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1 Discovery

The Desulfitobacterium strains have been isolated primarily as organohalide-respiring bacteria (OHRB) from organohalide-contaminated environments. The first Desulfitobacterium isolate was strain DCB-2 (for [aryl-] dechlorinating bacterium no. 2, currently known as Desulfitobacterium hafniense DCB-2) (Madsen and Licht 1992). Strain DCB-2 was isolated as a chlorinated phenol-degrading bacterium from a stable trichlorophenol (TCP)-dechlorinating consortium enriched from municipal digestor sludge in Copenhagen, Denmark. At that time, strain DCB-2 was reported to dehalogenate chlorophenols at the ortho and meta positions. This activity differed from that reported for Desulfomonile tiedjei DCB-1, which was the first isolate of the OHRB, dechlorinating only from the meta position (Shelton and Tiedje 1984; DeWeerd et al. 1990). Strain DCB-2 was later designated D. hafniense DCB-2 (Christiansen and Ahring 1996a). The species name hafniense reflects that Copenhagen was the place of isolation.

The nomenclature of the genus Desulfitobacterium was defined upon isolation of the second strain, D. dehalogenans JW/IU-DC1, in Athens, Georgia, USA (Utkin et al. 1994). The strain JW/IU-DC1 can dechlorinate at the ortho position of both chlorophenols and chlorophenylacetates (Utkin et al. 1994, 1995) and was the first Desulfitobacterium strain for which energy conservation through reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (3-Cl-4-OHPA) was shown (Mackiewicz and Wiegel 1998). The genus name Desulfitobacterium describes a rod-shaped bacterium that reduces sulfite. However, several Desulfitobacterium isolates that do not reduce sulfite have been reported, including D. metallireducens 853-15A and Desulfitobacterium sp. PR (Finneran et al. 2002; Ding et al. 2014). A total of 22 Desulfitobacterium strains exhibiting a wide dehalogenation spectrum have been isolated and characterized to date.

2 Isolation and Habitat

Desulfitobacterium strains are distributed worldwide, having been isolated from Denmark, the United States, Vietnam, The Netherlands, Germany, Canada, Japan, Finland, and Poland (Table 9.1). Most Desulfitobacterium strains have been isolated during the course of studies of reductive dechlorination processes. Therefore, the target organochlorines in each of these studies were generally used as the electron acceptors in enrichment cultures. A total of 15 Desulfitobacterium strains (D. hafniense strains DCB-2, TCE1, TCP-A, PCE-S, PCP-1, Y51; D. dehalogenans JW/IU-DC1; D. chlororespirans Co23; D. dichloroeliminans DCA1; and Desulfitobacterium. sp. strains Viet1, KBC1, PCE1, B31e3, JH1, PR) have been isolated from enrichment cultures containing the organochlorines tetrachloroethene (PCE), trichloroethene (TCE), 2,4,6-TCP, 2,3-dichlorophenol (2,3-DCP), pentachlorophenol (PCP), 3-Cl-4-OHPA, 1,2-dichloroethane (1,2-DCA), or 1,1,1-trichloroethane (Suyama et al. 2001; Gerritse et al. 1999; Breitenstein et al. 2001; Miller et al. 1997; Bouchard et al. 1996; Madsen and Licht 1992; Sanford et al. 1996; Löffler et al. 1997; Tsukagoshi et al. 2006; Utkin et al. 1994; Gerritse et al. 1996; De Wildeman et al. 2003; Yoshida et al. 2007; Fletcher et al. 2008; Ding et al. 2014). In contrast, six Desulfitobacterium strains (D. hafniense strains G2, DP7, and GBFH; Desulfitobacterium sp. RPf35Ei; D. metallireducens 853-15A; and D. aromaticivorans UKTL) were isolated in the absence of organohalides (Shelobolina et al. 2003; van de Pas et al. 2001b; Niggemyer et al. 2001; Kaksonen et al. 2004; Finneran et al. 2002; Kunapuli et al. 2010).

Table 9.1 Desulfitobacterium strains in order of the year they were first reported
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Desulfitobacterium strains have been isolated from a variety of anaerobic environments, including digestor sludge, sewage sludge, ditch sludge mixed with sewage, methanogenic granular sludge, freshwater sediment, river sediment, arsenic-contaminated sediments, uranium mill tailing site floodplain, active mines, compost soil, soil samples from crop fields, soil from a former coal gasification site, and human feces (Table 9.1). Studies indicate that Desulfitobacterium spp. are widely distributed in anaerobic terrestrial environments, but not in marine environments. Molecular ecological studies have confirmed the ubiquitous distribution of Desulfitobacterium spp. For example, in one study, Desulfitobacterium spp. were detected in 31 of 48 soil samples from Canada (mainly from Quebec), which included 24 samples from contaminated industrial sites (Lanthier et al. 2001). In addition, Desulfitobacterium spp. as well as other OHRB have been detected with a high frequency in samples from chloroethenes-contaminated sites (Davis et al. 2002; Yang et al. 2005; Yoshida et al. 2007; Dowideit et al. 2010; Rouzeau-Szynalski et al. 2011; Kranzioch et al. 2013). Thus, the Desulfitobacterium spp. are one of the most abundant OHRB in the natural environment.

