Anaerobic Fermentation of Glycerol in Paenibacillus macerans: Metabolic Pathways and Environmental Determinants

Strains.Wild-type P. macerans Northrop 234A (= LMG 13285 = N234A) was obtained from the Belgian Co-ordinated Collections of Microorganisms (BCCM/LMG, Gent, Belgium) and used throughout this study, unless otherwise specified. P. macerans strains B-394 (NRRL collection, Peoria, IL), ATCC 7068 (American Type Culture Collection, Manassas, VA), BKM B-51 (Bacillus Genetic Stock Center, Columbus, OH), B14029 (NRRL collection, Peoria, IL), and B-388 (NRRL collection, Peoria, IL) were also tested to determine their ability to ferment glycerol. E. coli K-12 wild-type strain MG1655 was obtained from the University of Wisconsin E. coli Genome Project (www.genome.wisc.edu ) (19). The strains were kept in 32.5% glycerol stocks at −80°C. Plates were prepared using Luria-Bertani (LB) medium containing 1.5% agar.

Culture medium and cultivation conditions.The minimal medium described by Schepers et al. (33) was used, with minor modifications. This medium contains (per liter [final volume]) 6.8 g KH₂PO₄, 3 g NH₄Cl, 1 g KCl, 0.5 g sodium citrate, 0.2 g MgSO₄·7H₂O, 30 mg MnSO₄·H₂O, 30 mg EDTA, 10 mg CaCl₂·2H₂O, 5 mg Na₂MoO₄, 5 mg FeSO₄·7H₂O, 5 mg H₃BO₃, 3 mg CoCl₂·6H₂O, 1 mg CuSO₄·5H₂O, 1 mg ZnSO₄·7H₂O, 1 mg nicotinic acid, 2 mg biotin, 2 mg p-aminobenzoic acid, and 2 mg thiamine hydrochloride. Unless otherwise specified, the medium was supplemented with 10 g/liter glycerol and 1 g/liter tryptone (final concentrations). “Low-phosphate and low-potassium” medium was prepared by replacing the KH₂PO₄ and KCl with 0.35 g NaH₂PO₄ per liter and 8.37 g MOPS (morpholinopropanesulfonic acid) per liter. All chemicals were obtained from Fisher Scientific (Pittsburg, PA) and Sigma Aldrich (St. Louis, MO).

Fermentations were conducted using a 750-ml fermentation system obtained from Ward's Natural Science (Rochester, NY) with a working volume of 500 ml and independent control of the temperature (37°C), pH, and stirrer speed. The pH was controlled at the desired values using a Jenco 3671 pH controller fitted with a Jenco 600p pH probe (Jenco, San Diego, CA). A base (2 M NaOH) for pH control was added by gravity flow using a pinch valve (Bio-Chem Inc., Boonton, NJ) connected to the pH controller. The stirrer speed was maintained at 200 rpm by using a Fisher Scientific Isotemp stirring plate (Pittsburg, PA). Anaerobic conditions were maintained by sparging the medium with ultra-high-purity argon (Matheson Tri-Gas, Inc., Houston, TX). Sterile conditions were achieved by using 0.2-μm and 0.45-μm HEPA filters (Millipore, Billerica, CA) in the inlet and outlet lines, respectively. Experiments conducted in tubes (as specified below) were carried out using 17-ml Hungate tubes (Bellco Glass, Inc., Vineland, NJ) that were modified by piercing the septa with two luer lock needles, one of which was used for oxygen-free argon sparging (20 gauge by 2 in.; Hamilton Company-USA, Reno, NV) and one of which was used for gas efflux (20 gauge by 8 in.; Hamilton Company-USA, Reno, NV). The contents were mixed by the rising gas bubbles. The working volume of these modified Hungate tubes was 10 ml.

Prior to use, the cultures (stored as glycerol stocks at −80°C) were streaked onto LB medium plates and incubated at 37°C. Single colonies were used to inoculate 17-ml modified Hungate tubes (see above) containing 10 ml of the medium described above supplemented with 10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter glycerol. The tubes were incubated at 37°C until the optical density at 550 nm was ∼0.4. An appropriate volume of each actively growing preculture was centrifuged, and the pellet was washed with the medium described above (lacking glycerol) and used to inoculate 500 ml of medium in the fermentor, using a target starting optical density at 550 of 0.05 nm.

