am grateful to W. C. Ghiorse for providing unpublished photographs.

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