l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Abstract

Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD⁺ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD₆₀₀]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (Δpfl mutant) expressing the ldhL1_LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.

INTRODUCTION

Biodiesel is one of the alternatives to fossil fuel that reduces greenhouse effects and is being produced worldwide (1, 2). Biodiesel is produced from triacylglycerol, which is a main component of vegetable oils and animal fats, by either chemical or enzymatic reactions (1, 3, 4). When biodiesel is produced by a chemical process, triacylglycerol is exposed to methanol under highly alkaline conditions to cleave the ester bond between glycerol and fatty acids, and the resulting methylated fatty acids are used as biodiesel (3). Consequently, this chemical process generates a by-product containing a large amount of crude glycerol, together with methanol and salts, that is called biodiesel waste. Microbial bioconversion processes to convert glycerol to other valuable materials, such as alcohols and organic acids, are being actively developed to reuse the crude glycerol included in biodiesel waste (5). When biodiesel waste contains impurities, such as methanol and residual fatty acids, it is pretreated to remove these impurities (6, 7) because raw biodiesel waste may be cytotoxic to producers (7, 8). However, cytotoxic substances have not been identified in biodiesel waste.

Many microorganisms can use glycerol as a carbon source (9, 10, 11). The bacterial glycerol metabolic pathway is known as a dehydrogenation pathway and a phosphorylation pathway (9). In the dehydrogenation pathway, glycerol is converted to dihydroxyacetone phosphate (DHAP) via dihydroxyacetone (DHA) by glycerol dehydrogenase and DHA kinase. Alternatively, glycerol is converted to DHAP via glycerol-3-phosphate (glycerol-3P) by glycerol kinase and glycerol-3P oxidase/dehydrogenase in the phosphorylation pathway. Enterococcus faecalis is a lactic acid bacterium that can use glycerol as a carbon source through one or both of the pathways described above (12, 13) and produces acetate, acetoin, ethanol, and lactate as end products (14). This indicates that E. faecalis is one of the bacteria that can convert crude glycerol to other valuable materials, such as ethanol and d/l-lactate. However, bioconversion processes for crude glycerol using E. faecalis have not been developed to date.

The glycerol consumption rate is an important factor for bioconversion efficiency from glycerol to other materials. Our previous study showed that the E. faecalis strain W11 consumes 100 mM glycerol within 16 h with or without oxygen (14), which is a higher rate than those of other E. faecalis strains (13). In addition, although the transformation efficiency of enterococci is strongly dependent on species and strains (15, 16), introduction of plasmid DNA into W11 by electroporation allowed W11 to express other genes and enabled the creation of a gene disruptant designated Δpfl (14). Thus, these backgrounds suggest the possibility that a bioconversion process using metabolically engineered W11 can convert glycerol to other materials at a high conversion efficiency.

The present study investigated the cytotoxicity of biodiesel waste and developed a bioconversion process for crude glycerol in biodiesel waste using E. faecalis. The oil component in biodiesel waste decreased the cellular NADH/NAD⁺ ratio and induced oxidative stress to cells, which decreased glycerol consumption by inhibition of the DHA kinase reaction in the dehydrogenation pathway. Utilizing glycerol under anaerobic conditions, E. faecalis W11 produced more ethanol than l-lactate. In contrast, our metabolic engineering combined the pflB gene disruptant for W11 (the Δpfl mutant) and the glycerol-inducible ldhL1_LP gene expression plasmid, which enabled the strain to produce up to 300 mM l-lactate from consumed glycerol/crude glycerol with a yield of >99% within 48 h under anaerobic conditions. Our study provides critical information and an approach for the development of a recycling process for crude glycerol in biodiesel waste.

MATERIALS AND METHODS

Strains and media.