Analytical methods.The optical density at 550 nm was determined and used to estimate the cell mass (1 optical density unit = 0.43 g [dry weight]/liter). After centrifugation, the supernatant was stored at −20°C before high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analysis. Glycerol, organic acids, acetone, ethanol, and hydrogen were quantified as previously described (13, 14).

NMR experiments.An experiment with 100% [U-¹³C]glycerol was conducted to assess the incorporation of glycerol into proteinogenic biomass and to verify that fermentation products originated from glycerol. This experiment was carried out as previously described (26), but the culture was harvested after 30 h. Cell pellets were prepared and analyzed to determine ¹³C enrichment using a one-dimensional (1D) proton spin echo with and without a concurrent 90° pulse on carbon (4, 26). 1D proton NMR spectroscopy was used to analyze the supernatants from the 30-h culture mentioned above and from experiments performed with unlabeled glycerol. The sample preparation and acquisition parameters used have been described previously (26). The resulting spectra were processed using the software packages FELIX 2001 (Accelrys Software Inc., Burlington, MA) and MestRe Nova 5.0.3 (Mestrelab Research SL, Santiago de Compostela, Spain).

Enzyme activities.For enzyme assays, cells were grown under specified culture conditions. Actively growing cells were harvested by centrifugation, washed twice with a saline solution (9 g/liter NaCl), and stored as cell pellets at −80°C until they were used. The pellets were resuspended in the same saline solution to obtain ∼1 mg (dry weight) of cells/ml and sonicated with a Branson 500 Sonifier (NJ) for 15 min at 4°C. The microtip configuration with a power rating of 7 and 50% pulse duration was used.

Glycerol kinase activity was assayed in a manner similar to that described by Hayashi and Lin (16) by measuring the change in absorbance at 340 nm and 25°C in a 1-ml reaction mixture containing 0.15 M glycine (pH 9), 11 mM MgCl₂, 0.27 M hydrazine, 1.2 mM NAD⁺, 5 mM ATP, 2 mM glycerol, 20 U of α-glycerolphosphate dehydrogenase, and 50 μl crude cell extract prepared as described above. Anaerobic G3PDH activity was measured with 50 μl crude cell extract as described previously (21), except that 33 mM, instead of 10 mM, glycerol 3-phosphate was used. The aerobic G3PDH activity was determined in the same way, except that flavines were omitted and sodium cyanide (10 μM) was included in the assay mixture. The assay mixtures were monitored spectrophotometrically at 570 nm. The extinction coefficient of reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was 17 mM⁻¹ cm⁻¹.

The activity of glyDH during the oxidation of glycerol was measured as described previously (15), with potassium carbonate (pH 9.5) as the buffer. Reductive glyDH activity (i.e., activity with hydroxyacetone [HA]) was measured using a similar mixture in which HA and NADH replaced glycerol and NAD⁺, respectively. Measurement of DHAK activity involved two different procedures for preparation of cell extracts, depending on whether the ATP- or PEP-dependent enzyme was assayed. Cell extracts for the ATP-dependent assay were prepared as described above, and a coupled assay for ATP-dependent conversion of DHA to DHAP and then NADH-dependent conversion of DHAP to glycerol 3-phosphate was performed as previously described (3) with 30 μl crude cell extract. The PEP-dependent DHAK activity was assayed using the method reported by Kornberg and Reeves (23), with minor modifications as previously described (43).

Alcohol dehydrogenase activity was determined by monitoring the NADH-dependent reduction of acetaldehyde at 340 nm as previously described (20) with 30 μl crude cell extract. Coenzyme A (CoA)-linked acetaldehyde dehydrogenase activity was assayed as previously reported (36) with 30 μl crude cell extract, using 20 mM dithiothreitol instead of 2-mercaptoethanol.

The activity of methylglyoxal (MG) synthase was determined using a colorimetric assay as described by Berrios-Rivera et al. (5). MG-reducing activities (e.g., activities of MG reductase and aldo-keto reductases) were measured using a 1-ml reaction mixture containing 10 mM MG, 0.1 mM NAD(P)H, and 100 mM potassium phosphate buffer (pH 7) (22). 1D proton NMR spectroscopy was used to identify the products of MG- and HA-reducing reactions. NMR measurements were obtained after cell debris was removed from a reaction mixture containing cell extract (30 μl), MG or HA (10 mM), coenzyme (1 mM NADH or 1 mM NADPH), buffer (100 mM potassium phosphate, pH 7.0), and D₂O. The NMR data were collected after 4 h of incubation at 25°C.

