Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Taxonomy, Historical and Current

Bottom sediments of hypersaline lakes and lagoons may support a rich community of anaerobic halophilic bacteria, as the solubility of oxygen in hypersaline brines is low and the amounts of organic matter available are often high (Oren 1988). It is therefore surprising that the first records of the isolation of obligatory anaerobic fermentative bacteria growing at salt concentrations of 10–20 % and higher were published only in the early 1980s, when Halanaerobium praevalens was isolated from the bottom sediments of Great Salt Lake, Utah (Zeikus 1983; Zeikus et al. 1983) and Halobacteroides halobius and Sporohalobacter lortetii were discovered in Dead Sea sediments (Oren 1983; Oren et al. 1984b). Halanaerobium praevalens probably resembles “Bacteroides halosmophilus,” isolated by Baumgartner (1937) from solar salt and from salted anchovies. Unfortunately no cultures of that isolate have been preserved.

Order Halanaerobiales corrig. Rainey and Zhilina 1995, 879VP (Validation List no. 55); Effective Publication: Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 193.

Hal.an.ae.ro.bi.a’les. N.L. neut. n. Halanaerobium, type genus of the order; suff. –ales, ending denoting an order; N.L. fem. pl. n. Halanaerobiales, the Halanaerobiaceae order.

Cells are rod-shaped and generally stain Gram-negative. Endospores are produced by some species. Strictly anaerobic. Oxidase negative and generally catalase negative. Most species ferment carbohydrates to products including acetate, ethanol, H2, and CO2. Some species may grow fermentatively on amino acids, and others have a homoacetogenic metabolism or may grow by anaerobic respiration on nitrate, trimethylamine.-oxide, selenate, arsenate, or Fe(III). Chemolithoautotrophic growth on H2 and elemental sulfur may also occur. Moderately halophilic. NaCl concentrations between 0.5 and 3.4 M are required for optimal growth, and no growth is observed below 0.3–1.7 M NaCl, depending on the species.

The mol% G+C of the DNA varies between 27 and 45.

Type Genus: Halanaerobium

The order Haloanaerobiales was created in 1995, based on 16S rRNA sequence comparisons. These resulted in a reclassification of the species of the former family Haloanaerobiaceae over two families: the Haloanaerobiaceae and the newly created family Halobacteroidaceae (Rainey et al. 1995). Physiologically the group is coherent, to the extent that, as yet, no aerobes or non-halophiles are known to cluster phylogenetically within the order.

The genus Halanaerobium (originally named Haloanaerobium and corrected in accordance with Rule 61 of the Bacteriological Code) (Oren 2000) is now the largest genus within the order (nine species and two subspecies). Based on 16S rRNA sequence comparisons (Rainey et al. 1995), a number of species formerly classified in other genera were transferred to this genus: the former Halobacteroides acetoethylicus (Rengpipat et al. 1988a) was reclassified as Halanaerobium acetethylicum (Oren 2000; Patel et al. 1995; Rainey et al. 1995), and the former Haloincola saccharolyticus (originally described under the name Haloincola saccharolytica) (Zhilina et al. 1992b) was renamed as Halanaerobium saccharolyticum, with two subspecies, saccharolyticum and senegalense (Cayol et al. 1994a; Oren 2000; Rainey et al. 1995). The genera Halobacteroides, Acetohalobium, Halanaerobacter, and Sporohalobacter, previously classified within the family Halanaerobiaceae, were transferred to the Halobacteroidaceae (Rainey et al. 1995).

At the time of writing (March 2012), 30 species had been described. The family Halanaerobiaceae currently has 4 genera with 12 species; the family Halobacteroidaceae contains 11 genera with 18 species (see Figs. 12.1 and 12.2 and Tables 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, and 12.7).

Fig. 12.1
figure 1figure 1

Phase-contrast micrographs of members of the Halanaerobiales: (a) Halanaerobium alcaliphilum; (b–d) young, senescent, and old cells of Halobacteroides halobius; (e) Acetohalobium arabaticum; (f) Natroniella acetigena; (g) Sporohalobacter lortetii; (h) Orenia marismortui; (i) Halothermothrix orenii. Figures were derived from Tsai et al. (1995), Oren et al. (1984b), Zavarzin et al. (1994), Zhilina et al. (1996), Oren (1983), and Cayol et al. (1994b), respectively; reproduced with permission

Full size image
Fig. 12.2
figure 2figure 2

Electron micrographs of members of the Halanaerobiales: (a) Halanaerobium lacusrosei; (b) Halanaerobium saccharolyticum; (c,d) Halobacteroides halobius; (e) Acetohalobium arabaticum; (f) Halothermothrix orenii; (g,h) Sporohalobacter lortetii; (i) Halanaerobium saccharolyticum subsp. senegalense. Figures were derived from Cayol et al. (1995), Zhilina et al. (1992b), Oren et al. (1984b), Zavarzin et al. (1994), Cayol et al. (1994b), Oren (1983), and Cayol et al. (1994a), respectively; reproduced with permission

Full size image
Table 12.1 Comparison of selected characteristics of members of the genus Halanaerobium
Full size table
Table 12.2 Comparison of selected characteristics of members of the monospecific genera Halocella, Halothermothrix, and Halarsenatibacter (family Halanaerobiaceae)
Full size table
Table 12.3 Comparison of selected characteristics of members of the genus Halobacteroides
Full size table
Table 12.4 Comparison of selected characteristics of members of the genus Halanaerobacter
Full size table
Table 12.5 Comparison of selected characteristics of members of the genus Orenia
Full size table
Table 12.6 Comparison of selected characteristics of members of the genus Natroniella
Full size table
Table 12.7 Comparison of selected characteristics of members of the monospecific genera Acetohalobium, Sporohalobacter, Fuchsiella, Halarsenatibacter, Halanaerobaculum, Halonatronum, Selenihalanaerobacter, and Halanaerocella (family Halobacteroidaceae)
Full size table

The group was earlier reviewed by Kivistö and Karp (2011); Lowe et al. (1993); Ollivier et al. (1994), and Oren (1986a, 1990, 1993a, b, 2006).

