1. Introduction
Solventogenic
Clostridia have been used to produce the commodity chemicals acetone, butanol, and ethanol from renewable feedstocks via the acetone, butanol, and ethanol (ABE) fermentation process [
1,
2]. The model solventogenic organism is
Clostridium acetobutylicum, which ferments a variety of carbohydrates that is prevalent in biomass [
3]. In batch culture, the organism’s metabolism proceeds from an initial acidogenic phase, producing mostly acetate and butyrate, to a solventogenic phase, where acetate and butyrate are reabsorbed, and along with a carbohydrate, are converted to acetone, butanol, and ethanol [
1]. The redox state of the organism is important for the relative distribution of acids and solvents produced [
4]. Hence, there have been numerous demonstrations of altering metabolic output by manipulation of electron flow via metabolic inhibitors, feedstock selection, and genetic engineering [
4,
5,
6,
7].
An important mechanism for maintaining redox balance in
C. acetobutylicum is the production of H
2 [
8]. Electron flow through H
2 in
Clostridium spp. is primarily controlled by two types of hydrogenases: H
2-evolving hydrogenases and H
2-uptake/respiratory hydrogenases [
9]. [FeFe]-hydrogenases are responsible for the production of H
2, whereas [NiFe]-hydrogenases are involved in H
2 oxidation and possibly the production of NADH/NAD(P)H [
9].
Internal production of H
2 by the [FeFe]-hydrogenase increases intracellular pH (via proton reduction) and it provides oxidized ferredoxin for central metabolism [
8,
10]. Oxidized ferredoxin is required for the major pyruvate decarboxylating enzyme, pyruvate ferredoxin oxidoreductase (PFOR), and the butyryl-CoA dehydrogenase (BCD), which is essential for butyrate and butanol production [
1,
11]. There are two HydA [FeFe]-hydrogenase genes in
C. acetobutylicum: HydA1, which is constitutively expressed, and HydA2, which is induced during solventogenesis [
10,
12]. While the HydA1 operates primarily in the direction of H
2 production during batch fermentations, the enzyme reaction is reversible and it is important for the H
2 dependent reduction of nitroaromatic compounds [
5,
10].
The [NiFe]-hydrogenase is the lesser studied hydrogenase of
Clostridia, pervasive among the different species, and is believed to be involved in H
2 uptake [
9]. An siRNA knock down of the [NiFe]-hydrogenase in
C. saccharoperbutylacetonicum N1, to our knowledge the only published study of
Clostridial [NiFe]-hydrogenase activity, showed a marked decrease in butanol production, thereby indicating physiological evidence of hydrogen uptake [
13]. A bioinformatics analysis of
Clostridial hydrogenases indicates that
C. acetobutylicum ATCC 824 encodes probable a group 1 [NiFe]-hydrogenase, and the maturation factors genes are located on both the chromosome and pSol megaplasmid [
9]. The predicted protein sequences are similar to the
C. saccharoperbutylacetonicum N1 [NiFe]-hydrogenase, and previous studies have shown
C. acetobutylicum mRNA expression from the corresponding genes is induced during solventogenesis [
12]. Increased expression during solvent phase suggests the
C. acetobutylicum [NiFe]-hydrogenase is important for solventogenic growth phase, but its exact role is not understood.
In this report, we begin to elucidate the function of C. acetobutylicum’s [NiFe]-hydrogenase and associated maturation factors. Interruption of the [NiFe]-hydrogenase maturation factor hypF homolog gene ca_c0810, and the subsequent gene in the operon, hypD homolog ca_c0811, inhibited hydrogenase activity, thereby indicating their role in maturation. The hypF/hypD mutant displayed altered metabolism, as consistent with a defect in hydrogen uptake, resulting in decreased ABE output and altered product ratios in early solventogenesis. Palladium nanoparticle formation at the site of hydrogen gas oxidation identified that the [NiFe]-hydrogenase is membrane localized with the catalytic subunit facing the extracellular side of the membrane. Calculated net reduced cofactor production over time for WT and mutant strains indicate a role in NADH production for the [NiFe]-hydrogenase during solventogenesis. Together with energetic calculations and sequence analysis, the results indicate the [NiFe]-hydrogenase likely couples H2 oxidation with electron transport and intracellular NAD+ reduction, thereby acting as a crude proton pump that conserves energy via hydrogen cycling.