Methods involving serial dilution or single colony isolation on agar plates or in agar shake cultures have been used to isolate Desulfitobacterium strains. In addition, flow cytometric sorting was utilized in the isolation of Desulfitobacterium sp. KBC1 (Tsukagoshi et al. 2006). Anaerobic media containing target organohalides in combination with an electron donor such as pyruvate works well when attempting to isolate organohalide-respiring Desulfitobacterium strains. Yeast extract can be used as a source of vitamins and trace elements when culturing Desulfitobacterium strains. Antimicrobial agents may also be useful for Desulfitobacterium enrichment, as evidenced by the isolation of D. hafniense DP7 using the antibacterial agent aztreonam (50 μg/L), which is a β-lactam compound that inhibits the growth of aerobic Gram-negative bacteria (van de Pas et al. 2001b). Sequence analyses identified a vancomycin resistance gene cluster in the genome of D. hafniense Y51 (Kalan et al. 2009; Kruse et al. 2014a). Thus, exploiting vancomycin resistance might also be useful for the enrichment and isolation of Desulfitobacterium strains. Pasteurization is also useful for the isolation of spore-forming Desulfitobacterium strains (see Sect. 9.3).

The Desulfitobacterium strains can be preserved as frozen or freeze-dried stocks (Spring and Rosenzweig 2006). For long-term storage, anaerobic 50 % glycerol stocks are kept at −80 °C or in liquid nitrogen. Glycerol stocks remain viable for at least 5 years when stored at −80 °C (authors’ unpublished data). The procedures used for manipulating the stocks under anaerobic conditions are important for the long-term preservation of Desulfitobacterium strains. Although freeze-drying is also suitable for long-term storage, several Desulfitobacterium strains are sensitive to the freeze-drying process and display low survival rates after lyophilization. For these strains, preparation of cell suspensions in freshly prepared medium supplemented with 5 % dimethyl sulfoxide (DMSO) as a cryoprotectant and subsequent storage in liquid nitrogen is recommended (Spring and Rosenzweig 2006).

3 Morphology, Physiology, and Growth Characteristics

Morphologically, the Desulfitobacterium strains appear as slightly curved rods (Fig. 9.1). The cells range from 2 to 7 μm in length and 0.3 to 1 μm in width (Table 9.2). Variation in cell length (exceeding 10 μm) has been reported for D. dichloroeliminans DCA1 (De Wildeman et al. 2003). In addition, a coupled cell structure was reported for Desulfitobacterium sp. PR (Ding et al. 2014), which may form filaments composed of up to 10 cells. Most Desulfitobacterium isolates are motile and have from 1 to 6 flagella. Nonmotile cells without flagella have been reported for D. hafniense PCP-1 (Bouchard et al. 1996). The motility characteristics of D. hafniense JH1 and Desulfitobacterium sp. PR have not been described, but these cells also do not have flagella (Fletcher et al. 2008; Ding et al. 2014).

Fig. 9.1
figure 1

Morphology of Desulfitobacterium strains. a–d Electron micrographs of negatively stained exponential-phase cells of Desulfitobacterium sp. PCE1. a Cell with four laterally attached flagella. b Ultrathin section revealing the thick Gram-positive cell wall. c S-layer surrounding the cell wall and d dividing long, curved cell of Desulfitobacterium sp. PCE1. Bars indicate 1 μm in (a) and (d) and 0.1 μm in (b) and (c). e Electron micrograph of negatively stained exponential-phase cells of D. hafniense Y51. Bar, 1 μm. f Phase contrast light micrograph of D. hafniense DP7. Bar, 10 μm. (g–h). g Scanning electron micrograph and h transmission electron micrograph of exponential-phase cells of Desulfitobacterium sp. KBC1. Bar, 1 μm. Photos were taken from Gerritse et al. (1996), van de Pas et al. (2001b), Furukawa et al. (2005), and Tsukagoshi et al. (2006) with permission

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Table 9.2 Physiological features of Desulfitobacterium strains
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The Desulfitobacterium strains are mesophilic and grow at neutral pH. The optimum growth temperature and pH are in the ranges of 25–40 °C and 6.5–7.8, respectively (Table 9.2). Ten Desulfitobacterium strains are capable of forming terminal endospores. The capability of forming spores is likely associated with ensuring survival during heat stress, as evidenced by the observation that D. hafniense strains DCB-2, PCP-1, and TCP-A survived pasteurization during the isolation process (Madsen and Licht 1992; Bouchard et al. 1996; Breitenstein et al. 2001); during the isolation of D. hafniense DCB-2, the dechlorinating enrichment consortium was incubated at 80 °C for 60 min (Madsen and Licht 1992). Other research has confirmed that D. chlororespirans Co23 and D. hafniense GBFH can survive heat treatment (Sanford et al. 1996; Niggemyer et al. 2001). On the other hand, the non-spore forming strains Desulfitobacterium sp. PCE1 and D. metallireducens 853-15A do not survive heat treatment (Gerritse et al. 1996; Finneran et al. 2002); incubation at 85 °C for 20 min irreversibly inhibits the growth of Desulfitobacterium sp. PCE1 (Gerritse et al. 1996).

Although the known Desulfitobacterium strains are strict anaerobes, they are slightly oxygen tolerant . For example, D. dehalogenans JW/IU-DC1 was shown to dechlorinate 3-Cl-4-OHPA under aerobic condition with 2 % air, indicating its tolerance for oxygen (Utkin et al. 1994). It has also been reported that D. hafniense strains DCB-2 and GBFH, D. dichloroeliminans DCA1, and Desulfitobacterium sp. strain PR could survive aerobic conditions for 24 h (Madsen and Licht 1992; Niggemyer et al. 2001; De Wildeman et al. 2003; Ding et al. 2014).