Phylogenetic Structure of the Family and Its Genera

Figure 12.3 shows a neighbor-joining phylogenetic tree of the type strains of the 31 species and subspecies of the order Halanaerobiales. It may be noted that Halobacteroides elegans does not cluster with Halobacteroides halobius, the type species of the genus, but with the species of the genus Halanaerobacter, suggesting that reclassification of H. elegans may be recommended. The family is associated with the low-G+C branch of the Firmicutes. The group forms a coherent cluster close to the bifurcation point that separates the Actinobacteria and the Bacillus/Clostridium group (Rainey et al. 1995; Tourova et al. 1995). The deep branching justifies classification in a separate order (Rainey et al. 1995). The order Halanaerobiales has been used as a paradigm to demonstrate the application of 16S rRNA gene sequencing and DNA-DNA hybridization in bacterial taxonomy (Tourova 2000). Two families were described: the Halanaerobiaceae (Oren et al. 1984a) and the Halobacteroidaceae (Rainey et al. 1995).

Fig. 12.3
figure 3figure 3

Neighbor-joining genealogy reconstruction of the 31 species and subspecies of the order Halanaerobiales present in the LTP_106 (Yarza et al. 2010). The tree was reconstructed by using a subset of sequences 767 type strains of Bacteria and Archaea to stabilize the tree topology. In addition, a 40 % conservational filter for the whole bacterial domain was used to remove hypervariable positions. Numbers in triangles denote number of taxa included. The bar indicates 1 % sequence divergence

Full size image

The species share a low content of G+C in their DNA, generally between 29 mol% and 34 mol%. Exceptions are the thermophilic Halothermothrix orenii with a G+C content of 37.9 mol% and the atypical, non-fermentative anaerobic respirer Halarsenatibacter silvermanii with 45 mol%.

Genome Analysis

At the time of writing (March 2012), three complete genome sequences of members of the Halanaerobiales had been published: the type strain of Halanaerobium praevalens (Ivanova et al. 2011), the thermophilic Halothermothrix orenii (Mijts and Patel 2001; Mavromatis et al. 2009), and a haloalkaliphilic hydrogen-producing strain known as “Halanaerobium hydrogenoformans,” earlier designated as “Halanaerobium sapolanicus” (Brown et al. 2011). This organism is not currently available from culture collections. Except for the genome sequence, little information is available about it beyond the fact that it was isolated from the alkaline hypersaline and sulfide-rich Soap Lake, Washington, USA, that it grows optimally at pH 11, 7 % NaCl, and 33 °C and that it produces acetate, formate, and H2 (Table 12.8).

Table 12.8 Properties of the sequenced genomes of members of the Halanaerobiales
Full size table

The three genomes are 2.3–2.6 Mbp in length and each contains four identical or nearly identical copies of the 16S rRNA gene. Analysis of the H. orenii gene showed a few features characteristic for Gram-negative bacteria such as a pathway for lipid A biosynthesis, outer membrane secretion proteins, and two copies of the chaperone OmpH, a periplasmic protein that helps to transport proteins to the outer membrane. There also are a number of sporulation-related genes. The main sporulation regulator Spo0A of bacilli and clostridia is present, but sporulation was never shown in this organism. Genes coding for the biosynthesis of organic osmotic solutes were not detected except for the finding of a gene for sucrose phosphate synthase, suggesting that sucrose can be formed and may possibly act as an osmotic solute (Chua et al. 2008; Mavromatis et al. 2009).

Comparative analysis of the three Halanaerobiales genomes did not show an unusually high content of acidic amino acids or a low content of basic amino acids in the encoded proteins. The apparent excess of acidic amino acids in the bulk protein of Halanaerobium praevalens, H. saccharolyticum, Halobacteroides halobius, Sporohalobacter lortetii, and Natroniella acetigena reported earlier (Detkova and Boltyanskaya 2006; Oren 1986b) is therefore due to the high content of glutamine and asparagine in their proteins, which yield glutamate and aspartate upon acid hydrolysis. The proteins of the Halanaerobiales, which are active in the presence of high intracellular KCl concentrations, do thus not possess the typical acidic signature of the “halophilic” proteins of the Archaea of the order Halobacteriales or of the extremely halophilic bacterium Salinibacter (Elevi Bardavid and Oren 2012).

Phages

No phages active on strains of Halanaerobiales have yet been described.

Phenotypic Analyses

General Comments

Members of the Halanaerobiales display a Gram-negative type of cell wall with an outer membrane and periplasmic space (Fig. 12.2). Meso-diaminopimelic acid was detected in the peptidoglycan of Halanaerobium saccharolyticum subsp. saccharolyticum (Zhilina et al. 1992b). Most species also show a negative Gram-stain reaction; however, Halanaerobium tunisiense and Halanaerocella petrolearia stain Gram-positive (Gales et al. 2011; Hedi et al. 2009).

Heat-resistant endospores are produced by a number of species of Halobacteroidaceae, including Sporohalobacter lortetii (Oren 1983), the three Orenia species (Mouné et al. 2000; Oren et al. 1987; Zhilina et al. 1999), and Natroniella acetigena (Zhilina et al. 1996). When initially isolated, Acetohalobium arabaticum produced spores, but sporulation was not observed during subsequent transfers (Zavarzin et al. 1994). Special conditions may be required for induction of endospore formation. Growth on solid media or in nutrient-poor liquid media enhances sporulation in certain species (Oren 1983; Oren et al. 1987). A phenotypic test which may be correlated with the phylogenetic position of the Halanaerobiaceae within the Firmicutes and with the ability to form endospores is the hydrolysis of the d-isomer of.′-benzoyl-arginine-.-nitroanilide (BAPA). Three representatives of the Halanaerobiales (Halobacteroides halobius, Halanaerobium praevalens, Orenia marismortui) were found to hydrolyze d-BAPA, while l-BAPA was not hydrolyzed. Sporohalobacter lortetii degraded neither of the BAPA stereoisomers (Oren et al. 1989).