2. Materials and Methods
2.1. Bacterial Strain Propagation
Clostridium acetobutylicum ATCC 824 (Wild type, or WT) was obtained from American Type Culture Collection (ATCC), and
Clostridium acetobutylicum M5 (M5), which lacks the pSol megaplasmid, was provided by the Papoutsakis laboratory [
14]. Wild type and derivative strains were maintained as anaerobic spore suspensions at room temperature in potato glucose medium (PGM) containing: 150.0 g·L
−1 potato (grated), 10.0 g·L
−1 glucose, 0.5 g·L
−1 (NH
4)
2SO
4, and 3.0 g·L
−1 CaCO
3 [
15]. The media was boiled for one hour, strained, and then autoclaved. M5 was maintained under anaerobic conditions on
Clostridial growth medium (CGM) agarose plates, as previously described [
6,
16].
C. acetobutylicum strains were propagated in CGM or P2 medium containing 6.0% glucose, as previously described [
12]. Cultures were grown in a Coy anaerobic chamber (Coy Lab Products) at 37 °C in an atmosphere of 5.0% H
2, 5.0% CO
2, and 90.0% N
2.
2.2. Construction of HypF/HypD Mutant
The
hypF annotated gene,
ca_c0810, was disrupted using a group II intron based system (ClosTron) [
17]. An intron insertion site was predicted at 153|154s in the sense sequence using the TargeTron Gene Knockout System website (MilliporeSigma, St. Louis, MO, USA) [
18]. The target insert was designed using the intron prediction primers, obtained from Biobasic, and cloned into pMTL007C-E2 (
Table S2) (GenBank accession no. HQ263410.1) [
19]. pMTL007C-E2:
CachypF was transformed into WT
C. acetobutylicum ATCC 824, mutants were screened as previously described, and then confirmed via PCR and southern blot [
20,
21] (see
Table S1 and Figure S1). One mutant was verified to have integration in
ca_c0810 and was designated CacATCC824-
hypF:CT(
ΔhypF/
hypD).
2.3. Construction of Complement Strains
The
ca_c0810/
ca_c0811 complement plasmid was designed using the phosphotransbutyrylase promoter (ptb) sequence followed by the full
ca_c0810/
ca_c0811 genetic sequence, position 935667 to 939040 on the WT
C. acetobutylicum ATCC 824 genome, and was synthesized by Biobasic (Genbank accession nos. NP_347446 and NC_003030.1). The sequence was cloned into the
AscI/
SbfI sites of pMTL007C-E2, retaining the
repH,
catP,
ColEI, and
traJ elements and designated pMTL007C-E2:
PtbhypFhypD. pMTL007C-E2:
PtbhypFhypD was transformed into the
ΔhypF/
hypD strain and control complement strains were created by the addition of an empty pMTL007C-E2 plasmid into
ΔhypF/
hypD and WT
C. acetobutylicum ATCC 824, as previously described [
21]. The complement strains were designated CacATCC824-
hypF:PtbhypFhypD (ΔhypF/
hypD:p-hypF/
hypD), CacATCC824-
hypF:pMTL(ΔhypF/hypD:pMTL), and CacATCC824
:pMTL(WT:pMTL).
2.4. Sample Collection for RNA Seq Data
Static planktonic cultures were grown in P2 medium containing 6.0% glucose, as previously described [
12]. Twenty milliliters of overnight cultures of WT
C. acetobutylicum and
ΔhypF/
hypD were grown to OD
600 of 0.8. Two milliliters of the overnight culture was then sub-cultured in triplicate to glass bottles containing 60.00 mL glucoseP2 medium. Two milliliters samples of planktonic cultures were collected at 24 h post sub-culture, which is the time point Liu et al. described to have the highest expression level of the genes of interest [
12]. Samples were treated with a final concentration of 30 µg·mL
−1 rifampicin, incubated for 10 min on ice, and then treated with RNA protect (Qiagen, Hilden, Germany), as previously described [
16].
