The Genera Thiobacillus, Thiomicrospira, and Thiosphaera

The Genera Thiobacillus, Thiomicrospira, and Thiosphaera J. GIJS KUENEN, LESLEY A. ROBERTSON, and OLLI H. TUOVINEN (Excerpts from CHAPTER 138 of "The Prokaryotes", edited by A. Balows et al., Vol. III, 2nd Edition, 1992)

A common feature of the members of these three genera is their ability to gain energy from the oxidation of reduced sulfur compounds (see Chapter 16). Nearly all of them are able to grow autotrophically, and about one-half can also grow organotrophically. Other inorganic sources of energy for some members of this group include hydrogen, ferrous iron, and other reduced metal ions. The ability to use organic compounds for carbon or energy varies among these bacteria. Many strains are highly specialized, and these obligate chemolithotrophs are only able to use carbon dioxide as their carbon source, and compounds such as sulfide or thiosulfate for energy. They can sometimes assimilate a small number of organic compounds, but only to a limited extent and in a restricted pattern (see Chapter 14). In contrast, the facultative chemolithotrophs are physiologically versatile and can grow autotrophically, heterotrophically, or mixotrophically (in the latter, inorganic and organic compounds are used simultaneously for energy and carbon). When grown as batch cultures on mixtures of these substrates, the versatile species often show diauxie, and the autotrophic pathways (e.g., the Calvin cycle for carbon dioxide fixation) are frequently repressed. At least one strain is a chemolithoheterotroph which can derive energy from the oxidation of reduced sulfur compounds, but cannot grow autotrophically because it lacks the Calvin cycle.

All species in the three genera are respiratory, being able to use oxygen as their terminal electron acceptor. A few can denitrify, and several appear to only be able to reduce nitrate to nitrite.

Habitats

In nature, the distribution of reduced inorganic sulfur compounds is only one of the factors governing the presence and activity of the colorless sulfur bacteria, and a complex of chemical, physical, and microbial interactions is almost

as important a controlling force. One crucial factor is the requirement for the simultaneous presence of an electron donor and electron acceptor (i.e., oxygen or nitrogen oxides). Sulfide is one of the most important natural substrates, and many of the colorless sulfur bacteria grow in the narrow zones and gradients where sulfide and oxygen coexist (Nelson and Jannasch, 1983). Such gradients can be found, for example, in stratified lakes and at the interface between aerobic water and an anaerobic sediment, but may be even more common in microniches in environments that contain anaerobic pockets (e.g., sediments and wet soils). Similarly, other gradients can influence the growth and selection of sulfur oxidizers. Thus, it has been shown that Thiobacillus species respond differently to varying redox potentials (Sokolova and Karavaiko, 1968; Timmer ten Hoor, 1977). The occurrence of acidophilic bacteria in apparently neutral environments indicates the existence of pH gradients associated with acidic microniches.

Sulfur oxidizers have been enriched and isolated from natural sites chosen by the obvious presence of inorganic sulfur or iron compounds (e.g., sediments from rivers, canals, estuaries and tidal flats, acid sulfate soil, (hot) acid springs, mine drainage effluent, hydrothermal vents, and wastewater treatment systems). The thiobacilli, especially the specialized chemolithotrophs, have long been viewed as one of the most important groups of organisms contributing to the transformation of sulfur in soils and sediments, although only a few systematic studies on their occurrence and distribution have been carried out. However, there is now evidence that, depending on the particular environmental conditions, the mixotrophic and heterotrophic sulfur oxidizers may be at least equally important in the overall conversion of reduced sulfur compounds to a more oxidized state (Guitonneau and Keiling, 1932; Kuenen, 1975; Mason and Kelly, 1988; Robertson et al., 1989a). In general, sulfur and iron oxidizers are ubiquitous in soil, sediments, and freshwater and marine environments. They grow over a wide range of pH values and temperatures (see Tables 1 and 2). Thus, it is relatively easy to find them in nature. An obvious factor that limits their growth is the availability of reduced inorganic sulfur compounds, a limitation that is especially important for the obligate autotrophs that completely depend on these compounds for growth. Despite their ability to incorporate some organic compounds into cell material, the contribution of the latter group to the breakdown of organic compounds in nature is probably negligible. Gottschal and Kuenen (1980) showed that the versatile species are generally favored in freshwater environments when the turnovers of available inorganic and organic substrates are approximately equivalent. When the inorganic substrates are dominant, obligate autotrophs should be favored and, similarly, heterotrophs will be favored when organic compounds make up the bulk of the available substrate (Fig. 1). However, this does not necessarily hold true for marine situations (Kuenen et al., 1985). Further work remains to be done to discover what other selective pressures are operative in the marine milieu.

Because many of the colorless sulfur bacteria produce sulfuric acid or ferric iron, they are often associated with the oxidative corrosion of concrete and pipes, and have been implicated in the corrosion of buildings and ancient monuments. Acid and metal pollution can also be a result of the activities of thiobacilli in mine wastes (Tuovinen and Kelly, 1972). On the more positive side, the production of acid can be used, for example, in leaching processes for the extraction of metals from poor ores that are unsuitable for extraction by conventional metallurgical methods. In addition, the pyrite-oxidizing capacity of T. ferrooxidans and related organisms has also been successfully exploited for the desulfurization of coal (Bos et al., 1988; Bos and Kuenen, 1990).