The bacterial strains and plasmids used in this study are listed in Table 1. de Man, Rogosa, and Sharpe (MRS) broth (Becton Dickinson, MD, USA), modified MRS (M-MRS) broth (1.6% nutrient broth [Becton Dickinson], 0.5% yeast extract [Becton Dickinson], 0.1% polysorbate 80, 0.2% ammonium citrate, 0.01% MgCl₂ · 6H₂O, 0.005% MnSO₄ · 7H₂O, 0.2 M K₂HPO₄, pH 8.0), M17 broth (Becton Dickinson), and Luria-Bertani (LB) medium (1% tryptone [Becton Dickinson], 0.5% yeast extract [Becton Dickinson], 0.5% NaCl) were used.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Descriptiona	Sourceb or reference
Strains
Enterococcus faecalis
W11	Wild type	IPOD
W11 (ldhL1+)	E. faecalis W11 harboring pPFL-ldhL1LP	This study
Δpfl	ΔpflB::Tcr	14
Δpfl (ldhL1+)	E. faecalis Δpfl harboring pPFL-ldhL1LP	This study
Escherichia coli DH10B	F− mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK λ− rpsL endA1 nupG	Laboratory stock
Lactobacillus pentosus NBRC 106467T	Wild type	NBRC
Plasmids
pHSG396	Cloning vector; Cmr	TaKaRa
pAM401	Shuttle vector for E. coli and E. faecalis; Cmr Tcr	ATCC
pAM-PpflB	Glycerol-inducible gene expression plasmid for E. faecalis; Cmr	This study
pPFL-ldhL1LP	Glycerol-inducible ldhL1LP gene expression plasmid for E. faecalis; Cmr	This study

^aCm, chloramphenicol; Tc, tetracycline.

^bIPOD, International Patent Organism Depository; NBRC, NITE Biological Resource Center.

Fractionation of biodiesel waste.

Biodiesel waste was obtained from Dowa Eco-System Co., Ltd. (Okayama, Japan). An equal quantity (wt/wt) of water was added to the biodiesel waste, and the pH of the biodiesel waste solution was decreased to less than 2.0 by adding 12 M HCl. After the biodiesel waste solution separated into oil and water, the oil fraction (upper layer) was removed using a separating funnel. The residual oil and insoluble precipitates in the water layer were completely removed by centrifugation (10,000 × g; 10 min; 20°C) or filtration, and the supernatant (or the filtrate) was used as a crude glycerol solution containing approximately 45% (wt/vol) crude glycerol. Chloroform extraction and freeze dehydration were performed to further purify the glycerol contained in the crude glycerol solution. After an equivalent amount (wt/wt) of chloroform was added to the crude glycerol solution, the solution was mixed well and centrifuged at 8,000 × g at 20°C for 5 min. The water fraction (upper layer) was collected, neutralized by KOH (solid), and freeze-dried by freeze dehydration. The freeze-dehydrated sample was centrifuged at 20,000 × g at 20°C for 10 min to remove deposited salts, and the supernatant was then used as a purified glycerol. On the other hand, the chloroform fraction was distilled using a rotary evaporator to remove the chloroform. The residual oil was mixed with the oil fraction described above and used as the oil component of biodiesel waste.

Culture conditions.

The E. faecalis strains were precultured in 20-ml test tubes containing 5 ml of MRS broth at 120 rpm at 30°C. To replace the carbon source contained in the MRS medium (Becton Dickinson), the main cultures were performed in M-MRS broth containing each carbon source. The precultures (0.4 ml) were transferred to 40 ml of M-MRS broth in a 200-ml Erlenmeyer flask stopped with Silicosen (Shin-Etsu Polymer Co. Ltd., Tokyo, Japan) and then cultured at 120 rpm and 30°C. When strains were cultured under anaerobic conditions, the headspace air in the flasks was replaced with nitrogen gas by purging for 15 min, and the flasks were sealed with butyl rubber stoppers. l-Lactate production by the strains was performed in a 500-ml Erlenmeyer flask. The precultures (1 ml) were transferred to 100 ml of M-MRS broth containing each carbon source and aerobically or anaerobically cultured as described above. To construct plasmids, Escherichia coli DH10B was cultured in LB medium at 37°C with vigorous agitation in the presence of appropriate antibiotics (chloramphenicol, 20 mg liter⁻¹, and tetracycline sulfate, 10 mg liter⁻¹).

Construction of a glycerol-inducible ldhL1 gene expression plasmid for E. faecalis.

