Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5802-8

BIOENERGY AND BIOFUELS

Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation Victor Ujor & Chidozie Victor Agu & Venkat Gopalan & Thaddeus Chukwuemeka Ezeji

Received: 22 March 2014 / Revised: 14 April 2014 / Accepted: 27 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Lignocellulose-derived microbial inhibitors such as furfural and 5-hydroxymethyl furfural adversely affect fermentation of lignocellulosic biomass hydrolysates to fuels and chemicals due to their toxicity on fermenting microbes. To harness the potential of lignocellulose as a cheap source of fermentable sugars, in situ detoxification of furfural and other lignocellulosederived microbial inhibitors is essential. To enhance in situ detoxification and tolerance of furfural by Clostridium beijerinckii NCIMB 8052 during acetone-butanolethanol (ABE) fermentation, the effect of glycerol on NADH/NADPH generation and ABE production by furfural (4, 5, and 6 g/L)-challenged cultures was investigated in this study. In all instances, beneficial outcomes were observed. For example, the fermentation medium supplemented with glycerol and subjected to 5 g/L furfural elicited up to 1.8- and 3-fold increases, respectively, in NADH and NADPH levels in C. beijerinckii 8052 relative to the control culture. These critical changes are the likely underpinnings for the glycerolmediated 2.3-fold increase in the rate of detoxification of 5 g/L furfural, substrate consumption, and ABE production compared to the unsupplemented medium. Collectively, these results demonstrate that increased intracellular NADH/NADPH in C. beijerinckii 8052 due to glycerol utilization engenders favorable effects on many V. Ujor : C. V. Agu : T. C. Ezeji (*) Department of Animal Sciences and Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, 305 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH 44691, USA e-mail: [email protected] V. Gopalan Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA

aspects of cellular metabolism, including enhanced furfural reduction and increased ABE production. Keywords Clostridium beijerinckii . Butanol . Furfural . Glycerol . NADH

Introduction Among other challenges, the generation of microbial inhibitory compounds during pretreatment of lignocellulosic biomass is a major bottleneck hampering large-scale utilization of lignocellulose-derived sugars as cheap fermentation substrates for the production of biofuels and bulk chemicals. Heat and acid-assisted deconstruction of lignocellulose release hexose and pentose sugars and dehydrate part of these sugars to generate 5-hydroxymethyl furfural (HMF) and furfural, respectively (Palmqvist and Hähn-Hägerdal 2000). These furanic aldehydes exert severe inhibitory effects on bacteria and yeasts, impairing growth and fermentation (Palmqvist and Hähn-Hägerdal 2000; Ezeji et al. 2007; Zhang et al. 2012; Allen et al. 2010). Furfural and HMF have been the focus of extensive research as model microbial inhibitory compounds, largely due to their diverse detrimental effects on cellular metabolism, including DNA damage, inhibition of glycolytic enzymes, disruption of cell membranes, and perturbation of redox balance (Palmqvist and Hähn-Hägerdal 2000; Almeida et al. 2007, 2008; Zhang and Ezeji 2013; Mills et al. 2009). Previously, we have demonstrated that the solventproducing clostridia, Clostridium beijerinckii BA101 (Ezeji et al. 2007) and Clostridium acetobutylicum ATCC 824 (Zhang et al. 2012) can reduce 2–3 g/L furfural and HMF to their corresponding less toxic alcohols during acetonebutanol-ethanol (ABE) fermentation, whereas higher concentrations of these aldehydes proved deleterious. The latter

