Reference Work Entry

The Prokaryotes

pp 555-588

Colorless Sulfur Bacteria

  • Gerard MuyzerAffiliated withDepartment of Biotechnology, Delft University of TechnologyDepartment of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam Email author 
  • , J. Gijs KuenenAffiliated withDepartment of Biotechnology, Delft University of Technology
  • , Lesley A. RobertsonAffiliated withDepartment of Biotechnology, Delft University of Technology

Abstract

Since its recognition in the late nineteenth century, the ability to gain metabolically useful energy from the oxidation of reduced sulfur compounds by bacteria has been regarded as of such significance that it has been used as a primary characteristic in taxonomy. Essentially, any Gram-negative rod that could grow with a reduced sulfur compound as its primary energy source was automatically called “Thiobacillus.” Similar bacteria with a spiral shape became “Thiomicrospira,” and so on. As research progressed over the years, this approach has become steadily less satisfactory, and the development of genetic methods for identification has finally confirmed that the ability to metabolize reduced sulfur compounds is of no more taxonomic significance than the utilization of any other specialized substrate.

This chapter describes the scientific stages taken to reach this point, reviews the reorganization that has been necessary among the colorless sulfur bacteria, and considers the fact that while the metabolic trait is of less taxonomic significance than previously believed, this grouping is important ecologically and should be retained.

Abstract

Since its recognition in the late nineteenth century, the ability to gain metabolically useful energy from the oxidation of reduced sulfur compounds by bacteria has been regarded as of such significance that it has been used as a primary characteristic in taxonomy. Essentially, any Gram-negative rod that could grow with a reduced sulfur compound as its primary energy source was automatically called “Thiobacillus.” Similar bacteria with a spiral shape became “Thiomicrospira,” and so on. As research progressed over the years, this approach has become steadily less satisfactory, and the development of genetic methods for identification has finally confirmed that the ability to metabolize reduced sulfur compounds is of no more taxonomic significance than the utilization of any other specialized substrate.

This chapter describes the scientific stages taken to reach this point, reviews the reorganization that has been necessary among the colorless sulfur bacteria, and considers the fact that while the metabolic trait is of less taxonomic significance than previously believed, this grouping is important ecologically and should be retained.

Introduction

The name “colorless sulfur bacteria” has been used since the time of Winogradsky to designate prokaryotes that are either able or believed to be able to use reduced sulfur compounds (e.g., sulfide, sulfur, and organic sulfides) as sources of energy for growth. In the late nineteenth century, and for much of the twentieth century, this property was considered to be taxonomically very significant even though the group includes microorganisms with very diverse morphological, physiological, and ecological properties and with equally diverse environmental requirements. Today, it is known that this group comprises a very heterogeneous collection of bacteria, many of which have little or no taxonomic relationship to each other. This is illustrated by Table 15.1a , which shows many of the genera identified as obligately chemolithoautotrophic colorless sulfur bacteria, because their primary metabolism involves the oxidation of inorganic energy sources and the fixation of CO2 (or CO). The oxidation of reduced sulfur compounds is a major feature of their metabolism, but many of them can also use other substrates such as H2 and transition metals. Table 15.1b shows genera that contain more versatile species which can grow heterotrophically and can also either grow chemolithoautotrophically or chemolithoheterotrophically on reduced sulfur compounds. There are other species such as Catenococcus thiocyclus (Sorokin 1992; Sorokin et al. 1996), which seem to gain a small amount of supplemental energy from one of the oxidation steps, or may use one of the reactions to detoxify a metabolite (e.g., sulfide). Over the last few years, there have also been reports that mitochondria from cells ranging from the gills of mussels to human guts are also able to generate energy from sulfide oxidation (Searcy 2006; Goubem et al 2007; Wendeberg et al. 2012). The ability to gain energy from the oxidation of reduced sulfur compounds is clearly widespread and has little taxonomic significance. That said, in ecological terms, the ability is important, and it is convenient to consider these bacteria as a group.
Table 15.1

(a) Obligately chemolithoautotrophic genera that can obtain energy for growth from one or more reduced sulfur compounds. Many can also use other substrates such as H2 and transition metals such as Fe(II). (b) Facultative chemolithoautotrophs and others able to gain energy from oxidizing reduced sulfur compounds

Genus name

Group

Optimum temperature °C

pH Optima

Source

Other

Publication establishing genus

(a)

Sulfuricella

β

22

7.5–8.0

Freshwater lake

Denitrifies NO 3 to N2

Kojima and Fukui (2010)

Thiobacillus

β

28–43

2.0–8.0

Ubiquitous

Some denitrify, others do not

Beijerinck (1904), Kelly and Wood (2000)

Thiobacter

β

50–55

6.5–7.0

Geothermal aquifer

 

Hirayama et al. (2005)

Acidithiobacillus

γ

25–45

2.0–3.5

Acid-mine drainage, sulfidic leachate

Can grow with Fe(II) as sole energy source

Kelly and Wood (2000)

Halothiobacillus

γ

28–40

6.5–8.0

Seawater at Naples

Halophilic, 0.4–1M NaCl

Kelly and Wood (2000)

Sulfurivirga

γ

50–55

6.0

Hydrothermal microbial mat

Uses thiosulfate and tetrathionate

Takai et al. (2006)

Thioalkalibacter

γ

30

88.0–10.2

Soda lakes

Optimum Na+ concentration 1.0 M

Banciu et al. (2008)

Thioalkalimicrobium

γ

Mesophilic

10.0

kenyan soda lakes

Grows at 0.2–4 M NaCl, some species denitrify

Sorokin et al. (2001)

Thioalkalispira

γ

30

10

Egyptian soda lake

Microaerobic, NO 3 only reduced to NO 2 , NaCl optimum 0.5 M

Sorokin et al. (2002)

Thioalkalivibrio

γ

40

10.0–10.2

Kenyan soda lakes

Requires NaCl, 0.3–4 M depending on species. carboxysomes

Sorokin et al. (2001)

Thiofaba

γ

45

6.5

Hot springs

 

Mori and Suziki (2008)

Thiohalobacter

γ

32

7.3–7.5

Hypersaline chloride-sulfate lakes

Can use SCN as substrate, requires NaCl (optimum 0.5 M)

Sorokin et al. (2010)

Thiohalomonas

γ

Mesophilic

7.3–8.2

Hypersaline lakes

Optimum NaCl 1.5–2.0M, denitrify, microaerobic

Sorokin et al. (2007)

Thiohalophilus

γ

32

7.3–7.5

Hypersaline chloride-sulfate lakes

NaCl optimum 0.5 M, can use SCN as energy source

Sorokin et al. (2007)

Thiohalorhabdus

γ

33–35

7.5–7.8

Hypersaline lake sediments

NaCl optimum 3 M

Sorokin et al. (2008)

Thiohalospira

γ

32–35

7.3–7.8

Hypersaline habitats

NaCl optimum 3 M, copious S4O 6 2− produced during S2O 3 2− oxidation

Sorokin et al. (2008)

Thiomicrospira

γ

28–30

6.5–8.0

Hydrothermal mud

Req NaCl up to 3 %

Kuenen and Veldkamp (1972)

Thioprofundum

γ

50

7.0

Deep sea hydrothermal vents

Anaerobic-microaerobic, NO 3 to N2 and N2O

Takai et al. (2009)

Thiovirga

γ

30–34

7.5

Wastewater treatment biofilm

Opt. NaCl 18 mM; max NaCl 180 mM carboxysomes

Ito et al. (2005)

Sulfuricurvum

10–35

7.0

Crude oil storage cavity

Denitrifies NO 3 , not NO 2 , microaerobic

Kodama and Watanabe (2004)

Sulfurimonas

.30

6.14

Hydrothermal, polychaetes’ nest

Denitrifies

Inagaki et al. 2003

Sulfurovum

28–30

6.5–7.0

Hydrothermal vent sediment

Denitrifies

Inagaki et al. (2004)

Thiovulum

<15

 

Sulfide/oxygen interface in water

Chemotactic for O2 and H2S, characteristic veils, chemotactic

Hinze (1913)

(b)

Starkeya

α

25–30

7.0

Soil

Requires additives such as biotin, autotrophic growth on formate

Kelly et al. (2000)

Thioclava

α

35

8.0

Near-shore hydrothermal

Optimum NaCl 35 g/l

Sorokin et al. (2005)

Sphaerotilus

β

20–30

6.5–7.5

Contaminated running water, activated sludge

Sheathed filaments

Kutzing (1833)

Sulfuritalea

β

25

6.7–6.9

Japanese freshwater lake

Autotrophic growth only anaerobically, denitrifies. H2 also energy source

Kojima and Fukui (2011)

Beggiatoa

γ

25–38

Neutrophilic

Sulfide/oxygen interface in sea and freshwater

Stores NO 3 for denitrification, can reduce intracellular So, filamentous

Trevisan (1842), Migula (1894)

Thermithiobacillus

γ

43–45

6.8–7.5

Thermal springs

Heterotrophic growth on complex media, not simple substrates

Kelly and Wood (2000)

Thiomargarita

γ

Moderate psychrophile

Neutrophilic

Sulfidic marine sediments off South America

Cells 100–200 μm, stores NO 3 and S2− in an intracellular vacuole, sheathed, not in pure culture

Schulz et al. (1999)

Thioploca

γ

Moderate psychrophile

Neutrophilic

Oxic/anoxic interface

Filamentous in a twisted braid, not in pure culture, gliding motility, stores NO 3 and S2− in an intracellular vacuole

Lauterborn (1907)

Thiothrix

γ

25–30

6.5–8.5

Sulfur springs and activated sludge plants

Filamentous, sheathed, may produce rosettes

Winogradsky (1888)

Acidianus

Archaea

90

2.0

Solfataras and marine hydrothermal vent systems

Aerobic So oxidation, anaerobic So reduction with H2

Segerer et al. (1986)

Sulfurisphaera

Archaea

84

2.0

Acid hot springs

Anaerobic growth with So as e acceptor

Kurosawa et al. (1998)

Sulfolobus

Archaea

75–80

2.0–3.0

Volcanic springs

Heterotrophic growth only with O2, anaerobic growth with So as e- acceptor

Brock et al. (1972)

Sulfurococcus

Archaea

40–80

Acidophilic

Hydrothermal

Also uses ferrous

Golovacheva et al. (1995)

Sulfurihydrogenibium

Aquificales

60–70

7.5

Hot aquifers

Some species use H2

Takai et al. (2003)

Sulfobacillus

Firmicute

38.5

1.5

Bioleaching stirred tanks

Facultatively anaerobic by ferric respiration

Johnson et al. (2008)

Thiospira

Unknown

Mesophilic

Neutrophilic

Sulfurous marine and freshwater

Type species not available, chemotactic with respect to O2

Visloukh (1914)

Aquaspirillum

Unknown

Mesophilic

Neutrophilic

Sulfurous marine and freshwater

Some Thiospira species may be members of this genus

Dubinina et al. (1993)

The colorless sulfur bacteria play an essential role in the oxidative side of the sulfur cycle (Fig. 15.1 ). Like all of the element cycles, the sulfur cycle has oxidative and reductive sides, which, in most ecosystems, are in balance. However, this balance does not always exist, and accumulations of intermediates such as sulfur, metallic sulfides, and hydrogen sulfide are often found. On the reductive side, sulfate (and sometimes elemental sulfur) functions as an electron acceptor in the metabolic pathways used by a wide range of anaerobic bacteria, leading to the production of sulfide. Conversely, on the oxidative side of the cycle, reduced sulfur compounds serve as electron donors for anaerobic, phototrophic bacteria or provide growth energy for the extremely diverse group of (generally) respiratory colorless sulfur bacteria. Common oxidation products of sulfide are elemental sulfur and sulfate (Fig. 15.1 ). The adjective “colorless” is used because of the lack of photopigments in these bacteria, although it should be noted that colonies and dense cultures could actually be pink or brown because of their high cytochrome content. This chapter will concentrate on the colorless sulfur bacteria, while the sulfate reducers and phototrophs will be discussed elsewhere. Taxonomic details about the various genera shown in Table 15.1a and b can be found in their respective chapters elsewhere in this handbook.
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Fig. 15.1

The sulfur cycle. The colorless sulfur bacteria are involved primarily in those steps in which S2− and So are oxidized with O2 or NO 3 (Adapted from Bos and Kuenen 1983)

As mentioned above, the ability to grown on reduced sulfur compounds was once regarded as taxonomically significant. In 2000, Kelly and Wood (2000) rationalized a situation in which obligate and facultative autotrophs, acidophiles and neutrophiles, and thermophiles and psychrophiles had all previously been included in the same genera, depending on the morphology of the cells involved (e.g., Thiobacillus, Thiomicrospira, Thiosphaera). They took into account the physiology and phylogeny of the various species and devised new names for the various genera that provided a bit more information about the species therein. For readers more accustomed to the historical names, Table 15.2 summarizes the changes to the names of the older colorless sulfur bacteria. To avoid confusion, the modern names will be used throughout this chapter.
Table 15.2

Summary of the reorganization of the colorless sulfur bacteria, showing the species whose names have changed; Tindicates a current type species

Subclass

Current name

Synonyms

α

Acidiphilium acidophilum

Thiobacillus acidophilus, Thiobacillus organoparus

α

Paracoccus pantotrophus

Thiosphaera pantotropha

α

Paracoccus versutus

Thiobacillus versutus, Thiobacillus rapidicrescens, Thiobacillus A2

α

Starkeya novella

Thiobacillus novellus

β

Thiobacillus thioparus T

Thiobacillus thiocyanoxidans, Bacterium thioparum

β

Thiomonas cuprina

Thiobacillus cuprinus

β

Thiomonas delicata

Thiobacillus delicatus

β

Thiomonas intermedia T

Thiobacillus intermedius

β

Thiomonas perometabolis

Thiobacillus perometabolis, Thiobacillus rubellus

β

Thiomonas thermosulfata

Thiobacillus thermosulfatus, Thiomonas thiosulfata

β

Unknown

Thiobacillus plumbophilus

γ

Acidithiobacillus albertensis

Thiobacillus albertis

γ

Acidithiobacillus caldus

Thiobacillus caldus

γ

Acidithiobacillus ferrooxidans

Thiobacillus ferrooxidans, Ferrobacillus ferrooxidans

γ

Acidithiobacillus thiooxidans T

Thiobacillus thiooxidans, Thiobacillus concretivorus, Thiobacillus kabobis, Thiobacillus thermitanus, Thiobacillus lobatus, Thiobacillus cretanus, Thiobacillus umbonatus