The pflB-pflA gene promoter region of W11 (P_pflB) amplified by PCR was cloned into XbaI-SalI-digested pAM401 to generate pAM-P_pflB (14). The Lactobacillus pentosus NBRC 106467^T l-lactate dehydrogenase gene (ldhL1_LP) lacking a translation start codon was amplified by PCR using KOD FX (Toyobo Co. Ltd., Osaka, Japan), the primers listed in Table 2, and total DNA as a template; digested with XhoI; and cloned into SalI-NruI-digested pAM-P_pflB to generate pPFL-ldhL1_LP. The plasmid was inserted into E. faecalis strains using the electroporation method, as described previously (14).

TABLE 2.

Primers used in this study

^aThe underlined nucleotides indicate restriction sites.

Ldh assay.

The E. faecalis strains were cultured in 40 ml of culture medium in 200-ml Erlenmeyer flasks, and the cell extract was prepared as described previously (14). The assay conditions for lactate dehydrogenase (Ldh) activity were a modification of the published method (17). The assay mixture contained 50 mM phosphate (pH 7.0) and 0.2 mM NADH, and the reaction was initiated by the addition of 10 mM pyruvate. When fructose 1,6-bisphosphate (FBP) was used as a cofactor for Ldh, a final concentration of 1 mM FBP (Sigma-Aldrich Co., St. Louis, MO) was added to the reaction mixture before the reaction was initiated. The Ldh reaction was monitored by measuring the absorbance at 340 nm at room temperature (25°C).

Reverse transcription (RT)-PCR.

Total RNA was purified using the RNeasy Protect Bacteria minikit (Qiagen, Hilden, Germany) with the RNase-Free DNase Set (Qiagen), and cDNA was synthesized using the QuantiTect Rev Transcription Kit (Qiagen). The specific amplicons for the 16S rRNA, glycerol dehydrogenase (gldA), DHA kinase (dhaK), glycerol kinase (glpK), glycerol-3P oxidase (glpO), alkyl hydroperoxide reductase (ahpC), thioredoxin peroxidase (tpx), and NADH peroxidase (npr) genes were amplified by PCR using cDNA as a template, the primers shown in Table 2, and KOD-Plus DNA polymerase (Toyobo).

Other methods.

Acetate, ethanol, formate, glycerol, l-lactate, and succinate in the culture media were quantified using an F-Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions, and the NADH/NAD⁺ ratio was measured as described previously (14). The optical density at 600 nm (OD₆₀₀) of cells cultured with biodiesel waste was measured in 0.2 M phosphate (pH 10) to solubilize the biodiesel waste. When the oil component was added to the culture medium, the cultures (1 ml) were centrifuged (20,000 × g; 10 min; 20°C) in 1.5-ml microtubes to remove the culture medium, including the oil. After the residual oil adhering to the microtube was removed with a paper towel, the cell pellets were resuspended in 1 ml of 0.2 M phosphate (pH 10), and the optical density at 600 nm was measured.

RESULTS

Growth with biodiesel waste.

Although strain W11 could grow in M-MRS broth containing 2% (wt/vol) biodiesel waste with or without oxygen, growth initiation was impeded for 20 h (Fig. 1A). In addition, the maximum culture density (OD₆₀₀) under anaerobic conditions was 8-fold lower than that under aerobic conditions (Fig. 1A). When biodiesel waste was added to M-MRS broth containing 100 mM glycerol, cell growth (OD₆₀₀) and glycerol consumption of W11 under anaerobic conditions were decreased with an increase in the biodiesel waste concentration (Fig. 1B), suggesting that the constituents within the biodiesel waste inhibited the anaerobic glycerol metabolism in W11. To investigate the growth-inhibitory factor, biodiesel waste was fractionated to crude glycerol, purified glycerol, and the oil component and added to the culture medium. The presence of 10% (wt/vol) crude glycerol slightly decreased the maximum culture density; however, the presence of more than 15% (wt/vol) crude glycerol significantly inhibited cell growth (Fig. 1C). Purified glycerol and glycerol (purity, 99%; Wako Pure Chemical Industries, Ltd., Osaka, Japan) inhibited cell growth when present at more than 15% (wt/vol) (Fig. 1C). In contrast, the oil component prepared from biodiesel waste inhibited cell growth when present at only 0.1% (wt/vol) (Fig. 1D). These results indicate that the oil component in biodiesel waste specifically inhibits the anaerobic glycerol metabolism in W11.

