Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

Background The C. bescii genome does not encode an acetaldehyde/alcohol dehydrogenase or an acetaldehyde dehydrogenase and no ethanol production is detected in this strain. The recent introduction of an NADH-dependent AdhE from C. thermocellum (Fig. 1a) in an ldh mutant of this strain resulted in production of ethanol from un-pretreated switchgrass, but the thermolability of the C. thermocellum AdhE at the optimum growth temperature of C. bescii (78 °C) meant that ethanol was not produced above 65 °C.Fig. 1 Proposed scheme for the pyruvate to ethanol pathway in C. thermocellum and T. pseudethanolicus 39E. a The C. thermocellum ethanol pathway. The red colored AdhE (Cthe_0423) is already expressed and tested in C. bescii [26]. b The T. pseudethanolicus 39E ethanol pathway. The green colored AdhE (Teth39_0206) and blue colored AdhB (Teth39_0218) are expressed and tested in C. bescii in this study Results The adhB and adhE genes from Thermoanaerobacter pseudethanolicus 39E, an anaerobic thermophile that produces ethanol as a major fermentation product at 70 °C, were cloned and expressed in an ldh deletion mutant of C. bescii. The engineered strains produced ethanol at 75 °C, near the ethanol boiling point. The AdhB expressing strain produced ethanol (1.4 mM on Avicel, 0.4 mM on switchgrass) as well as acetate (13.0 mM on Avicel, 15.7 mM on switchgrass). The AdhE expressing strain produced more ethanol (2.3 mM on Avicel, 1.6 mM on switchgrass) and reduced levels of acetate (12.3 mM on Avicel, 15.1 mM on switchgrass). These engineered strains produce cellulosic ethanol at the highest temperature of any microorganism to date. In addition, the addition of 40 mM MOPS to the growth medium increased the maximal growth yield of C. bescii by approximately twofold. Conclusions The utilization of thermostable enzymes will be critical to achieving high temperature CBP in bacteria such as C. bescii. The ability to produce ethanol at 75 °C, near its boiling point, raises the possibility that process optimization could allow in situ product removal of this end product to mitigate ethanol toxicity. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0346-4) contains supplementary material, which is available to authorized users.


Background
Production of renewable and sustainable biofuels from lignocellulosic biomass using thermophilic bacteria has attracted increasing attention because of the potential advantages of using high temperature fermentation, including improved kinetics, increased mixing rate, lower oxygen solubility, reduced risk of microbial contamination, reduced costs for cooling and heating during the bioconversion process, and providing the possibility of ethanol separation in continuous cultures [1][2][3][4][5][6]. Fermentative thermophilic anaerobic bacteria have been used to convert plant biomass into ethanol without added lignocellulose-degrading enzymes using a process called consolidated bioprocessing (CBP) [7][8][9]. Much of the CBP research to date has focused on Clostridium thermocellum [10][11][12], which grows at 60 °C and produces ethanol, acetate, lactate, formate, CO 2 , H 2 , and amino acids as major fermentation products [8,13]. Increased ethanol production and tolerance of this microorganism have been achieved by extensive metabolic engineering [14][15][16][17][18][19][20][21]. However, while C. thermocellum has the ability to deconstruct crystalline cellulose and catabolize hexose Open Access *Correspondence: janwest@uga.edu † Daehwan Chung and Minseok Cha contributed equally to this study 1 Department of Genetics, University of Georgia, Athens, GA, USA Full list of author information is available at the end of the article sugars, it cannot utilize hemicellulosic pentose sugars such as xylose and arabinose for growth and its optimal growth temperature is 60 °C [8,12]. To fully realize the potential of thermophilic CBP, several major improvements over the state of the art will be needed, including increased ethanol yield and titer, co-utilization of hemicellulosic sugars, and ethanol production near or above the boiling point of ethanol (~78 °C).
Caldicellulosiruptor bescii, previously known as "Anaerocellum thermophilum", is an anaerobic, Grampositive bacterium originally isolated from the hot springs of Kamchatka, Russia [22][23][24]. This microorganism has been reported to grow on cellulose up to 83 °C with an optimum temperature of 78 °C at pH 7 and is among the most thermophilic cellulolytic organisms discovered to date [23,24]. It is able to efficiently ferment crystalline cellulose, hemicellulosic sugars, xylan, and un-pretreated plant biomass, such as hardwood poplar, switchgrass and Napier grasses, producing acetate, lactate, and H 2 [12,23]. These characteristics could provide unique advantages over existing CBP approaches, avoiding costly lignocellulose pretreatment and allowing separation of ethanol from continuously growing cultures [25,26].