γ

Halothiobacillus halophilus

Thiobacillus halophilus

γ

Halothiobacillus neapolitanus T

Thiobacillus neapolitanus, Thiobacillus X

γ

Thermithiobacillus tepidarius T

Thiobacillus tepidarius

γ

Thiomicrospira thyasirae

Thiobacillus thyasiris

γ

Unknown

Thiobacillus prosperous

ε

Sulfurimonas denitrificans

Thiomicrospira denitrificans

Unknown

 

Thiobacillus capsulatus

As will be discussed later, the apparent similarity of the metabolic pathways for sulfur oxidation disguises a high level of variation in these pathways, indicating that the diversity among the colorless sulfur bacteria is probably due to convergent rather than divergent evolution. In addition to inorganic sulfur compounds, some species can also gain energy from the oxidation of other inorganic compounds such as hydrogen, ferrous iron, or even arsenic compounds. As well as differences in substrate range, there is also some variation in electron acceptor usage. Although most colorless sulfur-oxidizing bacteria require oxygen, some are able to grow anaerobically using nitrogen oxides (e.g., nitrate) as their terminal electron acceptor during denitrification. Others have been shown to use other oxides such as arsenate. One or two species of the genus Acidianus are capable of anaerobic metabolism by the reduction of sulfur (Segerer and Stetter 1989), during which organic compounds or hydrogen serves as electron donors. Acidithiobacillus ferrooxidans can reduce ferric iron under anaerobic conditions (Sugio et al. 1985). A somewhat exotic example of a sulfate reducer that might also be considered to be a colorless sulfur bacterium is Desulfovibrio sulfodismutans, which can grow anaerobically by the disproportionation of thiosulfate to sulfate and sulfide (Bak and Pfennig 1987). Some of the reactions that generate energy from inorganic reduced sulfur compounds using oxygen and nitrate as electron acceptors are shown in Table 15.3 .
Table 15.3

Examples of the reactions used by the colorless sulfur bacteria to gain energy for growth

\( {{\text{H}}_2}{\text{S}} + 2{{\text{O}}_2} \to {{\text{H}}_2}{\text{S}}{{\text{O}}_4} \)

\( 2{{\text{H}}_2}{\text{S}} + {{\text{O}}_2} \to 2{{\text{S}}^0} + 2{{\text{H}}_2}{\text{O}} \)

\( 2{{\text{S}}^0} + 3{{\text{O}}_2} + 2{{\text{H}}_2}{\text{O}} \to 2{{\text{H}}_2}{\text{S}}{{\text{O}}_4} \)

\( {\text{N}}{{\text{a}}_{{2}}}{{\text{S}}_{{2}}}{{\text{O}}_{{3}}} + 2{{\text{O}}_{{2}}} + {{\text{H}}_{{2}}}{\text{O}} \to {\text{N}}{{\text{a}}_{{2}}}{\text{SO}}_{4} + {{\text{H}}_{{2}}}{\text{S}}{{\text{O}}_4} \)

\( 4{\text{N}}{{\text{a}}_{{2}}}{{\text{S}}_{{2}}}{{\text{O}}_3} + {{\text{O}}_2} + 2{{\text{H}}_{{2}}}{\text{O}} \to 2{\text{Na}}{{\text{S}}_{{2}}}{{\text{O}}_{{3}}} + 4{\text{NaOH}} \)

\( 2{\text{N}}{{\text{a}}_{{2}}}{{\text{S}}_{{4}}}{{\text{O}}_{{6}}} + 7{{\text{O}}_2} + 6{{\text{H}}_{{2}}}{\text{O}} \to 2{\text{N}}{{\text{a}}_{{2}}}{\text{S}}{{\text{O}}_4} + 6{{\text{H}}_{{2}}}{\text{S}}{{\text{O}}_4} \)

\( 2{\text{KSCN}} + 4{{\text{O}}_2} + 4{{\text{H}}_{{2}}}{\text{O}} \to {\left( {{\text{N}}{{\text{H}}_4}} \right)_{{2}}}{\text{S}}{{\text{O}}_{{4}}} + {{\text{K}}_{{2}}}{\text{S}}{{\text{O}}_4} + 2{\text{C}}{{\text{O}}_{{2}}} \)

\( 5{{\text{H}}_{{2}}}{\text{S}} + 8{\text{KN}}{{\text{O}}_3} \to 4{{\text{K}}_{{2}}}{\text{S}}{{\text{O}}_4} + 4{{\text{N}}_2} + 4{{\text{H}}_{{2}}}{\text{O}} \)

\( 5{{\text{S}}^0} + 6{\text{KN}}{{\text{O}}_3} + 2{{\text{H}}_{{2}}}{\text{O}} \to 3{{\text{K}}_{{2}}}{\text{S}}{{\text{O}}_4} + 2{{\text{H}}_{{2}}}{\text{S}}{{\text{O}}_4} + 3{{\text{N}}_2} \)

In the following sections, we will first discuss the physiology of the colorless sulfur bacteria and then cover taxonomic aspects. This will be followed by a discussion of the habitats of the colorless sulfur bacteria, including artificial habitats, and finally some ways in which they are used. The chapter concludes with a brief section on the role of the colorless sulfur bacteria in the natural sulfur cycle, together with a description of the techniques available for the measurement of their activities.

Physiology

The great diversity of colorless sulfur bacteria should come as no surprise if it is remembered that the group encompasses Archaea as well as Bacteria, and that the latter group is also very diverse, including common pseudomonads and organisms that might be considered as “colorless blue green bacteria” such as species of Beggiatoa. Most of our knowledge of the physiology of these organisms comes from the study of the relatively limited number of bacteria that can be grown in the laboratory. This is particularly true of our understanding of the biochemistry of sulfur metabolism and, to a lesser extent, of carbon metabolism.

Although the biochemistry of the oxidation of sulfur compounds received much attention during the twentieth century, the pathways involved were not well understood. This was due, in particular, to the fact that the research was formulated around the hypothesis that there would be a single unifying enzymatic pathway for the oxidation of all reduced sulfur compounds. However, it is now clearly established that this is not the case. For example, the facultatively autotrophic Paracoccus versutus and the obligately autotrophic Thermithiobacillus tepidarius use two entirely different pathways (Fig. 15.2a , b ). Not only do the enzymes and electron carriers differ but also their location in the membranes of the two species appears to be different. This is, of course, important for the mechanism behind the generation of a proton motive force (PMF) in these organisms to drive the Calvin cycle. In most obligate and facultative autotrophs, the Calvin cycle serves as the route for carbon dioxide fixation. Some other species, including those from Sulfolobus and Hydrogenobacter, possess a carbon dioxide fixation pathway based on a reductive Calvin cycle (Segerer and Stetter 1989).
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Fig. 15.2

Pathways of oxidation of reduced sulfur compounds in two different organisms. (a) The periplasmic thiosulfate-oxidizing system of Paracoccus versutus as proposed by Kelly (1988a). The enzyme complex does not produce or metabolize polythionates such as tetrathionate. Thiosulfate is oxidized to sulfate without the formation of sulfur or other intermediates. Thiosulfate metabolism is initiated by its binding to enzyme A. In subsequent steps, sulfate is produced and released, while electrons are finally transferred to an aa 3-type of cytochrome oxidase. (b) The periplasmic and cytoplasmic metabolism of trithionate, thiosulfate, and tetrathionate by Thermithiobacillus tepidarius as proposed by Kelly (1988b). In contrast to the system shown in (a), tetrathionate appears to be an intermediate in the oxidation of both thiosulfate and trithionate. After an initial hydrolysis of trithionate, yielding thiosulfate and sulfate, the thiosulfate is oxidized to tetrathionate. Available evidence indicates a periplasmic location of these systems. Tetrathionate is believed to be transported into the cell and then oxidized to sulfite in the cytoplasm by an unknown mechanism. Sulfite dehydrogenase is responsible for the final oxidation to sulfate, in which cytochrome b may be involved. FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, HQNO 2- heptyl-4-quinolinol-1-oxide

Energy and Carbon Sources or Electron Donors

It has been common practice to subdivide the colorless sulfur bacteria in terms of their physiological type, as defined mainly by their carbon and energy metabolism. Table 15.4 defines these physiological types, which will be discussed briefly below. It should be remembered that some genera or species have not been studied in pure culture, and it is not yet certain to which of the physiological groups they belong.
Table 15.4

The different physiological types of colorless sulfur bacteriaa

 

Carbon source

Energy source

Physiological type

Inorganic

Organic

Inorganic

Organic

Obligate chemolithotroph

+b

+

Facultative chemolithotroph

+

+

+

+

Chemolithoheterotroph

+

+

+

Heterotroph

+

+

aCommonly used synonyms for chemolithotroph include chemolithoautotroph, autotroph, chemoautotroph, and lithotroph

b+, used by the group; -, not used by the group

Obligate Chemolithotrophs

These highly specialized bacteria require an inorganic source of energy and obtain their cell carbon from the fixation of carbon dioxide. As mentioned above, except in the case of the Archaea [which use a reductive carboxylic cycle (König and Stetter 1989)], the colorless sulfur bacteria do this by means of the Calvin cycle (e.g., Schlegel 1981). The citric acid cycle in these bacteria seems to be incomplete, and its enzymes probably serve a purely biosynthetic function. Despite their label as obligate autotrophs, it has been shown that many of these species can use small amounts of exogenous carbon compounds as a supplementary carbon source (Kuenen and Veldkamp 1973; Matin 1978) or can even ferment endogenous organic storage compounds such as glycogen (Beudeker et al. 1981; Kuenen and Beudeker 1982), but these are secondary metabolic activities, the organisms being primarily dependent on a lithotrophic energy source and carbon dioxide for autotrophic growth. Table 15.1a shows the genera that contain species which fall into this group.

Facultative Chemolithotrophs

These bacteria can grow either chemolithoautotrophically with an inorganic energy source and carbon dioxide or heterotrophically with complex organic compounds providing both carbon and energy, or mixotrophically. Mixotrophy is the simultaneous use of two or more different metabolic pathways for energy and carbon (Gottschal and Kuenen 1980). In the laboratory, mixotrophic growth is most easily observed during continuous culture on limiting mixtures of substrates. The term mixotrophy usually designates simultaneous growth on a mixture of autotrophic and heterotrophic substrates (e.g., on thiosulfate and acetate). However, the simultaneous use of any mixture of substrates requiring (partially) separate metabolic pathways or enzymes where diauxie or biphasic growth might occur in batch culture (e.g., glucose and lactose, succinate and glucose, iron and sulfur, hydrogen and sulfide, acetate and lactate) could be considered as mixotrophy. Table 15.1b lists genera that contain species able to grow on mixtures of reduced sulfur compounds and organic substrates. To some extent, the phototrophic sulfur-oxidizing bacteria might also be considered members of this group since most, if not all, of them are able to grow chemolithoautotrophically and mixotrophically on reduced sulfur compounds in the dark (Kuenen et al. 1985).

Chemolithoheterotrophs

This group of bacteria is characterized by an ability to generate energy from the oxidation of reduced sulfur compounds but which cannot fix carbon dioxide. It is not always obvious whether a species fits into this group as specialized growth conditions may be required both to establish that energy is generated and to exclude autotrophy. For example, for a long time, Thiomonas perometabolis was considered to be chemolithoheterotrophic but was then shown that under certain conditions, it can grow autotrophically (Katayama-Fujimura et al. 1984). However, other chemolithoheterotrophic species have been isolated, and a few strains have been well characterized (e.g., Tuttle et al. 1974; Gommers and Kuenen 1988). Some Beggiatoa strains may belong in this group (Larkin and Strohl 1983). As is clear from the example of Thiobacillus perometabolis, careful testing under a variety of conditions is necessary in order to discriminate chemolithoheterotrophs from the facultative autotrophs and the sulfur-oxidizing heterotrophs.

Sulfur-Oxidizing Chemoorganoheterotrophs

Some heterotrophic bacteria can oxidize reduced sulfur compounds but do not appear to derive energy from them. However, they may benefit from the reaction by the detoxification of metabolically produced hydrogen peroxide (e.g., some species of Beggiatoa, Macromonas, Thiobacterium, and Thiothrix) (Larkin and Strohl 1983; Dubinina and Grabovich 1984). The oxidation of thiosulfate to tetrathionate by many heterotrophic bacteria that do not seem to gain energy from the reaction is well documented (Tuttle and Jannasch 1972; Tuttle et al. 1974; Mason and Kelly 1988).

Electron Acceptors for Aerobic and Anaerobic Growth

Oxygen is almost universally used among the colorless sulfur bacteria, although the degree of aerobiosis that can be tolerated by different species varies. The response of some of the colorless sulfur bacteria to redox can be demonstrated by means of a spectrum as shown in Fig. 15.3 . Various colorless sulfur bacteria have different ways of growing or surviving anaerobically. One of the best studied is the use of nitrate or nitrite as a terminal electron acceptor, whereby the nitrogen oxides are reduced to nitrogen, a process termed denitrification. The ability to denitrify is not limited to any particular physiological type (see Table 15.1a , b ). A few species can only use part of the nitrification pathway. For example, Thiobacillus thioparus can only reduce nitrate to nitrite and requires the presence of a nitrite-reducing bacterium for anaerobic growth. Strictly speaking, of course, the latter reaction is not truly denitrification, but since the reaction still serves for electron transport and survival under anaerobic conditions, these species are appropriately included here.
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Fig. 15.3

A “spectrum” showing the response of five different species of colorless sulfur bacteria to redox. The position of each line indicates the range of conditions of redox under which the organism can grow (Based on Timmer ten Hoor 1977)

A few obligately chemolithotrophic sulfur bacteria carry out complete denitrification to nitrogen. Thiobacillus denitrificans is relatively versatile in being able to grow under fully aerobic conditions with oxygen and under fully anaerobic conditions with nitrate or nitrite (Aminuddin and Nicholas 1973; Ishaque and Aleem 1973). Sulfurimonas denitrificans is more fastidious. It grows well anaerobically with nitrate or nitrite but can only use oxygen for growth if its concentration is kept extremely low (i.e., below the detection level of normal oxygen electrodes) (Timmer ten Hoor 1975). These obligate autotrophs are far more efficient at anaerobic (denitrifying) growth on reduced sulfur compounds than the facultative species. For example, the facultative chemolithotroph Paracoccus pantotrophus has been found to retain its sulfur-oxidizing potential under denitrifying conditions, but its μmax while doing so is extremely low (approx. 0.015 h−1) compared with those of Thiobacillus denitrificans and Sulfurimonas denitrificans (0.06 h−1). Many other facultatively autotrophic bacteria lose their sulfur-oxidizing capacity in anaerobic cultures but are still able to denitrify using organic compounds, or even hydrogen. Among these are Paracoccus versutus and Paracoccus denitrificans (Taylor and Hoare 1969; Friedrich and Mitrenga 1981). Sulfide-dependent reduction of nitrate to N2 by Beggiatoa tufts was shown using 15N-labeled nitrate (Sweerts et al. 1990). Members of the genus Sulfuritalea are unusual in that they can only grow autotrophically under anaerobic conditions, when they denitrify (Kojima and Fukui 2011).

