Revealing formate production from carbon monoxide in wild type and mutants of Rnf- and Ech-containing acetogens,Acetobacterium woodii and Thermoanaerobacter kivui

Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H₂ plus CO₂ or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H₂-dependent CO₂ reductase and furthermore belong to different classes of ‘Rnf’- and ‘Ech-acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g⁻¹ h⁻¹ for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

Anaerobic biodegradation of methyl esters byAcetobacterium woodii andEubacterium limosum

Summary The ability ofAcetobacterium woodii andEubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H₂ requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO₂ allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Acetate synthesis from 2 CO2 in acetogenic bacteria: is carbon monoxide an intermediate?

Cultures of Acetobacterium woodii and Clostridium thermoaceticum growing on fructose or glucose, respectively, were found to produce small, but significant amounts of carbon monoxide. In the gas phase of the cultures up to 53 ppm CO were determined. The carbon monoxide production was completely inhibited by 1 mM cyanide. Cultures and cell suspensions of both acetogens incorporated ¹⁴CO specifically into the carboxyl group of acetate. This CO fixation into C₁ of acetate was unaffected by cyanide (1 mM). The findings are taken to indicate that CO (in a bound form) is the physiological precursor of the C₁ of acetate in acetate synthesis from CO₂. The cyanide inhibition experiments support the hypothesis that the cyanide-sensitive carbon monoxide dehydrogenase may serve to reduce CO₂ to CO rather than to incorporate the carbonyl into C₁ of acetate.     

Physiological changes in white lupin associated with variation in root-zone CO2 concentration and cluster-root P mobilization

White lupin (Lupinus albus L.) mobilizes insoluble soil phosphorus through exudation of organic acids from ‘cluster’ roots. Organic acid synthesis requires anaplerotic carbon derived from dark CO₂ fixation involving PEP-carboxylase. We tested the hypothesis that variation in root-zone CO₂ concentration would influence organic acid synthesis and thus P mobilization. Root-zone CO₂ concentrations and soil FePO₄ concentrations supplied to sand-grown white lupin (cv. Kiev Mutant) were varied. More biomass accumulated in plants supplied with 360 µL L⁻¹ CO₂ to the root-zone, compared with those aerated with either 100 or 6000 µL L⁻¹ CO₂. Increased FePO₄ in the sand resulted in greater leaf P concentrations, but root-zone [CO₂] did not influence leaf P concentration. Suppression of cluster-root development in plants supplied with 100 µL L⁻¹ root-zone CO₂ was correlated with increased leaf [P]. However, at both 360 and 6000 µL L⁻¹ CO₂, cluster-root development was suppressed only at the highest leaf P concentration. Phloem sap [P] was significantly increased by greater [FePO₄] in the sand, but was reduced with increased root-zone [CO₂], and this may have triggered increased cluster-root initiation. Succinate was the major organic acid (carboxylate) in the phloem sap (minor components included malate, citrate, fumarate) and was increased at greater [FePO₄], suggesting that this shoot-derived carboxylate might provide an important source of organic acids for root metabolism. Since cluster root development was inhibited by increasing concentrations of FePO₄ in the sand, it is possible that succinate was utilized for the functioning of the root-nodules.     

Electron availability in CO2, CO and H2 mixtures constrains flux distribution,energy management and product formation in Clostridium ljungdahlii

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO₂ footprint by converting industrial emissions containing CO₂, CO and H₂ into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO₂ + H₂, CO and CO + CO₂ + H₂ (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD⁺ provider for CO₂ + H_2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd_red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd_red formation when CO is used as substrate, whereas Fd_red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO₂ + H₂. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.     