Characterization of the cellular fatty acid composition is frequently used in microbial taxonomy. The fatty acid composition has been reported for D. dehalogenans JW/IU-DC1, D. hafniense strains DCB-2 and PCP-1, D. chlororespirans Co23, D. metallireducens 853-15A, and D. aromaticivorans UKTL. Spring and Rosenzweig (2006) reported that the major fatty acids in D. dehalogenans JW/IU-DC1, D. hafniense strains DCB-2 and PCP-1, D. chlororespirans Co23, and D. metallireducens 853-15A are 14:0 (4.0–22.8 %), 16:1 cis9 (6.4–13.0 %), 16:0 (4.4–25.6 %), and 18:1 cis11 (0.5–13.6 %). The fatty acid profiles of D. hafniense strains DCB-2 and PCP-1 are similar. In contrast, the fatty acid compositions of D. dehalogenans JW/IU-DC1 and D. metallireducens 853-15A differ. The characteristic fatty acids were identified as 16:0, 14:0, and 16:1 cis9 in D. dehalogenans JW/IU-DC1; 18:1 cis11 dimethylacetal, 18:1 cis1, and 16:1 cis9 in D. hafniense strains DCB-2 and PCP-1; 16:0, 16:1 cis9, and 14:0 in D. chlororespirans Co23; and 14:0, 16:1 cis9 dimethylacetal, and an unidentified fatty acid with an equivalent chain length of 13.52 in D. metallireducens 853-15A (Spring and Rosenzweig 2006). In addition, Kunapuli et al. (2010) investigated the fatty acid composition of D. aromaticivorans UKTL, with D. chlororespirans Co23 serving as a control, and found that strain UKTL contains 15:0 iso, but not 18:1ω7c fatty acids, in contrast to strain Co23. These data have to be used with a caution because fatty acid composition can change with factors such as medium composition and growth phase.

The cytochrome and quinone contents have been reported for several Desulfitobacterium strains. The D. hafniense strains DCB-2 and TCE1, D. metallireducens 853-15A, and Desulfitobacterium sp. PCE1 were shown to contain cytochrome c (Christiansen and Ahring 1996a; Gerritse et al. 1996, 1999; Finneran et al. 2002). Menaquinone-7 was identified as the primary menaquinone of D. hafniense TCP-A and D. aromaticivorans UKTL (Breitenstein et al. 2001; Kunapuli et al. 2010).

The Desulfitobacterium strains can also grow fermentatively; fermentative growth on pyruvate has been reported for most Desulfitobacterium isolates except for D. metallireducens 853-15A (Finneran et al. 2002) (Table 9.3). The fermentative growth yield of D. dehalogenans JW/IU-DC1 on pyruvate is approximately 14 g of dry cell weight per mole of pyruvate (van de Pas et al. 2001a). Tryptophan and serine also reportedly support the fermentative growth of D. hafniense DCB-2 and D. hafniense TCE1, respectively (Christiansen and Ahring 1996a; Gerritse et al. 1999).

Table 9.3 Primary metabolic features of Desulfitobacterium isolates
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Formate, lactate, and pyruvate generally serve as electron donors for Desulfitobacterium strains. In addition, most Desulfitobacterium strains exhibit O-demethylation activity. The O-demethylation was reported to be involved in the intermediary metabolism for methoxylated organochlorines such as tetrachloroguaiacol, tetrachloroveratrole, pentachloroanisole, and 3,5-dichloro-4-methoxyphenol in D. hafniense strains PCP-1 and DCB-2, D. chlororespirans Co23, and D. dehalogenans JW/IU-DC1 (Dennie et al. 1998; Milliken et al. 2004b). Then, the utilization of phenyl methyl ethers, vanillate and syringate, as electron donors via O-demethylation was reported for D. hafniense strains DCB-2, PCE-S, DP7, G2, PCP-1, TCP-A, and Y51, D. chlororespirans Co23, and D. dehalogenans JW/IU- 1 (Neumann et al. 2004; Mingo et al. 2014). Enzymes involved in O-demethylation have been biochemically characterized in D. hafniense strains DCB-2 and PCE-S (Kreher et al. 2008; Studenik et al. 2012). The methyl group from phenyl methyl ethers is transferred to tetrahydrofolate and considered to be further used as an electron donor via acetyl-CoA formation. Because phenyl methyl ethers are lignin decomposition products, Desulfitobacterium spp. are thought to be involved in biological lignin transformation. D. hafniense strains DCB-2 and PCE-S grown on fumarate and vanillate or syringate yield approximately 15 g of dry cell weight per mole of methyl moiety converted (Neumann et al. 2004).

Compounds capable of supporting the growth of Desulfitobacterium strains as electron acceptor include sulfite, thiosulfate, fumarate, nitrate, anthraquinone-2,6-disulfonate (AQDS, a humic acid analog), isethionate (2-hydroxyethanesulfonate), and cysteate (adenine-3-sulfonate). Desulfitobacterium strains generally cannot use sulfate and nitrite as electron acceptors with the exception that D. hafniense Y51 reduces sulfate (Suyama et al. 2001). Inorganic metals such as Fe(III), Mn(IV), Se(VI), As(V), and U(VI) can also be used as electron acceptors by Desulfitobacterium spp., and strains GBFH, G2, 853-15A, and UKTL have been isolated as metal reducers without demonstrable organohalide respiration. D. hafniense GBFH was isolated as an As(V)-reducing bacterium from arsenic-contaminated sediments (Niggemyer et al. 2001). D. hafniense G2 can utilize U(VI), Fe(III) and AQDS as electron acceptors (Shelobolina et al. 2003). D. metallireducens 853-15A can utilize AQDS, chelated Fe(III) (not crystalline Fe[III] oxide), humic acids, Mn(IV), colloidal sulfur, Se(IV), and Cr(VI) as electron acceptors (Finneran et al. 2002). D. aromaticivorans UKTL was isolated as an iron-reducing bacterium capable of anaerobic degradation of monoaromatic hydrocarbons, including toluene, phenol, and p-cresol (Kunapuli et al. 2010). The Desulfitobacterium strains initially isolated as OHRB also have the potential to reduce a variety of electron acceptors including metals (Table 9.3). Thus, the Desulfitobacterium spp. play important roles in the natural cycles of a variety of compounds other than organohalides.