Among the sulfur-oxidizing genera within the Archaea, Sulfolobus species appear to be the most dependent on oxygen, although some have been shown to use ferric iron or molybdate as electron acceptors under microaerobic conditions (Brock and Gustafson 1976; Brierley 1982). Members of the genera Sulfurisphaera and Acidianus are among those able to grow under anaerobic conditions, by using sulfur as their electron acceptor, thus making these bacteria both sulfur-oxidizing and sulfur-reducing, depending on the conditions (Segerer and Stetter 1989; Kurosawa et al. 1998). Nelson and Castenholz (1981) reported that some Beggiatoa species carry out an anaerobic reduction of intracellularly stored sulfur, using organic compounds such as acetate as electron donors. The ability of these organisms to oxidize sulfide to sulfur under aerobic conditions and then to reverse this reaction anaerobically would permit them to optimally profit from their habitat, where aerobic and anaerobic conditions frequently alternate. They may also actively migrate between aerobic and anaerobic zones.

Even apparently obligately aerobic strains may have mechanisms allowing them to survive during anaerobiosis for a limited length of time. Thus, Halothiobacillus neapolitanus, a species normally considered to be obligately respiratory, has been shown to be able to ferment internal reserves of polyglucose when confronted with anoxic conditions (Beudeker et al. 1981). As mentioned in the introduction, Thiobacillus ferrooxidans can use ferric iron as an electron acceptor.

Ecophysiology as a Function of pH, Temperature, and Nutrient Availability

Colorless sulfur bacteria have been found growing at pH 1.0 and pH 11.0, at 4 °C and 95 °C, and at dissolved oxygen concentrations ranging from air-saturation to anaerobiosis (Table 15.1a , b ). It is obvious that a combination of physical, chemical, and (eco) physiological factors will suit the ecological niche of the organism within a particular microbial community. A number of these will be considered here.

pH Range and Effects

The pH ranges of some of the genera of colorless sulfur bacteria are shown in Table 15.1a , b . Within these ranges, of course, species often have different pH optima. The outcome of competition for a substrate at different pH values will therefore be dictated to a large extent by the pH optima of the competing bacteria. Thus, Kuenen et al. (1977) found that at pH values above 7.5, Thiomicrospira pelophila dominated thiosulfate-limited chemostat cultures, whereas when the pH was below 6.5, Thiobacillus thioparus was able to outcompete the other for thiosulfate. At intermediate pH values, the outcome of the experiments was not reproducible, with varying levels of the two populations. Apparently, the substrate affinities of the two species were so similar that other, less well-controlled variables (e.g., iron concentration, minor amounts of wall growth, etc.) became important for the outcome of the competition. Similar pH effects have been observed in the competition between P. versutus and Thiobacillus neapolitanus (Smith and Kelly 1979).

The colorless sulfur bacteria that grow at neutral to slightly alkaline pH values are found in marine and freshwater sediments, soils, and wastewater treatment systems, to name but a few sources. Many of them have specialized in growth in the gradients where (anaerobic) sulfide-containing zones come into contact with air- or oxygen-containing water and will be discussed in the section on gradients. Some colorless sulfur bacteria are extreme acidophiles, able to grow at pH values as low as 1. This group includes mesophilic obligate and facultative autotrophs (e.g., Acidithiobacillus ferrooxidans and Acidithiobacillus acidophilus, respectively). The acidophilic colorless sulfur bacteria are abundant in locations such as acid mine-drainage water, and it is therefore interesting that many of them are also able to oxidize and gain energy from the oxidation of metals such as iron. Thus, Acidithiobacillus ferrooxidans is able to grow mixotrophically on the iron and sulfur components of pyrite (Arkestein 1980) or on mixtures of ferrous iron and tetrathionate, gaining energy from the iron- and sulfur-oxidizing reactions (Hazeu et al. 1986, 1988). There were a few reports of facultatively heterotrophic growth by Acidithiobacillus ferrooxidans (e.g., Shafia and Wilkinson 1969; Lundgren et al. 1964). However, it has since been shown that most of the Acidithiobacillus ferrooxidans cultures available from culture collections were contaminated with acidophilic facultative autotrophs and heterotrophs (Harrison 1984), including Acidithiobacillus acidophilus and Acidiphilium cryptum, and it is now generally accepted that Acidithiobacillus ferrooxidans is an obligate autotroph.

It has frequently been assumed that Acidithiobacillus ferrooxidans is one of the key species active in pyrite oxidation. In order to assess its likely significance for pyrite oxidation during coal desulfurization, Muyzer et al. (1987) used antibodies raised against Acidithiobacillus ferrooxidans for an immunofluorescent assay of slurries made from coal from different sources. Unsterilized and sterilized coal samples were inoculated with Acidithiobacillus ferrooxidans, with a mixed culture of pyrite-oxidizing bacteria from a coal-washing installation, and a mixture of the two. Despite the fact that a DNA-fluorescent stain indicated abundant microbial life in all of the slurries, the only sample in which a significant Acidithiobacillus ferrooxidans population was detected was the control, which had been sterilized and then inoculated with the pure culture of Acidithiobacillus ferrooxidans. It appears that in all other cases, other strains (which might include such species as Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, or Acidiphilium cryptum, to name but a few) were able to successfully outcompete Acidithiobacillus ferrooxidans for a niche in the consortium.

Temperature

As pointed out at the beginning of this section, colorless sulfur bacteria can be found growing at temperatures ranging from 4 °C to 95 °C. However, the majority of the frequently studied species are mesophilic. Thus, it is clear that the species discussed in this section should be regarded as indicative rather than definitive. As most of the examples discussed elsewhere in this chapter will be taken from mesophilic bacteria, most of this section will be dedicated to consideration of the thermophiles.

Thermophilic bacteria are generally associated with waters that have been geothermally heated. These range from warm springs, used for bathing since Roman times, through solfataras to submarine hydrothermal vents (e.g., Caldwell et al. 1976; le Roux et al. 1977; Jannasch 1985). The bacteria in this group can be subdivided into two groups, the moderate thermophiles (generally Bacteria), which grow over the range 45–55 °C, and the extreme thermophiles (generally Archaea), some of which can grow at temperatures approaching 100 °C.

Most of the moderately thermophilic groups are neutrophiles. However, there are also neutrophiles such as Thermothrix (Tx.) thiopara and Sulfurihydrogenibium, which have a higher optimum growth temperature (72 °C). These facultative autotrophs were found in neutral (pH 7.0), hot (74 °C) springs (Caldwell et al. 1976; Brannan and Caldwell 1980; Takai et al. 2003). Thiothrix thiopara forms macroscopic streamers as well as microscopic mats on the tufa. The streamers occur at the sulfide/oxygen interface (Caldwell et al. 1983), and the key role that oxygen plays in their development was demonstrated by means of a very simple experiment during which the surface of the hot spring was covered by a sheet of plastic to restrict entry of oxygen from the air. As a result, the dissolved oxygen dropped from 3 mg 1−1 to 0.1 mg 1−1, but other parameters, such as pH and temperature, were unaffected. The Thiothrix thiopara streamers then disappeared from their accustomed positions and reappeared at the edges of the sheet, where the sulfide/oxygen gradient had been reestablished.

The acidophilic Archaea represent the colorless sulfur bacteria among the hyperthermophiles. They are frequently found in association with sulfidic ores such as pyrite, chalcopyrite, and sphalerite. It has been suggested that the failure to find Sulfolobus species around hydrothermal vents, where Acidianus does occur, is due to the low salt tolerance of Sulfolobus species. Acidianus species can tolerate NaCl concentrations of up to 4 % (Stetter 1988). With growth temperatures between 60 °C and 95 °C, these species seem almost moderate in comparison to the growth temperatures of the sulfur-reducing Pyrobaculum and Pyrodictium species (74–110 °C).

Nutrient Availability and Ecological Niches

Of the physiological types shown in Table 15.4 , the obligate and facultative chemolithotrophs are the best known, having been the most extensively studied in pure and mixed cultures (e.g., Kelly and Kuenen 1984; Kuenen 1989; Kelly and Harrison 1989; Kuenen et al. 1985; Kuenen and Robertson 1989a, b). One of the most important environmental parameters affecting the selection of these bacteria in freshwater environments was found by Gottschal and Kuenen (1980) to be the relative turnover rates of inorganic and organic components in the available substrates (Fig. 15.4 ). Thus, if the available substrate in energy-limited systems is wholly or predominantly inorganic, obligate autotrophs such as Halothiobacillus neapolitanus will normally tend to dominate a community. Similarly, abundant organic substrates will generate communities dominated by heterotrophs. On mixed substrates, facultative autotrophs such as P. versutus or chemolithoheterotrophs will appear, depending on the ratio between the two types of substrate. If the substrate supply is predominantly organic, the sulfide-oxidizing heterotrophs or other heterotrophs will appear. This model was put to the test by means of a number of competition experiments in two- and three-membered mixed cultures of representatives from the physiological groups. In addition, a number of enrichment cultures inoculated from natural samples containing representatives of all of the physiological types were obtained. All of the experiments essentially showed that the predicted metabolic type became dominant (for examples, see Fig. 15.5a , b ). Although mathematical modeling predicted that in some cases pure cultures of only one metabolic type should be obtained, in practice, satellite populations of the others remained (Fig. 15.6 ). Clearly, secondary environmental or experimental conditions (e.g., excretion products such as glycolate, fluctuations in substrate or oxygen concentrations, and growth on the wall of the vessel) can result in deviations from the idealized model. It is obvious that a well-mixed chemostat is a model system that is rather remote from the common natural habitats of colorless sulfur bacteria, such as the sulfide/oxygen gradient in a sediment, and the results obtained can only demonstrate the principle. Moreover, the relative turnover rate of the organic and inorganic substrates is only one of the environmental parameters that determine the success of a particular species. Nevertheless, the use of this model (Fig. 15.4 ) has now clarified the situation, a practical consequence being that it has shown the way for the selective enrichment of facultatively autotrophic sulfur bacteria from freshwater.
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Fig. 15.4

A model to describe the selection of different physiological types by the ratio of inorganic to organic substrates supplied in the medium. This model may also hold for complex (seminatural) systems, where the relative turnover rates of the inorganic and organic compounds (or the ratio between the fluxes of these compounds) would determine the selection of different physiological types. For definitions of the various terms, see Table 15.4

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Fig. 15.5

The effect of organic or inorganic energy sources on competition. (a) The effect of different concentrations of organic substrates on the competition between Paracoccus versutus (Pa. versutus) and Halothiobacillus neapolitanus (H. neapolitanus) for growth-limiting thiosulfate in a continuous culture. The influent medium contained 40mM thiosulfate. During growth limitation by thiosulfate, it and the organic additives (where present) were used simultaneously by the mixed culture, and their actual concentrations in the chemostat were below the detection level. The graph shows the ratios of the two species at steady state. Open symbols, Pa. versutus; closed symbols, H. neapolitanus; circles, glycolate supplied; triangles, acetate supplied. (b) The effect of thiosulfate on the competition for acetate (10 mM) between Paracoccus versutus (Pa. versutus) and a heterotrophic spirillum called G7. For experimental details, see (a). Open symbols, spirillum G7; closed symbols, Pa. versutus (Based on Gottschal et al. 1979)

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Fig. 15.6

Competition for acetate and thiosulfate in a chemostat between an autotroph, Halothiobacillus neapolitanus (open triangles); a mixotroph, Paracoccus versutus (closed circles); and a heterotroph, spirillum G7 (open circles). The dotted lines indicate the results predicted from the model, the symbols indicate the actual results. The model held well for the extreme ratios of thiosulfate and acetate. However, although Pa. versutus dominated at intermediate ratios, as predicted, the other two types did not completely disappear. For the experimental details, see Fig. 15.5a . This model can be used for the selective enrichment of facultative autotrophs in chemostat cultures using an intermediate ratio of acetate and thiosulfate. (Based on Gottschal et al. 1981; Gottschal and Thingstad 1982)

Steady-state conditions are more common in artificial environments than in nature, and therefore in order to test the effect of substrate fluctuations on the selection of the three representative species used in the experiments discussed above (Figs. 15.5a , b , and 15.6 ), Gottschal et al. (1981) ran chemostat cultures alternating feeds of acetate and thiosulfate. In two-membered cultures, the mixotrophic P. versutus was able to maintain itself on the substrate not used by whichever obligate species was involved so that both species were subject to alternating periods of growth and starvation. However, in three-membered cultures, the two specialists were able to react more swiftly to the onset of substrate provision because of their constitutive enzymes, while the facultative species, which had to re-induce its autotrophic enzymes each time, disappeared. As with the steady-state experiments, when different mixtures of acetate and thiosulfate alternated, the outcome was determined by the concentrations involved. Enrichment cultures under this regime yielded a facultative autotroph that was able to avoid the need to induce its carbon dioxide fixation system by accumulating large amounts of PHB during the heterotrophic period.