Linearity of the reactions (protein concentration and time) was established for all preparations. All spectrophotometric measurements were obtained with a BioMate 5 spectrophotometer (Thermo Scientific, MA). The nonenzymatic rates were subtracted from the observed initial reaction rates. Enzyme activities are reported below in micromoles of substrate per minute per milligram of cell protein, and the values are averages for at least three cell preparations.

Anaerobic fermentation of glycerol by P. macerans.Despite previous reports of the inability of P. macerans to ferment glycerol (34), we found that several strains of this species can utilize glycerol in the absence of external electron acceptors. Commonly used strains, such as N234A, B-394, and ATCC 7068, all showed efficient growth and glycerol utilization (Table 1). Other strains that were tested but were unable to ferment glycerol included BKM B-51, B14029, and B-388. When the fermentation of glycerol by one of these strains, P. macerans N234A, is compared to that reported for E. coli MG1655 in a similar medium (13), it is evident that this metabolic process is much faster in P. macerans N234A; the rates of glycerol utilization and ethanol production were 7- and 12-fold higher, respectively. Since the conversion of glycerol to ethanol and other products via anaerobic fermentation has been proposed as a means of achieving economic viability for the biodiesel industry (42), the high metabolic rates exhibited by P. macerans N234A are of paramount importance.

To further investigate the fermentative metabolism of glycerol by strain N234A, we conducted experiments using a low-nutrient medium supplemented with 1 g/liter of tryptone instead of the 10 g/liter of tryptone and 5 g/liter of yeast extract used in the experiments described above. While tryptone supplementation was required to observe significant utilization of glycerol, rich supplements have been used in previous studies of the fermentative and respiratory metabolism of P. macerans (33, 39, 40). Figure 2A shows a typical fermentation profile for strain N234A in the low-nutrient medium. Similar results were obtained with the other strains described in Table 1 (data not shown). Exponential growth was observed for a period of 4 h with a maximum specific growth rate (μ_max) of 0.402 h⁻¹ (Fig. 2A, inset) (ln cell concentration = 0.402 t − 6.149, where t is time; R² = 0.9997). The μ_max for three independent fermentations was 0.40 ± 0.03 h⁻¹. This μ_max is about 10 times that reported for E. coli MG1655 in a similar medium and with similar culture conditions (26). The growth yield once the cells reached the stationary phase was 78.4 mg cells/g glycerol, which is about 2.4 times the yield reported for E. coli (26).

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FIG. 2.

Fermentation of glycerol by P. macerans N234A in minimal medium supplemented with 10 g/liter glycerol and 1 g/liter tryptone and identification of fermentation products by NMR spectroscopy. (A) Concentrations of glycerol (▪), cells (▴), ethanol (•), 1,2-PDO (⧫), and formic (*) and acetic (+) acids. (Inset) Log-linear plot of cell concentration. (B) 1D ¹H-NMR spectrum of the fermentation broth from a 16-h sample of the culture shown in panel A. (Inset) Magnification of the area of the spectrum where two peaks corresponding to the methyl protons of 1,2-PDO were found (doublet at 1.15 ppm).

The fermentation products identified via 1D ¹H-NMR spectroscopy include ethanol (two multiplets at 3.66 and 1.19 ppm) and acetic (1.93 ppm) and formic (8.46 ppm) acids (Fig. 2B). While succinic acid (2.444 ppm) was found in fermentations conducted at a basic pH (e.g., pH 8) (see below), no lactic acid was detected in the extracellular medium under any of the conditions evaluated. A doublet was observed in the spectra of late fermentation samples at a position with the same chemical shift as that of methyl protons of 1,2-PDO (doublet at 1.15 ppm) (Fig. 2B, inset). The same doublet has been found in the spectra of fermentation samples of E. coli (26), and upon further investigation via two-dimensional ¹H-¹H correlation spectroscopy NMR, the peaks were identified as originating from 1,2-PDO methyl protons (15). Therefore, we conclude that 1,2-PDO is a product of glycerol fermentation by strain N234A. While 1,2-PDO is a product of the fermentation of the 6-deoxyhexose sugars fucose and rhamnose (40), our findings represent the first report of 1,2-PDO synthesis during the metabolism of glycerol in P. macerans.