FIG 1.

Biodiesel waste metabolism in E. faecalis W11. (A) Growth of W11 cultured in M-MRS broth containing either 100 mM glycerol (● and ○) or 2% (wt/vol) biodiesel waste (▲ and △) under aerobic (● and ▲) or anaerobic (○ and △) conditions. (B) Growth and glycerol consumption of W11 cultured in M-MRS broth containing 100 mM glycerol with each concentration of biodiesel waste for 48 h under anaerobic conditions. ●, optical density at 600 nm; ○, residual glycerol concentration (%) in the culture medium. (C) Growth of W11 cultured in M-MRS broth containing 100 mM glycerol with each concentration of glycerol, crude glycerol, or purified glycerol for 48 h under anaerobic conditions. (D) Growth and glycerol consumption of W11 cultured in M-MRS broth containing 100 mM glycerol with each concentration of the oil component for 48 h under anaerobic conditions. ●, optical density at 600 nm; ○, residual glycerol concentration (%) in the culture medium. The experiments were performed at least three times. The error bars represent standard deviations.

Cytotoxicity of the oil component to anaerobic glycerol metabolism.

At the logarithmic growth phase, the growth of W11 cultured with glycerol under anaerobic conditions was inhibited to a greater extent by the oil component than under aerobic conditions (Fig. 2A and B). Similar to cell growth, the cellular NADH/NAD⁺ ratio in W11 exposed to the oil component under anaerobic conditions was decreased to a greater extent than under aerobic culture conditions (Fig. 2A and B). These results indicated that the oil component induces oxidative stress to cells and that the anaerobic glycerol metabolism is more sensitive to those oxidants than aerobic glycerol metabolism. RT-PCR showed that alkyl hydroperoxide reductase (AhpC) was induced by the oil component under anaerobic conditions, while the hydrogen peroxide (H₂O₂)-detoxifying enzyme NADH peroxidase (Npr) and thioredoxin peroxidase (Tpx) were not induced (Fig. 2C). In addition, tert-butyl hydroperoxide inhibited cell growth during anaerobic glycerol metabolism to a greater extent than cell growth during aerobic glycerol metabolism (Fig. 2D). These results strongly suggested that the cytotoxic substance in the oil component is an alkyl peroxide.

FIG 2.

Cytotoxicity of the oil component. (A and B) Growth (● and ○) and cellular NADH/NAD⁺ ratio (▲ and △) of E. faecalis W11 exposed to the oil component. W11 was cultured in M-MRS broth containing 100 mM glycerol under aerobic (A) or anaerobic (B) conditions. After culture for 6 h, a final concentration of 0.2% (wt/vol) of the oil component was added to the culture media (● and ▲), and the cultures were continued under the same conditions. Control cultures (○ and △) were continued without addition of the oil component. The experiments were performed at least three times. The error bars represent standard deviations. (C) RT-PCR to detect the ahpC, tpx, and npr transcripts in W11. The strain was cultured in M-MRS broth containing 100 mM glycerol under aerobic (left) or anaerobic (right) conditions. After culture for 6 h (0 h), a final concentration of 0.2% (wt/vol) of the oil component was added to the culture media, and the cultures were continued for 6 h (6 h, +). Control cultures were continued without addition of the oil component (6 h, −). DNA size markers (M) are shown on the left. (D) Effects of alkyl peroxide on E. faecalis growth. W11 was streaked on M-MRS broth in plates containing tert-butyl hydroperoxide at the indicated concentrations and cultured under aerobic or anaerobic conditions. All the plates contained 100 mM glycerol as a carbon source.