All members of the Halanaerobiales are strict anaerobes. They are oxidase negative, and most species lack catalase, Halarsenatibacter silvermanii being the only known exception. All members of the Halanaerobiaceae and most members of the Halobacteroidaceae obtain their energy by fermenting simple sugars (Tables 12.9 and 12.10). Halanaerobacter chitinivorans uses chitin, and Halocella cellulosilytica degrades cellulose. Fermentation products typically include acetate, H2, and CO2. Some strains produce in addition butyrate, lactate, propionate, and/or formate. Halarsenatibacter silvermanii lives by dissimilatory reduction of arsenate to arsenite, Fe(III) to Fe(II), and elemental sulfur to sulfide. Chemoautotrophic growth occurs with sulfide as the electron donor and arsenate as the electron acceptor (Switzer Blum et al. 2009). Within the family Halobacteroidaceae, the metabolic diversity is much greater than within the Halanaerobiaceae. Thus, there are species that ferment amino acids, either alone or by using the Stickland reaction. For example, Halanaerobacter salinarius and Halanaerobacter chitinivorans can use serine as an electron donor using the Stickland reaction while reducing glycine betaine, with the formation of acetate, trimethylamine, CO2, and NH3 (Mouné et al. 1999). Sporohalobacter lortetii is primarily an amino acid fermenter, and sugars are poorly used (Oren 1983). Anaerobic respiration also occurs, using different electron acceptors: Selenihalanaerobacter shriftii oxidizes glycerol or glucose by anaerobic respiration with nitrate, trimethylamine.-oxide, or selenate as electron acceptor (Switzer Blum et al. 2001a).

Table 12.9 Substrates used by the type strains of carbohydrate-fermenting species of Halanaerobiaceae
Full size table
Table 12.10 Substrates used by the type strains of carbohydrate-fermenting species of Halobacteroidaceae
Full size table

Acetohalobium arabaticum (neutrophilic), Natroniella acetigena (alkaliphilic), and Fuchsiella alkaliacetigena (alkaliphilic) have a homoacetogenic metabolism, producing acetate as the main end product of their energy metabolism. Acetohalobium arabaticum grows on H2 + CO2 or on carbon monoxide as a lithoautotroph, on trimethylamine as a methylotroph, and on other substrates (formate, glycine betaine, lactate, pyruvate, histidine, aspartate, glutamate, and asparagine) as an organotroph. Fuchsiella can also grow chemolithoautotrophically (Kevbrin et al. 1995; Zavarzin et al. 1994; Zhilina and Zavarzin 1990a, b; Zhilina et al. 1996, 2012).

Several species (Halanaerobium saccharolyticum, Halanaerobacter lacunarum, Halobacteroides halobius, Halobacteroides elegans) can use methanethiol as the sole source of assimilatory sulfur for growth and reduce elemental sulfur to sulfide (Kevbrin and Zavarzin 1992a; Zhilina et al. 1992a, b, 1997). Acetohalobium arabaticum slowly reduces sulfur to sulfide, but this was not accompanied by growth enhancement (Kevbrin and Zavarzin 1992b; Zavarzin et al. 1994). Natroniella acetigena can grow chemolithoautotrophically by oxidizing H2, using elemental sulfur as electron acceptor (Sorokin et al. 2011). Halanaerobium congolense uses thiosulfate and elemental sulfur as electron acceptors. Addition of thiosulfate or sulfur increased the growth yield sixfold and threefold, respectively, and growth rates were enhanced (Ravot et al. 1997). Thiosulfate reduction was also observed in Orenia marismortui and in Halanaerobium congolense (Oren et al. 1987; Ravot et al. 2005).

High concentrations of Na+, K+, and Cl−, high enough to be at least isotonic with the medium, were measured inside the cells of Halanaerobium praevalens, Halanaerobium acetethylicum, Halobacteroides halobius, and Natroniella acetigena (Detkova and Pusheva 2006; Oren 1986b; Oren et al. 1997; Rengpipat et al. 1988b). No organic osmotic solutes have been detected in the anaerobic halophilic bacteria (Oren 1986b; Oren et al. 1997; Rengpipat et al. 1988b), except in the case of Orenia salinaria, found to accumulate glycine betaine when grown in medium containing yeast extract (Mouné et al. 2000). The intracellular enzymatic machinery appears to be well adapted to function in the presence of high salt concentrations. The enzymes tested (including glyceraldehyde-3-phosphate dehydrogenase, NAD-linked alcohol dehydrogenase, pyruvate dehydrogenase, and methyl viologen-linked hydrogenase from Halanaerobium acetethylicum, the fatty acid synthetase complex of Halanaerobium praevalens, hydrogenase and CO dehydrogenase of Acetohalobium arabaticum, CO dehydrogenase of Natroniella acetigena) function better in the presence of molar concentrations of salts than in salt-free medium (Detkova and Boltyanskaya, 2006; Oren and Gurevich 1993; Pusheva and Detkova 1996; Pusheva et al. 1992; Rengpipat et al. 1988b; Zavarzin et al. 1994).

The Properties of the Genera and Species of Halanaerobiales

Information on the phenotypic properties of the genera and species of the Halanaerobiales, as summarized below, was derived from Cayol et al. 2009; Mesbah 2009; Oren 2009b, c, d, e, f; Oren et al. 2009; Rainey 2009; Zavarzin 2009; Zavarzin and Zhilina 2009a, b; and Zhilina et al. 2009 and from the original species descriptions.

Family Halanaerobiaceae corrig.

Oren, Paster and Woese 1984, 503VP (Validation List no. 16) (Effective Publication: Oren, Paster and Woese 1984a, 79).

Hal.an.ae.ro.bi.a.ce’ae N.L. neut. n. Halanaerobium, type genus of the family; suff. –aceae, ending to denote a family; N.L. fem. pl. n. Halanaerobiaceae, the Halanaerobium family.

Cells are rod-shaped and stain Gram-negative. Endospore formation never observed. Strictly anaerobic. Oxidase and catalase negative. Carbohydrates are fermented to products including acetate, ethanol, H2, and CO2. Moderately halophilic. NaCl concentrations between 1.7 and 2.6 M are required for optimal growth, and no growth is observed below 0.3–1.7 M NaCl, depending on the species.

Type Genus: Halanaerobium

Genus Halanaerobium corrig.

Zeikus, Hegge, Thompson, Phelps and Langworthy 1984, 503VP (Validation List no. 16), Emend. Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 197 (Effective publication: Zeikus, Hegge, Thompson, Phelps and Langworthy 1983, 232).