In soils, thiobacilli may sometimes be responsible for the solubilization of sulfur compounds, thus making sulfur available (as sulfate) for assimilation by other microorganisms and plants. The sulfur budget and the role of microorganisms in the sulfur cycle of cultivated soils is, of course, important for assessing fertility. In Australia, for example, thiobacilli are scarce in sulfur-deficient areas. Under suitable climatic conditions (e.g., in the tropics) rock phosphate pelleted with sulfur and seeded with thiobacilli has been shown to be a useful, slow-release source of phosphate and sulfate for soil fertilization (Swaby, 1975).

Marine thiobacilli have been isolated from oceans, hydrothermal vents, and coastal and tidal areas, and classified by reference to nonmarine cultures. Characteristically, some (but not all) marine forms have a requirement for (sodium) chloride levels comparable with the salinity of their natural environment. It is difficult to explain their presence in the open oceans - perhaps they are able to scavenge volatile sulfur compounds released from bottom sediments during the process of decomposition. At the hydrothermal vents, they are believed to be important producers of organic compounds at the start of the geochemically based food chain (Jannasch, 1985). However, as they are, on the whole, dependent on oxygen generated by photosynthesis for electron acceptors, they cannot, therefore, be considered "primary producers" in the strict sense of the term.

Physiology

By definition, all of the species described in this chapter can derive energy for growth from the oxidation of reduced sulfur compounds if provided with a terminal electron acceptor, but the conditions required (e.g., pH) may vary somewhat (Jackson et al., 1968). Moreover, the ability of all of the colorless sulfur bacteria to oxidize certain sulfur compounds has not been rigorously tested. Until this is done, the differentiation of the various species, especially within the genus Thiobacillus, on the basis of the oxidation of different sulfur compounds should be treated with caution. Some of the reactions can be summarized as follows:

H₂S + 20₂ ---->    H₂SO₄
2H₂S + 0₂ ---->    2S° + 2H₂0
2S° + 3O₂ + 2H₂0 ----> 2H₂SO₄
Na₂S₂0₃ + 20₂ + H₂0 ---->   Na₂SO₄ + H₂SO₄
4Na₂S₂0₃ + 0₂ + 2H₂0 ---->   2Na₂S₄0₆ + 4NaOH
2Na₂S₄0₆ + 7O₂ + 6H₂0 ---->   2Na₂SO₄ + 6H₂SO₄
2KSCN + 4O₂ + 4H₂0 ----> (NH₄)₂SO₄ + K₂S0₄ + 2CO₂
5H₂S + 8KNO₃ ----> 4K₂SO₄ + H₂SO₄ + 4N₂ + 4H₂0
5S° + 6KNO₃ + 2H₂0 ----> 3K₂SO₄ + 2H₂SO₄ + 3N₂

Moreover, some Thiobacillus species can derive energy from ferrous iron oxidation as follows:

2FeS₂ + 2H₂0 + 7O₂ ----> 2FeSO₄ + 2H₂SO₄

4FeSO₄ + 0₂ + 2H₂SO₄ ----> 2Fe₂(SO₄)₃ + 2H₂0

The complete oxidation of reduced sulfur compounds to sulfate is common to all of the obligate and facultatively autotrophic sulfur oxidizers, and the ability of a culture to lower its pH while growing on thiosulfate is one of the criteria that has been used for the detection of these bacteria. The pH often rises in cultures of heterotrophs able to partially oxidize inorganic sulfur compounds because of the formation of polythionates (e.g., tetrathionate from thiosulfate). It should be noted, however, that a lack of acid production is not a decisive criterion for the differentiation between the chemolithotrophic and chemoorganotrophic sulfur oxidizers because under unfavorable cultural conditions, even the chemolithotrophs accumulate intermediates in significant amounts. For example, it has been observed that T. ferrooxidans, when growing on thiosulfate, trithionate, or sulfide, produces sulfur which is not only detectable outside the cells, but also as a finely dispersed precipitate outside the cell membrane (Fig. 2; Hazeu et al., 1988). T. neapolitanus and Thiobacillus strain O both produce sulfur during growth on reduced sulfur compounds under various environmental stresses (e.g., oxygen tension), but do not appear to accumulate sulfur internally (Stefess and Kuenen, 1989).

The Genus Thiobacillus

All members of this genus are Gram-negative rods, some of which are motile. As mentioned above, classification is still, generally, based on morphological and physiological features. Inevitably, some strains have been studied more thoroughly then others. Detailed taxonomic descriptions can be found in Kelly and Harrison (1989). However, a few key points are summarized in Tables I and 2. For convenience these species have been separated into three groups: the neutrophilic specialists, the versatile species, and the acidophiles.

THIOBACILLUS FERROOXIDANS; SYNONYM FERROBACILLUS FERROOXIDANS; TYPE STRAIN ATCC 23270. T. ferrooxidans is an obligate autotroph which is able to derive energy from the oxidation of ferrous iron as well as reduced sulfur compounds. During growth on thiosulfate trithionate, tetrathionate, and sulfide, T. ferrooxidans has been observed to accumulate fine sulfur deposits (Fig.2) which are predominantly associated with the cell wall (Hazeu et al., l 988). Early reports of facultatively autotrophic T. ferrooxidans strains are now believed to have been due to the presence of facultatively autotrophic (see T. acidophilus) or heterotrophic contaminants, some of which have been difficult to remove as their presence can stimulate the growth of the autotroph (Harrison, 1984). The ability to oxidize Fe2+ is the key characteristic that is generally employed in isolation procedures. Initial enrichments with reduced sulfur compounds frequently produce mixtures of various acidophilic thiobacilli, whereas enrichment on Fe2+ can result in almost pure cultures of T. ferrooxidans.