Recently, new genetic tools have been successfully used for insertion, deletion, and heterologous/homologous expression of genes in C. bescii [26][27][28][29]. Like other near-maximal H 2 -yielding anaerobic microbes, C. bescii does not encode either AdhE (bi-functional acetaldehyde/alcohol dehydrogenase) or ALDH (acetaldehyde dehydrogenase) [30]; thus, no ethanol production is detected in this strain [23]. However, its chromosome encodes two AdhAs (alcohol dehydrogenase), Cbes0928 and Cbes0224 [31]. Introduction of a heterologous ethanol production pathway via expression of the NADHdependent AdhE from C. thermocellum (Fig. 1a) in a C. bescii ldh mutant strain resulted in production of ethanol from both model substrates and un-pretreated switchgrass [26]. Although neither the ethanol yield nor titer was sufficient to meet requirements for economic product recovery, the bioethanol production directly from real-world substrates opens the door to development of the next generation of CBP. In addition, because of the insufficient thermostability of the C. thermocellum AdhE, this engineered strain was only able to produce ethanol up to 65 °C [26], which is far below the C. bescii optimum growth temperature and the ethanol boiling temperature.
To overcome this issue, we sought to identify a more thermostable ethanol pathway to introduce into C. bescii. T. pseudethanolicus 39E is a Gram-positive, anaerobic thermophile that produces ethanol as a major fermentation product and grows up to 70 °C [32][33][34]. It encodes two bi-functional alcohol dehydrogenases, adhB and adhE [4,35]. The bi-functional acetyl-CoA thioesterase/ alcohol dehydrogenase (AdhB) is NADPH-dependent and proposed as a key terminal enzyme for ethanol production in T. pseudethanolicus based on microarray data [4,32]. The other bi-functional acetaldehyde/alcohol dehydrogenase (AdhE) showed NADH-dependent ALDH activity and weak NADPH-dependent ADH activity [32, Fig. 1 Proposed scheme for the pyruvate to ethanol pathway in C. thermocellum and T. pseudethanolicus 39E. a The C. thermocellum ethanol pathway. The red colored AdhE (Cthe_0423) is already expressed and tested in C. bescii [26]. b The T. pseudethanolicus 39E ethanol pathway. The green colored AdhE (Teth39_0206) and blue colored AdhB (Teth39_0218) are expressed and tested in C. bescii in this study 36]. Utilization of more thermostable enzymes such as these will be critical to achieving high temperature CBP in C. bescii. Here, we explore the use of these more thermostable enzymes from T. pseudethanolicus to develop ethanol production pathways that function near the optimal growth temperature in C. bescii. Further, we explore optimization of culture medium buffering capacity as a means of improving growth and fermentation in engineered C. bescii strains under batch conditions.