2.5. RNA Extraction, Purification, and rRNA Depletion
Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, with an additional homogenization and mechanical disruption step using a bead beater (BioSpec, Bartlesville, OK, USA) with Zirconia/Silica beads (BioSpec). RNA quality was assessed using the 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA), quantified with a Qubit fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA), and then stored at −80 °C prior to DNase treatment. DNA was removed using the TURBO DNA-free kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. RNA was again quantified and quality assessed, as stated above. Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit for Gram-Positive Bacteria (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. The quality of rRNA depleted samples was assessed once more prior to processing for sequencing library generation.
2.6. Sequencing Library Preparation
TruSeq Stranded mRNA Sample Preparation Kit (Illumina) was used to prepare the rRNA depleted RNA for sequencing, according to the manufacturer’s protocol. Libraries were quantified using the Kapa Library Quantification Kit (KapaBiosystems, Wilmington, MA, USA), according to the manufacturer’s instructions, then normalized and pooled for sequencing, according to the Denature and Dilute Libraries Guide for the NextSeq 500 (Illumina, San Diego, CA, USA). Pooled libraries were paired end sequenced on a NextSeq 500 (Illumina, San Diego, CA, USA).
2.7. RNA-Seq Data Analysis
Samples had an average of 20 million reads each, were assessed for quality using FastQC, and then trimmed to remove Illumina adaptors and low quality bases using Trimmomatic [
22]. Operon structure predictions were performed using Rockhopper [
23].
2.8. Hydrogenase Zymogram
Hydrogenase assay steps were performed under anaerobic conditions. Five milliliters cultures of
ΔhypF/
hypD, WT, M5, and
ΔhypF/
hypD:P-hypF/
hypD were grown in CGM containing 6.0% glucose at 37 °C to an OD
600 of 1.2. Cells were isolated via centrifugation (6000 rpm for 10 min in a Corning LSE centrifuge) and the resulting pellets were subsequently washed twice with 10 mL of PBS (MilliporeSigma, St. Louis, MO, USA), isolating cells between washes by centrifugation, as above. The pellets were then suspended in 800 µL CelLytic™ B Plus (MilliporeSigma, St. Louis, MO, USA), 1.0% dodecyl maltoside, and benzonase, and then subjected to 10 consecutive 15 s intervals of sonication on ice. The lysates were subjected to non-denaturing PAGE on Mini-PROTEAN
® TGX™ gels (Bio-Rad Laboratories Inc., Hercules, CA, USA). Hydrogenase activity was visualized by incubation of gel in 50.0 mM MOPs buffer (pH 8.0), 1.0 mM benzyl viologen (BV), and 2.0 mM 2,3,5,-triphenyltetrazolium chloride (TTC), as previously described by Pinske et al. [
24].
2.9. Growth and Metabolite Studies
Agitated and static planktonic culture studies were performed in CGM containing 5% glucose and grown in DasGip (Eppendorf, Hamburg, Germany) bioreactors (Eppendorf) for 48 h. Two hundred milliliters overnight cultures of WT and ΔhypF/hypD were grown to OD600 of 0.8 in CGM containing 5.0% glucose under anaerobic conditions. Thirty milliliters were then sub-cultured into 1 L of CGM containing 5.0% glucose in DasGip (Eppendorf, Hamburg, Germany) bioreactors under anaerobic conditions with Rushton impellors running at either 400 rpm or 0 rpm, in duplicate. The headspace of the bioreactors were flushed with N2. Samples for metabolite analysis were taken every 24 h. The samples were clarified via filtration through a 0.2 μm PES syringe filter (Corning, NY, USA) and stored at −20 °C.