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observation gains significance since furfural and HMF can reach ~6 and 3.5 g/L, respectively, in lignocellulosic hydrolysates (Klinke et al. 2004; Almeida et al. 2009). However, even at 2 g/L furfural/HMF, detoxification of furfural and HMF siphons reducing equivalents [NAD(P)H] in detoxifying microbial cells, thereby disrupting redox balance and hampering cofactor-dependent biosynthetic reactions (Zhang et al. 2012; Ask et al. 2013; Sávári et al. 2003; Palmqvist et al. 1999). These redox-associated stresses propagate effects on cellular metabolism (Hou et al. 2009; Almeida et al. 2008; Sávári et al. 2003; Nilsson et al. 2005). For instance, such stresses in solventogenic Clostridium species may cause an “acid crash” due to decreased acid reassimilation. While these observations suggest that cellular detoxification gains inevitably lead to solventogenic (ABE) losses, we reasoned that a steady supply of NAD(P)H in furaldehyde-challenged cultures through sustained reduction of the NAD(P)+ that accumulate during furfural/HMF reduction would facilitate efficient detoxification of lignocellulosic hydrolysates without compromising fermentation product formation. If successful, such in situ detoxification of furfural and other lignocellulose-derived microbial inhibitors will be a cost-effective strategy for the bioconversion of biomass-derived sugars to biofuels and chemicals (Almeida et al. 2008). Toward this goal, we first consider the catalytic repertoire and the cofactor status of the cell. The C. beijerinckii NCIMB 8052 (hereafter referred to as C. beijerinckii) genome has multiple aldo/keto reductases and alcohol dehydrogenases similar to those that participate in the detoxification of furfural and HMF by Saccharomyces cerevisiae (Gorsirch et al. 2006). Our transcriptomic data showed that the mRNA levels of multiple aldo/keto reductases and alcohol dehydrogenases in C. beijerinckii increased significantly in response to furfural challenge (Zhang and Ezeji 2013). We also observed upregulation of genes whose protein products are involved in redox homeostasis and purine and pyrimidine metabolism (Zhang and Ezeji 2013). Therefore, we rationalized that the gridlock to reduction of high concentrations of furaldehydes by C. beijerinckii is unlikely to be catalytic insufficiency (as was noted with S. cerevisiae) but likely a result of depletion of NADH/NADPH levels. A plausible strategy then for increasing intracellular generation of NAD(P)H, and thereby fully harnessing the furfuralreductive capacity of C. beijerinckii, is by supplementing the fermentation medium with glycerol, a more reduced substrate compared to glucose. Glycerol catabolism generates two additional moles of NADH relative to the consumption of a molar equivalent of glucose (Lin 1976; Neijssel et al. 1975). In fact, a significant increase in the production of butanolethanol with a concomitant decrease in acetone generation was observed when a C. acetobutylicum culture was grown in a mixture of glucose/glycerol (~1:2 molar ratio; Girbal and Soucaille 1994; Vasconlelos et al. 1994). High levels of

reducing equivalents originating from glycerol catabolism were believed to sustain the productivity of NADHdependent ethanol and butanol dehydrogenases (Girbal and Soucaille 1994; Vasconlelos et al. 1994; Girbal et al. 1995). Hence, it is reasonable to expect two NADH-related payoffs from supplementation of the fermentation broth with glycerol: expedient reduction of furfural to furfuryl alcohol during the acidogenic phase and subsequent robust ABE production post-furfural transformation. Our strategy was also motivated by the need for efficient bioconversion of cheap crude glycerol, which is experiencing a glut as the major by-product of biodiesel production (Johnson and Taconi 2007; OECDFAO 2010; Gerpen 2005). Approximately 10 kg of glycerol is generated for every ~114 L of biodiesel produced (Cavalheiro et al. 2009). With global production of biodiesel estimated to reach 41 billion L/year by 2019 (OECD-FAO 2010), it is critical to find viable options for managing this glut (Johnson and Taconi 2007). While others have pursued either fermentation or catalytic conversion of glycerol to chemicals such as 1,3-propanediol (Jun et al. 2010; González-Pajuelo et al. 2005), we have explored here glycerol supplementation of a glucose-based fermentation medium as a means of enhancing furfural detoxification through increased generation of NADH/NADPH. Hence, the aims of this C. beijerinckii-centered study were to (i) investigate the effect of supplementing the growth medium with glycerol on the reduction of furfural to the less toxic furfuryl alcohol; (ii) evaluate if glycerol catabolism is a viable strategy for ensuring a steady supply of reducing power during ABE fermentation in cultures challenged with furfural (4, 5, and 6 g/L); and (iii) correlate increased generation of NADH/NADPH in glycerol-supplemented cultures to the rate and efficiency of furfural detoxification and ABE production relative to control cultures.