This work was carried out on aerobic, freshwater chemostat cultures, and, as has been discussed before (Kelly and Kuenen 1984; Kuenen 1989; Kelly and Harrison 1989; Kuenen et al. 1985), marine enrichments are, for unknown reasons, generally less predictable. For example, mixotrophs did not form the dominant population in thiosulfate/acetate-limited marine cultures (Kuenen et al. 1985). Marine mixotrophs have been isolated, one of the earliest reports being a facultatively chemolithotrophic marine strain of Thiomonas intermedia from a thiosulfate-limited culture (Smith and Finazzo 1981). Similarly, an obligately autotrophic Thiomicrospira-type occurred in stable mixed cultures with a facultative Thioclava (Sorokin et al. 2005) when a facultative autotroph might have been expected.

Of course, factors other than the availability of electron donors can determine the type of population to be found in any given environment. For example, Kuenen et al. (1977) studied the effect of iron limitation and pH on the outcome of competition between two marine obligate autotrophs, Thiomicrospira pelophila and Thiobacillus thioparus. As can be seen from Fig. 15.7 , Thiomicrospira pelophila dominated mixed cultures of the two species at low iron concentrations, whereas Thiobacillus thioparus did better when iron was more abundant. One of the characteristics of Thiomicrospira pelophila is its tolerance of sulfide concentrations high enough to inhibit Thiobacillus species. It is likely that sulfide inhibition is caused by the reaction of the sulfide with available iron, forming insoluble ferrous sulfide and thus drastically reducing the concentration of iron.
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Fig. 15.7

The specific growth rates (μ) of Thiomicrospira pelophila and Thiobacillus thioparus as a function of the iron concentration in chemostat cultures at 25 oC. The graph was constructed from the results of competition experiments (at the growth rates indicated by the arrows at the y axis). The actual iron concentrations were not determined (From Kuenen et al. 1977)

Taxonomy

Many of the colorless sulfur bacteria were discovered in the early years of microbiology, at a time when scientists were relying mainly on morphological characteristics to identify their organisms. When Chester’s Manual of Determinative Bacteriology (a scanned copy of this forerunner of the famous Bergey’s Manual can now be downloaded from the Internet Archive; http://​www.​archive.​org) appeared in 1901, it was based almost entirely on morphology, staining reactions, and growth on different media. Only two of the genera listed in Table 15.2 were included – the morphologically distinctive Beggiatoa and Thiothrix. Even today, morphology and the ability to grow on particular substrates, such as the reduced sulfur compounds, are still regarded as taxonomically significant. Needless to say, this has caused a certain amount of confusion (see Table 15.2 for an overview of the genera involved and the most recent name changes). The problems associated with the identification of some colorless sulfur bacteria have been aggravated, because many of the bacteria involved are very specialized (e.g., obligate autotrophs), and consequently, the number of physiological traits that can be screened is limited. This has resulted in relatively trivial features being given undue weight during classification. Taxonomy is a way of establishing identities and relationships in an attempt to create a sense of order among the various forms of life on earth. In ecology, as in other applications of taxonomy, the precise identification of a particular species may not always be as relevant as an accurate description of its physiological characteristics, but the comparison and correlation of data from different sources become easier if one can be certain, or even reasonably sure, of the identities of the various bacteria involved. Changes in taxonomic practice largely reflect new developments in available technology as well as improvements in our understanding of which factors indicate relationships and which are merely resemblances. Taxonomic research into the colorless sulfur bacteria can thus be separated into four distinct, if overlapping phases (morphology, physiology, analytical taxonomy and phylogeny), which will be discussed sequentially here.

Morphology

The colorless sulfur bacteria include rods, spirals, cocci, filamentous cells, and it comes as no surprise to find that the first of them to be described, Beggiatoa (Trevisan 1842), is also one of the largest. Another morphologically distinct genus, Thiothrix, was described by Winogradsky in 1888, but it was not until 1904 that Beijerinck described the first of the smaller colorless sulfur bacteria, Thiobacillus thioparus. As may be seen from a survey of taxonomic manuals, a few genera are still based largely on morphological descriptions (e.g., Thiospira, Macromonas, Thiovulum), because pure cultures are either not available or have only recently been achieved.

In addition to cell size and shape, other morphological details that have been considered important are the appearance of inclusion bodies such as sulfur or poly ß-hydroxybutyrate (PHB), number and placement of flagella, colony size, colony form, and colony color. One of the dangers associated with too strong a reliance on such features is that all of them can vary depending on the growth conditions. As a single example of this problem, the faculatively autotrophic Paracoccus pantotrophus might be considered. When grown autotrophically on thiosulfate, it occurs as small cocci (0.7 × 0.9 mm), which are generally found singly or in pairs (Fig. 15.8a ). Cultivation in batch culture on rich media in which rapid growth will occur leads to a slightly larger, pleomorphic form (Fig. 15.8b ). In chemostat cultures on mineral medium with acetate, chains of cocci appear. The internal structure of Paracoccus pantotrophus also changes with its growth conditions. Thus, the normal appearance, with few inclusions, of a Gram-negative organism, which is found during substrate-limited chemostat culture (Fig. 15.8c ), gives way to cells with PHB granules and complex membranous structures (Fig. 15.8d ) when grown under oxygen or nitrogen-limited conditions, or in the presence of hydroxylamine. Cultivation on acetone or propan-2-ol results in the formation of large, crystalline structures (Fig. 15.8e ), while denitrifying growth on sulfide can result in the accumulation of a fine deposit of sulfur in the periplasm (Fig. 15.8f ). The colonial form of this species also varies, with off-white, translucent colonies being produced during growth on mineral medium with acetate, hydrogen, or thiosulfate, and larger, thicker, browner colonies being generated during growth on rich media. Long-term continuous cultivation at high growth rates can select for faster-growing variants, giving rise to much bigger colonies when the culture is transferred to plates. Species with flagellae can lose them if shaken or stirred too quickly.
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Fig. 15.8

Variations in the morphology of cells of Paracoccus pantotrophus in relation to growth conditions or substrates as seen under the electron microscope. (a) Aerobic, autotrophic growth on thiosulfate, Pt shadowed. (b) Aerobic, heterotrophic growth on a mixture of acetate, fructose, and yeast extract, Pt shadowed. (c) Thin section of cells from an acetate-limited, chemostat-grown culture, stained with ruthenium red to show the membrane structures. (d) Thin section of a cell from an aerobic, acetate-limited chemostat with hydroxylamine, stained with ruthenium red to show the membrane structures. The white bodies are PHB granules. (e) Thin section of an acetone-grown cell showing crystalline inclusions. (f) Thin section of an anaerobic (denitrifying) cell grown on sulfide and stained with silver to show the periplasmic deposits of sulfur. (Figure b from Robertson and Kuenen 1983b. Figure c from Bonnet-Smits et al. 1988. Figure f, courtesy of H. J. Nanninga. All electron microscopy courtesy of W. Batenberg.) All bars = 0.5 μm

Even the obligate autotrophs, which with their more limited range of growth conditions might appear to have less scope for variation, can produce substantial morphological changes. Thus, the number of carboxysomes formed by Halothiobacillus neapolitanus increases dramatically under CO2 limitation (Beudeker et al. 1980), and polyglucose inclusions appear under nitrogen limitation (Beudeker et al. 1981).

From all of this, it is clear that while valuable information can be gained from morphological studies on cells or colonies grown under well-defined conditions, this information should be used cautiously and in conjunction with other data.

Physiological Screening

As more pure cultures became available, it became possible to determine the physiological capabilities of different bacteria, and physiological criteria gradually became an integral part of the taxonomists’ toolbox (Kluyver and van Niel 1936). For the obligate autotrophs, these might include such tests as optimum pH and growth temperature, ability to denitrify, and (generally very limited) substrate range. In addition to these, the facultative autotrophs are generally subjected to the same range of tests used for heterotrophic bacteria, including oxidase, catalase and urease reactions, and the ability to grow on or generate acid from a range of substrates. An extensive study of the Thiobacillus species then available resulted in a numerical taxonomy analysis of the genus (Hutchinson et al. 1969) that recognized that “species” such as “Ferrobacillus ferrooxidans” and “Thiobacillus thiocyanoxidans” were actually strains of existing species (Acidithiobacillus ferrooxidans and Thiobacillus thioparus, respectively). The tests recommended by Hutchinson et al. (1969) for the identification of new Thiobacillus species included growth on sulfide, sulfur, thiocyanate, citrate, and nutrient broth, the amount of thiosulfate used, sulfur deposition, and the effect of inhibitors such as streptomycin, bacitracin, and ampicillin.

In many respects, the range of substrates on which an isolate is tested is defined by the interests of the research group. The reduced sulfur compounds are not included in standard test batteries, and the sulfur-oxidizing abilities of many bacteria were late in being discovered. For example, Friedrich and Mitrenga (1981) tested a number of hydrogen-oxidizing bacteria and found that many of them, including Paracoccus denitrificans and some Alcaligenes species, were able to grow autotrophically on thiosulfate. Attempts to use thiosulfate as an inhibitor of heterotrophic nitrification by a “Pseudomonas” species gave anomalous results, until it was realized that the culture was growing mixotrophically, using both the acetate supplied as the primary growth substrate and the thiosulfate intended as an inhibitor. Subsequent experiments revealed that this “Pseudomonas” species was also able to grow autotrophically using reduced sulfur compounds (Robertson et al. 1989).

A problem associated with the use of substrate ranges for taxonomic purposes is that it is difficult to determine how closely related bacteria with apparently similar enzyme systems are. Thus, possession of the Calvin cycle enzymes for carbon dioxide fixation or the denitrification pathway enzymes is not considered sufficient grounds for classifying the relevant bacteria into a single group, and it must be questioned whether the sulfur-oxidizing enzymes are a better indicator, especially since there appears to be several different pathways involved (Kelly 1988a, b) (see also Fig. 15.2 ).

Analytical Taxonomy

In many ways, the development of “analytical taxonomy” has been controlled by two factors – scientific knowledge and available technology. Often, as an analytical technique became available, someone, somewhere, tested it for taxonomic significance. This section will provide a few examples.

The determination of the %GC content of the DNA of bacterial isolates has been used for a long time to determine whether or not strains could be related. It is, to some extent, a negative test because, while widely differing %GC values can confirm that two strains are not related, matching %GC values do not guarantee that they are (see, for example, Kelly et al. 2005).

Cellular fatty acid analysis has been used in the taxonomy of the Thiobacilli (Agate and Vishniac 1973). Katayama-Fujimura et al. (1982) also included the analysis of ubiquinones and DNA base composition in their study. They initially subdivided the bacteria into groups based on whether they were obligately or facultatively autotrophic and then on the basis of their possession of menaquinone 8 or 10 (MK-8 or MK-10) and then used the fatty acid analysis to further examine each group. This led to a proposal for the grouping of the different strains, which is shown in Table 15.5 .
Table 15.5

Classification of species of colorless sulfur bacteria on the basis of their menaquinone and fatty acid composition

Autotrophy type

Menaquinone

Hydroxy fatty acid

Species

Group

Facultative

MK-10

None

Starkeya novella

I.1

Facultative

MK-10

3OH 10:0

Paracoccus versutus

I.1

Facultative

MK-10

3OH 14:0

Acidiphilium acidophilum

I.2

Facultative

MK-8

3OH 10:0

Thiobacillus delicatus

II

Facultative

MK-8

3OH 10:0, 3OH 12:0

Thiomonas perometabolis

II

Facultative

MK-8

3OH 10:0, 3OH 12:0

Thiomonas intermedia

II

Facultative

MK-8

3OH 10:0, 3OH 12:0

Thiobacillus denitrificans

III.1

Facultative

MK-8

3OH 10:0, 3OH 12:0

Thiobacillus thioparus

III.1

Facultative

MK-8

3OH 12:0

Halothiobacillus neapolitanus

III.2

Facultative

MK-8

3OH 14:0

Acidithiobacillus ferrooxidans

III.3

Facultative

MK-8

3OH 14:0

Acidithiobacillus thiooxidans

III.3

MK menaquinone. The number indicates the number of isoprenoid units. Groupings are as proposed by Katayama-Fujimura et al. (1982)

Some of the first publications to consider the Thiobacilli in relation to other colorless sulfur bacteria involved the phylogenetic analysis of the various species by comparison of their 5S rRNA sequences (Lane et al. 1985; Stahl et al. 1987). This work was then extended by the use of 16S rRNA analysis (Lane et al. 1992) and revealed that there are closer matches between some sulfur-oxidizing bacteria and other apparently unrelated strains such as Escherichia coli than between these and other sulfur oxidizers. Table 15.6 summarizes some of the results from the 5S and 16S rRNA comparisons.
Table 15.6

Classification of the colorless sulfur bacteria and examples of apparently related species (group “purple”), also termed Proteobacteria (Stackebrandt et al. 1988), as shown by 5S and partial 16S rRNA analysisa

Main group

Subgroup

Species

α

1

Acidiphilium acidophilus, Acidiphilium rubrum

1

Acidiphillium cryptum, Starkeya novella

2

Rhodobacter capsulatus, Paracoccus versutus

2

Paracoccus denitrificans

β

1

Thiobacillus denitrificans, Thiobacillus thioparus

1

Thiomonas intermedia, Thiomonas perometabolis

1

Rhodocyclus gelatinosus

1

Vitreoscilla

Borderline

  

Halothiobacillus neapolitanus, Chromatium vinosum

γ

1

Thiothrix nivea, Riftia symbionts

1

Thiomicrospira pelophila, Thiomicrospira L-12

1

Bathymodiolus symbionts

1

Other symbionts

1

Pseudomonas aeruginosa, Pseudomonas putida

1

Beggiatoa alba, Beggiatoa sp.