The concentrations of glycerol and fermentation products, as determined by HPLC, are shown in Fig. 2A. Ethanol, formate, and 1,2-PDO continuously accumulated in the extracellular medium as glycerol was consumed, although the formate concentration decreased after 16 h. Conversion of formate to CO₂ and hydrogen, catalyzed by the enzyme formate-hydrogen lyase (33, 39), appears to be responsible for the small amounts of formate present in the medium and the decrease in the formate concentration after 16 h. Acetate, on the other hand, exhibited a different profile; it rapidly accumulated during the first 8 h of fermentation, when very little glycerol had been consumed (Fig. 2A). This is in contrast to the findings for ethanol and 1,2-PDO, whose synthesis paralleled the consumption of glycerol. The decrease in the acetate concentration after 8 h was likely due to its conversion to acetone (with acetoacetate as an intermediate), a well-known pathway in P. macerans (33, 39). Based on the amount of acetate present at 8 h (0.192 g/liter) and the amount remaining at the end of the fermentation (0.046 g/liter), we calculated that 0.070 g/liter of acetone was produced (2 acetate → acetone + CO₂). Given the volatile nature of acetone (leading to significant evaporation at 37°C) and the small amounts produced, it was not surprising to find that the acetone concentration was below the limit of detection of our HPLC method. It is noteworthy that the amount of acetate produced during glycerol fermentation is only 1 to 5% (on a molar basis) of the amount of ethanol produced; both of these products originate from pyruvate and acetyl-CoA. Moreover, a significant fraction of the small amount of acetate was generated from tryptone components, as discussed below. The data mentioned above were used to conduct a fermentation balance analysis, which included both carbon and redox balances (Table 2). The amount of carbon recovered as fermentation products was close to 100% of the amount of carbon consumed as glycerol (3.061/3 = 1.02) (Table 2). Similarly, about 99% of the reducing equivalents generated during the fermentation of glycerol were captured in the synthesis of fermentation products (−1.985/−2 = 0.99) (Table 2). These data represent excellent closure of both redox and carbon balances.

No significant cell growth was observed when glycerol was omitted from the medium formulation (the cell concentration increased by less than 0.01 g/liter). However, the metabolism of tryptone components led to accumulation of 0.096 g/liter of acetate and 0.004 g/liter of ethanol in the extracellular medium. This amount of acetate is roughly 50% of the amount produced by the culture described in Fig. 2A, suggesting that tryptone components significantly contribute to acetate synthesis during glycerol fermentation. The amount of ethanol generated from tryptone metabolism, on the other hand, is only 0.14% of the total amount of ethanol produced during glycerol fermentation (Fig. 2A). No products were detected when both tryptone and glycerol were excluded from the medium.

Glycerol fermentation was also observed when the growth medium was supplemented with a mixture of proteinogenic amino acids at levels similar to those provided by tryptone supplementation (i.e., 0.175 g/liter of cells and 3.8 g/liter of glycerol fermented). However, amino acid supplementation resulted in a lower cell density and slower fermentation kinetics (specific growth rate, 0.17 h⁻¹) than those shown in Fig. 2A. Inclusion of citrate in the culture medium was beneficial but not required for glycerol fermentation, and the presence of citrate did not lead to synthesis of any fermentation product in the absence of tryptone and glycerol. It is noteworthy that citrate is a component of a standard minimal medium (commonly referred to as “Spizizen salts medium”) used to cultivate Bacillus species (2).

Identification of the origin of carbon in fermentation products and cellular components.Since glycerol metabolism requires supplementation of the medium with small amounts of tryptone, we conducted experiments to investigate whether fermentation products and cellular components are synthesized from glycerol or tryptone. To this end, cells were grown on 100% [U-¹³C]labeled glycerol, as described in Materials and Methods. Selected areas of the NMR spectrum of the supernatant of a 30-h sample from this culture are shown in Fig. 3. 1D ¹H-NMR spectroscopy was used in these experiments to distinguish between ¹³C and ¹²C atoms (see Materials and Methods for details). Since ¹³C is magnetic, protons attached to ¹³C atoms have two different chemical shifts due to the positive and negative energy levels of these atoms. Protons attached to ¹²C atoms have chemical shifts between those of ¹³C because all ¹²C atoms are in a neutral state. Thus, protons attached to ¹²C carbon atoms lead to a central peak structure flanked by two satellite peak structures that result from other protons that are attached to ¹³C atoms. The ratio of the area of ¹³C satellite peaks to the total area (expressed as a percentage) then reflects the ¹³C enrichment of the carbon atom.

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FIG. 3.

