Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum

The bacterial phylum Synergistetes is an under-represented phylogenetic cluster of Gram-negative, anaerobic, rod-shaped bacteria that are related to Synergistes jonesii (Jumas-Bilak et al., 2009). Members of this group have been isolated from a variety of environments including anaerobic digesters (LaPara et al., 2000; Wu et al., 2001), the termite hindgut (Ohkuma & Kudo, 1996), subgingival plaque (Munson et al., 2004) and petroleum reservoirs (Orphan et al., 2000; van der Kraan et al., 2010; Voordouw et al., 1996). The species in this group with validly published names display diverse physiological properties, but all currently cultivated strains catabolize amino acids (Jumas-Bilak et al., 2009; Vartoukian et al., 2007). It has been noted that these organisms, although underrepresented amongst cultivated taxa, are not uncultivable but are present at low cell numbers in microbial communities (Godon et al., 2005). The genus Anaerobaculum, comprising Anaerobaculum thermoterrenum (Rees et al., 1997) and Anaerobaculum mobile (Menes & Muxí, 2002), is a member of the phylum Synergistetes, but the ability of Anaerobaculum species to catabolize amino acids has not been studied. However, their capacity to produce hydrogen gas from sugars has been documented (Menes & Muxí, 2002).

Increasing global demand for fossil fuels and a need to decrease carbon dioxide emissions have driven research towards renewable, carbon-neutral energy sources. Hydrogen has the potential to be a sustainable alternative to some fossil fuels. It is a clean fuel that burns with no carbon dioxide emissions and the energy released can be easily converted to electricity by fuel cells (Davila-Vazquez et al., 2008). Hydrogen has an energy yield of 122 kJ g⁻¹, which is 2.75 times greater than that of hydrocarbon-derived fuels (Kapdan & Kargi, 2006). Presently, about 95 % of the world’s hydrogen is produced via steam reformation of natural gas, which does not lower net carbon dioxide emissions, and is resource- and energy-intensive (Lee et al., 2008). The production of hydrogen from renewable resources such as biomass or waste materials is being explored as a sustainable global fuel source. ‘Dark-fermentative’ bacteria can produce hydrogen from carbohydrates and other waste products (Angenent et al., 2004; Han & Shin, 2004; Li & Fang, 2007) without the need for light input (Nandi & Sengupta, 1998), according to the equation

The Gibbs’ free energy of formation given above was taken from Thauer et al. (1977), and is essentially the same as that given by Kengen et al. (2009).

Four hydrogen molecules can be produced per d-glucose molecule via this thermodynamically favourable reaction, with acetate and carbon dioxide as additional products. However, only a handful of organisms have been isolated that produce amounts of hydrogen close to this, such as Acetomicrobium faecalis (Winter et al., 1987), Anaerobaculum mobile (Menes & Muxí, 2002), Caldicellulosiruptor saccharolyticus (van Niel et al., 2002), Thermotoga maritima (Schröder et al., 1994) and Thermotoga elfii (van Niel et al., 2002). An objective of the United States Department of Energy is exploration of novel biological catalysts that can produce four hydrogen molecules per molecule of glucose (US Department of Energy, 2007).

The hydrogen-producing bacterium strain OS1^T isolated from oil production water is described in this study. On the basis of phenotypic and genotypic evidence, strain OS1^T is proposed to represent a new species of the genus Anaerobaculum. The description of the genus is also emended to include the amino acids catabolized by the three species reported to date. Strain OS1^T can produce almost four hydrogen molecules per molecule of d-glucose, which approaches the theoretical maximum via the aforementioned reaction. It also has the ability to produce hydrogen from a variety of amino acids and other organic acids.