Results and discussion
Higher buffering capacity of culture medium improves C. bescii growth The pH of cultures during growth is important for anaerobic microorganisms, particularly at higher growth yields [37,38]. C. bescii has a narrow optimal pH range, pH 6.8-7.3, in culture media in closed bottles [24,39] and the production of acetic and lactic acid have been shown to inhibit growth, especially at late growth phases [23,37]. While fed-batch cultivation with pH control in a fermentor, such as that used in industrial processes, can solve this problem, pH control in bench-scale genetic experiments is not practical [40]. An alternative approach is to use high initial buffering capacity to prevent excessive pH changes [41,42]. This has enabled higher growth yields and rates in other microorganisms [43,44]. To systematically investigate the influence of increased buffer concentrations on the growth yield, metabolite production, and final pH values of C. bescii fermentations; 3-(N-morpholino) propane sulfonic acid (MOPS) was used as a buffering reagent in addition to the 2.4 mM phosphate normally used in this medium supplemented with 1 % cellobiose as the carbon source [39]. Growth was tested with MOPS concentrations ranging from 0 to 200 mM to determine if pH was limiting bioconversion (Fig. 2). At 160 mM MOPS, growth was severely inhibited, and virtually no growth was observed at 200 mM MOPS. At lower MOPS concentrations, however, a clear improvement was observed. The maximal growth yield of C. bescii increased approximately twofold as measured by optical density at 680 nm (OD 680 ) when the MOPS concentration in the medium was increased from 0 to 40 mM (Fig. 2a). At 80 and 120 mM MOPS, a similar final OD was attained, but only after a lag period. Because C. bescii can grow at relatively high osmotic pressure (>550 mOsmol) [37,45], the longer lag phase at MOPS concentrations of 80 mM and above suggests a mechanism other than osmotic stress to explain this difference (Fig. 2a) [40,46]. The final pH of the culture medium increased from 4.5 to 5.8 as the MOPS concentration was increased from 0 to 120 mM (Fig. 2d). These results are consistent with previous results in pH-controlled fermenters; the decrease in pH during fermentation is a limiting factor for growth and limits the ability to reach higher cell densities [37]. Substrate conversion into products also increased with MOPS concentration (Fig. 2b-d). H 2 (Fig. 2d) and acetate production (Fig. 2b) showed a steady increase with increased buffer concentrations up to 120 mM MOPS. H 2 production increased 62 % (from 13.9 to 22.6 mM) and acetate production increased 149 % (from 7.2 to 17.9 mM) on 120 mM MOPS compared to the culture without MOPS. The maximum yield of lactate was obtained on 80 mM MOPS (Fig. 2c). In this analysis, 40 mM MOPS was sufficient to sustain exponential growth to maximal growth yield and the fastest doubling time (~2.3 h) with a final pH ~5.1. We, therefore, used 40 mM MOPS for subsequent analyses.

Heterologous expression of the thermostable AdhE and AdhB from T. pseudethanolicus in C. bescii
The T. pseudethanolicus adhE gene (bifunctional secondary ADH/aldehyde hydrogenase, Teth39_0206) and adhB gene (bifunctional secondary ADH/Acetyl-CoA thioesterase, Teth39_0218) were cloned from T. pseudethanolicus 39E under the transcriptional control of the C. bescii S-layer protein (Cbes2303) promoter (P S-layer ) [26,29] (Additional file 1: Figures S1, S2; Table 1). Both the P S-layer Teth39_0206 (adhE) and P S-layer Teth39_0218 (adhB) expression cassettes contain a Rho-independent transcription terminator and C-terminal 6X His-tag to facilitate protein purification and Western blotting. These genes were each inserted into the inter-cistronic region between Cbes0863 and Cbes0864 of the chromosome in C. bescii strain JWCB017 (ΔpyrFA Δldh) (Fig. 3a). Uracil prototrophic transformants were serially passaged as described [26] to allow segregation of merodiploids containing a mixture of the integrated adhE expression cassette and the wild-type genomes. This resulted in strains JWCB049 (ΔpyrFA Δldh CIS1:: P S-layer Teth39_0206), and JWCB054 (ΔpyrFA Δldh CIS1:: P S-layer Teth39_0218) ( Table 1). Verification of JWCB049 and JWCB054 was performed using PCR amplification with primers DC477 and DC478 (Fig. 3b), and sequencing the PCR products. The parent strain, JWCB017, produced the expected wild type 2.44-kb band, whereas amplification of JWCB049 and JWCB054 produced ~5.0-kb and ~ 3.5kb bands, respectively, indicating an insertion of the adhE and adhB expression cassettes within the targeted region (Fig. 3b). The previous deletion of the ldh gene in JWCB017, JWCB049, and JWCB054 was also confirmed (Additional file 1: Figure S3).
To test expression of the T. pseudethanolicus AdhE and AdhB proteins in C. bescii, JWCB049 and JWCB054 were grown in low osmolarity defined (LOD) medium [39] to mid-log phase at 75 °C (Fig. 3c). Cell extracts were then examined by Western hybridization using monoclonal   anti-His antibodies (Fig. 3c). An approximately 100 kDa protein from the AdhE strain (lane 2 in Fig. 3c) and an approximately 40 kDa protein from the AdhB strain (lane 3 in Fig. 3c) were detected, which correspond to the predicted molecular weights for the His-tagged proteins. No proteins were detected by the anti-His antibodies in the parental strain (JWCB017, lane 1 in Fig. 3c). Interestingly, the AdhE protein (lane 2) was less abundant than AdhB (lane 3). The AdhE protein contains a linker region between the ALDH and ADH domains [47] and this kind of linker region has been observed to be susceptible to proteolysis in various multi-domain proteins, which may explain the lower abundance of T. pseudethanolicus AdhE relative to AdhB in C. bescii. Alternatively, even small differences in T. pseudethanolicus codon usage could decrease translational efficiency in C. bescii, which would suggest codon optimization as a future strategy to increase expression.