Hal.an.ae.ro’bium. Gr. n. hals halos, salt; Gr. pref. an, not; Gr. n. aer, air; Gr. n. bios, life; N.L. neut. n. Halanaerobium, salt organism which grows in the absence of air.

Cells rod-shaped, nonmotile or motile by peritrichous flagella, generally staining Gram-negative. Strictly anaerobic, chemoorganotrophic with fermentative metabolism. Carbohydrates are fermented with production of acetate, H2, and CO2; in some species, ethanol, formate, propionate, butyrate, and lactate are found in addition. Thiosulfate and elemental sulfur may be used as electron acceptors in certain species. Halophilic, growing optimally at NaCl concentrations around 1.7–2.5 M and requiring a minimum of 0.3–1.7 M NaCl for growth. Neutral or slightly alkaline pH values are preferred. Endospore formation never observed.

Type Species: Halanaerobium praevalens

The main features of members of the genus Halanaerobium, updated for March 2012, are listed in Table 12.1.

Genus Halocella

Simankova, Chernych, Osipov and Zavarzin 1994, 182VP (Validation List no. 48) (Effective publication: Simankova, Chernych, Osipov and Zavarzin 1993, 389).

Ha.lo.cel’la. Gr. n. hals halos, salt; L. fem. n. cella, a store-room and in biology a cell; N.L. fem. n. Halocella, salt cell.

Cells are straight or slightly curved rods, non-sporulating, and motile by means of peritrichous flagella. Cell wall of Gram-negative structure. Obligately anaerobic. Moderately halophilic. Ferment carbohydrates, including cellulose, producing acetate, ethanol, lactate, H2, and CO2. Peptides and amino acids are not utilized.

Type Species: Halocella cellulosilytica

Genus Halothermothrix

Cayol, Ollivier, Prensier, Guezennec and Garcia 1994b, 538VP

Ha.lo.ther’mo.thrix. Gr. n. hals halos, salt; Gr. adj. thermos, hot; Gr. fem. n. thrix, hair; N.L. fem. n. Halothermothrix, a thermophilic (fermentative) hair-shaped halophile.

Long rod-shaped bacteria with cells that are 0.4–0.6 × 10–20 μm, occurring mainly singly. Motile by peritrichous flagella. Non-sporulating. Gram stain-negative. Strictly anaerobic. Chemoorganotrophic; ferment carbohydrates to acetate, ethanol, H2, and CO2. NaCl and yeast extract are required for growth. Thermophilic.

Type Species: Halothermothrix orenii

Genus Halarsenatibacter

Switzer Blum, Han, Lanoil, Saltikov, White, Tabita, Langley, Beveridge, Jahnke and Oremland 2010, 1985VP (Validation List no. 135) (Effective publication: Switzer Blum, Han, Lanoil, Saltikov, White, Tabita, Langley, Beveridge, Jahnke and Oremland 2009, 1958).

Hal.ar.se.na.ti.bac’ter. Gr. n. hals halos, salt; N.L. n. arsenas -atis, arsenate; N.L. masc. n. bacter, rod; N.L. masc. n. Halarsenatibacter, halophilic arsenate-utilizing rod.

Gram-negative, motile, strictly anaerobic, slightly curved rods (3.0 by 0.5 μm). Motility achieved by a pair of flagella located along the side of the organism. Extremely halophilic, growing between 20 % and 35 % salt with an optimum at salt saturation. Alkaliphilic. A limited number of organic substrates support growth, including a few sugars and organic acids but not fatty acids or amino acids. Fermentative growth or microaerophilic growth not observed. Growth is by dissimilatory (respiratory) reduction of arsenate to arsenite, Fe(III) to Fe(II), and elemental sulfur to sulfide. Chemoautotrophic growth occurs with sulfide as the electron donor and arsenate as the electron acceptor. Catalase positive.

Type Species: Halarsenatibacter silvermanii

The main features of members of the monospecific genera Halocella, Halothermothrix, and Halarsenatibacter, updated for March 2012, are listed in Table 12.2.

Family Halobacteroidaceae

Zhilina and Rainey 1995, 879VP (Validation List no. 55) (Effective publication: Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 193).

Ha.lo.bac.te.ro.i.da.ce’ae. N.L. masc. n. Halobacteroides, type genus of the family; suff. –aceae, ending to denote a family; N.L. fem. pl. n. Halobacteroidaceae, the Halobacteroides family.

Cells are rod-shaped and stain Gram-negative. Endospores produced by some species. Strictly anaerobic. Oxidase and generally catalase negative. Most species ferment carbohydrates to products including acetate, ethanol, H2, and CO2. Some species may grow fermentatively on amino acids; others have a homoacetogenic metabolism or grow by anaerobic respiration while reducing nitrate, trimethylamine.-oxide, selenate, or arsenate or chemolithoautotrophically on H2 and elemental sulfur. Moderately halophilic. NaCl concentrations between 1.7 and 2.5 M are required for optimal growth, and no growth is observed below 0.3–1.7 M NaCl, depending on the species.

Type Genus: Halobacteroides

Genus Halobacteroides

Oren, Weisburg, Kessel and Woese 1984, 355VP (Effective publication: Oren, Weisburg, Kessel and Woese 1984, 68).

Ha.lo.bac.te.ro’i.des. Greek n. hals halos, salt; N.L. masc. n. bacter, a staff or rod; L. suff. –oides (from Gr. suff. eides, from Gr. n. eidos, that which is seen, form, shape, figure; N.L. masc. n. Halobacteroides, rod-like salt organism.

Cells are long, thin, often flexible rods and motile by peritrichous flagella, staining Gram-negative. Endospores may be formed. Strictly anaerobic, chemoorganotrophic with fermentative metabolism. Carbohydrates are fermented with production of acetate, ethanol, H2, and CO2. Halophilic, growing optimally at NaCl concentrations around 1.7–2.6 M and requiring a minimum of 1.2–1.7 M NaCl for growth.

Type Species: Halobacteroides halobius

The main features of members of the genus Halobacteroides, updated for March 2012, are listed in Table 12.3.

Genus Halanaerobacter

Liaw and Mah 1996, 362VP (Validation List no. 56), Emend. Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 197; Emend. Mouné, Manac’h, Hirschler, Caumette, Willison and Matheron 1999, 109 (Effective publication: Liaw and Mah 1992, 265).