Sessile culture studies were performed in comparison to static planktonic cultures in P2 medium containing 6.0% glucose in glass bottles, as previously described [
12]. Twenty milliliters overnight cultures of WT and
ΔhypF/
hypD were grown to OD
600 of 0.8. Two milliliters were then sub-cultured into glass bottles under anaerobic conditions (without N
2 headspace flushing) containing 60.0 mL P2 medium, with either 2 g of autoclaved cotton balls for sessile studies, or without cotton balls for static planktonic culture studies, both in triplicate. The complement strains,
ΔhypF/
hypD:p-hypF/
hypD,
ΔhypF/
hypD:pMTL, and WT:pMTL were grown overnight in similar conditions, except the overnight cultures contained final concentrations of 30 µg·mL
−1 chloramphenicol for all strains and 40 µg·mL
−1 for the
ΔhypF/
hypD:p-hypF/
hypD and
ΔhypF/
hypD:pMTL strains. Two milliliters of the overnight complement strains were then sub-cultured into glass bottles under anaerobic conditions (without N
2 headspace flushing) containing 60.0 mL P2 medium and a final concentration of 30 µg·mL
−1 chloramphenicol, with either 2 g of autoclaved cotton balls for sessile studies, or without cotton balls for static planktonic culture studies, all in triplicate. The cultures were spiked with a final concentration of 30 µg·mL
−1 of chloramphenicol every 48 h to maintain the plasmids. Samples for metabolite analysis were taken every 24 h for 144 h. Metabolite samples were clarified via filtration through a 0.2 μm PES syringe filter (Corning) and then stored at −20 °C. HPLC analysis of samples for metabolite concentrations was performed on an Agilent 1200, as previously described [
6,
25]. Samples were injected into the HPLC system, eluted isocratically, and quantified using an external calibration curve of pure known components as standards.
2.10. Reduction of Palladium
To determine location of the NiFe hydrogenases, 20 mL WT
C. acetobutylicum ATCC 824, heat killed (1 h at 80 °C) WT
C. acetobutylicum ATCC 824, pSol null
C. acetobutylicum M5, and [NiFe]-hydrogenase mutant
C. acetobutylicum Δ810/811 grown to OD
600 of 1.5 in 6.0% glucose CGM were centrifuged for 15 min at 6000 RPM (Corning LSE centrifuge) in anaerobic conditions, and then the supernatant was removed. The pellets were washed twice with 20 mL of 20 mM MOPS buffer (pH 7.2) and re-suspended with 20 mL of the same buffer. The resting cells were sparged with oxygen free nitrogen (OFN) for 10 min. Two millimolar Pd(II) solution was made by dissolving sodium tetrachloropalladate (Na
2PdCl
4) in 0.01 M HNO
3, pH 2.3 [
26]. The Pd(II) solution was degassed for 10 min, then flushed with OFN for 10 min. The Pd(II) solution was added to the suspension of resting cells to give a mass ration of 1:4 Pd(II): dry biomass. The mixture was incubated for 30 min at 37 °C, with occasional shaking to allow for initial absorption of Pd(II). Pd(II) reduction was initiated by flushing the mixture with H
2 gas for 2 h at 37 °C.
2.11. Sample Preparation for TEM
The Pd loaded bacteria were rinsed twice with 20 mL distilled water, fixed in 1 mL of 2.5% (w/v) glutaraldehyde for 30 min, centrifuged, resuspended in 0.5 mL 0.1 M phosphate buffer (pH 7), and stained in a final concentration of 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7) for 60 min. The bacteria after staining were centrifuged and the pellets were freeze-dried. The dried bacterial samples were embedded in epoxy resin (low viscosity embedding kit) (Electron Microscopy Sciences, Hatfield, PA, USA) and cured overnight at 60 °C in the oven. Samples for TEM were prepared using a Leica EM UC7 ultramicrotome with a Diatome 35° wet diamond knife (Leica Microsystems, Buffalo Grove, IL, USA). Sections were cut from the embedded samples at room temperature to a thickness of approximately 200 nm. Sections were then coated with a 2 nm carbon film using a Leica Microsystems EM ACE600 high vacuum sputter coater equipped with a carbon thread coater (Leica Microsystems, Buffalo Grove, IL, USA).