2

Escherichia coli, Salmonella, Proteus, Vibrio

γb

3

Thermithiobacillus tepidarius, Acidithiobacillus ferrooxidans

3

Acidithiobacillus albertensis, Acidithiobacillus thiooxidans

Delta

 

Thiovulum, Campylobacter, Wolinella

Atypical strains have been omitted for the sake of simplicity

aAdapted from Lane et al. 1992 and Harrison 1989

bThe organisms shown here as “γ3" were originally shown as being on a ß side branch, but improved techniques have now shown them to be in group γ (Kelly et al. 2000)

If the initial separation into obligate and facultative autotrophs employed by Katayama-Fujimura et al. (1982) is removed, the results shown in Tables 15.5 and 15.6 apparently supported each other. Thus, groups I.1 and I.2 from the menaquinone/fatty acid analysis apparently correspond to group alpha from the 16S rRNA, groups II and III-1 with group beta-1, and groups III-2 and III-3 with beta-2. The range of bacteria subjected to the menaquinone/fatty analysis was much smaller than that in the 5S and 16S rRNA survey. Comparison of the results in these tables with those showing the eventual outcome of the reorganization of the colorless sulfur bacteria shows some agreements among the obligate autotrophs. However, among the facultative species, the gulf between presumed related species (e.g., group 1.1. in Table 15.5 ) has widened, and they are now believed to be separate genera. While chemotaxonomy and phylogeny might provide more reliable tools for the classification of these bacteria than physiological or morphological observations, the results are simply the best that can be obtained with current knowledge and technology. Microbial taxonomy has always been mutable. Phylogenic analyses of nucleic acids and proteins has revealed that physiological similarities are frequently coincidental rather than accurate indicators of relationships between microorganisms. Multilocus sequence analysis, rather than 16S rRNA analysis, has recently been gaining popularity. However, the pitfalls of relying on any single analytical system have been reviewed by Kämpher and Glaeser (2012).

Phylogeny of Colorless Sulfur Bacteria

Although novel opinions about defining prokaryotic species (Gevers et al. 2005) are numerous, 16S rRNA sequence analysis is still popular for the determination of the phylogenetic affiliation of species. Different databases giving rRNA sequences, such as the Ribosomal Database Project (RDP; Cole et al. 2009), the SILVA database (Pruesse et al. 2007), GreenGenes (DeSantis et al. 2006), EzTaxon (Chun et al. 2007), and the All-Species Living Tree Project (Yarza et al. 2010) are currently available. The principle of phylogenetic analysis (Felsenstein 2004) is simple, sequences are aligned to each other, and a phylogenetic tree is calculated using different algorithms (neighbor joining (Saitou and Nei 1988), maximum parsimony, maximum likelihood (Saitou and Nei 1988) and evolutionary models (e.g., Kimura 1980). Special programs, such as ARB (Ludwich et al. 2004), are available for this purpose, but different tools to create phylogenetic trees are also incorporated in some of the rRNA databases mentioned above.

Based on comparative analysis of 16S rRNA sequences, the known colorless sulfur bacteria are, at the time of writing, grouped into four phylogenetic lineages, three within the Bacteria and one within the Archaea (Fig. 15.9 ). Most of the colorless sulfur bacteria belong to the phylum Proteobacteria, in particular the class Gammaproteobacteria. The group named Thiomicrospira also includes four Thioalkalimicrobium species (Thioalkalimicrobium aerophilum, Thioalkalimicrobium microaerophilum, Thioalkalimicrobium sibiricum, and Thioalkalimicrobium cyclicum), which are closely related to Thiomicrospira pelophila and Thiomicrospira thyasirae. Other groups of colorless sulfur bacteria within the Gammaproteobacteria are Thiothrix (nine species), Halothiobacillus (four species), Thiohalomonas (two species), Thiohalospira (two species), Thioalkalivibrio (nine species), and the Acidithiobacillaceae (five species). Candidatus Thiomargarita namibiensis, which has not been isolated in pure culture, is related to Thioploca ingrica and Beggiatoa alba. Only a few of the colorless sulfur bacteria belong to the other classes within the Proteobacteria; five Thiobacillus spp. (Thiobacillus denitrificans, Thiobacillus thioparus, Thiobacillus thiophilus, Thiobacillus aquaesulis, and Thiobacillus subterraneus) as well as Sulfuricella denitrificans (Kojima and Fukui 2010) are grouped within the Betaproteobacteria. Starkeya koreensis, Starkeya novella, and Thioclava pacifica (Sorokin et al. 2005) belong to the Alphaproteobacteria, and Sulfurimonas autotrophica, Sulfurimonas denitrificans, Sulfurimonas paralvinella, and Sulfurovum lithotrophicum, grouped as Sulfurimonas, are belonging together with Thiovulum to the Epsilonproteobacteria. So far, there are no colorless sulfur bacteria known that belong to the Deltaproteobacteria. In addition to the Proteobacteria, five species belong to the genus Sulfobacillus within the phylum Firmicutes, and five others are belonging to the genus Sulfurihydrogenibium within the phylum Aquificae. Within the Archaea, six Sulfolobus species, four Acidianus species, and Sulfurisphaera ohwakuensis belong to the family Sulfolobaceae within the phylum Crenarchaeota.
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Fig. 15.9

Phylogenetic tree based on nearly complete 16S ribosomal RNA (rRNA) sequences of described colorless sulfur bacteria. The sequences were obtained from “The All-Species Living Tree” project (LTP release 106; Yarza et al. 2010), which contains high-quality, curated 16S rRNA sequences of type strains. The tree was created using ARB software. The number within the collapsed clusters indicates the number of different species within a particular group. The scale bar indicates 10 % sequence difference

Comparative 16S rRNA sequence analysis is not only commonly used to determine the phylogenetic position of novel species, it is also used to reclassify the phylogenetic position of existing species or to solve different taxonomic problems. Recently, Boden et al. (2011) performed a phylogenetic assessment of 12 strains of Thiobacillus thioparus present in different culture collections in order to check whether the so-called Starkey type strain deposited in the different collections was identical, to obtain a definitive reference 16S rRNA sequence, and to check of other strains labeled as Thiobacillus thioparus were well-founded examples of the species. They found that four examples of the Starkey type strain were identical, and so they could obtain a definitive reference sequence. Comparative sequence analysis subsequently showed that 6 strains were correctly affiliated to Thiobacillus thioparus, but that two strains were wrongly named and had to be renamed as Halothiobacillus neapolitanus and Thermithiobacillus tepidarius, respectively.

Apart from the use of 16S rRNA sequences, the phylogeny of sulfur-oxidizing bacteria, including colorless sulfur bacteria, has also been studied using the soxB gene (Meyer et al. 2007), the aprBA genes (Meyer and Kuever 2007b), and RuBisCO (Tourova et al. 2006). Although in general the phylogenies based on these genes are similar to the ones inferred from 16S rRNA sequence data, there might be differences due to lateral gene transfer (see Meyer and Kuever 2007a).

Molecular Methods to Study the Diversity of Colorless Sulfur Bacteria in Natural Habitats

Detection of Colorless Sulfur Bacteria Using Molecular Markers

It is now well recognized that less than 1 % of all bacteria in nature can be isolated in pure cultures. In order to study the diversity and activity of microbial communities and the dynamics of their community members, it is necessary to use molecular techniques (e.g., polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS)) that were originally developed in molecular biology and medicine. Thus far, most of the bacteria, including the colorless sulfur bacteria, have been detected and identified by using the 16S rRNA approach where DNA fragments PCR-amplified with primers targeting the 16S rRNA gene of Bacteria or Archaea were sequenced after cloning or DGGE analysis. By using this approach, Hubert et al. (2011) could show a massive dominance of sequences (87 %) closely related to the chemolithoautotrophic sulfur-oxidizing bacterium Sulfuricurvum kujiense in formation waters from a Canadian oil sand reservoir. Lansén et al. (2011) used the 16S rRNA approach to detect members belonging to the epsilonproteobacterial genera Sulfurimonas and Sulfurovum in microbial communities of the Jan Mayen hydrothermal vent field. Yang et al. (2011) detected the dominance of members affiliated to the genus Sulfurihydrogenibium (Aquificales) in hydrothermal vents of Yellowstone Lake with temperatures of 50–60 °C, while members related to the gammaproteobacterium sulfur oxidizer Thiovirga were more dominant in vents with lower temperatures.

To quantify the number of cells and to study their spatial distribution, fluorescence in situ hybridization (FISH) is used. The principle of FISH is straightforward. Bacterial cells are fixed in paraformaldehyde and dehydrated in ethanol. Subsequently, the cells are hybridized with oligonucleotide probes labeled with different fluorescent dyes and visualized with an epifluorescence microscope (Amann and Fuchs 2008). Maestre et al. (2010) used 16S rRNA gene libraries and FISH to study the bacterial community in a laboratory-scale biotrickling filter treating high loads of H2S and found that most of the community members were affiliated to Thiothrix, Thiobacillus, and Sulfurimonas denitrificans. A similar approach was used by Okabe and coworkers (2007) to follow the succession of sulfur-oxidizing bacteria (SOxB) in microbial communities involved in concrete corrosion of sewer systems. They found that six different phylotypes of SOxB were present and that their abundance shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans.

Another method for quantifying the number of bacteria is quantitative PCR (qPCR). Reigstad et al. (2011) used qPCR of 16S rRNA genes to study microbial communities in thermal springs on Svalbard and found that bacteria closely related to Thiothrix and Sulfurovum were the most dominant constituents of these communities. Liu et al. (2006) designed specific primers targeting the 16S rRNA genes of different microorganisms involved in bioleaching, including the colorless sulfur bacteria Sulfolobus, Sulfobacillus, and Acidithiobacillus caldus, and used these primers in a qPCR to rapidly detect and quantify these organisms in bioleaching processes. A combination of different molecular methods (qPCR, FISH, CARD-FISH) and most probable number (MPN) cultivation techniques was used by Kock and Schippers (2008) to quantitatively analyze microbial communities from three different sulfidic mine waste tailing dumps. They found that Acidithiobacillus spp. dominated over Leptospirillum spp. and that Sulfobacillus spp. were generally less abundant.

Apart from the use of 16S rRNA genes as molecular markers, functional genes were also used to study colorless sulfur bacteria in their natural habitat. Meyer and Kuever (2007b) used aprA as a functional marker to study the diversity of sulfate-reducing and sulfur-oxidizing prokaryotes in different samples from the Caribbean Sea and found a dominance of putative chemolithoheterotrophic sulfur-oxidizing Alphaproteobacteria in non-hydrothermal sediments and in the water column and chemolithoautotrophic sulfur-oxidizing Beta- and Gammaproteobacteria on the surface of volcanic manganese crusts. Sorokin and coworkers used RuBisCO and ATP lyase genes to study the diversity of sulfur-oxidizing bacteria in hypersaline (Tourova et al. 2010) and haloalkaline (Kovaleva et al. 2011) lakes. Chen and coworkers (2007) used both 16S rRNA and sulfur oxygenase reductase (SOR) genes as molecular markers to study the microorganisms in bioreactors treating gold-bearing concentrates.

Natural Habitats of Colorless Sulfur Bacteria

As may be deduced from the range of physiological characteristics discussed above, the colorless sulfur bacteria, in one form or another, are to be found in almost every life-supporting environment where reduced sulfur compounds are found. Indeed, where they are very active, the reduced sulfur compounds may not reach detectable levels. Because the range of habitats is so wide, the principles underlying the selection of colorless sulfur bacteria in selected situations will be discussed below. The following section will then deal more generally with the role of the colorless sulfur-oxidizing bacteria in the sulfur cycle. This discussion of habitats is not intended to be exhaustive.

In natural habitats, the reduced sulfur compounds available tend to be either sulfides (including metallic ores) or sulfur. Thanks to the activities of sulfate-reducing bacteria, especially in anoxic sediments, hydrogen sulfide is very commonly available, and some algal and cyanobacterial mats have been shown to generate organic sulfides (e.g., Andreae and Barnard 1984). One of the main factors that bacteria growing on hydrogen sulfide have to contend with is the chemical reaction between sulfide and oxygen, and therefore the colorless sulfur bacteria are frequently found in the gradients at the interface between anoxic sulfide-containing areas and aerobic waters and sediments. There, at very low oxygen and sulfide concentrations, they can effectively compete with the spontaneous chemical oxidation reaction. Of course, the rate of chemical oxidation of metal sulfides with oxygen is very low at acid pH levels, and the acidophilic bacteria need not, therefore, grow predominantly in gradients, as their neutrophilic counterparts must. The same holds for deposits of elemental sulfur, which does not react spontaneously with oxygen at a significant rate at any pH. Another habitat in which sulfide-oxidizing bacteria appear to be of some importance is in the complex communities of prokaryotes and eukaryotes around hydrothermal vents, where the sulfide is geologically rather than biologically generated. As well as free-living species such as Thiomicrospira crunogena (Jannasch 1985), it has been shown that many invertebrates have symbiotic colorless sulfur bacteria, and this can itself be regarded as a distinct habitat (Cavanaugh et al. 1981). A third example of a type of habitat for these bacteria that is becoming steadily more common is that associated with human activities, largely in connection with waste treatment and industrial leaching of ores for (heavy) metal recovery.

Gradients in Aquatic Systems and Sediments

Sulfide/oxygen gradients occur in stratified water bodies, as well as in soils and sediments. Such gradients can range in size from a few hundred micrometers thick in a microbial mat or surface sediment to several meters in a stratified body of water (Sorokin 1970, 1972; Jørgensen et al. 1979). These gradients can sometimes be distinguished with the naked eye. For example, Thiovulum sp. grows as a fine white veil at the interface between sulfide and oxygen (Jørgensen 1988). Wirsen and Jannasch (1978), studying the effect of the sulfide/oxygen gradient on the formation of these veils in continuous flow cultures, observed that the veils dispersed within minutes of the cessation of the flow of seawater through the culture vessel, and formed again once the flow was resumed, indicating chemotaxis of the swarming form of Thiovulum toward critical concentrations of oxygen and sulfide.