E. faecalis utilizes glycerol through the dehydrogenation pathway and/or the phosphorylation pathway (13). Enzymes for the dehydrogenation pathway are encoded by the gldA (glycerol dehydrogenase) and dhaK (DHA kinase) genes, while glycerol kinase and glycerol-3P oxidase, as enzymes for the phosphorylation pathway, are encoded by the glpK and glpO genes, respectively (12, 13). RT-PCR showed that genes of both pathways were expressed in W11 during aerobic glycerol metabolism (Fig. 3A). In contrast, during anaerobic glycerol metabolism, although gldA and dhaK gene expression was detected in W11, glpK and glpO gene expression was not detected (Fig. 3A). These results indicated that W11 utilized glycerol through the dehydrogenation pathway and the phosphorylation pathway under aerobic conditions, while W11 utilized glycerol only through the dehydrogenation pathway under anaerobic conditions. Regardless of whether the conditions were aerobic or anaerobic, the growth of W11 cultured with DHA, which is a product of glycerol dehydrogenase in the dehydrogenation pathway, was significantly inhibited by the oil component compared with the growth of W11 cultured with glucose or pyruvate (Fig. 3B and C), suggesting that the DHA kinase reaction in the dehydrogenation pathway was sensitive to alkyl peroxides.

FIG 3.

Comparison of cytotoxicities of the oil component with W11 cultured with various carbon sources. (A) RT-PCR to detect the gldA, dhaK, glpK, and glpO transcripts in W11 cultured in M-MRS broth containing 100 mM glycerol for 6 h (L) and 12 h (S) under aerobic (left) or anaerobic (right) conditions. L, logarithmic phase; S, stationary phase. DNA size markers (M) are shown on the left. (B and C) Glucose, DHA, and pyruvate metabolism in E. faecalis W11 exposed to 1% (wt/vol) of the oil component (●, ▲, and ■). W11 was cultured in M-MRS broth containing either 50 mM glucose (● and ○), 100 mM DHA (▲ and △), or 100 mM pyruvate (■ and □) under aerobic (B) or anaerobic (C) conditions. The open symbols represent control culture without the oil component. The experiment was performed at least three times. The error bars represent standard deviations.

Expression of the ldhL1 gene by the glycerol-inducible gene expression system.

E. faecalis W11 utilizing glycerol under anaerobic conditions produced ethanol and formate rather than l-lactate (14). The l-lactate dehydrogenase (LdhL) activity level in cells affects the l-lactate production yield (18). To predominate over pyruvate metabolism by the LdhL reaction, an L. pentosus LdhL1 gene (ldhL1_LP) expression plasmid, pPFL-ldhL1_LP, based on pAM-P_pflB and having a glycerol-inducible gene promoter for E. faecalis, was created and then introduced into W11 to create W11 (ldhL1⁺). When these strains were cultured with glucose, the Ldh activities of W11 and W11 (ldhL1⁺) were not very different (Fig. 4A), which indicates that the pflB-pflA gene promoter did not strongly respond to the presence of glucose. On the other hand, the Ldh activity of W11 utilizing glycerol under anaerobic conditions was 4.1-fold lower than that under aerobic conditions, whereas the Ldh activity of W11 (ldhL1⁺) under anaerobic conditions remained more than half of the activity under aerobic conditions (Fig. 4A), which was 3.5-fold higher than the Ldh activity of W11 in anaerobic culture. When FBP was not added to the assay mixture, Ldh activity levels of W11 cultured with glycerol were essentially undetectable, whereas for W11 (ldhL1⁺) cultured with glycerol, Ldh activities of 4.5 ± 0.4 and 3.2 ± 0.4 μmol mg⁻¹ min⁻¹ were detected under aerobic and anaerobic conditions, respectively (Fig. 4B). Because LdhL1 of L. pentosus does not require FBP for the Ldh reaction (19), 66% (aerobic culture) and 83% (anaerobic culture) of Ldh activity in W11 (ldhL1⁺) cultured with glycerol was equivalent to LdhL1_LP activity. These results indicate that the pflB-pflA gene promoter is activated during glycerol metabolism and is suitable for l-lactate production from glycerol.

FIG 4.