Methods Microorganism and culture conditions C. beijerinckii used in this study was obtained from the American Type Culture Collection (Manassas, VA) as C. beijerinckii ATCC 51743. Laboratory stocks were maintained as spore suspensions in sterile, double-distilled water at 4 °C. Spores were heat-shocked at 75 °C for 10 min, cooled on ice, and then inoculated into anoxic tryptone-glucose-yeast extract (TGY) broth for 12 to 14 h for inoculum generation as previously described (Ezeji et al. 2003). Fermentation was conducted in loosely capped 250 mL Pyrex culture bottles at 35 ± 1 °C. To facilitate comparisons with earlier reports

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(Girbal and Soucaille 1994; Vasconlelos et al. 1994), we used the previously employed glucose:glycerol molar ratio of ~1:2 (36 g/l glucose: 36.1 g/L glycerol), while control cultures contained only glucose (60 g/L) in standard P2 medium containing mineral, buffer, and vitamin additives as described elsewhere (Ezeji et al. 2003). Furfural was pulse-fed to both control and test cultures at 10 h of fermentation to final concentrations of 4, 5, and 6 g/L. All cultures were buffered with 2-(N-morpholino)ethanesulfonic acid (MES; 7 g/L) and grown in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI) with a modified atmosphere of 82 % N2, 15 % CO2, and 3 % H2. All experiments were conducted in triplicate. NAD+/NADH and NADP+/NADPH assays Cultures of C. beijerinckii were grown in either glucose- or glucose + glycerol-based P2 media, and furfural (5 g/L) was pulse-fed at 10 h of fermentation. Test samples (5 mL) were collected at 10, 16, 48, and 60 h. Assays were conducted with NAD+/NADH and NADP+/NADPH kits (Sigma, St. Louis, MO) according to the manufacturer’s protocol. Cell pellets were lysed with a Qiagen Tissue Lyser LT (Qiagen Hilden, Germany) at 50 oscillations/s for 3 min in NAD+/NADH and NADP+/ NADPH extraction buffers (provided in the assay kit), and the resulting lysate was used to quantitatively assay for NAD+/ NADH and NADP+/NADPH with the aid of colorimetric indicators at 450 nm. Spectrophotometric measurements were made using an iMarkTM microplate reader (Bio-Rad, Hercules, CA). Analytical methods Cell concentration was monitored by measuring optical density at 600 nm (OD600) using a DU® spectrophotometer (Beckman Coulter Inc., Brea, CA). Changes in the concentrations of furfural and furfuryl alcohol were determined by monitoring absorbance at 276 and 220 nm, respectively, using the same spectrophotometer. Additional confirmation by HPLC was performed using the Waters 2796 Bioseparations Module equipped with a photodiode array (PDA) detector (Waters, Milford, MA) and a 3.5-μm Xbridge C18, 150-mm × 4.6-mm column (Waters, Milford, MA) as previously described (Zhang et al. 2012). The concentrations of the fermentation products including acetone, butanol, ethanol, acetic acid, and butyric acid were determined using a 7890A Agilent gas chromatograph (Agilent Technologies Inc., Wilmington, DE) equipped with a flame ionization detector and a 30-m (length) × 320-μm (internal diameter) × 0.5-μm (HP-INNOWax film) J × W 19091N-213 capillary column. Nitrogen was used as carrier gas, while the inlet and detector were maintained at 250 and 300 °C, respectively. The oven temperature was programmed to span from 60 to 200 °C in 20 °C/min increments, with a 5-