2.12. TEM Imaging of Samples
The scanning transmission electron microscopy (STEM) mode of a JEOL JEM-2100F TEM (JEOL USA Inc., Peabody, MA, USA) equipped with a Gatan 806 high-angle annular dark field (HAADF) detector (Gatan, Pleasanton, CA, USA) was used to collect STEM-HAADF images from each sample. The microscope was operated at 200 kV, with a spot size of 0.2 nm, and a 40 μm condenser aperture that gave a convergence angle of 8.3 mrad. The STEM-HAADF collection angle range was 48–148 mrad. Gatan Microscopy Suite version 1.85 with DigiScan was used to collect the images.
2.13. Energy Filtered TEM
Energy Filtered TEM (EFTEM) jump ratio and elemental map images were acquired from C-K, O-K, and Pd-L edges using Gatan’s suggested energy windows. For each filtered image, ten images were acquired at a hardware binned value of 8 to give 256 × 256 pixel images at an exposure that minimized drift effects. Individual image exposures were typically 5 s.
2.14. Reduction Potential Calculations
Total reaction potentials for a range of external pH values were calculated by summing the potentials of individual oxidoreduction reactions and electron transport across the membrane. All of the calculations assumed standard conditions and an H
2 concentration of 1 bar. Previously determined pH-dependent membrane potentials, proton motive force (pmf), and intracellular vs. extracellular pH values were utilized [
27]. Reduction potentials for NAD
+/NADH and NADP
+/NADPH were calculated for solvent phase cells using the Nernst equation and intracellular concentrations reported by Amador-Noguez et al. [
28]. Electron transport values were obtained using the previously reported membrane potentials and assumed transport of two electrons [
27]. Additional details of the calculations are available in the
supplemental material Figure S4.
4. Discussion
Control of electron flow is essential to engineer metabolism for the production of desired outputs [
39]. Key enzymes for maintaining redox balance are the hydrogenases, which can direct electron flow to proton reduction. Hydrogenases can produce and/or consume H
2, depending on metabolic state and/or the electron carrying cofactors used. In
C. acetobutylicum, the majority of hydrogenase studies have focused on the [FeFe]-hydrogenases, which are important for ferredoxin recycling and the reduction of xenobiotic compounds [
1,
5]. The saccharolytic nature of
C. acetobutylicum and the associated high metabolic flux through PFOR leads to a strong demand for ferredoxin oxidation by the [FeFe]-hydrogenase in growth conditions important for solvent formation [
40]. While it has been demonstrated that the [FeFe]-hydrogenase oxidizes H
2 in vitro, the physiologically relevant need for ferredoxin oxidation indicates that its primary role is H
2 production [
10]. During acidogenesis, the H
2/CO
2 ratio is approximately 1.2, but this decreases to ~0.7 during solventogenesis as a result of rerouting the electron flow from H
2 to alcohol formation [
5,
6]. Reduction of carbohydrates and carboxylic acids to alcohols during solventogenesis changes the requirements for reduced cofactors, increasing the demand for NADH and NADPH.
A bioinformatics based classification of
Clostridia hydrogenases indicated
C. acetobutylicum encodes a probable group 1 membrane associated [NiFe]-hydrogenase implicated in hydrogen uptake [
9]. Increased transcript levels for the
C. acetobutylicum [NiFe]-hydrogenase and maturation genes were observed during late solventogenesis in sessile cells, and evidence of H
2 uptake by the [NiFe]-hydrogenase in a related solventogenic organism indicated a solvent phase role for the
C. acetobutylicum [NiFe]-hydrogenase [
12,
13].