Lactate dehydrogenase activity in E. faecalis strains. (A) Ldh activity in W11 and W11 (ldhL1⁺) cultured in M-MRS broth containing either 50 mM glucose (Glc) or 100 mM glycerol (Gly) for 8 h (glucose) or 16 h (glycerol) under aerobic (filled bars) or anaerobic (open bars) conditions. (B) FBP-independent Ldh activity in W11 and W11 (ldhL1⁺) cultured in M-MRS broth containing 100 mM glycerol for 16 h under aerobic (filled bars) or anaerobic (open bars) conditions. The data shown are the mean values of the results of three experiments. The error bars represent standard errors. P < 0.05.

l-Lactate productivity by E. faecalis strains.

Strains were cultured in M-MRS broth containing 100 mM glycerol or crude glycerol, and their l-lactate productivities were investigated. Some studies showed that E. faecalis needs exogenous fumarate to utilize glycerol under anaerobic conditions (12, 20), but the cell growth (OD₆₀₀) of W11 was only slightly affected by exogenous fumarate, as previously reported (14) (Fig. 5A). However, the concentrations of l-lactate produced from glycerol/crude glycerol by W11 in the presence of fumarate were up to 3-fold higher than those produced by W11 in the absence of fumarate (Fig. 5B). In contrast, the supply of fumarate to strains expressing the ldlL1_LP gene significantly increased not only l-lactate production yields, but also cell growth (Fig. 5A and B). In the presence of more than 50 mM fumarate, W11 (ldhL1⁺) could produce up to 85 to 89 mM l-lactate from 100 mM glycerol or crude glycerol; furthermore, Δpfl (ldhL1⁺), which cannot produce acetyl-coenzyme A (CoA) from pyruvate via Pfl, could produce up to 101 to 104 mM l-lactate from 100 mM glycerol or crude glycerol (Fig. 5B). Regardless of whether W11, W11 (ldhL1⁺), or Δpfl (ldhL1⁺) was used, all the added fumarate was reduced to succinate; this succinate then accumulated in the culture media (Fig. 6A). When fumarate was absent, W11 and W11 (ldhL1⁺), which can produce acetyl-CoA from pyruvate by Pfl, produced ethanol rather than l-lactate (Fig. 6B). Addition of fumarate increased the production yield of acetate (W11) or l-lactate [W11 (ldhL1⁺)] instead of ethanol (Fig. 5B and 6C). These results indicated that fumarate downregulates ethanol fermentation during anaerobic glycerol metabolism and enables strains to metabolize pyruvate to other products.

FIG 5.

Effects of exogenous fumarate on cell growth and l-lactate productivity. Shown are the growth (A) and l-lactate production yields (B) of E. faecalis W11, W11 (ldhL1⁺), and Δpfl (ldhL1⁺) cultured with glycerol (left) or crude glycerol (right) in the presence of fumarate. The strains were cultured in M-MRS broth containing either 100 mM glycerol or crude glycerol with each concentration of fumarate for 60 h under anaerobic conditions. The data shown are the mean values of the results of three experiments. The error bars represent standard errors.

FIG 6.

Concentrations of succinate (A), ethanol (B), and acetate (C) produced by E. faecalis W11, W11 (ldhL1⁺), and Δpfl (ldhL1⁺). The strains were cultured in M-MRS broth containing 100 mM glycerol with each concentration of fumarate for 60 h under anaerobic conditions. The data shown are the mean values of the results of three experiments. The error bars represent standard errors.

Production of l-lactate from crude glycerol by E. faecalis Δpfl (ldhL1⁺).

E. faecalis Δpfl (ldhL1⁺) cultured in M-MRS broth containing 0.1 to 1 M crude glycerol with 50 mol% fumarate could produce up to 300 mM l-lactate from crude glycerol with a yield of >99%, and the cell growth (OD₆₀₀) reached the maximum in M-MRS broth containing 200 to 300 mM crude glycerol (Fig. 7A). In the M-MRS broth containing 300 mM either glycerol or crude glycerol with 150 mM fumarate, the glycerol consumption rate and l-lactate productivity of Δpfl (ldhL1⁺) were 8.2 mM h⁻¹ (0.75 g liter⁻¹ h⁻¹) and 8.1 mM h⁻¹ (0.73 g liter⁻¹ h⁻¹), respectively (Fig. 7B), which are the same or higher than those of the other engineered strains (21). The maximum glycerol consumption rate and l-lactate productivity were achieved between 12 and 18 h and reached 20 mM h⁻¹ (1.8 g liter⁻¹ h⁻¹) and 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹), respectively (Fig. 7B). These results indicate that Δpfl (ldhL1⁺) could completely convert up to 300 mM crude glycerol to l-lactate within 48 h without optimization of culture conditions, such as adjustment of the pH.