4 Phylogeny

The genus Desulfitobacterium belongs to the phylum Firmicutes , class Clostridia , order Clostridiales , and family Peptococcaceae (Lupa and Wiegel 2009). Members of the family Peptococcaceae are anaerobes, and the Desulfitobacterium spp. which are strictly anaerobic. However, in contradiction to the name Peptococcaceae, the known Desulfitobacterium strains are not cocci, but curved rods throughout all growth phases, similar to some other Peptococcaceae genera (e.g., Dehalobacter; see Chap. 8) (Fig. 9.1). This apparent contradiction derives from the fact that the family Peptococcaceae was composed of three genera of cocci (Peptococcus, Peptostreptococcus, and Ruminococcus) when it was proposed (Rogosa 1971). As the phylum Firmicutes is composed of Gram-positive bacteria, the Desulfitobacterium spp. are recognized as Gram positive. However, Gram stain analyses determined that five isolates, including D. hafniense strains DCB-2, PCP-1, and Y51, D. chlororespirans Co23, and Desulfitobacterium sp. B31e3, are actually staining Gram negative (Bouchard et al. 1996; Christiansen and Ahring 1996a; Sanford et al. 1996; Suyama et al. 2001) (Table 9.2). In the case of D. hafniense PCP-1, the cells stain Gram negative, but observation of ultrathin cross section of a strain PCP-1 by electron microscopic analysis indicated that this strain is Gram positive. The thick Gram-positive cell wall structure is also evident in ultrathin sections of Desulfitobacterium sp. PCE1 (Fig. 9.1b). Thus, it appears that staining is insufficient for determining whether Desulfitobacterium strains are Gram positive or Gram negative. On the other hand, the cell envelope architecture can give a clearer definition than the staining properties (Sutcliffe 2010). From this point of view, the Desulfitobacterium spp. should be recognized as monoderm that have an envelope with one membrane.

The phylogenetic relationships of Desulfitobacterium isolates based on 16S rRNA gene sequences are shown in Fig. 9.2. The Desulfitobacterium isolates are classified into six species: D. hafniense, D. dehalogenans, D. dichloroeliminans, D. chlororespirans, D. metallireducens, and D. aromaticivorans. Currently, no species name has been assigned to six strains: Viet1, PCE1, RPf35Ei, KBC1, B31e3, and PR. The D. hafniense strains PCP-1, DP7, TCP-A, G2, and TCE1 were previously classified as D. frappieri, but the species name frappieri is no longer used based on detailed 16S rRNA gene analyses. As a result, D. frappieri has been reclassified as D. hafniense. This confusion can be attributed to the size of the 16S rRNA gene in D. hafniense PCP-1 (formerly D. frappieri PCP-1) (Bouchard et al. 1996, reviewed in Villemur et al. 2006). The 16S rRNA gene of strain PCP-1 is 1655-bp long, whereas that of D. hafniense DCB-2 is 1530-bp long. It is now known that the Desulfitobacterium genome encodes multiple 16S rRNA genes and shows intra-genomic heterogeneity (Villemur et al. 2007). Denaturing gradient gel electrophoresis (DGGE) analysis indicated that 16S rRNA gene copy numbers in Desulfitobacterium strains vary from 2 to 7. Heterogeneity with respect to 16S rRNA gene copies is caused by 100- to 200-bp insertions in the 5′ region, the region that differs between strains PCP-1 and DCB-2. The 16S rRNA gene of strain PCP-1 has a 128-nt insertion that is absent in the 16S rRNA gene of strain DCB-2 (Bouchard et al. 1996). The insertions are predicted to form an energetically stable loop when they are transcribed. In addition, reverse transcriptase-PCR analyses have demonstrated that most of the observed insertions in the 16S rRNA gene of Desulfitobacterium strains are excised from the mature 16S rRNA transcripts. Although such insertion sequences are rarely observed in bacterial 16S rRNA genes, they are also found in several other bacterial genera, such as Desulfotomaculum and Anaerospora (Patel et al. 1992; Woo et al. 2005).

Fig. 9.2
figure 2

Phylogenetic relationships of Desulfitobacterium isolates based on 16S rRNA gene sequences. Sequence accession numbers are indicated in parentheses. Branch supporting values (%) were evaluated with 1000 bootstrap replications. The tree was constructed using the neighbor-joining method based on an alignment of almost-complete 16S rRNA gene sequences. The 100- to 200-bp insertion sequence harboring the 5′ end (Villemur et al. 2007) was excluded using the complete gap deletion option in MEGA version 6 software (Tamura et al. 2013). Desulfosporosinus orientis and Dehalobacter restrictus PER-K23 were used as outgroups. Multiple copies of 16S rRNA gene sequences of D. hafniense strains Y51 and DCB-2, D. dehalogenans JW/IU-DC-1, D. dichloroeliminans DCA1, D. metallireducens 853-15A, and Dehalobacter restrictus PER-K23 were identified based on genome information. In those cases, locus tags are indicated in the parentheses. Different species are highlighted in different colors. The scale bar represents 10 % estimated sequence divergence

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5 Reductive Dehalogenation Characteristics

The reductive dehalogenation activities of Desulfitobacterium isolates are summarized in Table 9.4. The reductive dehalogenation spectrum of the strains does not correlate with their phylogenetic relationships and seems to depend heavily upon the enzyme reductive dehalogenase (RDase), which is a terminal reductase in the organohalide respiratory pathway expressed in each Desulfitobacterium isolate (Hug et al. 2013).