Strain OS1^T was isolated from oil production water collected from Alaska, USA, as part of a study of the microbiology of a North Slope oil facility (Duncan et al., 2009). A most probable number (MPN) enumeration of general heterotrophs was conducted at 50 °C using anaerobic half-strength tryptic soy broth (TSB; Difco) with 1 % NaCl and 100 % nitrogen gas phase (Balch & Wolfe, 1976). Strain OS1^T was the dominant culturable heterotroph, present in the production water at 2.3 cells ml⁻¹ (MPN). The dominant culturable hydrogen oxidizer, Methanothermobacter thermautotrophicus, was present at the same cell density (Duncan et al., 2009). The Hungate agar roll-tube method was used for isolation (Hungate, 1969). Colonies on anaerobic roll tubes were small, circular, smooth and yellowish in colour. Exponential-phase cells grown on d-glucose were used for transmission electron microscopy. Cells were fixed with 1 % glutaraldehyde, spread onto carbon-coated Formvar grids and stained with 0.5 % phosphotungstate (pH 7.0). Cells were photographed using a JEOL JEM 2000 FX transmission electron microscope. Cells were Gram-negative, non-flagellated, non-motile, non-spore-forming rods, 1.7–2.7×0.4–0.5 µm, that occurred singly (see Fig. S1, available in IJSEM Online).

Strain OS1^T was cultivated routinely at 55 °C on anaerobic TSB plus 1 % NaCl. Cells grown under these conditions can be lyophilized for long-term preservation, provided adequate attention is given to the maintenance of anaerobic conditions. The temperature and NaCl range and optima were determined using TSB as the growth medium. Growth (optical density) was measured spectrophotometrically at 600 nm using a Spectronic 20D (Thermo Spectronic). Strain OS1^T grew optimally at 55 °C (range 40–65 °C) with 1 % NaCl (range 0.8–7 %). For determination of the pH range and optimum, TSB was amended with the following buffers (at 10 g l⁻¹): HOMOPIPES, pH 4 and 5; MES, pH 6 and 6.5; TES, pH 7 and 7.5; TAPSO, pH 8; TAPS, pH 8.5; CAPSO, pH 9 and 9.5 (Tanner, 2007). The final pH was adjusted at 55 °C. Strain OS1^T grew optimally at pH 7.5 (range pH 6–9). A mineral medium was developed for routine growth, containing (l⁻¹) 10 ml mineral solution (Tanner, 2007), 10 ml vitamin solution (Tanner, 2007), 10 ml trace metal solution (Tanner, 2007), 10 g NaCl, 2 g yeast extract (Difco) and 3 g d-glucose. The medium was buffered with 10 g TES at pH 7.5. The medium was prepared using strict anaerobic technique under a final gas phase of 100 % nitrogen (Balch & Wolfe, 1976).

Nitrate reduction and sulfide production were measured using CHEMetrics test kits (CHEMetrics, Inc). Nitrate, l-cystine, sulfate, thiosulfate and sulfite were added to the medium from sterile stock solutions. Elemental sulfur was added and sterilized as described previously (Rees et al., 1997). Crotonate reduction was measured by HPLC using a Shimadzu LC-20AT with an SPD-20A UV/Vis detector equipped with an Aminex HPX-87H ion exclusion column (Bio-Rad) using 0.001 M H₂SO₄ as the mobile phase at a flow rate of 0.9 ml min⁻¹. Strain OS1^T reduced elemental sulfur, thiosulfate and l-cystine to sulfide when grown in anaerobic TSB plus 1 % NaCl. Nitrate, sulfite and sulfate reduction were not observed. Strain OS1^T reduced crotonate to butyrate in the presence of glucose but did not ferment crotonate alone. Sulfur and thiosulfate reduction, as well as crotonate reduction in the presence of d-glucose, seems to be a universal phenotype of the genus Anaerobaculum (Menes & Muxí, 2002; Rees et al., 1997).