C. acetobutylicum is unique because the hydrogenase genes and maturation factor genes are located on different genetic elements [
9]: the chromosome and pSol megaplasmid, respectively (
Figure 1). In the current study, the [NiFe]-maturation pathway was disrupted via interruption of
ca_c0810,
hypF, using the ClosTron type II intron system. A likely consequence of the
hypF mutation was reduced HypD expression because the
hypD gene is downstream of
hypF in a polycistronic operon. Zymograms were used to show hydrogenase activity was similarly reduced in the
ΔhypF/
hypD mutant and M5 strain, providing evidence that chromosomally encoded HypF and HypD are important for the maturation of the megaplasmid encoded [NiFe]-hydrogenase. Furthermore, maturation of [FeFe]-hydrogenases requires a different set of maturation factors, so a disruption of [NiFe]-maturation was not expected to alter functional [FeFe]-hydrogenase production.
Acid and ABE formation were examined under several growth conditions to begin elucidating the [NiFe}-hydrogenase’s metabolic role. The first condition comprised cultures subject to agitation in bioreactors, where the headspace was flushed with N
2. Under these growth conditions, levels of dissolved H
2 would be comparatively lower than other conditions examined, reducing impacts from potential loss of H
2 uptake in the
ΔhypF/
hypD mutant [
6]. WT and the
ΔhypF/
hypD mutant in the first condition produced very little solvents and similar amounts of acetate and butyrate, indicating the [NiFe]-hydrogenase was not necessary for acidogenesis. The second condition examined planktonic cultures in bioreactors with zero agitation, where the headspace was flushed with N
2. This would lead to relatively higher dissolved H
2 levels as compared to agitated cultures. Under these conditions, the [NiFe]-hydrogenase appeared to be important for solvent production, since the
ΔhypF/
hypD mutant produced fewer solvents than the WT after 24 h, as shown in
Table 1. A third condition, static planktonic growth in defined medium in an anaerobic chamber without N
2 headspace flushing, exacerbated this effect. In the static planktonic conditions the
ΔhypF/
hypD mutant produced higher butyrate and lower butanol levels when compared to WT, thereby indicating the mutant was defective in reduction of butyrate to butanol or was less tolerant to butanol accumulation. The reduced capacity to produce butanol could be due to low NADH and NADPH availability, as reduction of butyryl-CoA to butanol is thought to require one NADH and one NADPH [
1]. The apparent lack of reduced cofactor availability in the
ΔhypF/
hypD mutant during static planktonic growth indicates that the [NiFe]-hydrogenase is important for reducing NAD
+ and/or NADP
+ via hydrogen uptake.
To further test if the [NiFe]-hydrogenase was important for solvent formation, metabolite output was examined under a fourth growth condition consisting of sessile cultures grown in a defined medium in an anaerobic chamber without N
2 headspace flushing. A recent study showed that growth in sessile conditions enhanced butanol production and that the [NiFe]-hydrogenase genes were induced during these conditions [
41]. The
ΔhypF/
hypD mutant exhibited delayed growth, as indicated by the small changes in metabolite concentrations between 0 and 24 h shown in
Figure 3. The
ΔhypF/
hypD mutant was ultimately able to recover, and during early solventogenesis the mutant had a higher acetone to butanol ratio than the WT, which is consistent with the inability to uptake H
2 for the reduction of butyryl-CoA to butanol. This is in agreement with a recent study showing a lack of reduced NAD(P)H availability increased acetone output in solvent phase [
42]. Solventogenesis stalled in the sessile
ΔhypF/
hypD mutant cultures between 48 h and 72 h, but resumed thereafter, presumably due to a shift in metabolism. During the solventogenic stall there was an increase in calculated net NADH formed from acid production when normalized to glucose consumption, suggesting there was compensation due to disruption of an NADH production pathway in the
ΔhypF/
hypD mutant. In the latter stages of solventogenesis of the sessile
ΔhypF/
hypD mutant cultures there was an increase in butanol, but not acetone or ethanol, and there was little to no net uptake of acetate and butyrate. This altered output/uptake profile suggests the
ΔhypF/
hypD mutant has a different electron flow for maintaining redox balance when compared to WT.