Table 9.4 Dehalogenation activity of Desulfitobacterium strains
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The OHRB exhibits two types of reductive dehalogenation: hydrogenolysis and dihaloelimination (Mohn and Tiedje 1992; Holliger et al. 1998; Smidt and de Vos, 2004). In hydrogenolysis , the halogen substituents of alkyl or aryl halides are replaced with hydrogen atoms. In contrast, in dihaloelimination , two halogen substituents of alkyl halides are removed from adjacent carbon atoms. Both types of reductive dehalogenation have been observed in Desulfitobacterium strains.

Most Desulfitobacterium strains can dechlorinate chloroaliphatic and/or chloroaromatic compounds. In addition, several Desulfitobacterium strains were found to dehalogenate organobromine and organoiodine compounds. However, to date, no dehalogenation activity has been reported for four Desulfitobacterium strains: D. hafniense DP7 and GBFH, Desulfitobacterium sp. RPf35Ei, and D. aromaticivorans UKTL. Of these strains, D. hafniense DP7 was characterized as a non-dechlorinator. Strain DP7 was isolated from human feces (nonpolluted environment) and does not dechlorinate monochlorophenols, 2,3-, 2,4-, 2,5-, and 2,6-DCPs, PCP, PCE, 3-Cl-4-OHPA, 2,3,5,6-tetrachloro-4-methoxyphenol, or tetrachlorohydroquinone (van de Pas et al. 2001b; Milliken et al. 2004a).

5.1 Dehalogenation of Chloroaliphatic Compounds

As it is one of the most common environmental pollutants, bacterial dechlorination of chloroethenes has been widely studied. The Desulfitobacterium spp. identified to date do not dechlorinate DCE or vinyl chloride and can be classified into one of two categories: PCE to cis-DCE dechlorinators or PCE to TCE dechlorinators (Table 9.4). The former category includes D. hafniense strains TCE1, PCE-S, Y51, G2, JH1, D. metallireducens 853-15A, Desulfitobacterium sp. B31e3, D. hafniense DCB-2, and D. dehalogenans JW/IU-DC1, whereas the latter includes D. hafniense PCP-1 and Desulfitobacterium sp. strains Viet1, PCE1, and KBC1. In the case of strains DCB-2 and JW/IU-DC1, they dechlorinate PCE to TCE slowly via cometabolic processes, which do not provide energy for growth, when the cells are cultured in the presence of 3-Cl-4-OHPA as an inducer (Gerritse et al. 1999). In addition, D. hafniense TCP-A and Desulfitobacterium sp. PR also dechlorinate PCE to TCE slowly, but the reaction does not require inducers (Breitenstein et al. 2001; Ding et al. 2014).

The unique reaction utilized by Desulfitobacterium strains for the dechlorination of chloroaliphatic compounds is as follow. D. hafniense Y51 dechlorinates hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane (Suyama et al. 2001). D. dichloroeliminans DCA1 can dechlorinate 1,1,2-trichloroethane, 1,2-DCA, meso and DL stereoisomers of 2,3-dichlorobutane, 1,2-dichlorobutane, and 1,2-dichloropropane (De Wildeman et al. 2003). Strain DCA1 dechlorinate these compounds via the dichloroelimination system. Desulfitobacterium sp. PR reductively dechlorinates both 1,1,1-trichloroethane and chloroform (Ding et al. 2014). Strain PR can dechlorinate 1,1,1-trichloroethane completely to monochloroethane and dechlorinates chloroform (trichloromethane) to predominantly carbon dichloride (dichloromethane) and trace amounts of monochloromethane. Efficient degradation of chloroform has been reported only in strain PR among the Desulfitobacterium strains described to date.

The PCE/TCE RDase PceA has thus far been purified and characterized in D. hafniense strains PCE-S, Y51, and TCE1 (Miller et al. 1998; Suyama et al. 2002; Maillard et al. 2003). The PceA-encoding gene is found in the conserved pceA-pceB-pceC-pceT gene cluster in D. hafniense strains TCE1 and Y51. PceB is predicted to be a membrane anchor protein for PceA. The pceT gene encodes a trigger factor protein that binds to the twin arginine translocation (Tat) signal sequence of PceA (Morita et al. 2009; Maillard et al. 2011). The degradation gene cluster is sometimes located on a mobile element , and in fact, the pce gene cluster forms a catabolic transposon in D. hafniense strains TCE1 and Y51 (Maillard et al. 2005; Futagami et al. 2006a, b; Duret et al. 2012).

The activity of the PCE RDase PrdA of Desulfitobacterium sp. KBC1 is controlled at the transcriptional level (Tsukagoshi et al. 2006). Transcription of the prdA gene in strain KBC1 is induced by PCE. In contrast, the cultivation conditions reportedly affect PceA activity in D. hafniense Y51, without transcriptional regulation. Because the pceA gene is located on the transposable element in strain Y51, the activity of PceA decreases when cells are grown in the absence of an organochlorine substrate due to loss of the pce gene cluster through transposition (Reinhold et al. 2012). Vitamin B12 (a corrinoid) also reportedly affects the stability of the pce genes (Reinhold et al. 2012). Corrinoids play a significant role in regulating RDase catalytic activity as cofactors. Exogenous vitamin B12 hampers the transposition of the pce gene cluster, although the exogenous vitamin B12 does not appear to be incorporated into the PceA precursor. In addition, when strain Y51 is grown in the absence of an organochlorine substrate, the intracellular corrinoid level decreases and the PceA precursor forms catalytically inactive and corrinoid-free aggregates.