Genomic DNA was isolated from strain OS1^T using a modification of the Marmur procedure (Johnson, 1994). The G+C content of the genomic DNA was measured by HPLC, as described previously (Mesbah et al., 1989), with modifications described previously (Allen et al., 2008). 16S rRNA gene sequencing was conducted using DNA as a template for PCR amplification using the universal primers 27f, 357f, 704f, 926f, 907r and 1492r corresponding to the Escherichia coli numbering system (Johnson, 1994). Sequencing was performed by the Oklahoma Medical Research Foundation (Oklahoma City, OK, USA). Primer sequences were aligned using the Sequencher software suite (Gene Codes Corporation), which resulted in a 1444-base contiguous DNA sequence. The phylogenetic position of strain OS1^T was assessed via maximum-likelihood analysis (Olsen et al., 1994). A consensus tree was generated by bootstrapping at values greater than 90 % confidence (Felsenstein, 1985) (Fig. 1). The topology and major branching points of the phylogenetic tree were confirmed by neighbour-joining and maximum-parsimony analyses in the arb software suite (Ludwig et al., 2004) (not shown).

16S rRNA gene sequence analysis indicated that strain OS1^T was a member of the genus Anaerobaculum (Fig. 1). A. thermoterrenum and A. mobile are currently the two described members of this genus (Menes & Muxí, 2002; Rees et al., 1997), which are members of the phylum Synergistetes (Hugenholtz et al., 2009; Vartoukian et al., 2007). The 16S rRNA gene of strain OS1^T was 99.7 % similar to that of the type strain of A. thermoterrenum and 97.8 % similar to that of the type strain of A. mobile. It may not be surprising to find a close relative to A. thermoterrenum in oil production water, because the type strain of this species was isolated from a similar environment (Rees et al., 1997). Culture-independent surveys have shown the presence of Anaerobaculum strains in petroleum reservoirs (GenBank accession numbers GU357467 and EU573105) (Gieg et al., 2010; Kaster et al., 2009), mining wastewater (DQ256300) (Gihring et al., 2006), a solid waste digestor (EF559029) (Li et al., 2009) and methanogenic reactors (AB234001, FN563242, FN563270 and AB274508) (Krakat et al., 2011; Sasaki et al., 2006, 2007). A. mobile was isolated from an anaerobic wastewater treatment lagoon (Menes & Muxí, 2002). This suggests that strains of Anaerobaculum may be present in many anaerobic environments, particularly thermophilic ones. The G+C content of the genomic DNA from strain OS1^T was 46.6 mol%, compared with 44 mol% for A. thermoterrenum (Rees et al., 1997) and 51.5 mol% for A. mobile (Menes & Muxí, 2002).

DNA–DNA reassociation analysis was necessary for taxonomic placement of strain OS1^T, and was conducted in the laboratory of Dr Peter Schumann at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) as described by De Ley et al. (1970), with the modifications described by Escara & Hutton (1980) and Huß et al. (1983). Analysis was performed using a model 2600 spectrophotometer equipped with a model 2257-R thermoprogrammer and plotter (Gilford Instrument Laboratories). Rates of renaturation were computed with the program transfer.bas (Jahnke, 1992). A DNA–DNA reassociation value of 68±4 % was observed when strain OS1^T was hybridized to A. thermoterrenum DSM 13490^T, which is close to the threshold value of 70 % for the definition of species (Stackebrandt & Ebers, 2006), suggesting that strain OS1^T may belong to a species distinct from A. thermoterrenum.

A. thermoterrenum DSM 13490^T and A. mobile DSM 13181^T were obtained from the DSMZ and used in characterization assays. The basal medium used for routine growth of these strains was the mineral medium described above. Growth experiments were conducted in triplicate. All substrates were added from sterile anaerobic stock solutions to a final concentration of 3 g l⁻¹ before inoculation. d-Glucose-grown cells were used as the inoculum for substrate tests. Growth and end-product formation were compared with substrate-unamended controls. Chemicals used in this work were obtained from Sigma-Aldrich unless otherwise noted. Growth was measured spectrophotometrically at 600 nm (Spectronic 20D) (Balch & Wolfe, 1976). Phenotypic characteristics of strain OS1^T are listed in the species description and in Table 1. Several of these substrates were not tested in the original descriptions of A. thermoterrenum (Rees et al., 1997) and A. mobile (Menes & Muxí, 2002), nor was the ability to utilize amino acids as substrates, a characteristic of members of the phylum Synergistetes (Vartoukian et al., 2007). The list of substrates examined for the genus Anaerobaculum, as well as a comparison of the phenotypes of A. thermoterrenum DSM 13490^T, A. mobile DSM 13181^T and strain OS1^T, is given in Table 1.