min hold at 200 °C. The injection volume of samples was 1 μl, with a split ratio of 10:1. Glucose concentrations were measured by using a 3,5dinitrosalicylic acid-based assay (Miller 1959). Additional validation by HPLC was performed with a Waters 2796 Bioseparations Module equipped with Evaporative Light Scattering Detector (ELSD; Waters, Milford, MA) and a 9-μm Aminex HPX-87P 300-mm × 7.8-mm column maintained at 65 °C, in series with a 4.6-mm ID × 3-cm-long Aminex deashing guard column (Bio-Rad, Hercules, CA) as previously described (Zhang et al. 2012). HPLC grade water was used as the mobile phase. Glycerol levels were quantified by HPLC using the Waters 2796 Bioseparations Module equipped with a PDA detector (Waters, Milford, MA) and an Alltech Prevail Carbohydrates ES 250 mm × 4.6 mm × 5 μm column (Grace, Deerfield, IL). The analysis was performed between 210 and 400 nm, with a step size of 1.2 nm. Samples were eluted using a linear gradient of acetonitrile [85 % (v/v)] and water [15 % (v/v)].

Results Biotransformation of furfural to furfuryl alcohol by C. beijerinckii The reduction of 4, 5, and 6 g/L furfural to furfuryl alcohol by C. beijerinckii was evaluated in media containing either glucose (control) or a 2:1 molar mixture of glycerol and glucose (test). The biotransformation profiles are depicted in Fig. 1. To allow considerable buildup of C. beijerinckii cells prior to the furfural challenge, the fermentation was pulsefed with furfural only at 10 h in all cases. At 4 g/L furfural, both test and control cultures reduced all the furfural in the medium by 16 h. However, the rate of furfural reduction was significantly faster in glycerol-containing cultures, which reduced 76 % of the added furfural 2 h after addition at the rate of 1.52 g/L h, nearly 2-fold more than the rate observed for the control cultures which reduced only 42 % of the 4 g/L furfural in the same time period at 0.84 g/L h (Fig. 1a). In fact, this attribute is even more evident from examining the reduction profiles of cultures treated with 5 g/L (Fig. 1b) and 6 g/L (Fig. 1c) furfural. Although both glycerol- and non-glycerol-containing cultures reduced 5 g/L furfural to furfuryl alcohol, the rate of detoxification was considerably faster in glycerol-supplemented cultures, which achieved full detoxification in 6 h, compared to 14 h for cultures containing only glucose (Fig. 1b). Furfural was transformed at the rate of 0.83 g/L h by C. beijerinckii grown in glycerol-supplemented medium as opposed to a rate of 0.36 g/L h in the control glucose medium. C. beijerinckii grown in the glucose-alone medium reduced only 44 % (2.7 g/L) of the 6-g/L furfural at a rate of 0.19 g/L h,

Appl Microbiol Biotechnol Fig. 1 Furfural and furfuryl alcohol profiles of glycerolcontaining and nonglycerolcontaining (only glucose) cultures of C. beijerinckii. Furfural and furfuryl alcohol levels in cultures treated with 4 (a), 5 (b), and 6 (c) g/L furfural at 10 h. Glu glucose, Gly glycerol, F furfural, FA furfuryl alcohol. Error bars represent standard deviations of means (n = 3)

Since furfural limits cell growth, one measure of its toxicity is decreased optical density at 600 nm. The OD600 of the glycerol-containing cultures increased 35, 11, and 4 % after treatment with 4, 5, and 6 g/L furfural, respectively; this contrasts with 18, 2, and 5 % brief increases observed with the glucose cultures (Fig. 2). It is worth noting that the glycerol-containing culture continues to grow (albeit briefly) after addition of 4 and 5 g/L furfural, while the glucose culture (control) shows an immediate cessation. At 6 g/L furfural, both cultures were affected adversely.