In order to determine the [NiFe]-hydrogenase’s location, EFTEM Pd elemental mapping was used to identify locations of Pd(0) nanoparticles that were formed by
C. acetobutylicum strains using H
2 gas as an electron donor. Pd(II) would be reduced to palladium(0) nanoparticles at the catalytic site of H
2 oxidation and thus identify the location of the hydrogenase [
43]. Comparisons of the EFTEM mapping results between the strains indicated that the [NiFe]-hydrogenase is membrane localized with the catalytic subunit facing the extracellular side of membrane. This is evidenced by the decrease of Pd(0) nanoparticle deposits on the
ΔhypF/
hypD and M5 pSol megaplasmid null strain, when compared to wild type.
It was not possible to definitively determine the electron acceptor of the [NiFe]-hydrogenase based upon sequence analysis, but the putative heme binding protein encoded by
ca_p0144 is a strong candidate. A common mechanism for hydrogen uptake [NiFe]-hydrogenases is reduction of a cytochrome during H
2 oxidation [
44]. The reduced cytochrome then serves as an electron shuttle to move electrons from the hydrogenase to other enzyme systems. The location of the [NiFe]-hydrogenase on the extracellular face of the membrane suggests that a mechanism likely exists for transfer of electrons to and intracellular enzyme system. Based upon previously measured cellular parameters, it was calculated that NAD
+ reduction could be coupled to membrane transport of two electrons. The calculations were performed assuming 1 bar of H
2. Planktonic cultures can be supersaturated for H
2, resulting in an increase of reaction potentials, which could make some of the reactions, that were calculated to be slightly below zero, favorable [
45].
Evidence presented above shows it is likely the [NiFe]-hydrogenase that allows for extracellular H
2 oxidation coupled to reduction of an intracellular electron acceptor. This activity, combined with the [FeFe]-hydrogenase actvity, allows for hydrogen cycling, which could serve as a crude proton pump (see
Figure 5B), similar to the mechanism of hydrogen cycling characterized in
Desulfovibrio [
38]. Butanol has been shown to disrupt the pmf of
C. acetobutylicum, and the [NiFe]-hydrogenase could counter this disruption during solvent production [
46]. In this scenario, the [NiFe]-hydrogenase could improve the efficiency of pmf maintenance through hydrogen cycling while providing the NADH needed for alcohol production. It is plausible that disruption of hydrogenase maturation, resulting in an inability to maintain pmf, was responsible for the initial stalling in solvent phase growth of the sessile
ΔhypF/
hypD mutant cultures.
During solventogenesis the [NiFe]-hydrogenase could generate the required NADH, but an NADPH source would still be required for butanol dehydrogenase. NADPH could be supplied by the ferredoxin:NAD(P) reductase. A recent report indicated augmented ferredoxin:NAD(P) reductase expression during the solvent phase increases relative butanol output, presumably by redirecting electron flow from H
2 production [
41,
47]. An alternative NADPH source could be NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) [
48]. A previous study showed that the
gapN and [NiFe]-hydrogenase genes were induced during growth in sessile cultures, while genes for the glyceraldehyde-3-phosphate and phosphoglycerate kinase were repressed [
12]. This suggests the route through glycolysis could be altered to compensate for increased NADPH demand. Calculations of reduction potentials using previously measured NAD
+/NADH, NADP/NADP
+, and internal pH values (see
supplemental information) indicated the physiologic reduction potential is ~−260 mV for NAD
+/NADH and ~−320 mV for NADP/NADP
+. Due to this disparity, it is plausible that the cells have separate mechanisms to recycle the two cofactors.
This study demonstrates, to the best of our knowledge, the first evidence of hydrogen cycling as a potential energy conserving mechanism in Clostridium. C. acetobutylicum encodes for a group 4 [NiFe]-hydrogenase, not a group 1, that participates in hydrogen cycling to maintain the pmf for energy conservation and most likely produce NADH. The work highlights the need to improve knowledge of electron flow and membrane energetics in C. acetobutylicum in order to guide engineering strategies for directing product formation. Additionally, understanding how the ΔhypF/hypD mutant re-routed its metabolism during the latter phases of solventogenesis, when the major output was butanol, could lead to new strategies for the selective production of butanol.