FIG 7.

l-Lactate production by E. faecalis Δpfl (ldhL1⁺) from glycerol or crude glycerol. (A) l-Lactate production rate from glycerol (filled bars) and cell growth (open bars) of Δpfl (ldhL1⁺) cultured in M-MRS broth containing 0.1 to 1 M crude glycerol for 60 h under anaerobic conditions. The culture medium contained 50 mol% fumarate of the initial glycerol concentration. (B) l-Lactate production of Δpfl (ldhL1⁺) cultured in M-MRS broth containing either 300 mM glycerol (●, ▲, and ■) or 300 mM crude glycerol (○, △, and □) under anaerobic conditions. The culture media contained 150 mM fumarate. ● and ○, optical density at 600 nm; ▲ and △, concentration of residual glycerol; ■ and □, concentration of l-lactate in culture media. The experiments were performed at least three times. The error bars represent standard deviations.

DISCUSSION

Although various microbiological approaches have been proposed to reuse crude glycerol in biodiesel waste (5), their physiological effects have scarcely been investigated. In this study, the alkyl peroxides included in the oil component of biodiesel waste were found to cause oxidative damage to cells (Fig. 1C and D and 2A and B). The acylglycerols, which are the main constituent of the oil component, generate lipid peroxides known as reactive oxygen species (ROS), which cause oxidative stress to cells (22, 23). That is, alkyl peroxides, as cytotoxic substances, are probably lipid peroxides. Many microorganisms, including E. faecalis, have a peroxide detoxification system (23–27). Therefore, the growth lag in the presence of the oil component was considered the time required for adaptation to oxidative stress caused by lipid peroxides.

Compared with anaerobic conditions, the oil component strongly inhibited cell growth when cultured with DHA under aerobic conditions (Fig. 3B and C). During aerobic glycerol metabolism, the glycerol-3P oxidase (GlpO) reaction in the phosphorylation pathway generates hydrogen peroxide (9, 12). In addition, the NADH oxidase (Nox) reaction, which is important for reoxidation of NADH (28), may generate hydrogen peroxide as a reaction product. Hydrogen peroxide (H₂O₂) generates hydroxyl radicals by a Fenton reaction, and the hydroxyl radicals and oxygen molecule accelerate the generation of lipid peroxide through a lipid peroxidation (29). Although W11 cultured under aerobic conditions upregulated the overall gene expression of peroxiredoxins (AhpC, Tpx, and Npr) compared with W11 cultured under anaerobic conditions (Fig. 2C), those detoxifications might be incomplete (27), i.e., because residual hydrogen peroxide accelerated lipid peroxidation, aerobic DHA metabolism might be more strongly inhibited by the resulting lipid peroxide than anaerobic DHA metabolism.

The bacterial LdhLs (NAD-dependent LdhL) include an FBP-dependent LdhL and an FBP-independent LdhL (30), and LdhL1 of E. faecalis is the FBP-dependent LdhL (31). The activity of FBP-dependent Ldh is highly dependent on FBP produced from fructose 6-phosphate by 6-phosphofructokinase (Pfk) using ATP in glycolysis (31); however FBP is not produced in the glycerol metabolic pathway because glycerol is converted to DHAP and then enters glycolysis (9). W11 expressing the ldhL1_LP gene could have LdhL activity under anaerobic conditions in the absence of FBP and could produce more l-lactate from glycerol than W11 (Fig. 4B and 5B). Thus, introduction of an FBP-independent LdhL, such as LdhL1 of L. pentosus, to E. faecalis effectively enhanced l-lactate production from pyruvate during glycerol metabolism (when no FBP is produced).

