Energetics and Application of Heterotrophy in Acetogenic Bacteria

Abstract

Acetogenic bacteria are a diverse group of strictly anaerobic bacteria that utilize the Wood-Ljungdahl pathway for CO2 fixation and energy conservation. These microorganisms play an important part in the global carbon cycle and are a key component of the anaerobic food web. Their most prominent metabolic feature is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates. However, most members also show an outstanding metabolic flexibility for utilizing a vast variety of different substrates. In contrast to autotrophic growth, which is hardly competitive, metabolic flexibility is seen as a key ability of acetogens to compete in ecosystems and might explain the almost-ubiquitous distribution of acetogenic bacteria in anoxic environments. This review covers the latest findings with respect to the heterotrophic metabolism of acetogenic bacteria, including utilization of carbohydrates, lactate, and different alcohols, especially in the model acetogen Acetobacterium woodii Modularity of metabolism, a key concept of pathway design in synthetic biology, together with electron bifurcation, to overcome energetic barriers, appears to be the basis for the amazing substrate spectrum. At the same time, acetogens depend on only a relatively small number of enzymes to expand the substrate spectrum. We will discuss the energetic advantages of coupling CO2 reduction to fermentations that exploit otherwise-inaccessible substrates and the ecological advantages, as well as the biotechnological applications of the heterotrophic metabolism of acetogens.

FIG 1

The Wood-Ljungdahl pathway of acetogenic bacteria. [H], redox equivalent (one electron + one proton); THF, tetrahydrofolate; CoFe-SP, corrinoid iron-sulfur protein. In A. woodii, the utilized redox equivalents are H₂ for the formate dehydrogenase step, NADH for the methylene-THF dehydrogenase and methylene-THF reductase steps, and reduced ferredoxin for the CO dehydrogenase/acetyl-CoA synthase step.

FIG 2

The heterotrophic metabolism of A. woodii, the model organism of the Rnf-dependent acetogens, is composed of well-defined modules. (A) Most heterotrophic substrates (e.g., glucose, lactate, ethanol) are oxidized to the level of acetyl-CoA by transferring the electrons to NAD⁺ and ferredoxin. Acetyl-CoA is converted to acetate, generating 1 ATP by SLP. (B) The module responsible for redox balancing between modules A and C. This is achieved either by a soluble hydrogenase using flavin-based electron bifurcation or by a membrane-bound Rnf complex. The latter is coupled to ATP synthesis via an electrochemical sodium ion gradient and a Na⁺-dependent F₁F_O ATP synthase/ATPase. Note that some acetogens have Ech instead of Rnf, and in both, Ech- and Rnf-containing species, the coupling ion may be Na⁺ or H⁺. (C) The WLP functions as an electron sink to reoxidize the electron carriers by reducing CO₂ to acetate. Fd_red, reduced ferredoxin (reduced by 2 electrons).

FIG 3

Carbohydrate (A) and lactate (B) metabolism of A. woodii. The inset in panel B represents the energetics (ΔG^0′ profile for the conversion of the corresponding substrates to the products/intermediates) of the oxidation of lactate to acetyl-CoA not coupled (blue) or coupled (orange) to flavin-based electron bifurcation. Blue line, lactate is oxidized with NAD⁺ as the electron acceptor, followed by ferredoxin-dependent oxidation of pyruvate. Orange line, lactate is oxidized with NAD⁺ as the electron acceptor, coupled to NAD⁺ reduction by reduced ferredoxin by electron bifurcation, and pyruvate is oxidized with ferredoxin as the electron acceptor. EMP, Embden-Meyerhof-Parnas pathway (glycolysis); Fd_red, reduced ferredoxin (reduced by 2 electrons).

FIG 4

Ethanol (A) and 2,3-butanediol (B) metabolism of A. woodii. The inset in panel A represents the energetics (ΔG^0′ profile for the conversion of the corresponding substrates to the products/intermediates) of the oxidation of ethanol to acetyl-CoA. Both ethanol and acetaldehyde are oxidized with NAD⁺ as the electron acceptor. AdhE, bifunctional acetaldehyde/ethanol dehydrogenase; Fd_red, reduced ferredoxin (reduced by 2 electrons).

FIG 5

Role of acetogens in the anaerobic food web. The methanogenic degradation in sulfate-poor environments, such as freshwater sediments, is characterized by initial hydrolysis of polymers followed by the conversion of monomers by primary fermenting bacteria. Alcohols, short-chain fatty acids, and other intermediary products are converted by secondary fermenting bacteria (syntrophs) to acetate, H₂, and CO₂ before these substrates are used by methanogenic archaea for methanogenesis. Under low hydrogen pressure in a well-balanced fermentation, the major routes are labeled with a B. Higher hydrogen pressures lead to an increased flux through the routes marked with an A. Conversions where acetogens can participate are shown in orange.

FIG 6

Synthetic biology approach to increase the yield coefficient of ethanol formation. Coupling the WLP to the formation of ethanol from glucose can increase the theoretical maximum yield from 2 to 3 molecules of ethanol formed per glucose molecule. The necessary energy is provided by SLP and chemiosmotic energy conservation via the Rnf complex. Electrons can be provided, for example, by molecular hydrogen or by direct electron uptake from a cathode. EMP, Embden-Meyerhof-Parnas pathway (glycolysis); Fd_red, reduced ferredoxin (reduced by 2 electrons).