1D ¹H-NMR spectra of the fermentation broth from a 30-h culture grown on 100% [U-¹³C]glycerol. (A) Region of the spectrum corresponding to the ¹³C and ¹²C signals of ethanol and acetic and formic acids. Central ¹²C peaks are flanked by two satellite peak structures resulting from protons attached to the ¹³C atoms. The ratio of the area of each peak to the total area (i.e., sum of all peaks) (expressed as a percentage) is indicated. (B) Region of the spectrum where ¹³C satellites and ¹²C signals of 1,2-PDO methyl protons are located. The peaks indicated by arrows represent ¹³C satellite signals. (Inset) Magnification of the area where the ¹²C methyl proton signals should be located.

As Fig. 3A shows, the level of ¹³C enrichment in ethanol and formic acid approached 100%, demonstrating that most carbon atoms in these products originated from glycerol. ¹³C enrichment was also observed in the case of acetic acid, although only about 64% of the carbon in this product originated from glycerol (Fig. 3A). In the case of acetate, the calculations assume that the area of the satellite peak structures located to the left of the ¹²C central peak (which overlaps with other peaks) is equal to the area of the satellite peak structures visible to the right of the ¹²C peak. The large fraction of unlabeled carbon in acetate results from the contribution of tryptone components to acetate synthesis, as reported above. Acetate accumulates only during the first 8 h of fermentation, when very little glycerol is consumed and therefore a large fraction of the carbon in this product originates from tryptone components (Fig. 2A) (note as well that experiments performed in the absence of glycerol showed that synthesis of significant amounts of acetate occurred). As fermentation of glycerol occurs after 8 h, most of the carbon is channeled to ethanol (Fig. 2A). It then follows that the fraction of carbon originating from tryptone (i.e., unlabeled carbon) should be much larger in acetate than in ethanol; i.e., most of the carbon in ethanol originates from glycerol, which is ¹³C labeled, while almost 50% of the carbon in acetate originates from tryptone, which is not labeled.

The area of the spectrum where the doublet corresponding to 1,2-PDO methyl protons was observed is shown separately in Fig. 3B. While the ¹³C signals were identified, no signal was observed at the ¹²C positions. The inset in Fig. 3B shows a magnification of the area of the spectrum where a doublet due to the protons attached to ¹²C carbons should appear. When this inset is compared to the inset in Fig. 2B, which shows the ¹²C doublet observed when unlabeled (naturally labeled) glycerol was used, it is evident that 1,2-PDO is synthesized exclusively from glycerol.

Also in the experiment described above, the ¹³C enrichment of proteinogenic biomass was assessed to determine whether glycerol is used in the synthesis of cell mass. For this purpose, the ¹³C enrichment of proteinogenic amino acids in cells grown on [U-¹³C]glycerol was compared to that in a reference culture in which cells were grown on unlabeled glycerol. The cells from the two cultures were hydrolyzed to obtain a cocktail of their proteinogenic amino acids, which was subsequently analyzed using NMR. ¹³C enrichment was determined using a 1D proton spin echo with and without a concurrent 90° pulse on carbon as described in Materials and Methods. The 90° pulse on carbon refocused the ¹³C carbon atoms, thereby suppressing the ¹³C satellites arising due to proton-carbon spin coupling. The nucleus of ¹²C is nonmagnetic; thus, protons attached to ¹²C do not experience any differences in the two situations. Therefore, ¹³C satellite peaks could be easily identified upon comparison of the spectra obtained using these two methods. The 1D NMR spectra obtained for the two samples (with and without labeled carbon) contained small ¹³C satellite peaks, which in several cases were hidden below bigger peaks. However, many of the small peaks were well resolved, with visible ¹³C satellites. Parts of the NMR spectra depicting two of these resolved areas for a sample in which ¹³C-labeled glycerol was used are shown in Fig. 4. The amino acid carbon atoms shown are threonine-γ (left panels) and alanine-β (right panels) atoms. In the case of unlabeled glycerol, the ¹³C satellite peaks accounted for about 1% of the total signal (data not shown), which is approximately the natural abundance of this isotope. However, when 100% [U-¹³C]glycerol was used (Fig. 4, top panels), the ¹³C satellites accounted for about 22% of the total signal, indicating that about 20% of these amino acids in the biomass originated from glycerol. Although not shown here due to space limitations, similar spectra were obtained for many carbons corresponding to other amino acids in proteinogenic biomass.

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FIG. 4.

NMR spectra of proteinogenic amino acids in cell biomass obtained from an experiment performed with 100% [U-¹³C]glycerol. The identity of ¹³C satellites as peaks arising due to labeled carbon was confirmed by performing a ¹³C decoupled experiment in which the ¹³C signals were suppressed (bottom panels). The peaks indicated by arrows represent the incorporation of labeled carbon into threonine-γ (left panels) and alanine-β (right panels). (Insets) Magnifications of areas of interest in the spectra.