8.8 %, respectively) in fermentations with glycerolsupplemented medium (Fig. 3b; Table 1). We noted more impressive gains when we tested 5 and 6 g/L. At 5 g/L furfural, both butanol and ABE concentrations in the glycerol-supplemented culture were 2.3-fold higher than titers produced in the control (Fig. 3c; Table 1). Although 6 g/L furfural impaired ABE productivity in both cultures (Fig. 3d; Table 1), the effect was more severe in the control relative to C. beijerinckii grown in glycerol-supplemented medium. In fact, 2.3- and 1.6-fold increases in butanol and total ABE levels, respectively, were observed in cultures grown on glycerol-supplemented medium compared to those grown on glucose alone (Fig. 3d; Table 1). Further, furfural challenge elicited substantial accumulation of acids in the control cultures. Overall, glycerol supplementation enhanced acid reassimilation by C. beijerinckii, even without the addition of furfural (Figs. 4a and 6a). Although only acetic acid accumulation (37 % increase) was observed with 4 g/L furfural treatment in the control glucose culture compared to the glycerol-containing cultures, both acetic and butyric acid concentrations increased when subjected to 5 and 6 g/L furfural (Figs. 4 and 5). With 5 g/L furfural, acetate and butyrate concentrations were at least 29 and 27 % higher, respectively (Figs. 4c and 5c), in the control cultures relative to cultures grown on glycerol-supplemented medium. In cultures treated with 6 g/L furfural, the control fermentations exhibited at least 18 and 33 % increases in acetate and butyrate concentrations in the fermentation broth, respectively (Figs. 4d and 5d), compared to the glycerol-supplemented fermentations.

ABE and acid profiles with and without glycerol supplementation

Acetone/butanol ratios as another indicator of altered metabolic flux

Similar to furfural reduction, supplementation of the fermentation medium with glycerol significantly enhanced butanol, total ABE production, and yield at all concentrations of furfural tested (Fig. 3; Table 1). Although C. beijerinckii grown in both glycerol-supplemented and unsupplemented media effectively reduced 4 g/L furfural to furfuryl alcohol, final butanol and total ABE titers were marginally higher (8.4 and

Acetone-butanol ratio increased for both test and control cultures with an increase in furfural concentration (0–6 g/L). However, these increases were more significant in the control fermentations performed with glucose alone. Acetone/butanol ratios were 0.38, 0.46, 0.6, and 1.2, respectively, in cultures of C. beijerinckii cultivated on the control medium and challenged with 0, 4, 5, and 6 g/L furfural (Fig. 3e). In contrast,

leaving 3.3 g/L furfural in the medium for the rest of the fermentation. Conversely, in glycerol-supplemented medium, 98 % (~5.9 out of 6 g/L) of furfural was reduced to furfuryl alcohol at 24 h at a rate of 0.42 g/L h. By 36 h, furfural was undetected in the medium, while a corresponding 6-g/L furfuryl alcohol was observed (Fig. 1c). While the glucose-alone control culture could cope with 4 and 5 g/L furfural and even ensure near-complete transformation to furfuryl alcohol (albeit with some other adverse effects, see below), less than 50 % of the 6-g/L furfural was detoxified. These results highlight the need for empirical fermentation studies to ascertain the concentration threshold for effective detoxification of inhibitors, present either individually or in combinations as is the case in biomass hydrolysates. Growth of glycerol-supplemented and unsupplemented cultures in the presence of furfural

Appl Microbiol Biotechnol Fig. 2 Optical densities of glycerol-containing and onlyglucose cultures challenged with 0 (a), 4 (b), 5 (c), and 6 (d) g/L furfural at 10 h. Arrows indicate time of addition of furfural. Error bars represent standard deviations of means (n = 3)