Engineered C. bescii strains produce ethanol from cellobiose at 75 °C
In previous work to engineer ethanol production in C. bescii, mutation of the lactate dehydrogenase gene (ldh) was obtained in the C. bescii chromosome [48] and an adhE gene from C. thermocellum was introduced [26]. Deletion of the ldh gene resulted in a strain, JWCB017 that produced no lactate and more acetate and hydrogen than the wild type [49]. Addition of the adhE gene to the ldh mutant strain resulted in a strain that produced ethanol and less acetate, presumably because carbon was channeled from acetate to ethanol [26]. The introduction of new fermentation pathways could affect growth and product formation due to potential redox imbalances.
To investigate the functionality of T. pseudethanolicus AdhE and AdhB in C. bescii, strains were characterized in LOD medium supplemented with 40 mM MOPS and 1 % (wt/vol) cellobiose. We first compared growth rate of the strains expressing AdhE (JWCB049) and AdhB (JWCB054) to the parent strain (JWCB017) at 75 °C (Fig. 4). There was no obvious difference in growth rate, with calculated doubling times of 2.0, 1.9, and 1.9 h for JWCB017, JWCB049 and JWCB054, respectively. Further, all three strains reached similar maximal optical densities (~0.8 at OD 680 nm ) after 15 h incubation (Fig. 4a), suggesting that heterologous expression of these genes is not detrimental to C. bescii. Fermentation products were also monitored into the stationary growth phase by high performance liquid chromatography (HPLC) and gas chromatography (Fig. 4b-d), and final product yields were calculated ( Table 2). As expected, none of the strains produced lactate because they contain a deletion of the lactate dehydrogenase gene. C. bescii JWCB017 (ΔpyrFA Δldh) produced 17.0 mM acetate, 23.9 mM H 2 , and no ethanol (Fig. 4b, c), representing 12.9 % conversion of cellobiose into acetate and 91 % carbon recovery overall without accounting for microbial cell mass. The AdhB expressing strain JWCB054 produced 1.4 mM ethanol in addition to 15.5 mM acetate and 23.2 mM H 2 from cellobiose ( Fig. 4b-d), representing 11.6 % conversion of cellobiose into acetate and 1.1 % into ethanol with an overall 88 % carbon recovery ( Table 2). The AdhE expressing strain JWCB049 produced approximately twice as much ethanol (2.9 mM) as strain JWCB054 and showed reduced production of acetate (14.1 mM) and H 2 (22.5 mM) (Fig. 4b, c; Table 2) compared to the parental stain JWCB017, indicating redirection of carbon flow from pyruvate away from acetate and toward ethanol. JWCB049 showed 10.7 % conversion of cellobiose into acetate and 2.2 % to ethanol with 81 % overall carbon recovery. Interestingly, H 2 production is reduced in strain JWCB049, but not in strain JWCB054. This may be explained by the fact that H 2 production in C. bescii is likely catalyzed by an NADH-and ferredoxin-dependent electron bifurcating [FeFe] hydrogenase (Cbes1297-1299) [50]. Thus, the AdhE pathway, which utilizes one NADH and one NADPH, partially competes with the hydrogenase for electrons, while the strictly NADPH-dependent AdhB does not.

Engineered C. bescii strains produce ethanol from Avicel and lignocellulose at 75 °C
To demonstrate the utility of this ethanol-producing pathway for high temperature CBP, we also investigated the ability of the engineered strains to produce ethanol from the model substrate crystalline cellulose (2 % Avicel) and the real-world cellulosic substrate switchgrass (2 %). Results were similar to those observed for cellobiose (Fig. 5). As expected, parent strain JWCB017 produced acetate (13.8 mM from Avicel, 16.1 mM from switchgrass) but no ethanol. The AdhB expressing strain, JWCB054, produced ethanol (1.4 mM on Avicel, 0.4 mM on switchgrass) as well as acetate (13.0 mM on Avicel, 15.7 mM on switchgrass). The AdhE expressing strain, JWCB049, produced more ethanol (2.3 mM on Avicel, 1.6 mM on switchgrass) than strain JWCB054 and reduced levels of acetate (12.3 mM on Avicel, 15.1 mM on switchgrass). To date, these strains represent the maximum temperature at which cellulosic ethanol production has been engineered into a microorganism.