Hal.an.ae.ro.bac’ter. Gr. n. hals halos, salt; Gr. pref. an, not; Gr. n. aer aeros, air; N.L. masc. n. bacter, rod; N.L. masc. n. Halanaerobacter, salt rod which grows in the absence of air.

Cells are rod-shaped or slightly curved, flexible, and motile by means of peritrichous flagella. Gram-stain-negative. Strictly anaerobic. Chemoorganotrophic with fermentative metabolism; some strains can utilize amino acids in the Stickland reaction or with hydrogen as electron donor. Carbohydrates are fermented with production of acetate, H2, and CO2. In some species, ethanol, propionate, formate, and isobutyrate are also formed. Elemental sulfur can be used as electron acceptor in certain species. Halophilic; optimal growth occurs at NaCl concentrations around 2.0–3.0 M. Cells require a minimum of 0.5–1.6 M NaCl for growth. Neutral to slightly alkaline pH values required for optimal growth. Mesophilic to slightly thermotolerant. Endospores not observed. Short degenerate cells and spheroplasts occur in stationary phase.

Type Species: Halanaerobacter chitinivorans

The main features of members of the genus Halanaerobacter, updated for March 2012, are listed in Table 12.4.

Genus Orenia

Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 880VP (Validation List no. 55) (Effective publication: Rainey, Zhilina, Boulygina, Stackebrandt, Tourova and Zavarzin 1995, 197).

O.re’ni.a. N.L. fem. n. Orenia, named after Aharon Oren, an Israeli microbiologist.

Rods, 2.5–13 μm in length with rounded ends. Gram-stain-negative. Motile by peritrichous flagella. Spores are round, terminal, or subterminal. Gas vesicles detected in some species. Forms spheroplasts. Strictly anaerobic. Halophilic; optimum NaCl concentration for growth 3–12 %; no growth below 2 % or above 25 %. Mesophilic to slightly thermophilic. Chemoorganotrophic. End products of glucose fermentation include H2, CO2, lactate, acetate, butyrate and ethanol.

Type Species: Orenia marismortui

The main features of members of the genus Orenia, updated for March 2012, are listed in Table 12.5.

Genus Natroniella

Zhilina, Zavarzin, Detkova and Rainey 1996, 1189VP (Validation List no. 59); Emend. Sorokin, Detkova and Muyzer 2011, 94 (Effective publication: Zhilina, Zavarzin, Detkova and Rainey 1996b, 324).

Na.tro.ni.el’la. N.L. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. fem. n. Natroniella, organism growing in soda deposits.

Flexible rods, motile by peritrichous flagella. Spores may be formed. Cell wall has Gram-negative structure. Strictly anaerobic. Possesses a respiratory type of homoacetogenic metabolism. Extremely alkaliphilic, developing in soda brines at pH 9–10. Halophilic, growing at 1.7–4.4 M NaCl. Obligately dependent on Na+, Cl−, and CO3 2− ions. Mesophilic. Chemoorganotrophic: some organic acids, amino acids, and alcohols are fermented. Acetate is the product of fermentation. Some representatives have obligate sulfur-dependent respiratory metabolism and are able to grow autotrophically or with acetate as an electron donor with sulfur serving as an electron acceptor.

Type Species: Natroniella acetigena

The main features of members of the genus Natroniella, updated for March 2012, are listed in Table 12.6.

Genus Acetohalobium

Zhilina and Zavarzin 1990, 470VP (Validation List no. 35) (Effective publication: Zhilina and Zavarzin 1990b, 747).

A.ce.to.ha.lo’bi.um. L. n. acetum, vinegar; Gr. n. hals halos, salt; Gr. n. bios, life; N.L. neut. n. Acetohalobium, acetate-producing organism living in salt.

Rod-shaped cells. Motile with 1–2 subterminal flagella. Multiplication by binary fission is by constriction rather than septation. Gram-negative wall structure. Thermoresistant endospores formed by some strains. Strictly anaerobic. Possess a respiratory type of homoacetogenic metabolism. Extremely halophilic, growing at 1.7–4 M NaCl. Neutrophilic. Mesophilic Metabolism variable; lithoheterotrophic, utilizing H2, formate, and carbon monoxide; methylotrophic, utilizing methylamines and betaine; or chemoorganotrophic, fermenting some amino acids and organic acids. Acetate is the end product with all substrates utilized.

Type Species: Acetohalobium arabaticum

Genus Sporohalobacter

Oren, Pohla and Stackebrandt 1988, 136VP (Validation List no. 24) (Effective publication: Oren, Pohla and Stackebrandt 1987, 239).

Spo.ro.ha.lo.bac’ter. Gr. n. spora, seed; Greek n. hals halos, salt; N.L. n. bacter a staff or rod; N.L. masc. n. Sporohalobacter spore-producing salt rod.

Gram-negative rod-shaped cells, motile by peritrichous flagella. Halophilic, growing optimally at 1.4–1.5 M NaCl and requiring minimum 0.7 M NaCl for growth. Temperature optimum about 40 °C. Strictly anaerobic. Ferments amino acids with production of acetate, propionate and other acids, H2, and CO2. Sugars poorly used. Endospores produced. Gas vesicles are attached to the endospores in the single species described.

Type Species: Sporohalobacter lortetii

Genus Fuchsiella

Zhilina, Zavarzina, Panteleeva, Osipov, Kostrikina, Tourova and Zavarzin 2012, 1671VP.

Fuch.si.el’la. N.L. gem. dim. n. Fuchsiella, named in the honor of Prof. Georg Fuchs (Freiburg, Germany), who made a most serious contribution to our understanding of multiple pathways of CO2 assimilation by microorganisms.

Gram-negative, spore-forming rods, motile by peritrichous flagella. Obligatory anaerobic. Obligately alkaliphilic and natronophilic. Performing homoacetogenic metabolism of a restricted number of compounds. Able to grow chemolithoautotrophically with H2 + CO2. Few organic compounds are metabolized with external electron acceptors.

Type Species: Fuchsiella alkaliacetigena

Genus Halanaerobaculum

Hedi, Fardeau, Sadfi, Boudabous, Ollivier and Cayol 2009, 923VP (Effective publication: Hedi, Fardeau, Sadfi, Boudabous, Ollivier and Cayol 2009, 317).