As shown in Table 1, strain OS1^T can be readily differentiated from species of Anaerobaculum by several phenotypic characteristics, such as the ability to utilize malonate, maltose and inulin and the inability to catabolize l-malate, glycerol and starch. Strain OS1^T had an absolute requirement for NaCl for growth, which the other two species do not share. In addition, strain OS1^T grew in medium containing up to 7 % NaCl, which is much higher than the maxima of 2.0 and 1.5 % observed for cultures of A. thermoterrenum (Rees et al., 1997) and A. mobile (Menes & Muxí, 2002), respectively.

Cellular fatty acid methyl ester (FAME) and polar lipid analyses were conducted on strain OS1^T, A. thermoterrenum DSM 13490^T and A. mobile DSM 13181^T. Cells were grown at 55 °C in anaerobic TSB supplemented with 1 % NaCl containing a 100 % nitrogen gas phase. FAME and polar lipid analyses were carried out by the Identification Service of the DSMZ and Dr B. J. Tindall (DSMZ). The fatty acid profiles for all three strains are shown in Table 2. FAME analysis showed that all three strains contained iso-C_15 : 0 and iso-C_11 : 0, with iso-C_15 : 0 as the predominant fatty acid. Strain OS1^T contained both iso-C_13 : 0 and iso-C_13 : 0 3-OH. However, A. thermoterrenum DSM 13490^T did not have iso-C_13 : 0 3-OH and A. mobile DSM 13181^T did not have iso-C_13 : 0. Polar lipid analysis results are summarized in Table S1. Polar lipid analysis showed that all three strains contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids (PL1–PL3) and four unknown aminophospholipids (PN1–PN4). Strain OS1^T and A. thermoterrenum DSM 13490^T were shown to contain phospholipids PL4 and PL5, but these were not detected in A. mobile DSM 13181^T.

Both A. thermoterrenum and A. mobile produced hydrogen, acetate and carbon dioxide when grown in pure culture on d-glucose. A. mobile was shown to grow syntrophically with a methanogen using d-glucose as the substrate (Menes & Muxí, 2002). Syntrophic studies were conducted using strain OS1^T and the hydrogen-oxidizing methanogen M. thermautotrophicus DSM 1053^T. The syntrophic medium was the mineral medium described above, but 3 g NaHCO₃ was added after adjustment to pH 7.5. All syntrophic and pure-culture fermentation studies were carried out in 160 ml crimp-sealed serum bottles with 10 ml medium at 55 °C with d-glucose (3 g l⁻¹) as the substrate. Acetate was measured with a Shimadzu GC-8A equipped with a flame-ionization detector and a 2 m glass column packed with an 80/120 Carbopak B-DA/4 % Carbowax resin 20M (Supelco) with helium as the carrier gas at a flow rate of 35 ml min⁻¹. Methane, carbon dioxide and hydrogen were measured with a Shimadzu GC-8A GC equipped with a thermal conductivity detector and a Porapak Q (2 m stainless steel) column (Alltech) using nitrogen as the carrier gas at a flow rate of 30 ml min⁻¹. Results from the pure and co-culture work are presented in Table 3. Strain OS1^T, A. thermoterrenum DSM 13490^T and A. mobile DSM 13181^T all produced close to four hydrogen molecules per molecule of d-glucose and one molecule of methane per molecule of d-glucose when grown syntrophically with a hydrogen-oxidizing methanogen. In pure culture, strain OS1^T produced up to 3.1–4.5 mmol hydrogen (l culture)⁻¹ using clarified raw sewage as the substrate (Maune & Tanner, 2008).

On the basis of the genotypic and phenotypic data presented, strain OS1^T is proposed to represent a novel species of the genus Anaerobaculum, for which the name Anaerobaculum hydrogeniformans sp. nov. is proposed.