Genetic diversity of hydrogen-producing bacteria in an acidophilic ethanol-H2-coproducing system, analyzed using the [Fe]-hydrogenase gene

Abstract

Hydrogen gas (H2) produced by bacterial fermentation of biomass can be a sustainable energy source. The ability to produce H2 gas during anaerobic fermentation was previously thought to be restricted to a few species within the genera Clostridium and Enterobacter. This work reports genomic evidence for the presence of novel H2-producing bacteria (HPB) in acidophilic ethanol-H2-coproducing communities that were enriched using molasses wastewater. The majority of the enriched dominant populations in the acidophilic ethanol-H2-coproducing system were affiliated with low-G+C-content gram-positive bacteria, Bacteroidetes, and Actinobacteria, based on the 16S rRNA gene. However, PCR primers designed to specifically target bacterial hydA yielded 17 unique hydA sequences whose amino acid sequences differed from those of known HPB. The putative ethanol-H2-coproducing bacteria comprised 11 novel phylotypes closely related to Ethanoligenens harbinense, Clostridium thermocellum, and Clostridium saccharoperbutylacetonicum. Furthermore, analysis of the alcohol dehydrogenase isoenzyme also pointed to an E. harbinense-like organism, which is known to have a high conversion rate of carbohydrate to H2 and ethanol. We also found six novel HPB that were associated with lactate-, propionate-, and butyrate-oxidizing bacteria in the acidophilic H2-producing sludge. Thus, the microbial ecology of mesophilic and acidophilic H2 fermentation involves many other bacteria in addition to Clostridium and Enterobacter.

FIG. 1.

Fermentation products in the two reactors. (A and B) H₂ production in reactors A and B, respectively. •, H₂ production rate; ○, H₂ conversion efficiency. (C and D) Concentrations of VFAs and alcohol and pHs in reactors A and B, respectively. ○, acetic acid; ▪, propionic acid; □, butyric acid; ▴, valeric acid; •, ethanol; ▵, lactate; ▿, pH. Error bars, means ± standard deviations obtained from three replications.

FIG. 2.

DGGE profiles of the PCR-amplified V3 regions of the 16S rRNA genes in the microbial communities from two reactors. (A) Reactor A; (B) reactor B. Lanes are labeled with the time of sampling. Arrows indicate the DGGE bands selected for cloning and sequencing.

FIG. 3.

Collector's curves of observed and estimated phylotype richness of 16S rRNA gene clone libraries by the DOTUR program. (A) Reactor A; (B) reactor B. Estimator curves include observed OTUs (bottom), Chao1 (middle), and ACE (abundance-based coverage estimator) (top). Phylotypes were defined using the 99% OTU cutoff.

FIG. 4.

Phylogenetic tree derived from 16S rRNA gene clone libraries. Each row represents a different OTU (phylotype). The phyla are color coded as follows, from top to bottom: green, low-G+C-content gram-positive bacteria; blue, Actinobacteria; black, unclassified; yellow, Alphaproteobacteria; red, Bacteroidetes. The relative abundances of phylotypes from the 16S rRNA gene clone libraries of the two reactors are shown on the right in grayscale values. Letters above the abundance graph correspond to two different enrichments.

FIG. 5.

Relative abundances of sequences from the 16S rRNA gene libraries of two reactors (A and B). The sequence frequencies are grouped according to phylum. “Unknown” sequences are unclassified.

FIG. 6.

DGGE profiles of partial hydA genes in the microbial communities from two reactors (A and B, respectively). Lanes are labeled with the time of sampling. Arrows with numbers indicate the DGGE bands selected for cloning and sequencing. The initial RNA template was standardized for RT-PCR. The PCR product was loaded for every lane and was not standardized.

FIG. 7.

Phylogenetic tree derived from alignments of partial amino acid sequences of [Fe]-H₂ases, including the sequences of DGGE bands and clone libraries and sequences from the database. GenBank accession numbers are given in parentheses. Bootstrap values of >50% for neighbor joining are shown (percentages of 1,000 resamplings). Bar indicates 1% divergence. ○, sequences from the hydA clone library of reactor A; •, sequences from the clone library of reactor B; □, bands excised from the DGGE gel (see Fig. 6). Numbers after the hyphen (b1 to b5) represent bands. Different clusters of hydA derived from the two reactors are highlighted by different colors.

FIG. 8.

Clone libraries of partial hydA genes from reactors A and B. Bar graph shows percentages of Clostridium thermocellum-like (C. t), Syntrophomonas wolfei-like (S. w), Ethanoligenens harbinense-like (E. h), Megasphaera elsdenii-like (M. e), Clostridium saccharoperbutylacetonicum-like (C. s), and Syntrophobacter fumaroxidans-like (S. f) organisms in each library. The number of clones is given in parentheses above each bar.

FIG. 9.

Zymograms of ADHs from anaerobic sludge. (A) Reactor A; (B) reactor B. Lanes are labeled with the time of sampling. Roman numerals on the left represent zymotypes. The specificities of ADHs were defined by their activities toward the substrate ethanol.