On all substrates, cofactor specificity of the ethanol synthesis pathway likely limits ethanol formation. Previous work focused on expression of C. thermocellum adhE in C. bescii [26], which utilizes NADH as an electron donor for both acetyl-CoA reduction to acetaldehyde (ALDH activity) and acetaldehyde reduction to ethanol (ADH activity). The T. pseudethanolicus AdhB and AdhE enzymes, on the other hand, both utilize NADPH for ADH activity, and AdhB also utilizes NADPH for its Acetyl-CoA thioesterase activity (Fig. 1b). While diverse pathways for NADPH generation are known, the mechanism by which C. bescii generates NADPH is not known. C. bescii does not encode an obvious Glucose-6-phosphate dehydrogenase, suggesting it does not utilize the non-oxidative Pentose Phosphate Pathway (PPP), nor does it encode a malate dehydrogenase that would be necessary for a "malate shunt" as is seen in C. thermocellum [51]. C. bescii does encode a putative NADH:Ferredoxin:NADP + oxidoreductase (NfnAB; Athe_0644-0645) [31,52], that would take electrons from NADH and reduced ferredoxin to generate two NADPH, but the in vivo importance of NfnAB in C. bescii is unknown.
We hypothesize that low electron flux through NADPH could be limiting ethanol production. The AdhE pathway requires one NADPH per ethanol, while the AdhB pathway requires two NADPH per ethanol (Fig. 1b). If NADPH were limiting, we would expect the AdhE pathway to produce twice as much ethanol as the AdhB pathway. Indeed, this is exactly what is observed. The ethanol yield per mole of consumed cellobiose in the adhE expression strain was twice that of the adhB expression strain (0.54 and 0.28 mol ethanol/mol cellobiose for the AdhE and AdhB strains, respectively) ( Table 2). Furthermore, both strains produced substantially less ethanol compared to our previous study in which we expressed the NADH-dependent C. thermocellum AdhE (15.3 mM) [26], again supporting the idea that NADPH availability is limiting ethanol production when utilizing these NADPH-dependent pathways in C. bescii. Future  engineering efforts to increase NADPH availability or alter cofactor specificity have the potential to further increase ethanol yields. By producing ethanol at 75 °C, this work lays the foundations for further metabolic engineering and process optimization that could lead to in situ ethanol removal during fermentation to enhance recovery and decrease product toxicity.

Conclusions
To gain the most value from high temperature CBP, both C5 and C6 sugars will need to be converted into fuel at high yield and titer at a temperature near or above the boiling point of ethanol. Here we report metabolically engineered strains that produce ethanol in vivo near the ethanol boiling point by introducing thermostable AdhB and AdhE proteins from T. pseudethanolicus into C. bescii. The yield and titer of ethanol are still low, but the prospect of further strain modification to improve NADPH availability or alter co-factor specificity of the enzymes has the potential to further improve upon this strain. Further, this work provides insights into mechanisms of redox balancing within C. bescii that will assist future metabolic engineering efforts.