The genus Beggiatoa contains marine and freshwater species that are typical of life at the aerobic/anaerobic interface. Dense mats of almost axenic cultures of Beggiatoa on sulfide-containing sediments are frequently observed, especially in marine sediments where sulfide production rates can be very high. These mats are characterized by very steep oxygen and sulfide gradients over a few mm (Jørgensen 1982, 1988). Since Beggiatoa oxidizes the sulfide at a very high rate, the overlying aerobic water is effectively “protected” from diffusion of toxic sulfide. The typical conditions for growth in this type of mat have been very difficult to reproduce in the laboratory. Indeed, they are so specialized that it was only when available techniques had improved sufficiently to allow in vitro cultivation on sulfide/oxygen gradients that the autotrophic potential of marine strains was established unambiguously (Nelson and Jannasch 1983; Nelson 1988). The Beggiatoa cells were cultured in closed tubes using a layer of very soft (0.2 %) agar over a sulfide-containing plug of harder (1.5 %) agar, thus allowing the formation of an upward sulfide gradient. Diffusion from a headspace containing air contributed a downward oxygen gradient. The Beggiatoa colony grew as a “plate” that was less than 1 mm thick at the point where the two gradients overlapped. The very rapid oxidation of sulfide allowed the organisms to maintain an extremely low concentration of the two substrates. As a result, chemical oxidation of sulfide was insignificant. The turnover time for sulfide and oxygen was only 3 s in Beggiatoa gradients, whereas the half-life of these two substances in sterile controls was about 20 min. Enzyme analysis and the fixation of 14CO2 by these cells confirmed that they were capable of autotrophic growth. The situation regarding freshwater strains is not so clear-cut. Schmidt et al. (1987) showed sulfide oxidation rates for a freshwater strain comparable to those obtained with the marine strain discussed above.

Another well-known place where gradients occur is within phototrophic mats. Jørgensen and des Marais (1986) studied the zonation around a cyanobacterial mat growing in a hypersaline pond and found that a band of Beggiatoa occurred 1.5 mm below the cyanobacteria. The photosynthetic activity of the cyanobacteria generated sufficient oxygen to produce an oxygen peak with a maximum of 1 mM at the cyanobacterial band. A steep downward gradient of oxygen overlapped a sulfide gradient at the point where the Beggiatoa were growing. In an earlier study, Jørgensen (1982) described the diurnal changes in the sulfide and oxygen gradients and the microbial community to be found in a sulfuretum (a microbial mat in which the total turnover of inorganic and organic compounds is heavily dominated by the sulfur cycle) on the surface of sediment. It was observed that the mixture of cyanobacteria, phototrophic sulfur bacteria, and Beggiatoa was stratified and that the relative positions of the three populations among the strata were governed by the level of photosynthetically generated oxygen (Fig. 15.10 ). During the night, when the oxygen had been depleted and the oxygen boundary extended to the surface of the sediment, the phototrophic Chromatium was found at the surface. However, once photosynthesis began, with the onset of daylight, oxygen began to build up in the sediment, and the Chromatium followed the sulfide boundary down, remaining within the anaerobic part of the sediment. The Beggiatoa population tended to move with the sulfide/oxygen interface, except during the night when this was in the stagnant water above the surface of the sediment. As Beggiatoa is only motile by means of a gliding action, it is restricted to the solid phase.
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Fig. 15.10

Diurnal cycle of oxygen and sulfide distribution and of microbial zonation in a marine sulfuretum. The zero line in each box indicates the interface between the sediment and the overlaying water phase. The dominant genera at each stratum are indicated in each box. Diatoms were primarily seen among the Oscillatoria. In addition to diurnal changes in light, oxygen, and sulfide, another important factor was that the Beggiatoa which are gliding bacteria could not move out of the sediment, whereas Chromatium, which is also motile, was able to move into the water phase above (From Jørgensen 1982)

Other conspicuous colorless sulfur bacteria such as Thiothrix, Thioploca, and Achromatium have all been encountered as typical organisms in such gradients. Furthermore, mixed cultures of Thiobacillus-like bacteria sampled from sulfide/oxygen gradients and showing active sulfide-dependent carbon dioxide fixation clearly exhibit chemotaxis toward the interface when transferred to artificial sulfide/oxygen gradients in the laboratory (J. G. Kuenen, unpublished observations).

Hydrothermal Vents

An interesting extension of the model for the selection of freshwater colorless sulfur bacteria discussed above is to be found in the results of research on the mesophilic bacterial communities found around the different hydrothermal vents (see Jannasch 1985, 1988 for early reviews). These vents are a result of the movements of the tectonic plates of the Earth’s crust. Seawater penetrates deep under the sea floor and is heated geothermally, reaching temperatures as high as 1,200 °C. Under these conditions, it reacts with and dissolves various reduced chemicals before being forced to the surface again as hydrothermal fluid, which contains sulfide, CO2, and methane, as well as various metals and hydrogen. The type of vent that occurs depends very much on the overlying geology and can be at least partially separated into “bare lava” and “warm” systems. In the bare lava vents, the pressurized hydrothermal fluid reaches the surface of the sea floor at temperatures around 350 °C. As it issues from the vents, it reacts with chemicals in the seawater, forming precipitates that often accumulate as “chimneys.” Because the formation of metal sulfides gives the fluid issuing from these chimneys the appearance of smoke, they have become known as “black smokers.” The “warm” vents, on the other hand, are the result of the hydrothermal fluid percolating through sediments on its way to the surface, and the solution tends to be much cooler (<25 °C) and to have substantial organic content when it reaches the seawater. The waters around these vents support dense communities of bacteria able to grow on the geothermally generated reduced compounds such as the sulfides and methane. Obligately autotrophic sulfur bacteria, especially Thiomicrospira species (Ruby et al. 1981; Ruby and Jannasch 1982), have been found associated with the areas around black smokers where the organic turnover is relatively low, while facultative autotrophs such as Beggiatoa appear to dominate around the sedimented vents where the organic turnover is much higher (Jannasch 1988). For a time, it was believed that Beggiatoa mats rarely exceeded 1 mm in thickness (Nelson 1988). However, observations at the Guaymas Basin hydrothermal vents, where the hydrothermal fluid in some areas percolates though 400 m of sediment before reaching the surface, revealed mats of Beggiatoa up to 60 cm thick (Nelson et al. 1989). The communities around these vents appeared to be made up of three strains of Beggiatoa that had widely differing cell widths (115–122 μm, 40–42 μm, and 24–32 μm). The narrowest of these dominated, almost to the point of a monoculture, in the thickest layers, which were apparently associated with colonies of the vestimentiferan tube worms (see section “Symbiosis” below). In addition to sulfide concentration and temperature, the authors suggested that organic excretion compounds from the worms may be an important factor in the development of these mats. Beggiatoa mats are also strongly affected by the patchy nature of hydrothermal seeps (Lloyd et al. 2010) and the rate of pore water flow (de Beer et al. 2006) since these control the supply rates for electron donors and acceptors to the mat.

Symbiosis

Geologists studying areas of volcanic activity on the seabed (1,800–3,700 m below the surface) were surprised to find that not only were there dense, free-living bacterial populations associated with the vents, but that these permanently dark areas were also occupied by an extensive community of invertebrates (and also, in some areas, fish), most of which were previously unknown (Corliss et al. 1979). Despite the density of the bacterial community, it was difficult to see how a food chain based entirely on suspended bacteria as a source of prey could support the considerable population of very large tube worms, clams, and other invertebrates. Investigation of the anatomy of the tube worms (Riftia pachyptila) revealed that they do not have an alimentary tract but instead possess a large (more than half the weight of the worm) body of tissue, the trophosome, which is very rich in blood vessels. Examination of this tissue under the electron microscope revealed that it also contained a dense, intracellular community of bacteria (Cavanaugh et al. 1981; Cavanaugh 1983a). The trophosome had already been shown to contain the enzymes necessary for chemoautotrophic growth on reduced sulfur compounds. These enzymes did not occur elsewhere in the tissues of the worm (Felbeck 1981; Felbeck et al. 1981) and were presumably derived from the bacteria. This was the first example of prokaryotic/eukaryotic symbiosis in which the hosts rely on organic compounds excreted by the bacteria. The blood of the tube worms carries sulfide as well as oxygen from the gills to the trophosome and has a special sulfide-binding protein that prevents sulfide toxicity. Endosymbionts were then found in a range of vent faunas including the giant white clams (Calyptogena magnifica) and were not limited to sulfide-oxidizing bacteria, since methylotrophs have also been found (Jannasch 1988). 5S and 16S rRNA analysis indicated a relatively close relationship between the endosymbionts and members of the genus Thiomicrospira (Lane et al. 1985, 1992), which, as mentioned above, is one of the best-represented genera among the free-living bacterial community at the vents (Ruby et al. 1981; Ruby and Jannasch 1982; Jannasch 1988).

Once the occurrence of endosymbiotic bacteria in the animals of the hydrothermal vents had been accepted, many more occurrences were recognized in more mundane locations, including sewage outfalls and sulfide-rich sediments (e.g., Southward 1986; Dando and Southward 1986). Many of the animals associated with symbionts resemble Riftia pachyptila in that they completely lack a mouth and digestive system, whereas others may have only small guts and feeding appendages (Cavanaugh 1983a, b). Not all of them have a specialized organ like the trophosome, and many endosymbionts appear to be associated with the gills of the eukaryotic host. For example, intracellular colorless sulfur bacteria have been found in the gill tissues of bivalves such as Solemya velum (Cavanaugh 1983b) and Thyasira flexuosa (Wood and Kelly 1989). The description of a novel Thiobacillus-like species, Thiomicrospira thyasiridae (Wood and Kelly 1989), from the gill tissue of Thyasira flexuosa was probably the first report of the isolation of one of these symbionts.

Smith et al. (1989) illustrated the effect that a localized deposit of organic material in an otherwise oligotrophic environment can have on the indigenous community. The skeleton of a 20-m-long whale at a depth of 1,240 m on the seabed in the Santa Catalina basin was not only covered with mats of Beggiatoa resembling Beggiatoa gigantea, but it also supported six metazoan species, at least four of which are known in other locations to contain endosymbionts. As well as vent species (Vesicomya gigas and Calyptogena pacifica), others organisms known from anoxic sediments (Lucinoma annulata) and rotting wood (Idasola washingtonia) were also observed. None of these prokaryotic or eukaryotic species had been observed in this area before. It was found that the pore water under the skeleton contained around 20 mM sulfide, and the samples of whale-bone that were recovered were found to be rich in oil and smelled strongly of sulfide. It would appear from the apparent ages of some of the mollusks present that a single whale carcass is sufficient to support these sulfide-dependent communities for several years.

Man-made Habitats and Application of Sulfur Bacteria

Man-made environments such as the bioreactors used for industrial wastewater treatment have provided habitats for bacteria that impose selective parameters not necessarily found in nature. Thus, substrates tend to be more abundant and conditions are generally more stable than in most natural situations. Two categories of artificial habitat where colorless sulfur bacteria are particularly important are wastewater treatment bioreactors and those associated with various leaching activities. Examples of other artificial habitats include industrial sulfur deposits or dumps, mining operations that expose sulfidic ores or sulfur to water or air, coal storage sites, and, last but not least, systems (including sewage treatment plants) containing various amounts of reduced sulfur compounds.

Waste Treatment

Reduced sulfur compounds can occur in industrial wastes in a variety of forms and from a variety of sources. For example, sulfide is an inevitable by-product of sulfate reduction associated with methanogenesis (if the effluent from which the methane is being generated contains significant amounts of sulfate) and the oil and gas industries. Thiosulfate and thiocyanate make up a substantial amount of the chemical content of photographic film processing waste, and some papermaking processes generate both inorganic and organic sulfides. Of course, the amount of reduced sulfur compounds generated from industrial processes pales into insignificance when the quantity generated from animal wastes is considered.

Reduced sulfur compounds present a problem both environmentally, because of their toxicity, and socially, because of their odor. If large amounts of sulfide are released into natural waters, this can result in oxygen depletion, either because of the oxygen demand for biological oxidation or, in the absence of suitable bacteria, by spontaneous chemical oxidation. Many water treatment plants impose surcharges for the treatment of such effluent because it can disturb the microbial community in the bioreactors, and there is obviously considerable pressure on companies to treat their effluent on the site. There are both chemical and physical methods of removing hydrogen sulfide from effluent; these include the use of ion-exchange resins, absorption with aqueous or organic solvents, and chemical oxidation (Gommers 1988). Many of these simply transfer the problem to another waste stream or involve expensive or complex processes, and they are all expensive, especially for the removal of the last traces of sulfide compounds.

Colorless sulfur bacteria occur in many sewage treatment systems and, in fact, are inadvertently used to oxidize reduced sulfur compounds in the wastewater. In some cases, this can lead to problems, such as the “bulking” caused by Thiothrix. The deliberate use of biological treatment of sulfide-containing waste using colorless sulfur bacteria has become commonplace. The end products (sulfur or sulfate) are not hazardous, and sulfate can be discharged directly into the sea or into brackish estuaries (which already are so high in sulfate that the discharge is insignificant). Moreover, biological treatment systems can be based on existing reactor designs (e.g., fluidized and packed bed reactors) and require very little in the way of new technology.

Another advantage of a biological process is that it can be combined with the treatment of other problems in an effluent. For example, the effluent of a methane reactor will contain ammonia in addition to sulfide. If the ammonia is then converted to nitrate or nitrite by aerobic, nitrifying bacteria, the resulting effluent can then be recycled to provide the electron acceptor for a sulfide-oxidizing reactor immediately after the methane reactor. The microbiological investigation of such a sulfide-oxidizing, denitrifying reactor revealed the presence of large numbers of facultatively autotrophic colorless sulfur bacteria, which could oxidize sulfide to sulfate while reducing nitrate to nitrogen gas (Robertson and Kuenen 1983a). In addition to the removal of nitrogen compounds, other advantages associated with the use of denitrifying bacteria rather than aerobic ones include lower production of both biomass and acid.

Combined Sulfide Oxidation and Denitrification

A denitrifying, sulfide -oxidizing reactor system was patented by a Dutch company, Gist-brocades (now a branch of DSM), for the posttreatment of effluent from methane-producing reactors (Patent number E.P.A.0051 888). Studies on a laboratory-scale model of this reactor, running on artificial wastewater, revealed that sulfide (2–3 kg S/m3/day), acetate (4–6 kg S/m3/day), and nitrate (5 kg S/m3/day) were all effectively removed (Gommers et al. 1988a). The rate-limiting step in the reactor proved to be the oxidation of sulfur to sulfate and, under most loads, the biomass had an overcapacity for both the oxidation of sulfide to sulfur and the conversion of acetate (Gommers et al. 1988b). During experiments in which nitrate depletion occurred, it became evident that in the absence of nitrate, at least one member of the bacterial community was able to reduce any available sulfur, thus illustrating the need for careful monitoring of the electron donor/electron acceptor ratios in such reactors (Gommers et al. 1988b).