5.2 Dehalogenation of Chloroaromatic Compounds

The dechlorination of chlorophenols and 3-Cl-4-OHPA has also been well studied. Dechlorination of these chloroaromatic compounds has been reported in D. hafniense strains DCB-2 and TCP-A, D. dehalogenans strains JW/IU-DC1 and XZ-1, Desulfitobacterium sp. strains Viet1, PCE1, and KBC1, D. chlororespirans Co23, and D. metallireducens 853-15A (Table 9.4). The spectrum of haloaromatic compound dechlorination has been characterized most thoroughly in D. hafniense PCP-1 and D. dehalogenans JW/IU-DC1 (reviewed in Villemur 2013), e.g., D. hafniense PCP-1 can dechlorinate the ortho, meta, and para positions of chlorophenols, in this strain, PCP is converted to 3-chlorophenol via 2,3,4,5-tetrachlorophenol, 3,4,5-TCP, and 3,5-DCP (Bouchard et al. 1996). In strain PCP-1, ortho-dechlorination of PCP to 3,4,5-TCP proceeds rapidly, and after sufficient accumulation, 3,4,5-DCP is meta- and para-dechlorinated to 3,5-DCP and 3-chlorophenol. Thus, two different RDases are involved in PCP dechlorination in strain PCP-1. Ortho-dechlorination activity is induced by PCP, 2,4,6-TCP, 2,3,4-TCP, 2,3,5-TCP, 2,6-DCP, and 2,4-DCP, whereas meta- and para-dechlorination activities are induced by 3,4,5-TCP and 3,5-DCP (reviewed in Villemur 2013).

In general, RDases preferentially target highly halogenated compounds as substrates for reductive attack over compounds exhibiting lower degrees of halogenation. For example, to date, no DCE- or vinyl chloride-respiring Desulfitobacterium strains have been isolated, as mentioned above (see Sect. 9.5.1). In the case of chlorophenols, nine strains have been tested for dechlorination of monochlorophenols, but only Desulfitobacterium sp. PCE1 was found to dechlorinate 2-chlorophenol efficiently (Gerritse et al. 1996). Dechlorination of 2,3,5-trichlorohydroquinone has been reported only in strain PCE1.

With respect to chloroaromatic compounds other than chlorophenols and 3-Cl-4-OHPA, D. hafniense PCP-1 dechlorinates organochlorides containing a phenyl or pyridine ring substituted by hydroxyl, nitro, methoxy, or amino groups (Bouchard et al. 1996; Dennie et al. 1998). Polychlorinated nitrobenzene is dechlorinated after reduction of the nitro group to an amino group. In addition, D. dehalogenans strains JW/IU-DC1 and XZ-1 and D. hafniense strain PCP-1 were reported to dechlorinate chlorinated hydroxybiphenyls such as 3,5-dichloro-4-hydroxybiphenyl (Dennie et al. 1998; Wiegel et al. 1999).

The enzyme chlorophenol-RDase (CprA) has been purified and characterized in D. hafniense strains PCP-1 and DCB-2, D. dehalogenans JW/IU-DC1, Desulfitobacterium sp. PCE1, and D. chlororespirans Co23 (Christiansen et al. 1998; van de Pas et al. 1999, 2001a; Krasotkina et al. 2001; Löffler et al. 1996; Boyer et al. 2003; Thibodeau et al. 2004; Bisaillon et al. 2010). These enzymes exhibit features similar to the RDases PceA, such as the presence of a Tat signal sequence, two Fe–S clusters, and a corrinoid cofactor. However, the chlorophenol-RDase CrdA purified from D. hafniense PCP-1 exhibits different features (Boyer et al. 2003). CrdA ortho-dechlorinates PCP and 2,4,6-TCP. Like other RDases, CrdA contains a corrinoid cofactor, but this enzyme also has a LysM domain that may be involved in binding to peptidoglycan (Bateman et al. 2000, reviewed in Villemur 2013). The crdA gene has been found in D. hafniense strains DCB-2, DP7, TCP-1, Y51 and TCE1, Desulfitobacterium sp. PCE1, D. dehalogenans JW/IU-DC1, and D. chlororespirans Co23 (Gauthier et al. 2006; Nonaka et al. 2006). Transcription of crdA in D. hafniense Y51 was shown to be upregulated 225-fold by TCE (Peng et al. 2012), although strain Y51 has not been tested for chlorophenol dechlorination activity.

The larger components of the RDase gene cluster have been identified in the upstream and downstream region of the cprA gene in D. dehalogenans JW/IU-DC1. This gene cluster consists of cprA, cprB, cprC, cprD, cprT, cprK, cprZ, and cprE (Smidt et al. 2000). In contrast to the pce gene cluster, the cpr cluster encodes the transcriptional regulator CprK, a member of the CRP-FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family. Transcription of the cprA gene is strictly regulated by CprK (Smidt et al. 2000; Gábor et al. 2008). CprK is the most thoroughly studied transcriptional regulator in the OHRB (see Chap. 15).

5.3 Dehalogenation of Organobromine and Organoiodine Compounds

Little information is available regarding the reductive dehalogenation of organobromine and organoiodine compounds as compared with organochlorine compounds (Table 9.4). D. dehalogenans JW/IU-DC1 can debrominate 2-bromo-4-chlorophenol, 2-bromo-4-methylphenol, 2,6-dibromophenol, and 2-bromophenol (Utkin et al. 1995). D. hafniense PCP-1 can debrominate 2,4,6-tribromophenol into 4-bromophenol in the absence of inducers (Dennie et al. 1998). D. chlororespirans Co23 can debrominate 2,4,6-tribromophenol, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (Sanford et al. 1996; Cupples et al. 2005). 2,4,6-tribromophenol is debrominated into 4-bromophenol by strain Co23 (Sanford et al. 1996). Bromoxynil and ioxynil are herbicides used for the postemergence control of dicotyledonous weeds in cereal crops. Bromoxynil is stoichiometrically converted into 4-cyanophenol and the metabolite 3,5-dibromo-4-hydroxybenzoate, which is then converted to 4-hydroxybenzoate. On the other hand, ioxynil is stoichiometrically converted into 4-cyanophenol in the presence of 3-chloro-4-hydroxybenzoate as an inducer. Strain Co23 can utilize bromoxynil and 3,5-dibromo-4-hydroxybenzoate as growth-supporting electron acceptors (Cupples et al. 2005). D. hafniense PCE-S can debrominate and grow on cis- and trans-dibromoethenes (Ye et al. 2010). Strain PCE-S converts cis- and trans-dibromoethenes to vinyl bromide and ethene. The PceA of strain PCE-S was initially purified as a PCE/TCE RDase (Miller et al. 1998), but the enzyme was later found to also catalyze the debromination of tribromoethene, cis- and trans-dibromoethenes (Ye et al. 2010).