Growth ofClostridium thermoaceticum on H2/CO2 or CO as energy source

The type strain Fontaine ofClostridium thermoaceticum proliferated on H₂/CO₂ as energy source and was culturally adapted to grow on 100% CO in the headspace. The doubling times at 55°C on CO or H₂/CO₂ were 16 and 18 h, respectively. Under these conditions, the substrate-product transformation stoichiometries observed were: 4H₂+2.1CO₂→0.9 acetate and 4CO→2CO₂+1.1 acetate. It is concluded thatC. thermoaceticum has a single carbon growth physiology.     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Ruminococcus hydrogenotrophicus sp. nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces

A new H₂/CO₂-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject. The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli. The isolates grew autotrophically by metabolizing H₂/CO₂ to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds. The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate. The G+C content of DNA of strain S5a33 was 45.2 mol%. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed. The type strain of R. hydrogenotrophicus is S5a33 (DSM 10507). Furthermore, H₂/CO₂ acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus). Received: 11 April 1996 / Accepted: 11 June 1996     

Mixotrophy in the termite gut acetogen,Sporomusa termitida

Cell suspensions of H₂/CO₂-grown Sporomusa termitida catalyzed an H₂-supported synthesis of acetate from CO₂ at rates of about 1 mol acetate x h^-1 x mg protein^-1. Cells pre-grown on methanol, mannitol, lactate, or glycine also displayed H₂-supported acetogenesis from CO₂, although at rates 5–85% that of H₂/CO₂-grown cells. With methanol-grown cell suspensions: the presence of methanol greatly stimulated the rate of H₂-supported conversion of ¹⁴CO₂ to ¹⁴C-acetate (which became labeled mainly in the COOH-group); and like-wise the presence of H₂ stimulated the conversion of ¹⁴CH₃OH+CO₂ to ¹⁴C-acetate (which became labeled mainlyan the CH₃-group). Analogous stimulatory effects were observed for cell suspensions pre-grown on methanol + CO₂+H₂. Furthermore, when H₂ (+CO₂) was included as a growth substrate with either methanol or lactate: both substrates were used simultaneously; there was no diauxie in the growth of cells or in acetate production; and the molar growth yield of S. termitida was close to that predicted from summation of the yields observed when grown with each substrate alone. These data indicated that S. termitida can grow by mixotrophy, i.e. by the simultaneous use of H₂/CO₂ and organic compounds for energy. Results are discussed in light of the ability of H₂/CO₂ acetogens to outprocess methanogens in H₂ consumption in the hindgut fermentation of wood-feeding termites.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

Ordered PtPb/Pt Core/Shell Nanodisks as Highly Active,Selective, and Stable Catalysts for Methanol Reformation to H2

Herein, we report on unique bimetallic PtPb/Pt core/shell nanodisks consisting of structurally ordered PtPb hexagonal nanoplates as the core and the well‐organized Pt as the shell, as extremely active and selective catalysts towards CH₃OH reformation. We found that the created Pt‐Pb nanodisks/C show the composition‐dependent activity with the optimized PtPb_0.56 nanodisks/C being the most active for the CH₃OH reformation to H₂, 5.1 times higher than those of the commercial Pt/C. Significantly, only very limited carbon monoxide (CO) is produced during the CH₃OH reformation, which is crucial for the practical application in fuel cells. The PtPb_0.56 nanodisks/C is also more active for CH₃OH reformation than PtPb hexagonal nanoplates/C and PtPb_0.58 nanoparticles/C. X‐ray photoelectron spectroscopy (XPS) results reveal that the high ratio of Pt (0) to Pt (II) in Pt‐Pb nanodisks/C enhances the CH₃OH reformation to H₂, while the high content of Pb (0) is beneficial for decrease the CO production. Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) of CO adsorption shows that Pt‐Pb nanodisks can promote the activation of CO molecules by forming the carboxylate (CO₂^δ?) intermediates, leading to the low CO production.     

Bioprocess strategies for enhancing biomolecules productivity in Chlorella fusca LEB 111 using CO2 a carbon source

The aim of this study was to evaluate the influence of different carbon dioxide (CO₂) concentrations on the distribution of carbon forms in the culture medium and the biomass production and biomolecules productivity of the strain Chlorella fusca LEB 111. In this study, experiments were carried out in which C. fusca cultures were exposed to different CO₂ concentrations, 0.03% (0.08 ml_CO2 ml_medium⁻¹ days⁻¹), 5% (0.18 ml_CO2 ml_medium⁻¹ days⁻¹), and 15% vol/vol CO₂ (0.54 ml_CO2 ml_medium⁻¹ days⁻¹). Among the carbon chemical species distributions in the culture medium, bicarbonate was predominant (94.2–98.9%), with the highest quantitative percentage in the experiment receiving a 15% CO₂ injection. C. fusca LEB 111 cultivated with 15% CO₂ showed the highest biomass productivity (194.3 mg L⁻¹ days⁻¹) and CO₂ fixation rate (390.9 mg L⁻¹ days⁻¹). The carbohydrate productivity in the culture that received 15% CO₂ was 46.2% higher than the value verified for the culture with the addition of CO₂ from the air (0.03% CO₂). In addition, CO₂ concentration providing increases of 0.03–15% to C. fusca cultures resulted in a 31.6% increase in the lipid productivity. These results showed that C. fusca can be used for CO₂ bioconversion and for producing biomass with potential applications for biofuels and bioproducts.     