Fig. 3 Butanol concentrations in cultures of C. beijerinckii challenged with 0 (a), 4 (b), 5 (c), and 6 (d) g/L furfural and the corresponding acetone/butanol (e) ratios in media containing glucose or glucose + glycerol. Arrows indicate time of addition of furfural. Error bars represent standard deviations of means (n = 3)

Appl Microbiol Biotechnol Table 1 Total ABE and butanol yields/productivities and residual substrates in glycerol-supplemented and unsupplemented cultures of C. beijerinckii challenged with different concentrations of furfural Furfural Substrates concentration (g/L)

4 5 6

Glucose + glycerol Glucose Glucose + glycerol Glucose Glucose + glycerol Glucose

Butanol

ABE

Residual substrates

Concentration Yield (g/g) (g/L)

Productivity Concentration Yield (g/g) (g/L h) (g/L)

Productivity Glucose (g/L h) (g/L)

Glycerol (g/L)

9.9 ± 0.3 9.1 ± 0.5 6.8 ± 0.7 2.9 ± 0.8 3.1 ± 0.3 1.3 ± 0.1

0.20 ± 0.00 0.18 ± 0.01 0.14 ± 0.00 0.05 ± 0.00 0.05 ± 0.01 0.02 ± 0.01

0.3 ± 0.01 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.01 0.1 ± 0.0 0.05 ± 0.00

17.1 ± 0.1 N/A 20.9 ± 0.4 N/A 28.3 ± 0.1 N/A

0.21 ± 0.00 0.23 ± 0.01 0.18 ± 0.01 0.14 ± 0.01 0.17 ± 0.00 0.12 ± 0.00

14.8 ± 0.5 13.5 ± 0.8 10.7 ± 0.9 4.7 ± 1.5 5.5 ± 0.3 2.9 ± 0.13

0.31 ± 0.01 0.35 ± 0.01 0.29 ± 0.01 0.22 ± 0.00 0.30 ± 0.01 0.27 ± 0.01

0.0 ± 0.0 16.6 ± 0.7 5.7 ± 0.3 39.5 ± 0.1 25.2 ± 0.2 49.1 ± 0.1

N/A not applicable

the acetone-butanol ratios were 0.36, 0.48, 0.53, and 0.75 for glycerol-supplemented fermentations challenged with 0, 4, 5, and 6 g/L furfural, respectively (Fig. 3e). Therefore, acetone/ butanol ratio was up to 1.6-fold higher in cultures of C. beijerinckii grown on glucose alone relative to cultures cultivated on glycerol-supplemented medium. Fig. 4 Acetic acid concentrations in cultures of C. beijerinckii following treatment with 0 (a), 4 (b), 5 (c), and 6 (d) g/L furfural in media containing glucose or glucose + glycerol. Arrows indicate time of addition of furfural. Error bars represent standard deviations of means (n = 3)

Intracellular levels of NAD+, NADH, NADP+, and NADPH For the NADH/NAD + and NADPH/NADP + assays, C. beijerinckii was grown in P2 medium supplemented with glycerol or in standard P2 medium as control, and furfural (5 g/L) was pulse-fed at 10 h of fermentation.

Appl Microbiol Biotechnol Fig. 5 Butyric acid levels in cultures of C. beijerinckii treated with 0 (a), 4 (b), 5 (c), and 6 (d) g/L of furfural in fermentations containing glucose and a mixture of glucose and glycerol. Arrows indicate time of addition of furfural. Error bars represent standard deviations of means (n = 3)