Defluorination has not been observed by Desulfitobacterium spp. or any other OHRB. Desulfitobacterium dehalogenans JW/IU-DC1 does not defluorinate 3-fluoro-4-hydroxyphenylacetate (Utkin et al. 1995). Desulfitobacterium hafniense PCP-1 does not defluorinate 2,4,6-trifluorophenol or 2,3,5-trifluorophenol (Dennie et al. 1998), and D. chlororespirans Co23 does not grow in medium containing 2-fluorophenol as an electron acceptor (Sanford et al. 1996).

6 Bioremediation

The isolation of and ecological studies pertaining to Desulfitobacterium spp. has demonstrated that these organisms play a significant role in the natural attenuation of organohalide contaminants such as chloroethenes (see Sect. 9.2). For the bioremediation of chloroethenes, however, it is noteworthy that the genus Dehalococcoides is known as a key player. This is due to their ability to respire with DCE and vinyl chloride, a potential that has thus far been found solely in the genus Dehalococcoides (see Chap. 6).

Desulfitobacterium strains have been examined in both laboratory- and field-scale bioremediation studies. For example, the applicability of bioaugmentation with D. hafniense strains PCP-1 and Y51, D. dehalogenans JW/IU-DC1, or D. dichloroeliminans DCA1 for the degradation of PCP, PCE, 3-Cl-4-OHPA, and 1,2-DCA, respectively, has been demonstrated (El Fantroussi et al. 1997; Beaudet et al. 1998; Lanthier et al. 2000; Lee et al. 2001; De Wildeman et al. 2004). In addition, bioaugmentation using strain DCA1 for the removal of 1,2-DCA at an industrial site in Belgium was also demonstrated (Maes et al. 2006). The success of bioremediation efforts, however, depends largely on environmental factors, such as soil composition (reviewed in Villemur et al. 2006). For example, Lanthier et al. (2000) reported the dechlorination of PCP in a PCP-amended soil rich in organic matter, but no dechlorination was observed in sandy soil amended with PCP, highlighting the impact of factors such as organic matter and toxicity associated with other pollutants on the viability of OHRB. Geochemical properties such as redox conditions and pH also significantly impact the success of in situ bioremediation efforts.

Desulfitobacterium strains have also been used in investigations aimed at increasing bioremediation efficiency. The effect of PCE dense nonaqueous phase liquid on dechlorination by Desulfitobacterium strains Viet1 and PCE1 was examined via mathematical modeling (Amos et al. 2007; Huang and Becker 2011). Strain Viet1 was used to investigate the environmental fate of 2,4-DCP sequestered by the aquatic plant Lemna minor (Tront et al. 2006). In the environment, aquatic plants are known to take up and sequester various organohalines, such as chlorophenols (Newman and Reynolds 2004).

Desulfitobacterium strains have been utilized in bioreactors for treating organohaline-contaminated wastes. For example, D. hafniense strains DCB-2 and PCP-1 were inoculated into an up-flow anaerobic-sludge bed reactor used for PCP degradation (Christiansen and Ahring 1996b). This PCP degrading bioreactor using strain PCP-1 is one of the most thoroughly studied systems of its type (e.g., the localization of strain PCP-1 in the reactor granules and biofilm was demonstrated by fluorescence in situ hybridization) (Tartakovsky et al. 1999; Lanthier et al. 2000, 2002, 2005). Degradation of 2,4,6-TCP in anaerobic granular sludge bioaugmented with D. hafniense strains PCP-1 and TCP-A and D. chlororespirans Co23 has also been reported (Puyol et al. 2011). The Desulfitobacterium spp. were also found to be enriched in a deiodination bioreactor for 5-amino-2,4,6-triiodoisophthalic acid (a core structure of X-ray contrast media) (Lecouturier et al. 2003). These studies have demonstrated that Desulfitobacterium spp. are key components in dechlorination bioreactors.

Desulfitobacterium spp. can be employed not only for organohalide detoxification but also for the removal of metals. The solubility of a metal significantly affects its mobility in the environment. Therefore, altering a metal contaminant’s solubility through bioreduction is one approach for removing it. For example, several reports have described metal bioremediation strategies employing Desulfitobacterium strains to reduce soluble U(VI) to insoluble U(IV) (Fletcher et al. 2010; Boyanov et al. 2011).

Tools such as real-time PCR targeting the 16S rRNA and RDase genes of OHRB, and stable-isotope fractionation analysis are important for monitoring bioremediation efforts and evaluating their success. These tools were also developed for Desulfitobacterium spp. as one of important class of OHRB (see Chaps. 25 and 26).

7 Genomic Features

7.1 Basic Genome Facts

Complete genome sequences have been published for D. hafniense strains Y51 and DCB-2 and D. dehalogenans JW/IU-DC1 (Nonaka et al. 2006; Kim et al. 2012; Kruse et al. 2014b). In addition, the complete genome sequence for D. dichloroeliminans DCA1 and draft genome sequences for D. hafniense strains PCE-S (Goris et al. 2015), PCP-1, TCP-A, and DP7, D. metallireducens 853-15A, and Desulfitobacterium sp. PCE1 are available in the National Center for Biotechnology Information (NCBI) database.