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO₂) fixation by toxic metals such as cadmium – classified as a priority pollutant – could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO₂ and labelled ¹³C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO₂ fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (μXRF)/micro X-ray absorption near-edge structure (μXANES) at Cd L_III-edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the ¹³C/¹²C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO₂ decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.     

The interaction of D-galactonic acid with CrVI and CrIII. Structure,stability and physical properties of CrIII—aldonate complexes

The redox reaction between D-galactonic acid and potassium chromate yields ((lyxonateH₋₁)(galactonateH₋₁)Cr(OH₂))K·H₂O_n, with both aldonate molecules acting as bidentate ligands with the carboxylate and one alkoxo function as the donor sites. The shift of the CO₂^{poststaggered−} stretching vibration towards lower frequencies upon coordination and the high value of Δv indicate that the carboxylate acts as a monodentate donor site. Magnetic susceptibility data for the compound in the temperature range 3–300 K exhibit a drop in the effective magnetic moment with temperature below 70 K, which is indicative of antiferromagnetic interactions between the Cr^III centres. The molar magnetic susceptibility versus temperature plot could be fitted with the Fisher Hamiltonian for the case of infinite chains, equation-modified for the presence of monomeric species. The EPR and UV-Vis spectroscopic studies reveal that, in solution, the complex retains the distorted octahedral local coordination geometry. The ((lyxonateH₋₁)(galactonateH₋₁)Cr(OH₂))K_n dissociates slowly in aqueous solution but faster at high [H⁺], because of the rapid protonation of the alkoxo bridges linking the monomeric units. The potentiometric evaluation of the closely related binary system Cr^III-d-galactonate shows that the (Cr(galactonateH_−n)₂)^{1 − 2n} complexes are the major species in the 4–12 pH range, when a 1:2 Cr^III:ligand ratio is used. ¹³C NMR reveals that theCO₂^{poststaggered−} group is one of the coordination sites of the ligand.     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Cladode development for Opuntia ficus-indica (Cactaceae) under current and doubled CO2 concentrations

Morphological and anatomical changes for first-order daughter cladodes (flattened stem segments) of a prickly pear cactus, Opuntia ficus-indica, were monitored to determine the effects of a doubled atmospheric CO₂ concentration on their development and mature form. For daughter cladodes developing in controlled environment chambers for 60 d, maximal elongation rates were similar under a photosynthetic photon flux density (PPFD) of 6 mol m⁻² d⁻¹ and a CO₂ concentration of 370 μl liter⁻¹, an increased PPFD (10 mol m⁻² d⁻¹), and an increased PPFD and a doubled CO₂ concentration. These maximal rates, however, occurred at 20, 15, and 12 d, respectively. The maximal relative growth rate under the doubled CO₂ concentration was about twice that under the other conditions. For cladodes at 60 d as well as after 4 and 16 mo in open-top chambers, doubling the CO₂ concentration had no effect on final length or width. At 4 mo, cladodes under doubled C0₂ were 27% thicker, perhaps allowing the earlier production of second-order daughter cladodes. The chlorenchyma was then 31% thicker and composed of longer cells. At 16 mo, the difference in cladode thickness diminished, but the chlorenchyma remained thicker under doubled CO₂, which may contribute to greater net CO₂ uptake for O. ficus-indica under elevated CO₂ concentrations. Two other persistent differences were a 20% lower stomatal frequency and a 30% thicker cuticle with more epicuticular wax for cladodes under doubled CO₂, both of which may help reduce transpirational water loss.     

Metabolic selectivity and growth of<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis> in continuous culture under elevated hydrostatic pressure

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10² relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H₂, CO₂) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.