The use of tryptone supplementation also raised the question of whether glycerol utilization is truly fermentative or whether compounds present in tryptone, or generated from it, serve as electron acceptors. The almost exclusive synthesis of the reduced product ethanol (Fig. 2A and Table 2) is a strong indication that glycerol is in fact metabolized in a fermentative manner; otherwise, the presence of an electron acceptor would consume the reducing equivalents generated from glycerol, and significant amounts of oxidized products (such as acetate) would be produced instead of ethanol. The excellent closure of the redox balance (∼99%) (Table 2) is another indication that glycerol metabolism is mediated by fermentative pathways. More direct evidence of the fermentative nature of this metabolic process was provided by the results of experiments in which cell growth and glycerol utilization were observed despite the use of the inhibitors cyanide and azide, which block general respiratory processes (data not shown).

Metabolic routes involved in the conversion of glycerol to glycolytic intermediates and the synthesis of fermentation products by P. macerans N234A.Two pathways have been reported to mediate the microbial conversion of glycerol to the glycolytic intermediate DHAP (6, 24, 42) (Fig. 1). In an attempt to identify whether these pathways mediate glycerol utilization in P. macerans N234A, we measured the activities of relevant enzymes in crude cell extracts obtained from a 16-h sample of the culture shown in Fig. 2A. The results indicate that the glycerol kinase-G3PDH route was inactive, as no activity was detected for these enzymes (Table 3). Both anaerobic G3PDH activity and aerobic G3PDH activity were assayed. On the other hand, significant activity was observed for glyDH and DHAK, indicating that the glyDH-DHAK route mediates the utilization of glycerol in P. macerans N234A (Table 3). These activities were not present in cultures grown on LB medium but were detected when glycerol was included in the cultures (Table 4), suggesting that they could be induced by this carbon source. The glyDH activity was found to be NAD⁺ specific, as it was not detectable when NADP⁺ was used as a cofactor. The four types of bacterial glyDHs reported to date can be differentiated on the basis of their inducibility by glycerol, DHA, and HA (9). The glyDH activity detected in P. macerans N234A extracts was induced by both glycerol and HA, but not by DHA (Table 4). Type II glyDHs from enteric bacteria are induced by glycerol and HA (9); therefore, the glyDH of P. macerans N234A appears to be a type II enzyme. The DHA generated by the action of glyDH was converted to DHAP by the action of both ATP- and PEP-dependent DHAKs, as inferred from the enzyme assays (Table 3). Both enzymes were induced by glycerol, but DHA was able to induce only the ATP-dependent activity (Table 4). Taken together, these results agree with current models for the microbial metabolism of glycerol in which the glyDH-DHAK pathway is associated with the fermentative utilization of this carbon source (Fig. 1) (6, 15, 24, 42). These activities were not found in strains unable to ferment glycerol (data not shown).

The pathways involved in the synthesis of the fermentation product 1,2-PDO were investigated using enzyme activity measurements, characterization of enzymatic reactions via NMR, and supplementation of intermediates in the postulated pathways. Based on previous studies of bacteria, the pathways that could mediate the synthesis of 1,2-PDO from DHAP are summarized in Fig. 5A (1, 8, 12, 15, 22, 25, 30, 40). DHAP is a glycolytic intermediate generated during the utilization of glycerol via the glyDH-DHAK route discussed above. Since the synthesis of MG from DHAP is a common step in the 1,2-PDO pathway, regardless of the branch used for the conversion of MG to 1,2-PDO (Fig. 5A), MG synthase was assayed, and significant activity of this enzyme was found (Table 3). MG synthase was not detected in the absence of glycerol (Table 4), indicating that it could be induced by this carbon source. When cell extracts were assayed for MG-reducing enzymes, significant activity in the presence of either NADH or NADPH was found (Table 3). Since these activities could be involved in the conversion of MG to either HA or lactaldehyde, 1D ¹H-NMR spectroscopy was used to characterize the reaction(s). Figure 5B shows that the product of the MG-reducing activities was indeed HA (no lactaldehyde was detected). Additionally, cells fermenting glycerol exhibited significant HA-reducing activity (Table 3). The enzyme catalyzing this conversion was able to use NADH and NADPH as cofactor. Further characterization of the reaction(s) using 1D ¹H-NMR spectroscopy showed that the product of HA reduction was 1,2-PDO (Fig. 5C). Both MG-reducing and HA-reducing activities were induced by glycerol (Table 4). It is noteworthy that these activities were not found in strains unable to ferment glycerol (data not shown).

