Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-)  + 2 NAD(+)  → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.

The thermophilic acetogenic bacterium Thermoanaerobacter kivui, previously described not to use carbon monoxide as a carbon and energy source, was adapted to grow on CO. This was achieved by using a preculture grown on H 2 plus CO 2 and by increasing the CO concentration in small, 10% increments. T. kivui was finally able to grow within a 100% CO atmosphere. Growth on CO was found in complex and mineral media, and vitamins were not required. Carbon monoxide consumption was accompanied by acetate and hydrogen production. Cells also grew on synthesis gas (syngas) with the simultaneous use of CO and H 2 coupled to acetate production. CO oxidation in resting cells was coupled to hydrogen and acetate production and accompanied by the synthesis of ATP. A protonophore abolished ATP synthesis but stimulated H 2 production, which is consistent with a chemiosmotic mechanism of ATP synthesis. Hydrogenase activity was highest in crude extracts of CO-grown cells, and carbon monoxide dehydrogenase (CODH) activity was highest in H 2 -plus-CO 2 -or CO-grown cells. The genome of T. kivui harbors two CODH gene clusters, and both CODH proteins were present in crude extracts, but one CODH was more prevalent in crude extracts from CO-grown cells. Carbon monoxide is a colorless, odorless gas, which is toxic to most organisms in trace amounts. However, some organisms can use carbon monoxide as an electron and carbon source (1, 2). These organisms are aerobic carboxydotrophic bacteria such as Oligotropha carboxydovorans (3), phototrophic purple sulfur bacteria such as Rhodospirillum rubrum (4), hydrogenogenic bacteria and archaea such as Thermosinus carboxydivorans (5) or Thermococcus sp. strain AM4 (6), or some organisms that employ the Wood-Ljungdahl pathway (WLP), such as methanogens (7-9) or acetogens (10-13). Acetogenic bacteria use the WLP for CO 2 fixation to acetate. This pathway is considered one of the most ancient biochemical pathways for CO 2 fixation, as it combines two essential features: CO 2 fixation and the synthesis of ATP (14, 15). The use of carbon monoxide as an electron donor for the WLP has the advantage of providing extremely low potential electrons for reducing cellular electron carriers with a CO 2 /CO reduction potential of Ϫ520 mV (16).The production of third-generation biofuels from carbon dioxide, molecular hydrogen, and/or carbon monoxide as catalyzed by, for example, acetogenic bacteria is a promising alternative for existing biofuel production routes from renewable sources (17-19). However, it is still in its infancy with respect to knowledge on the biochemistry and bioenergetics of CO oxidation and CO 2 reduction in many acetogens. Several acetogenic representatives have been shown to use CO as the sole energy source, such as Butyribacterium methylotrophicum (CO strain) (20), Eubacterium limosum (21), Blautia producta (22), Clostridium thermoautotrophicum (11), Moorella thermoacetica (12), and Clostridium ljungdahlii (23). The extent of CO tolerance and consumption varies greatly between different spec...

BackgroundAcetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2 + CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.ResultsThe genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G + C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2 + CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).ConclusionsThe thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2 + CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.