Complete Genome Sequence of the Haloalkaliphilic, Hydrogen-Producing Bacterium Halanaerobium hydrogeniformans

Abstract

Halanaerobium hydrogenoformans is an alkaliphilic bacterium capable of biohydrogen production at pH 11 and 7% (wt/vol) salt. We present the 2.6-Mb genome sequence to provide insights into its physiology and potential for bioenergy applications.

GENOME ANNOUNCEMENT

Hydrogen has numerous applications as a clean energy carrier with water and heat as by-products. For industrial purposes, it is currently produced abiotically from natural gas and coal (9). Microorganisms are being explored for chemical and biofuel production, including future biohydrogen generation at industrial scales. Growth in biofuel production will most likely be derived from lignocellulosic biomass. However, pretreatment of biomass, required so that the sugars can be accessible, is carried out with regimens ranging from alkaline to acidic conditions, often including elevated temperatures, with each step adding to the final product cost (6).

Halanaerobium hydrogeniformans (formerly Halanaerobium sapolanicus) was isolated from haloalkaline (pH ∼10, 15- to 140-g/liter NaCl), anaerobic sediments of Soap Lake, WA, with extraordinarily high sulfide concentrations of up to 10 g/liter (1, 3, 8, 12). It is an obligately anaerobic, Gram-negative, nonmotile, nonsporulating, elongated rod. It can utilize a range of C₅ and C₆ sugars with optimal growth at pH 11, 7% (wt/vol) NaCl, and 33°C, producing acetate, formate, and hydrogen as major metabolic end products. The genome sequence for H. hydrogenoformans was determined to improve assessment of its metabolic and bioenergy potential, particularly toward improving alkaline or haloalkaline pretreatment regimens for robust hydrogen production by this bacterium.

The H. hydrogeniformans genome sequence was determined through a combination of Illumina (1a) and 454 (7) technologies. We constructed and sequenced an Illumina GAii shotgun library which generated 27,639,916 reads totaling 2,100 Mb, a 454 Titanium standard library generated from 77,351 reads, and a paired-end 454 library with an average insert size of 10.607 ± 2.651 kb that generated 160,293 reads totaling 82.3 Mb of 454 data. A total of 486 additional reactions and 6 shatter libraries were necessary to close gaps and to raise the finished sequence quality. Methods for determining the genome sequence were previously described (5), and this is a “finished” genome (2).

The total genome size is 2,613,116 bp, with final assembly based on 52.2 Mb of 454 draft data providing an average 21.5× genome coverage and 463 Mb of Illumina draft data providing an average 178× genome coverage. The genome is 33.1% G+C and contains 2,295 candidate protein-encoding gene models. The genome contains four separate rRNA operons, each containing a 5S, a 16S, and a 23S rRNA gene, with 99.9 to 100% identity between 16S rRNA genes. The closest significant 16S rRNA gene matches (GenBank accession number GQ215697) were to Halanaerobium sp. strain AN-B15B (97.2% similarity, AM157647.1), Halanaerobium praevalens DSM 2228 (97%, AB022035.1), Halanaerobium sp. strain KT-2/3-3 (96.9%, AJ309519.1), and Halanaerobium acetethylicum (96.4%, NR_036958.1) (4, 10, 11). All comparative species are physiologically different as they are neutrophilic. The Halanaerobium hydrogeniformans genome sequence will enable a range of studies into halotolerance and alkaliphily, will facilitate a better understanding of the potential of this organism for bioenergy applications, and will expand our knowledge of the physiology of this genus.

Nucleotide sequence accession number.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP002304. The version described in this paper is the first version, CP002304.