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FIG. 5.

Investigation of the pathways involved in the synthesis of 1,2-PDO from DHAP. (A) Enzymes reported to mediate the synthesis of 1,2-PDO in bacteria. The thick lines indicate the route used by P. macerans N234A for 1,2-PDO synthesis during glycerol fermentation, as inferred from our studies (see text, Tables 3 and 4, and Fig. 5B and 5C). MGS, MG synthase; AKR, aldo-keto reductase; MGR, MG reductase; 1,2 PDOR, 1,2-PDO reductase. (B) 1D ¹H-NMR characterization of the reactions involved in the reduction of MG to HA or lactaldehyde (see panel A). The lower and upper spectra are the spectra for the initial and final (4-h) samples, respectively. The arrows pointing down and the asterisk indicate MG and HA peaks, respectively. The arrow pointing up indicates the peak for acetate, an impurity in MG. No lactaldehyde was found as a product of this activity. (C) 1D ¹H-NMR characterization of the reaction(s) converting HA to 1,2-PDO, catalyzed by glyDH (see panel A). The lower and upper spectra are spectra for the initial and final (4-h) samples, respectively. The arrow and the asterisk indicate HA and 1,2-PDO peaks, respectively.

The results described above suggest that the synthesis of 1,2-PDO during glycerol fermentation occurs through the conversion of DHAP to MG to HA to 1,2-PDO (Fig. 5A). An experiment in which amplification of the 1,2-PDO pathway allowed glycerol fermentation in the absence of rich supplements provided evidence of the important role of this pathway: by supplementing the growth medium with 18.3 mM HA (1.36 g/liter), glycerol fermentation was observed in the absence of tryptone. In 48 h the cells grew to a concentration of 0.23 g/liter, fermented 3.88 g/liter of glycerol, and produced 1.27 g/liter 1,2-PDO, 1.50 g/liter ethanol, 0.038 g/liter acetate, and 0.20 g/liter acetone. A carbon balance analysis in this experiment yielded 99% recovery of carbon in cell mass and fermentation products. These results suggest that the synthesis of 1,2-PDO facilitates fermentation of glycerol in P. macerans N234A. The conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents and has been proposed to facilitate glycerol fermentation in E. coli by enabling redox balance in the absence of external electron acceptors (15, 26).

1,3-PDO, a structural isomer of 1,2-PDO, is a known metabolic product that enables fermentative utilization of glycerol in enteric bacteria (6, 24, 42). 1,3-PDO is synthesized in these microorganisms via a pathway that involves the reduction of the intermediate 3-HPA to 1,3-PDO by an NADH-linked 1,3-PDODH (Fig. 1A). We did not find 1,3-PDO in the supernatant of P. macerans N234A cultures, and 1,3-PDODH activity was not detected in cell extracts (Table 3).

The almost homoethanologenic nature of glycerol fermentation (Fig. 2 and 3) reflects the highly reduced state of carbon in glycerol and suggests a central role for this pathway. Ethanol is synthesized in microorganisms through the reduction of acetaldehyde, a reaction catalyzed by alcohol dehydrogenase (27, 31). Acetaldehyde, in turn, originates from either the oxidation of pyruvate (catalyzed by pyruvate decarboxylase [PDC]) or the reduction of acetyl-CoA (catalyzed by acetaldehyde dehydrogenase) (11, 27, 31). In previous studies, the dissimilation of pyruvate in P. macerans strains during fermentative metabolism of sugars was mediated by pyruvate formate-lyase (PFL), an enzyme that converts pyruvate to acetyl-CoA and formate (35, 39, 40). Therefore, we postulated that the route to ethanol synthesis should involve the consecutive reduction of acetyl-CoA to acetaldehyde to ethanol. To verify this hypothesis, we assayed cell extracts of P. macerans N234A grown on glycerol for acetaldehyde and alcohol dehydrogenase activities. Both activities were present at significant levels (Table 3).

Effect of culture conditions and medium composition on the fermentative metabolism of glycerol.The fermentative metabolism of glycerol by P. macerans N234A is negatively affected by neutral and alkaline pH (Fig. 6). Both cell growth and glycerol utilization at pH 7 were less than one-half those at pH 6. Even more surprising was the observation that no significant cell growth or glycerol consumption was observed at pH 8. A relationship between pH and the ability to ferment glycerol has recently been reported for E. coli and attributed to several factors (13, 15, 26), which are examined in detail below.