The complete genomes of Desulfitobacterium strains vary widely in size, ranging from 3.62 to 5.73 Mbp, with similar GC content, ranging from 44.2 to 47.5 % (Table 9.5). No plasmids have been identified by genomic analyses. The number of coding sequences (CDSs) identified ranges from 3340 to 5060, which is consistent with the differences in genome size. As described in Sect. 9.4, multiple copies of rRNA operons (in the range from 2 to 7) are found in Desulfitobacterium genomes.

Table 9.5 Desulfitobacterium complete genome information
Full size table

The number of RDase genes differs in each strain (ranging from 2 to 7), which explains the differences in their dehalogenation spectra. Analyses of the D. hafniense strains Y51 and DCB-2 genomes revealed the existence of versatile terminal reductases, such as DMSO reductase superfamily proteins, sulfite reductase, nitrate reductase, and fumarate reductase, in addition to RDases (Nonaka et al. 2006; Kim et al. 2012). This finding is consistent with the growth phenotype of the Desulfitobacterium strains, with the exception that the nitrate reductase gene cluster (Nap system) was not identified in the genome of strain DCB-2, even though it can grow using nitrate as an electron acceptor.

As the Desulfitobacterium strains are slightly oxygen tolerant, the genomes of D. hafniense strains Y51 and DCB-2 encode putative catalases, superoxide dismutases, and rubrerythrin-rubredoxin enzymes that may be involved in imparting oxygen tolerance (Nonaka et al. 2006; Kim et al. 2012). In addition, consistent with its ability to form spores, genes involved in spore formation are present in strain DCB-2 (Kim et al. 2012).

7.2 Functional Genes and Metabolic Pathways

Genomic information has enabled the prediction of metabolic pathways in Desulfitobacterium strains. Central metabolic pathways for D. hafniense strains Y51 and DCB-2 have been proposed (Nonaka et al. 2006; Kim et al. 2012). Both strains Y51 and DCB-2 have a functional Embden-Meyerhof-Parnas pathway. On the other hand, the tricarboxylic acid cycle is characterized by the absence of 2-oxoglutarate dehydrogenase and the anaplerotic glyoxylate bypass, indicating an incomplete cycle. The reductive acetyl-CoA pathway (Wood-Ljungdahl pathway) for carbon dioxide fixation has also been found in both strains Y51 and DCB-2. Moreover, autotrophic growth of strain DCB-2 in a carbon dioxide fixation medium was confirmed (Kim et al. 2012).

Genome analyses of D. hafniense DCB-2 confirmed the presence of the complete synthetic pathways for the cofactors flavin adenine dinucleotide, nicotinamide adenine dinucleotide, menaquinone, heme, and cobalamin (Kim et al. 2012). The cobalamin biosynthetic pathway has also been identified in D. hafniense strains Y51 and TCE1 (Nonaka et al. 2006; Choudhary et al. 2013). Genomic analyses also revealed the presence of diverse cobalamin riboswitches that may be involved in regulating corrinoid metabolism in these D. hafniense strains (Choudhary et al. 2013). In addition, 14 putative ABC-type corrinoid transporter-encoding genes were identified in the genome of D. hafniense Y51 (Nonaka et al. 2006), but exogenous corrinoid may not be incorporated into the PceA precursor in strain Y51 (Reinhold et al. 2012). Menaquinone is believed to serve as an electron carrier in organohalide respiration. Electron paramagnetic resonance spectroscopy, visible spectroscopy, and proteomic analyses of D. dehalogenans JW/IU-DC1 suggested that electrons are transferred from menaquinones to RDase CprA via an as yet unidentified membrane complex and potentially by an extracellular flavoprotein (Kruse et al. 2014b).

7.3 Protein Characterization Based on Genome Data

Genomic data have also enabled other “omics” studies. For example, high expression of the unusual rhodanese protein PhsE during respiration with PCE in D. hafniense TCE1 as revealed by a proteomic study led to the characterization of this protein (Prat et al. 2011, 2012). PhsE was shown to have two unusual rhodanese domains that usually function to bind sulfane sulfur and catalyze sulfur transfer. The expression of PhsE was found to be induced in the presence of sulfide in the medium and is believed to play a role in sulfur homeostasis. In addition, genomic studies have also opened the door to the discovery of new scientific knowledge from perspectives other than organohalide respiration. For example, the crystal structure of Dhaf4260 from D. hafniense DCB-2 was determined as the first representative of the Pfam family PF04016 (DUF364) (DUF: domains of unknown function) in the course of a study aimed at enhancing knowledge regarding the structures of proteins of unknown biological function (Miller et al. 2010). Moreover, DSY3156 (MtgB) from D. hafniense Y51 was characterized as a nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family and was the first glycine betaine:corrinoid methyltransferase characterized (Ticak et al. 2014).

Desulfitobacterium strains are among the ideal model microorganisms for the study of organohalide respiration. Molecular biology tools will become increasingly important in future studies of Desulfitobacterium spp. Transposon mutagenesis using Tn916 and gene disruption using a thermosensitive plasmid have been used in studies of D. dehalogenans JW/IU-DC1, and functional heterologous expression of RDase in D. hafniense strains Y51 and DCB-2 in the Gram-negative gamma-proteobacterium Shimwellia blattae ATCC 33,430 has been demonstrated using molecular tools (Smidt et al. 1999, 2001; Mac Nelly et al. 2014). Recent sequencing efforts of further Desulfitobacterium genomes will also attract researchers to the study of Desulfitobacterium species from perspectives other than organohalide respiration. Combining molecular biology tools with current genomic information will reveal novel insights into the genus Desulfitobacterium.