The facultatively autotrophic species Paracoccus pantotrophus was isolated from a denitrifying, sulfide-oxidizing uidized bed reactor that was supplied with approximately equivalent amounts of organic and inorganic substrates (Robertson and Kuenen 1983b), and it initially appeared that the selection of a facultative bacterium would lend support to the model described for the ecological niches of aerobic, freshwater sulfur-oxidizing bacteria (Fig. 15.4 ). However, subsequent attempts to isolate obligate autotrophs from a laboratory-scale model of this system that was being fed with an exclusively inorganic feed also resulted in the isolation of facultative autotrophs (M. Verbeek, W. Bijleveld, L. A. Robertson, and J. G. Kuenen, unpublished observations). It is not clear whether obligate autotrophs were present in the inoculum, or the isolation techniques employed were inadequate for any obligate autotrophs present (although they were adequate for the cultivation of known obligate autotrophs), or whether growth in a biofilm in this type of reactor poses an additional selective pressure that favors facultatively autotrophic bacteria. Subsequent work has shown that a number of sulfide oxidizers from a wastewater system required cultivation on special membrane filters with sulfide gas before isolated colonies could be obtained (Visser et al. 1997). The same basic idea of using denitrifying colorless sulfur bacteria was employed in a method proposed by Sublette and Sylvester (1987) for removing H2S from gas streams by passing them through a reactor containing Thiobacillus denitrificans. The bacteria were first immobilized by coculturing with floc-forming heterotrophs after the authors demonstrated that the presence of the heterotroph had no effect on the sulfide oxidation rate of Thiobacillus denitrificans.

Removal of Sulfide as Elemental Sulfur

As already mentioned, sulfate-containing effluents can be discharged into the sea without significantly increasing the sulfur budget. However, the same is not true if the effluent is discharged into a body of freshwater. To overcome this problem, recovery as elemental sulfur, an intermediate in the oxidation of sulfide to sulfate, would be more appropriate. Research has shown that certain Thiobacillus-like bacteria are more inclined to produce sulfur than other species and that both the dissolved oxygen and the sulfide concentration play an important part in determining whether sulfur or sulfate is the primary end product during sulfide oxidation. Both electron acceptor limitation and high sulfide loads favor sulfur production (Stefess and Kuenen 1989). A pilot plant based on this principle, using a mixed bacterial biofilm reactor to treat the effluent from a paper mill, was developed in the Netherlands (Buisman 1989; Janssen et al 2009).

Removal of Organic Sulfides

A problem frequently encountered during the alkaline pulping of wood is the production of organic sulfides such as methyl mercaptan and dimethyl sulfide. Alkaline pulping is done in order to improve the yield and quality of pulp derived from conifers to be used primarily in the manufacture of paper. Organic sulfides are toxic at even lower concentrations than hydrogen sulfide and have a very low threshold odor. Despite their toxicity, it has proved possible to grow bacteria on high concentrations of organic sulfides by using substrate-limited chemostats (Suylen et al. 1986; Kanagawa and Kelly 1986; Smith and Kelly 1988a, b, c). That the ability to oxidize these compounds may be widespread is suggested by the observation that the dominant organism in one set of experiments was a Hyphomicrobium species that was later shown to be able to grow as a facultative chemolithotroph on organic sulfur compounds in pure culture (Suylen and Kuenen 1986; Suylen et al. 1986), whereas the key organism in the other series was a strain of Thiobacillus thioparus, an obligate autotroph (Kanagawa and Kelly 1986). Immobilized cells of Thiobacillus thioparus strain TK-m have been successfully used on the laboratory scale to deodorize gases containing methyl mercaptan, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide (Kanagawa and Mikami 1989; Tanji et al. 1989).

All of the colorless sulfur bacteria mentioned thus far are beneficial in wastewater treatment. However, in oxidation tanks fed with sulfide-containing waste water, the filamentous Thiothrix species can cause problems because they are associated with the phenomenon known as “bulking”; this occurs when bacterial aggregations that usually settle easily become loose and flocculent. This can result in blockages or loss of the biomass from the reactor.

Leaching-Associated Activities

Acidophilic bacteria are used in the recovery of metals from poor ores by leaching, and their potential use in the desulfurization of coal was extensively studied in the later decades of the twentieth century. To some extent, coal desulfurization and microbial leaching are the same process, in that in both cases sulfidic ores are oxidized, using similar organisms. However, the desired end products are different, and they are thus generally discussed separately. The aim of coal desulfurization is to produce a solid product (coal) that is as free of sulfur (including sulfur-containing precipitates) as possible, and it is therefore necessary to convert reduced sulfur compounds to soluble forms. In leaching, it is metal recovery that is important, and the presence of jarosite (MFe3(SO4)2OH5, where M is a monovalent cation such as Na+ or K+) and other precipitates in the solid waste is not relevant (although it may constitute an environmental problem around the leaching heaps).

Bacterial leaching is used in the recovery of metals from ores that are too poor for conventional metallurgical extraction methods (Kelly and Tuovinen 1972; Brierley and Lockwood 1977; Brierley 1982; Ehrlich and Brierley (1990)). Combinations of Acidithiobacillus ferrooxidans and either A. thiooxidans or Acidiphilium acidophilum and Leptospirillum ferrooxidans have been associated with the degradation of pyrite (FeS2) and chalcopyrite (CuFeS2). The leaching reactions may involve the direct bacterial oxidation of the sulfide ores with oxygen and/or an indirect process during which ferric ions produced by the bacterial oxidation of ferrous iron are used to chemically oxidize the sulfide ores. The ferric ions are thereby reduced to ferrous iron, which, in turn, can be recycled by the bacteria. During this process, other metallic ions such as cupric copper dissolve. Other metals that have been extracted using processes that involve bacteria include zinc, uranium, lead, gold, molybdenum, and, especially, copper. Dump leaching operations, which are frequently used to extract copper, can be fairly primitive, involving the creation of ore dumps, often in valleys or old open pit mines. As water percolates through the heaped rocks, bacterial activity releases the metals into solution. This solution is then collected in catch basins, the metals recovered, and the liquids recycled to the top of the dump. A somewhat better controlled system is known as heap leaching. During this process, the ore-bearing rocks are crushed to promote contact with the acidified water, and the heaps are built on impermeable bases that prevent seepage into the soil beneath. Aeration systems can be built into the heaps. As mineral reserves become depleted and demand increases, it is becoming economically attractive to extract even small amounts of metals in poor ores and spoilage heaps, technological improvements should increase the efficiency of microbial leaching processes and lessen their environmental impact. Bioleaching of other potential sources such as mine tailings, contaminated sediments, and even sewage sludge is also attracting attention (Liu et al. 2008; Seidel et al. 2006; Pathak et al. 2009).

Research into the use of the pyrite-oxidizing abilities of bacteria such as Acidithiobacillus ferrooxidans and Sulfolobus species for the removal of sulfur compounds from coal before it is burned, thus reducing sulfur emission into the atmosphere, has been carried out at a number of centers in the last few decades. It has been shown that such a process could be effective, especially for low-sulfur coals, using consortia of mesophilic bacteria (Bos et al. 1988; Bos and Kuenen 1990). Laboratory studies have shown that an optimal process requires two steps. First, a mixed-flow inoculation step, where a fairly dense population of bacteria already growing on pyrite can be brought into contact with fresh, finely ground coal at a pH suitable for growth (around pH 1.8). This inoculation step would then be followed by the use of plug-flow reactors, where the bulk of the pyrite oxidation would take place. At the end of the process, the process water can be recirculated, as can some of the biomass-bearing coal particles, to serve as the inoculum for the fresh coal. A plant design, involving a cascade of Pachuca tanks (Fig. 15.11 ), was devised for this type of system (Bos et al. 1988). Pachuca tanks (in their simplest form, an inverted cone with aeration at the narrowest point, at the bottom of the tank) are particularly suitable for this type of process because the upflow of air into the tanks not only provides the bacterial community with the oxygen and carbon dioxide necessary, but it also keeps the slurry well-mixed, without any need for complex and expensive stirring mechanisms.
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Fig. 15.11

Simplified scheme for the microbiological desulfurization of coal in Pachuca tanks (biodesulfurization reactors). After grinding and mixing with water, the coal slurry contains particles less than 100 μm in diameter at a concentration of 20 % (w/v). At this particle size, virtually all of the pyrite crystals become accessible to microbial leaching. The total leaching process requires about 10 days. More than 95 % of the inorganic sulfur is removed, but little or no organic sulfur is degraded

Corrosion

Together with the sulfate-reducing bacteria, many of the sulfuric-acid-producing bacteria, and in particular the acidophiles, have been implicated in many corrosion problems. Indeed a strain of Acidithiobacillus thiooxidans isolated from a corroding concrete pipe was originally known as Thiobacillus concretivorus (i.e., concrete-eating). In sewage pipes with an aerobic headspace, sulfide may be produced in the anaerobic water phase and then be transferred to the film of water on the aerobic part of the pipe where it may be oxidized to sulfuric acid. In order to dissolve the carbonates in concrete, the pH need only be below 5.0–5.5 and such a pH can be generated by either neutrophilic or acidophilic bacteria. The activities of the acidophiles may also be responsible for steel pipe corrosion (Kuenen and Bos 1988) as well as many of the pollution problems associated with the acid runoff from mine spoil heaps. These environmental problems are not only associated with the low pH of the water, but also with the toxic concentrations of heavy metals that they may contain. In addition, acidic water containing ferric sulfate may generate precipitates of jarosite, and these can block drainage pipes and cover stream and river sediments.

The Role of Colorless Sulfur-Oxidizing Bacteria in the Sulfur Cycle

Although much is known about the physiology and occurrence of colorless sulfur bacteria (especially with the advent of gene detection and DNA analysis methods), less is known about the quantitative aspects of their activity in nature. Many of the reasons for this are difficulties commonly associated with field work (e.g., heterogeneous samples, unstable gradients, low concentrations of substrates) and are therefore outside the scope of this chapter, but a few difficulties are uniquely associated with the colorless sulfur bacteria.

Commonly used methods for estimating the activity of sulfur-oxidizing bacteria in the field include cell counts, oxidation of (radiolabeled) substrate (sulfide, thiosulfate, or sulfur), product formation (especially sulfuric acid, since this causes pH changes), and 14CO2 fixation. Other, more specific techniques include the measurement of substrate-dependent respiration and immunofluorescent microscopy.

Cell Counts

With some of the more conspicuous bacteria (e.g., Beggiatoa, Thiovulum), it is possible to obtain a rough estimate of numbers based on direct cell counts. However, most of the colorless sulfur bacteria require cultivation before they can be counted. The choice of media and substrates for most probable number (MPN) estimates or direct plate counts is especially difficult for the colorless sulfur bacteria. The most obvious problem is that outside the chemostat there is no way of selectively growing facultative autotrophs or chemolithoheterotrophs. They must first be isolated on autotrophic or heterotrophic media, respectively, and then screened for sulfur-oxidizing capacity. In addition, low recovery efficiency can be a problem with both plate counts and dilution series. Two other problems are associated with the obligate autotrophs. Firstly, thiosulfate is frequently used as an energy source in solid media, but this is not always the most suitable energy source. For some bacteria, agar plates containing colloidal sulfur may be more appropriate, while other bacteria may require sulfide. The use of solid sulfide media can present technical problems with regard to toxicity and instability unless one of the less-soluble nontoxic sulfides (e.g., calcium sulfide) is used. Secondly, some autotrophic species do not give distinct colonies on agar, and moreover, the acidophiles may be inhibited by organic compounds resulting from chemical acid hydrolysis of the agar itself at their required growth pH values. To overcome these agar-associated problems, other techniques, such as the use of silica gel plates or floating filters (de Bruyn et al. 1990; Visser et al 1997), may be more appropriate. Some of the sulfur-oxidizers may have a requirement for an unidentified growth factor such as a vitamin or mineral.

Activity Measurements

Data on the rates of sulfide oxidation in natural systems are scattered and somewhat variable, possibly because of the difficulty of accurate sampling as well as the reactivity of the compounds involved.

Once cell numbers have been estimated with a degree of confidence, they can only be used to provide an idea of the potential activity of colorless sulfur-oxidizing bacteria within that particular ecosystem. The measurement of substrate transformations (i.e., utilization or accumulation), preferably in situ, can be used as a measure of actual activity. A major problem associated with the use and measurement of many reduced sulfur compounds, especially sulfide and sulfite, is that they are chemically very reactive and are readily oxidized spontaneously by oxygen. Appropriate controls can, to some extent, overcome this problem, but it must be remembered that in nature biological and chemical reactions compete, and equilibrium reactions causing the exchange of radiolabel in reduced sulfur compounds mean that extra caution must be used in the interpretation of results. Moreover, chemical oxidation rates are influenced by many of the environmental parameters that also affect biological activity (e.g., pH, temperature, chemical constitution of the solutions involved). In a few cases, where dominant populations of known colorless sulfur bacteria occur (e.g., Sulfolobus in solfataras, Beggiatoa mats), rough estimates have been made of the activity of these organisms. Mosser et al. (1973) found rates for sulfur oxidation to sulfate of 67 and 190 g m−2day−1 for mats of Sulfolobus acidocaldarius growing in two hot pools (Moose Pool and Sulfur Cauldron, Yellowstone National Park, respectively). In the Black Sea, a maximum rate of 710 nmol l−1day−1 was observed by Sorokin (1970). For extended discussions of sulfur oxidation rates in nature, the reader is referred to Kuenen (1975) and to Jørgensen (1988).