Strains expressing the ldhL1 gene required exogenous fumarate to grow to the same level as W11 (Fig. 5A). Glycerol metabolism via the dehydrogenation pathway generates two NADH molecules per pyruvate, so E. faecalis W11 utilizing glycerol under anaerobic conditions performs ethanol fermentation rather than lactate fermentation to oxidize two NADH molecules (14) (Fig. 5B and 6B). Accordingly, the lactate fermentation-enhanced strains, particularly the pfl gene disruptant, need an NADH oxidation pathway other than Pfl-ethanol fermentation to avoid reductive stress caused by excess NADH. Although the detailed mechanisms are unclear, fumarate reductase (Fdh) reactions are accompanied by NADH oxidation (32). Therefore, fumarate reduction assisted the oxidization of cellular NADH and enabled the lactate fermentation-enhanced strains to utilize glycerol without ethanol fermentation under anaerobic conditions.

E. faecalis Δpfl (ldhL1⁺) could not produce more than 300 mM l-lactate even if the strain was cultured with more than 400 mM glycerol (Fig. 7A). This suggests that the strain could not consume more than 300 mM glycerol under anaerobic conditions. Because glycerol metabolism by lactic acid bacteria is inhibited under acidic conditions (33), the culture medium was neutralized during the l-lactate production process (Fig. 8). E. faecalis Δpfl (ldhL1⁺) cultured under aerobic conditions could consume 500 mM crude glycerol in neutralized culture medium (Fig. 8A), whereas E. faecalis Δpfl (ldhL1⁺) cultured under anaerobic conditions could not (Fig. 8B). E. faecalis Δpfl (ldhL1⁺) at stationary phase consumed glycerol under aerobic conditions but did not consume glycerol under anaerobic conditions (Fig. 8), indicating that glycerol consumption under anaerobic conditions is dependent on cell growth. E. faecalis Δpfl cultured with glycerol showed constant glycerol dehydrogenase activity, regardless of whether the culture was aerobic or anaerobic (14), indicating that the strain can metabolize glycerol if glycerol is transported into the cell. Considering these results, glycerol consumption and l-lactate production yields are limited by downregulation of glycerol uptake, although the underlying mechanism remains unclear.

FIG 8.

Effect of neutralization on glycerol metabolism of E. faecalis Δpfl (ldhL1⁺). The strain was cultured in M-MRS broth containing 500 mM crude glycerol with 200 mM fumarate under aerobic (A) or anaerobic (B) conditions, and the culture medium was neutralized to pH 7.0 with 5 M KOH (●, ▲, ■, ⬥). The open symbols represent control culture without neutralization. ● and ○, optical density at 600 nm; ▲ and △, concentration of residual glycerol; ■ and □, concentration of acetate (A) or ethanol (B); ⧫ and ◇, concentration of l-lactate in culture media. Acetoin and d-lactate were not detected at more than 1 mM. (Top) pH of the culture medium with (●) and without (○) neutralization. The arrows indicate neutralization points. The experiments were performed at least three times. The error bars represent standard deviations.

l-Lactate (l-lactic acid) is an important compound for both the food and chemical industries because l-lactate is used not only as a food additive, but also as a material for the biopolymer polylactic acid (PLA) (34, 35). Recently, it was reported that metabolically engineered E. coli produced up to 50 g liter⁻¹ l-lactate from crude glycerol, with a yield of 93% (36), and the Rhizopus oryzae wild-type strain could produce 48 g liter⁻¹ l-lactate from 75 g liter⁻¹ crude glycerol in the presence of supplemental nutrients (37). In contrast, E. faecalis Δpfl (ldhL1⁺) could convert 300 mM (27 g liter⁻¹) crude glycerol to l-lactate with a yield of >99%. Therefore, if E. faecalis Δpfl (ldhL1⁺) can utilize more than 300 mM crude glycerol under anaerobic conditions, E. faecalis Δpfl (ldhL1⁺) may become the most efficient bacterial strain to produce l-lactate from crude glycerol.