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FIG. 6.

Effect of pH, carbon dioxide, and concentrations of potassium and phosphate on cell growth (filled bars) and glycerol fermentation (open bars) by P. macerans N234A. The basal medium used in this study contained 6.8 g/liter KH₂PO₄ and 1 g/liter KCl (see Materials and Methods) (“high potassium and phosphates”). A low-potassium and low-phosphate medium was prepared by replacing KH₂PO₄ and KCl in the basal medium with 0.35 g/liter of Na₂HPO₄ and 8.36 g/liter of MOPS (“low potassium and phosphates”). Experiments were conducted at 37°C, using the media described above supplemented with 10 g/liter glycerol and 1 g/liter tryptone. The gas atmosphere and pH used are indicated. The bars indicate the means and the error bars indicate the standard deviations for three samples taken once the cultures reached the stationary phase. Differences in cell growth and glycerol utilization discussed in the text were significant at P values of ≤0.037 (Student's t test).

One reason for the absence of glycerol fermentation at pH 8 could be the limited availability of CO₂ under alkaline conditions, which in turn could be caused by the low activity at this pH of CO₂-generating enzymes, such as formate-hydrogen lyase (13). While CO₂ could be generated by conversion of acetoacetate to acetone, this pathway appears to be inactive during glycerol fermentation, as we did not find acetone in the extracellular medium. This agrees with previous studies that reported no acetone production by P. macerans under alkaline conditions (39). The CO₂ limitation hypothesis was investigated by providing a gas atmosphere containing 20% CO₂ (with the balance argon) while controlling the pH by addition of base (Fig. 6). At pH 8 the cells even transitioned from a non-glycerol-fermenting state in an argon atmosphere to a glycerol-fermenting state in a CO₂-enriched atmosphere.

Another reason for the negative impact of alkaline conditions could be that a basic pH, in combination with high levels of potassium and phosphate, has a negative impact on the activity of two key pathways involved in the fermentative utilization of glycerol, namely the 1,2-PDO and glyDH-DHAK pathways (15). While evidence for this originates from studies conducted with E. coli, these pathways are active in P. macerans N234A as well (Tables 3 and 4 and Fig. 5). The use of a medium with low levels of phosphate and potassium had a beneficial effect on glycerol fermentation at pH 7 and 8 (Fig. 6). Low levels of both phosphate and potassium were required to observe this improvement. Interestingly, the use of a CO₂-enriched atmosphere in combination with a low-phosphate and low-potassium medium did not lead to further improvements in cell growth or glycerol utilization (Fig. 6). Glycerol fermentation at pH 6 was not affected by addition of CO₂ or by the concentrations of phosphate and potassium (data not shown).

Since the use of a CO₂-enriched atmosphere and a low-phosphate and low-potassium medium allowed fermentation of glycerol under basic conditions, we examined the effect of pH on the distribution of fermentation products (Fig. 7). While at pH 6 the fermentation broth contained almost exclusively ethanol, the use of alkaline conditions led to a slight decrease in the ethanol yield, along with the production of significant amounts of formic, acetic, and succinic acids (Fig. 7). Differences in organic acid yields at different pHs have also been reported for other P. macerans strains during glucose fermentation (39). However, the shift from almost homoethanologenic fermentation to the mixed-acid type of fermentation is unique to the fermentation of glycerol. To illustrate this, it is helpful to compare the ratio of organic acids to ethanol (on a weight basis), which changed from 0.04 at pH 6 to 1.6 at pH 8, a 37-fold difference. The higher biomass yield under alkaline conditions was accompanied by a higher 1,2-PDO yield, which further supports the enabling role of 1,2-PDO synthesis. Another important finding was the identification of succinic acid as a product of glycerol fermentation under alkaline conditions (Fig. 7). Previous studies of the fermentation of glucose, xylose, and deoxy sugars by P. macerans strains did not report succinic acid as a product (33, 39, 40).

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FIG. 7.

Effect of pH on product and biomass yields. Experiments were conducted using a low-potassium and low-phosphate medium (see the legend to Fig. 6 and the text for details). The data are for pH 6 (black bars), pH 7 (gray bars), and pH 8 (open bars). The bars indicate the means and the error bars indicate the standard deviations for three samples taken once the cultures reached the stationary phase. Differences in product and biomass yields discussed in the text were significant at P values of ≤0.045 (Student's t test).