Hal.an.ae.ro.ba’cu.lum. Gr. n. hals halos, salt; Gr. pref. an-, not; Gr. n. aer aeros, air; L. neut. n. baculum, stick; N.L. neut. n. Halanaerobaculum, salt stick not living in air.

Cells are Gram-negative, nonmotile, non-sporulating rods appearing singly, in pairs, or occasionally as long chains, halophilic, obligate anaerobes. Metabolize only carbohydrates. Grow at NaCl concentrations ranging from 14 to 30 %. The end products from glucose fermentation are butyrate, lactate, acetate, H2, and CO2.

Type Species: Halanaerobaculum tunisiense

Genus Halonatronum

Zhilina, Garnova, Tourova, Kostrikina and Zavarzin 2001, 263VP (Validation List no. 79) (Effective publication: Zhilina, Garnova, Tourova, Kostrikina and Zavarzin 2001a, 70).

Ha.lo.na.tro’num. Gr. n. hals halos salt; N.L. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. neut. n. Halonatronum, an organism growing with salt and soda.

Cells are rod-shaped, flexible, and motile by peritrichous flagella. The cell wall has a Gram-negative structure. Strictly anaerobic, chemoorganotrophic with fermentative metabolism. Carbohydrates, including soluble polysaccharides, are fermented to acetate, ethanol, formate, H2, and CO2. Halophilic and alkaliphilic. Endospores produced.

Type Species: Halonatronum saccharophilum

Genus Selenihalanaerobacter

Switzer Blum, Stolz, Oren and Oremland 2001, 1229VP (Validation List no. 81) (Effective publication: Switzer Blum, Stolz, Oren and Oremland 2001, 217).

Se.le.ni.hal.an.ae.ro.bac’ter. N.L. n. selenium (from Gr. n. selênê, the moon), selenium, element 34; Gr. n. hals halos, salt; Gr. pref. an, not; Gr. n. aer aeros, air; N.L. masc. n. bacter, a staff or rod; N.L. masc. n. Selenihalanaerobacter, the salty anaerobic selenium rod.

Gram-negative rod-shaped cells, nonmotile. Halophilic, growing optimally at 3.6 M NaCl and requiring minimum 1.7 M NaCl for growth. Temperature optimum about 38 °C. Strictly anaerobic. Grows by anaerobic respiration on organic electron donors, using selenate and other electron acceptors. Fermentative growth not observed. Endospores not produced.

Type Species: Selenihalanaerobacter shriftii

Genus Halanaerocella

(Effective publication: Gales, Chehider, Joulian, Battaglia-Brunet, Cayol, Postec, Borgomano, Neria-Gonzalez, Lomans, Ollivier and Alazard 2011, 570; the name is yet to be validated).

Hal.an.ae.ro.cel’la. Gr. h. hals halos, salt; Gr. pref. an, not; Gr. n. aer, air; L. fem. n. cella, a store-room and in biology a cell; N.L. fem. n. Halanaerocella, salt cell not living in air.

Cells stain Gram-positive, nonmotile, non-sporulating rods occurring singly, in pairs, or occasionally as long chains. Obligate anaerobe metabolizing only carbohydrates. The end products from glucose fermentation are lactate, ethanol, acetate, formate, H2, and CO2.

The Type Species is Halanaerocella petrolearia.

The main features of members of the monospecific genera Acetohalobium, Halanaerobacter, Sporohalobacter, Fuchsiella, Halanaerobaculum, Halonatronum, Selenihalanaerobacter, and Halanaerocella (updated for March 2012), are listed in Table 12.7.

Isolation, Enrichment, and Maintenance Procedures

Any anoxic reducing medium containing high salt concentrations (5–25 %) and containing a suitable carbon source is a potential enrichment and growth medium for members of the Halanaerobiales. A variety of such media have been used for isolation and cultivation. Table 12.11 presents a selection. Most species grow as fermenters on simple sugars. Although most species are not extremely sensitive to molecular oxygen, strict anaerobic techniques should be used, including boiling the media under nitrogen or nitrogen-CO2 (80:20) and adding reducing agents such as cysteine, dithionite, or ascorbate + thioglycollate to the boiled media. Protocols for the preparation of media were compiled by Oren (2006); details can be found in the original species descriptions. For the enrichment of thermophiles such as Halothermothrix, the incubation temperature should be adjusted to that of the natural environment. More specialized media have been designed for the cultivation of amino acid fermenting, homoacetogenic, selenate- and arsenate-respiring members, and other atypical organisms belonging to the order. For the isolation of Selenihalanaerobacter, selenate is the preferred electron acceptor, because nitrate and trimethylamine.-oxide also enable anaerobic growth of a variety of facultative anaerobes belonging to other orders.

Table 12.11 Media for the growth of members of selected members of the Halanaerobiales (all values in g/L, unless stated otherwise). Additional information can be found in the original species description papers and in the website of the Deutsche Sammlung von Mikroorganismen und Zellkulturen: http://www.dsmz.de
Full size table

The formation of heat-resistant endospores has been exploited in a selective enrichment procedure for Halobacteroides halobius-like bacteria, based on negative selection by pasteurization of the inoculum for 10–20 min at 80–100 °C (Oren 1987). In view of the number of endospore-forming genera within the family (Halobacteroides, Orenia, Sporohalobacter, Acetohalobium, Natroniella), such an enrichment strategy could be useful for the isolation of other novel members.

Maintenance

Many species of Halobacteroidaceae, notably the species of the genera Halobacteroides (Oren et al. 1984b; Zhilina et al. 1997), Orenia (Oren et al. 1987; Mouné et al. 2000; Zhilina et al. 1999), Haloanaerobacter (Liaw and Mah 1992; Zhilina et al. 1992; Mouné et al. 1999), Halonatronum (Zhilina et al. 2001), and Natroniella (Zhilina et al. 1996), easily undergo autolysis, generating spherical degeneration forms (Fig. 12.1 c, d). Lysis starts at the end of the exponential growth phase, especially at relatively high growth temperatures. One possibility to avoid death of such cultures is the use of media with a reduced nutrient content and lower growth temperatures (15–25 °C). Weekly transfers may then suffice to maintain viable cultures. Long-term preservation is by freezing anaerobic suspensions in 20 % glycerol at −80 °C (Rengpipat et al. 1988a), by lyophilization, or by storage in liquid nitrogen.