Another problem is that the sulfur-oxidizing heterotrophs may also contribute to the turnover of reduced sulfur compounds at natural sites. In some cases, 14CO2 fixation can be used to eliminate this, but in many locations where mixotrophs or chemolithoheterotrophs are involved, CO2 may not be the primary source of carbon. This type of experiment could, therefore, sometimes result in underestimates if it is not used in tandem with other measurements. An associated problem is that the specific activity of a given species can vary. For example, Beudeker et al. (1980) found that, when grown under carbon dioxide limitation, the ribulose bisphosphate carboxylase (RuBisCO) activity in Halothiobacillus neapolitanus was 240 nmol min−1mg protein−1. If, however, thiosulfate was the limiting factor, the enzyme level fell to 72 nmol min−1mg protein−1. Other substrate conversion rates can also vary, especially among species. Thus, it has been found that Thiobacillus denitrificans and Sulfurimonas denitrificans oxidize thiosulfate at rates of 0.86 and 2.9 mM thiosulfate g C−1h−1, respectively (Timmer ten Hoor 1977).

A combination of CO2 fixation and oxygen and hydrogen sulfide analysis was used to measure microbial activity in Saelenvaan Lake in Norway. As can be seen from Fig. 15.12 , a peak of CO2 fixation was found to coincide with the very narrow zone where oxygen and sulfide coexisted. It should be noted that the sampling technique was critical for the success of these experiments. A special sampling device with an inlet that removes water from a horizontal area of the column at 1–2 cm intervals (Jørgensen et al. 1979) was necessary if a less accurate device was used, the very narrow CO2 fixation zone could not be seen because of dilution by the surrounding water.
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Fig. 15.12

Profiles of CO2 fixation, dissolved oxygen, and dissolved hydrogen sulfide concentrations in Saelenvaan Lake (Norway) sampled at 5 a.m. on 15 August 1978. The high resolution was due to the use of a special sampling device connected to a pump. CO2 fixation rates (top horizontal axis) were obtained using 14CO2 injected into dark bottles, which were incubated in situ. The left vertical axis is depth in m. The bottom horizontal axis is the concentration of dissolved gas in μmol l−2. Triangles, μmol CO2 l Eh−1; squares, μmol oxygen liter1; circles, μmol hydrogen sulfide liter–1 (From Kelly and Kuenen 1984)

Those working on the ecosystems around the hydrothermal vents have, of course, severe difficulties to overcome in making in situ measurements, especially since a new variable, pressure, must be considered (Jannasch 1985). In order to measure the activity of autotrophic bacteria at these sites, 14CO2 fixation was measured in syringes incubated on the seabed (approximately 250 atm, 3 °C) and on board ship (1 atm) a 3 °C and 23 °C. Little or no difference was found between the two samples incubated at 3 °C, and the bacteria responsible for the 14CO2 fixation were thus obviously barotolerant rather than barophilic. Moreover, 14CO2 fixation sharply increased if thiosulfate was added, or when the samples were incubated at 23 °C, indicating that mesophilic colorless sulfur bacteria were responsible (Tuttle et al. 1983; Wirsen et al. 1986).

Inferring the Activity of Colorless Sulfur Bacteria in Their Natural Habitat with Microelectrodes

A technique that has been used with some success in the study of in situ bacterial biofilms and immobilization for biotechnology employs the use of microelectrodes with a tip of 1 μm of less that can be progressively moved through a biolayer, gradually registering the gradients present. The slope of the gradient, combined with data on the diffusion coefficient for the substrate measured, can provide direct information on the flux and turnover of substrates and thus can give accurate information on in situ activities. The microelectrodes are frequently linked to a computer that not only controls the rate of passage of the electrode tip through the sediment or biofilm but also records and calculates the results (e.g., Revsbech et al. 1986). Among others, oxygen, pH, sulfide, carbon dioxide, and N2O microelectrodes have been used, but the use of some (e.g., sulfide, CO2) is limited by their low sensitivity at commonly used pH values. However, the oxygen electrode has been extensively used, especially in systems where photosynthesis is involved and oxygen supply can easily be controlled by modifying the availability of light (e.g., Jensen and Revsbech 1989; Revsbech and Ward 1984). The construction of microelectrodes suitable for microbiology, and their use in various ecosystems, was extensively reviewed by Revsbech and Jørgensen (1986), but they are now commercially available (e.g., see www.​unisense.​com). Their use, in conjunction with some of the other methods mentioned above, provides a means of measuring actual activities in gradients, rather than potential activities in in vitro cultures.

Microelectrodes, alone or in combination with other approaches, have been used in several studies to infer the in situ physiology of the colorless sulfur bacterium Beggiatoa. Schulz-Vogt and coworkers, for instance, used microelectrodes for oxygen, sulfide, nitrate, and pH to study the chemotactic response of freshwater Beggiatoa on different concentrations of nitrate (Kamp et al. (2006). Preisler et al. (2007) used similar microsensors to study the ecological niche of nitrate-storing Beggiatoa in coastal sediments and their contribution in the removal of sulfide. Hinck and coworkers (2007) used a combination of different microsensors, stable isotopes, and molecular techniques to study the dial cycling of Beggiatoa in hypersaline microbial mats.

Similar microsensor studies were carried out for other colorless sulfur bacteria. Zopfi et al. (2001) used microelectrodes for oxygen and nitrate to study nitrate and sulfur storage in Thioploca species collected from the Bay of Concepción, Chile. They found positive chemotaxis of Thioploca after addition of nitrate and nitrite. In a subsequent study, Jørgensen and coworkers (Høgslund et al. 2009) used a combination of microelectrodes, quantitative microautoradiography, and 15N-stable isotopes to infer the in situ physiology of Thioploca. From their results they concluded that Thioploca is well adapted to a fluctuating environment by accumulation and transportation of nitrate and sulfur.

Inferring the Activity of Colorless Sulfur Bacteria in Their Natural Habitat with Molecular Methods

Currently, different molecular approaches are being used to infer the in situ activity of microorganisms in general and colorless sulfur bacteria in particular. One of the approaches is MAR-FISH, which is a combination of microautoradiography (MAR) and fluorescence in situ hybridization (FISH). In this approach, a mixed microbial community is briefly incubated with a radioactive labeled substrate such as [14C]-acetate. Subsequently, samples are taken at different time intervals and fixed in paraformaldehyde. The specimens are incubated with fluorescently labeled probes and covered with a photographic emulsion. After development of the photograph, the specimens can be seen under the microscope as the bacteria that took up the labeled substrate are covered with silver grains. The identity of these bacteria can be determined by fluorescence in situ hybridization. Nielsen and coworkers used MAR-FISH to study the in situ physiology of Thiothrix in wastewater treatment plants. They could demonstrate that Thiothrix was very versatile, being able to consume acetate and/or bicarbonate under heterotrophic, mixotrophic, and chemolithoautotrophic conditions (Nielsen et al. 2000). In a later study, they developed quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) to determine the quantitative uptake of specific substrates by Thiothrix in activated sludge and found that the substrate affinity (Ks) for acetate was 2.4 μM (Nielsen et al. 2003).

To increase the fluorescence in in situ hybridization experiments, CARD-FISH (catalyzed reported deposition-fluorescence in situ hybridization) was used. In this approach, the oligonucleotide probe, which is labeled with the enzyme horseradish peroxidase, catalyzes the deposition of fluorescently labeled tyramide molecules at the site of the probe hybridization, resulting in a strongly enhanced fluorescent signal (Amann and Fuchs 2008).

By using 16S rRNA gene cloning, qPCR, and a combination of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with microautoradiography after incubation with [14C]-bicarbonate, Grote and coworkers (2007, 2008) found that Epsilonproteobacteria, and in particular those closely related to Sulfurimonas, were the main organisms responsible for CO2-fixation in the sulfidic waters of the pelagic redoxclines of the Baltic and Black Seas.

Another way to measure activity in situ is the use of substrates labeled with stable isotopes, such as [13C]-acetate. As a consequence, all molecules in the bacterium that consumed the substrate will be labeled with the “heavy” carbon. The nucleic acids are extracted, and the 13C-labeled DNA (“heavy” fraction) can be separated from 12C-labeled DNA (“light” fraction) by cesium chloride or cesium trifluoroacetate gradient centrifugation, because of their difference in mass. Subsequently, the separated DNAs can be further analyzed using PCR amplification combined with cloning, DGGE, or NGS. The approach is known as DNA-SIP (DNA-stable isotope probing) or RNA-SIP (see Neufeld et al. (2007) for an overview).

By using rRNA-based stable isotope probing (RNA-SIP) on water samples incubated with [13C]-labeled bicarbonate, they could reveal that 2 Gammaproteobacteria and Sulfurimonas were responsible for feeding the microbial food web in the redoxcline of the central Baltic Sea (Glaubitz et al. 2009).

By using a combination of 13C labeling, FISH and secondary ion mass spectrometry (SIMS), Dattagupta et al. (2009) could show a novel symbiosis between the chemolithoautotrophic Thiothrix sp. and the freshwater cave amphipod Niphargus ictus and demonstrated that Thiothrix sp. was growing autotrophically.

Zhang and coworkers (2005) used confocal laser-scanning microscopy, lipid biomarkers, stable carbon isotopes, and 16S rRNA gene sequencing to infer carbon cycling within Beggiatoa-dominated microbial mats associated with gas hydrates and cold seeps in the Gulf of Mexico.

Recently, Wendeberg and coworkers (2012) studied in situ gene expression of pmoA (encoding subunit A of the particular methane monooxygenase) and aprA (encoding the subunit A of the dissimilatory adenosine-5′-phosphosulfate reductase) in methane- and sulfur-oxidizing symbionts of the hydrothermal vent mussel Bathymodiolus puteoserpentis. They found the highest mRNA expression levels at the ciliated epithelium of the gills, indicating a rapid response of the cells to incoming seawater rich in methane, reduced sulfur compounds and oxygen.

Conclusion

New insights into the pathways of sulfur metabolism in the colorless sulfur bacteria have done away with the old unifying concept of sulfur metabolism, as it is now clear that there are diverse pathways in the organisms investigated thus far. Due to the revolution in DNA sequencing, the genomes of many different colorless sulfur bacteria (e.g., Acidithiobacillus, Sulfolobus, Sulfurihydrogenibium, Sulfurimonas, Sulfuricurvum, Thioalkalimicrobium, Thioalkalivibrio, Thiobacillus, Thiomicrospira) are currently available or underway (see the Genomes OnLine Database (www.​genomesonline.​org; Pagani et al. 2012). With these sequences in hand it will now be possible to study the evolution and ecophysiology, including the pathways in sulfur metabolism, of colorless sulfur bacteria in great detail. Whitaker and coworkers, for instance, studied the biogeographical structure of the pan-genome of Sulfolobus islandicus by comparative analysis of seven S. islandicus genomes from three different locations (Reno et al. 2009). They found that there was no gene flow between the geographically isolated populations.

Beller and coworkers (2006) used whole genome transcriptomics to study thiosulfate oxidation by Thiobacillus denitrificans under aerobic versus denitrifying conditions. Genes that were upregulated under aerobic conditions were siderophore-related genes, cytochrome cbb3 oxidase genes, genes (cbbl, cbbS) encoding form I RuBisCO, and chaperone genes, while genes upregulated under denitrifying conditions included nar, nir, and nor, genes (cbbM) encoding form II RuBisCO, and genes involved in the oxidation of sulfur compounds (i.e., sqr and dsrC). Yamamoto et al. (2010) used transcriptomics to study the sulfur metabolism in Sulfurovum sp. NBC37-1.

In addition to whole genome sequencing of isolates, the use of the so-called meta-omics approach (i.e., metagenomics (e.g., Tringe et al. 2005), metatranscriptomics (e.g., Stewart et al., 2012), and metaproteomics (e.g., Siggins et al. 2012) makes it possible to study colorless sulfur bacteria that resist being isolated in pure culture.

Although the “meta-omics” approach is very powerful, it is often difficult to obtain complete genomes from microbial communities that contain many different microorganisms. To circumvent this limitation, single cells can be isolated from environmental samples by micromanipulation or other means and their genomes can subsequently be sequenced (e.g., Yilmaz and Singh 2011; Martinez-Garcia et al. 2012). Mussman and coworkers (2007) used this approach to sequence the genome of single filaments of Beggiatoa. From these genomes, they could reconstruct pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirming the chemolithoautotrophic physiology of Beggiatoa. Recently, Salman et al. (2011) handpicked individual cells of the large colorless sulfur bacteria Thiomargarita namibiensis, Thioploca araucae, and Thioploca chileae and sequenced their 16S rRNA genes and internal transcribed spacer (ITS) regions to determine their classification. However, a logical next step would be to sequence their complete genomes.

In addition, high-throughput cultivation techniques and methods mimicking environmental parameters more accurately, such as gradient systems and in situ cultivation, are currently used to increase the success rate of isolation of bacteria in general and colorless sulfur bacteria in particular (see Alain and Querellou (2009) for an overview). Isolation of strains in pure culture is still essential to obtain a comprehensive understanding of the (eco)physiology of the bacteria.

Since the writing of the previous edition of this chapter, one important question has been answered – should the colorless sulfur bacteria still be considered a taxonomic group? As discussed throughout this paper, the use as a taxonomic criterion of the ability to gain energy from the oxidation of inorganic reduced sulfur compounds has resulted in the definition of a very heterogeneous group, collectively known as the colorless sulfur bacteria. The possession of the relevant pathways for growth on reduced sulfur compounds is of no greater taxonomic relevance than the ability to use the Calvin cycle or to grow on hydrogen. As the results obtained with RNA and DNA analysis, have confirmed, we are seeing the result of evolutionary convergence towards the (eco)physiological properties encountered in many of the colorless sulfur bacteria. The extreme heterogeneity of the group is further emphasized as other long-known bacteria are tested and found to also possess the properties of colorless sulfur bacteria. Indeed, the common lack of a test for thiosulfate or sulfide oxidation in routine taxonomic screening has meant that the sulfur-oxidizing potential of species of genera such as Paracoccus, Pseudomonas and Alcaligenes are only now being recognized.

That said, despite their morphological and phylogenetic diversity, the colorless sulfur bacteria present a coherent picture in physiological terms. As it is generally the physiological specifications of an organism that define its ecological significance, the reclassification of the colorless sulfur bacteria may present something of a microbiological dilemma because new taxonomic relationships bear little relation to the ecophysiological activities of the organisms. Thus, in spite of the reallocation of species among different genera, research can only profit from the retaining of physiological as well as taxonomic groupings, such as the sulfate reducers, nitrogen fixers, denitrifiers, and colorless sulfur bacteria.

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