Strains, media and culture conditions
C. bescii and E. coli strains and plasmids used in this study are listed in Table 1. All C. bescii strains were grown anaerobically in liquid or on solid media in low osmolality defined (LOD) medium [39], bottles with five gassing cycles with argon. For uracil auxotrophs, the LOD medium was supplemented with 40 µM uracil. E. coli strain DH5α was used for plasmid DNA construction and preparation. Standard techniques for E. coli were performed as described [53]. E. coli cells were grown in LB broth supplemented with apramycin (50 μg/mL) and plasmid DNA was isolated using a Qia-  Fig. 1a; Additional file 1: Figure S1) was constructed by inserting the Teth39_0206 open reading frame into pDCW142 [26], which contains the regulatory region of Cbes2303, a C-terminal 6X Histidine-tag and a Rho-independent transcription terminator. The 6.3 kb DNA fragment was amplified with primers DC464 containing a BamHI cut site and DC466 containing a SphI cut site using pDCW142 as a template. A 2.62 kb DNA fragment containing the coding sequence of Teth39_0206 was amplified with DC597 containing a BamHI site and DC598 containing a SphI site using T. pseudethanolicus 39E genomic DNA as a template. The two linear DNA fragments were then digested with BamHI and SphI, and ligated to construct pDCW180 (8.9 kb) (Additional file 1: Figure S1). Plasmid pDCW183 (Additional file 1: Figure  S2) is identical to pDCW180 except for the cloning of Teth39_0218. To make this change, a 1.06-kb DNA fragment containing the coding sequence of Teth39_0218 was amplified by PCR using DC621 (with BamHI site) and DC622 (with SphI site) using T. pseudethanolicus 39E genomic DNA as a template. The 6.3-kb of backbone fragment was amplified from pDCW142 [26] by PCR using DC619 (with BamHI site) and DC620 (with SphI site). DNA sequences of the primers are shown in Additional file 1: Table S1. E. coli strain DH5α cells were transformed by electroporation in a 2-mm gap cuvette at 2.5 V and transformants were selected for apramycin resistance. The sequences of all plasmids were verified by Automatic sequencing (Macrogen USA, Rockville, MD, USA). All plasmids are available on request.

C. bescii transformation, screening, purification, and sequence verification of engineered strains
To construct strain JWCB049, one microgram of M.Cbe1 methylated pDCW180 DNA was used to electrotransform JWCB017 (C. bescii ΔpyrFA Δldh) as described [28] and cells were plated onto solid LOD medium. Uracil prototrophic transformants were selected and used to inoculate into liquid medium for subsequent genomic DNA extraction and confirmation of the pDCW180 knock-in into the targeted chromosome via PCR screening. The insertion was targeted to base pair coordinates 962290 to 964604 in the C. bescii chromosome, designated CIS1 (chromosomal integration site one). Confirmed transformants were inoculated into nonselective liquid defined medium, with 40 μM uracil, and incubated overnight at 75 °C to allow loop-out of the plasmid prior to plating onto 5-FOA (8 mM) containing solid medium. After the initial screening, transformants containing the expected knock-in were further purified by one additional passage under selection on solid medium and screened a second time by PCR to check for segregation of the P S-layer Teth39_0206 insertion. The insertion of Teth39_0206 at the targeted chromosome region was verified by PCR amplification and sequence analysis using primers DC462, DC463, DC477, DC478 DC599, DC600, DC601 and DC602. A PCR product was generated from genomic DNA using primers DC477 and DC478 targeting outside the homologous regions used to construct the knock-in. Construction of JWCB054 was the same as JWCB049 except that pDCW183 (Table 1) was used to electrotransform JWCB017. All primers and sequences used in this study are listed in Additional file 1: Table S1.

Analytical techniques for determining fermentation end products
On the fermentation experiments, cellobiose, glucose, acetate, lactate, and ethanol, were monitored and analyzed on an Agilent 1200 Infinity HPLC system (Agilent Tech., Santa Clara, CA, USA) fitted with a Micro-Guard Cation H+ guard column and an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). All separations were performed under isocratic conditions with a temperature of 50 °C and a flow rate of 0.6 mL/min in 5.0 mM H 2 SO 4 , and then passed through a refractive index detector (Agilent 1200 Infinity Refractive Index Detector; Agilent Tech., Santa Clara, CA, USA). The total peak areas were integrated and compared to peak areas and retention times of known standards for each compound of interest. For H 2 production at the end of the fermentation, H 2 was measured using a GC8A Gas chromatograph (Shimadzu, Kyoto, Japan). Gas separations were performed on a Molecular Sieve packed column 5A (80/100 mesh; Alltech, Columbia, MD, USA) with a 70 °C oven temperature and detection with a Thermal Conductivity Detector at 120 °C. The samples were prepared from the headspace of stoppered 125-mL serum-bottle cultures using a gastight glass syringe (Hamilton, Reno, NV). The standards were also prepared from identical bottles with the same medium with known amounts of H 2 in the headspace. H 2 was calculated as moles produced per liter liquid phase to facilitate comparison to soluble fermentation products. Based on ideal gas law, pure H 2 is assumed as 1 mol of H 2 at 298 k (25 °C) and 1 atmosphere in 24.5 litters [54].