Ecology

Species belonging to the Halanaerobiales can probably be found in any hypersaline anaerobic environment where simple sugars are available or other substrates metabolized by the members of the order. Representatives have been isolated from Great Salt Lake, Utah (Tsai et al. 1995; Zeikus et al. 1983); Salton Sea, California (Shiba 1991; Shiba and Horikoshi 1988; Shiba et al. 1989); Searles Lake, California (Switzer Blum et al. 2009), the Dead Sea (Oren 1983; Oren et al. 1984b, 1987; Switzer Blum et al. 2001); and a hypersaline sulfur spring on the shore of the Dead Sea (Oren 1989); from the alkaline (pH 10.2) hypersaline lake in Magadi, Kenya—shown to harbor a varied anaerobic community, including cellulolytic, proteolytic, saccharolytic, and homoacetogenic bacteria (Shiba and Horikoshi 1988; Zhilina and Zavarzin 1994; Zhilina et al. 1996, 2001)—Big Soda Lake, Nevada (Shiba and Horikoshi 1988; Shiba et al. 1989), soda lakes in Russia (Sorokin et al. 2011; Zhilina et al. 2012), and hypersaline lakes and lagoons in the Crimea (Simankova et al. 1993; Zhilina and Zavarzin 1990b; Zhilina et al. 1991, 1992b) and Senegal (Cayol et al. 1994a, 1995); and from the hypersaline lakes in Tunisia (Cayol et al. 1994b; Hedi et al. 2009; Mezghani et al. 2012) and saltern evaporation ponds in California (Liaw and Mah 1992) and France (Mouné et al. 1999, 2000). Brines associated with oil wells and petroleum reservoirs also yielded a number of interesting species (Bhupathiraju et al. 1991, 1993, 1994, 1999; Gales et al. 2011; Ravot et al. 1997; Rengpipat et al. 1988a). They may also be present in salted fermented foods (Kobayashi et al. 2000a, b). 16S rRNA sequences of yet uncultured organisms affiliated with the Halanaerobiales are often recovered in clone libraries prepared from DNA extracted from anaerobic hypersaline environments such as sediments of saltern evaporation ponds (Mouné et al. 2003; Sørensen et al. 2005) and also from anaerobic brines in the depths of the Red Sea (Eder et al. 2001).

The ability to use glycerol, glucosylglycerol, trehalose, cellulose, and chitin may be of particular ecological importance. The first three compounds are accumulated at high concentrations as organic osmotic solutes by aerobic photosynthetic halophilic microorganisms inhabiting salt lakes: glycerol in the green unicellular alga Dunaliella and glucosylglycerol and trehalose in a variety of cyanobacteria. Such compounds may then be available to the anaerobic bacterial community in the bottom sediments of these lakes. Halanaerobium saccharolyticum and Halanaerobium lacusrosei ferment glycerol (Cayol et al. 1994a, 1995; Zhilina et al. 1992). Glycerol oxidation by anaerobic halophiles may be markedly improved through interspecies hydrogen transfer when grown in coculture with H2-consuming sulfate-reducing bacteria (Cayol et al. 1995). Halanaerobium saccharolyticum was isolated from a cyanobacterial mat dominated by Coleofasciculus (Microcoleus) chthonoplastes, covering the bottom of a hypersaline lagoon in the Crimea; its ability to use glucosylglycerol, the osmotic solute produced by the cyanobacteria, may be of great ecological importance (Zhilina and Zavarzin 1991). The same organism also degrades trehalose, produced by other cyanobacteria for similar purposes (Zhilina et al. 1992b).

The hypersaline lagoons of the Crimea also contain large masses of dead macroalgae (Cladophora). Such environments show high cellulolytic activity. The optimum salt concentration for cellulose decomposition was 15 %, and decomposition was possible up to 25 % salt (Siman’kova and Zavarzin 1992). The cellulose-degrading Halocella cellulosilytica was isolated from this habitat (Simankova et al. 1993), and its cellulase complex was characterized in part (Bolobova et al. 1992). Another biopolymer that may be available in large quantities in hypersaline lakes is chitin, derived from the brine shrimp Artemia and from larvae of the brine fly which are often abundant in such environments. Evolution of gas bubbles was observed from the sediment of a Californian saltern containing massive amounts of dead brine shrimp. Two strains of Halanaerobacter chitinivorans were isolated from this saltern, of which only one grew on chitin (Liaw and Mah 1992). Another substrate that may be available abundantly in hypersaline environments is glycine betaine. This compound is produced as an osmotic solute by the most halophilic among the cyanobacteria and by halophilic anoxygenic photosynthetic bacteria such as Halorhodospira species. Glycine betaine is fermented to acetate and trimethylamine by Halanaerobium alcaliphilum isolated from Great Salt Lake (Tsai et al. 1995), and by the (non-saccharolytic) Acetohalobium arabaticum. The latter species produces only minor amounts of trimethylamine as most is converted to acetate (Zhilina and Zavarzin 1990b). Glycine betaine can also be used as an electron acceptor in the Stickland reaction by Halanaerobacter salinarius and Halanaerobacter chitinivorans, with H2 or serine as electron donor (Mouné et al. 1999).

Quantitative data on the occurrence of members of the Halanaerobiales in hypersaline anoxic environments are scarce. Halanaerobium praevalens was reported to be present in Great Salt Lake surface sediment in numbers of up to 108 per mL sediment (Zeikus 1983; Zeikus et al. 1983), while 103–105 Halobacteroides cells were counted per mL of Dead Sea sediment (Oren et al. 1984b). Up to 107–109 anaerobic halophilic cellulolytic bacteria were enumerated per mL sediment in lagoons of the Arabat strait (Siman’kova and Zavarzin 1992), and up to 4.6 × 103 anaerobic halophiles were counted in anaerobic brines associated with an oil reservoir in Oklahoma (Bhupathiraju et al. 1991, 1993). The few data available prove that these anaerobic halophiles may form a significant component of the ecosystem in anaerobic hypersaline sediments.

Pathogenicity, Clinical Relevance

All members of the Halanaerobiales are moderately halophilic and do not grow at low salt concentrations. Accordingly, no pathogens are found within the group.

Sensitivity to antibiotics was tested in some species. Within the family Halanaerobiaceae, Halanaerobium salsuginis, H. kushneri, and H. lacusrosei were reported to be sensitive to penicillin, chloramphenicol, and tetracycline. An alkaliphilic member of the genus H. alcaliphilum resists low concentrations of antibiotics but is inhibited by 200 μg/mL penicillin, 400 μg/mL cycloserine, and 1,000 μg/mL streptomycin. Halocella cellulosilytica is inhibited by streptomycin, penicillin, vancomycin, rifampicin, and bacitracin; Halarsenatibacter silvermanii is sensitive to vancomycin, kanamycin, penicillin, and tetracycline.

Members of the Halobacteroidaceae tested for antibiotics sensitivity include Halobacteroides halobius (forming large spheres in the presence of penicillin, also sensitive to chloramphenicol and bacitracin), Halanaerobacter chitinivorans (inhibited by chloramphenicol, but not by 100 μg/mL cycloserine, penicillin, streptomycin, or tetracycline), H. salinarius (sensitive to chloramphenicol, erythromycin, kanamycin, and tetracycline), Orenia marismortui (sensitive to penicillin, bacitracin, novobiocin, erythromycin, polymyxin, and chloramphenicol, but not to streptomycin), O. salinaria (sensitive to chloramphenicol, erythromycin, and tetracycline but resistant to kanamycin), and Fuchsiella alkaliacetigena (sensitive to vancomycin, novobiocin, and rifampicin).

Application

Use in Food Fermentations

Halanaerobium fermentans was isolated from “fugunoko nukaduke,” a traditional Japanese food prepared from fermented salted puffer fish ovaries. Puffer fish ovaries are salted for at least 6 months, and the ovaries are then fermented naturally with rice bran, fish sauce, and koji for several years. H. fermentans may be one of the main bacteria involved in the fermentation process (Kobayashi et al. 2000a). Halophilic anaerobes identified as Halanaerobium praevalens (based on 16S rRNA sequence and DNA-DNA hybridization) or H. alcaliphilum, producing acetate, butyrate, and propionate, were isolated from canned Swedish fermented herrings (“surströmming”) (Kobayashi et al. 2000b). Members of the genus Halanaerobium may thus be involved in the manufacturing of traditional fermented food products.

Industrial Fermentation for Hydrogen and Acetate

The use of anaerobic halophilic bacteria in the industrial fermentation of complex organic matter and the production of organic solvents has been proposed (Lowe et al. 1993; Wise 1987), but any such applications are still in an experimental stage. Recently it was proposed to use Halanaerobium saccharolyticum subsp. saccharolyticum and subsp. senegalense for the industrial production of hydrogen from glycerol formed as by-product of the biodiesel industry. The highest H2 yield (1.6 mol H2/mol glycerol) was obtained with H. saccharolyticum subsp. senegalense grown at 15 % salt. H. saccharolyticum subsp. saccharolyticum produced less H2 (0.6 mol/mol glycerol) but also yielded 1,3-propanediol (up to 0.49 mol/mol glycerol) as a valuable by-product (Kivistö et al. 2010). Halocella cellulosilytica is a cellulose degrader (Simankova et al. 1993), but its biotechnological potential for cellulose degradation at high salt concentrations has not yet been exploited.

Enhanced Oil Recovery

Several species of Halanaerobium (H. salsuginis, H. acetethylicum, H. kushneri, H. congolense) and Halanaerocella petrolearia were isolated from brines associated with oil reservoirs (Bhupathiraju et al. 1994, 1999; Gales et al. 2011; Ravot et al. 1997; Rengpipat et al. 1988a). Such bacteria may be applied for microbially enhanced oil recovery from oil reservoirs by plugging of porous reservoirs and by anaerobically metabolizing nutrients with the production of useful products such as gases, biosurfactants, and polymers under the environmental conditions that exist in the reservoirs (Bhupathiraju et al. 1991).

Treatment of Saline Wastewater

Treatment of saline wastewater in an anaerobic packed bed reactor inoculated with Halanaerobium lacusrosei was explored, using model wastewaters with glucose as carbon source applying a gradual increase in salinity from 0 to 5 % or from 3 to 10%. Glucose removal at 70 % efficiency was claimed at 3 % salt (Kapdan and Erten 2007; Kapdan and Boylan 2009). As H. lacusrosei does not grow below 6 % salt and has its optimum at 20 % salt (Cayol et al. 1995), it is not clear to what extent the glucose degradation observed was indeed effected by Halanaerobium.

Nitrosubstituted aromatic compounds such as nitrobenzene, nitrophenols, 2,4-dinitrophenol, and 2,4-dinitroaniline are reduced to the amino derivatives by Halanaerobium praevalens and by Orenia marismortui (Oren et al. 1991).

Enzymes

Several enzymes from members of the Halanaerobiales have been cloned, purified, and characterized. One such enzyme is the rhodanese-like protein (thiosulfate: cyanide sulfurtransferase; EC 2.8.1.1) of Halanaerobium congolense (Ravot et al. 2005). Halothermothrix orenii has become a popular object of such studies because of the prospect of enzymes that function both at high salinity and at high temperature. A few such enzymes have been crystallized to study their structure: α-amylase AmyA (EC 3.2.1.1) (optimum activity at 65 °C in 5 % NaCl, with significant activity at 25 % NaCl) (Li et al. 2002; Mijts and Patel 2002), α-amylase AmyB (Tan et al. 2008), ribokinase (EC 2.7.1.15) (Kori et al. 2012), sucrose phosphate synthase (EC 2.4.1.14) (Chua et al. 2008; Huynh et al. 2005), fructokinase (EC 2.7.1.4) (Chua et al. 2010), and class II 5-enopyruvylshikimate-3-phosphate synthase (EC 2.5.1.19). The latter protein, a key enzyme in the synthesis of aromatic amino acids, when expressed in Arabidopsis plants bestowed resistance to glyphosate herbicides (Tian et al. 2012).