We also measured the levels of these cofactors in glycerol-supplemented and unsupplemented media without furfural challenge. Glycerol supplementation significantly enhanced NADH and NADPH regeneration (Fig. 6a, d). Following furfural challenge, NADH and NADPH levels, respectively, in C. beijerinckii grown in glycerol-supplemented medium increased 1.4- and 2.2-fold at 16 h, 1.8- and 3-fold at 48 h, and 1.6- and 2.7-fold at 60 h relative to C. beijerinckii grown in medium containing only glucose as carbon source. Similarly, in C. beijerinckii grown in glycerol-supplemented medium unchallenged with furfural (0 g/L), NADH and NADPH increased up to 1.7- and 1.4-fold, respectively (at 48 h), relative to C. beijerinckii grown in medium with no glycerol (Fig. 6a, d). Further, at 16, 48, and 60 h fermentation, the NADH/NAD+ ratio increased 2.4-, 5-, and 1.3-fold, respectively, while the NADPH/ NADP+ ratio increased 2.6-, 3.6-, and 3.9-fold, respectively, in the furfural-challenged C. beijerinckii grown on glycerol-supplemented relative to glucose-alone medium.

Substrate utilization by C. beijerinckii Substrate utilization trends are in agreement with the ABE levels and furfural detoxification profiles observed for test and control fermentation media. Overall, we observed a higher substrate consumption rate and increased substrate utilization by C. beijerinckii grown in glycerol-supplemented medium than the control medium with no glycerol (Fig. 7). With 4 g/L furfural and the mixed substrate medium, glucose was consumed at the rate of 0.98 g/L h within the first 24 h, during which furfural was transformed, and at an overall rate of 0.76 g/L h. In fact, at the end of fermentation, all of the glucose (36 g/L) in the medium was consumed in addition to 19 g/L of glycerol (for a total substrate consumption of 55 g/L; Fig. 7a). In contrast, C. beijerinckii grown on control medium consumed glucose at the rate of 0.73 g/L h within the first 24 h of fermentation and at an overall rate of 0.60 g/L h. Out of 60 g/L glucose, 43.45 g/L was consumed. Even more striking and instructive trends are observed at 5 and 6 g/L furfural. The effect of furfural supplementation on substrate utilization was more pronounced at 5 g/L furfural, where a 2.3-fold

Appl Microbiol Biotechnol Fig. 6 Intracellular levels of NADH (a), NAD+ (b), NADPH (d), NADP+ (e), and NADH/NAD+ (c) and NADPH/ NADP+ (f) ratios in furfural (5 g/L)-treated and untreated cultures of C. beijerinckii cultivated on glucose and glucose + glycerol. Error bars represent standard deviations of means (n = 3)

increase in substrate utilization was observed in glycerolsupplemented cultures, when compared to cultures grown on glucose alone (Fig. 7b). At this concentration of furfural, glucose consumption in the control cultures proceeded at the rate of 0.70 g/L h during exposure to furfural (24 h) and at an overall rate of 0.63 g/L h. A total of 30 g/L glucose and 15.24 g/L glycerol were consumed by C. beijerinckii grown on glycerol-supplemented medium. In C. beijerinckii grown on the control medium, the rates of glucose utilization were 0.48 and 0.28 g/L h during furfural transformation (24 h) and for the entire duration of fermentation (72 h), respectively. A total of ~21 g/L glucose was consumed by C. beijerinckii grown on the control medium (Fig. 7b). With 6 g/L furfural, total substrate consumption was 1.7-fold higher in glycerol-

supplemented medium (7.8 g/L glycerol + 10.9 g/L glucose) than in glucose-alone medium (10.9 g/L glucose; Fig. 7c). Notably, most of the glucose (about 65 %) metabolized in the control fermentation was consumed pre-furfural challenge. As a result, the overall rate of glucose consumption was 0.15 g/L h in both sets of cultures with 6 g/L furfural challenge. The ratio of glycerol to glucose utilization was 0.7 with 6 g/L furfural, compared to ~0.5 at both 4 and 5 g/L furfural.

Fig. 7 Glucose and glycerol utilization in cultures of C. beijerinckii grown on a mixture of glucose and glycerol relative to cultures grown on glucose alone. Substrate utilization was measured at various times in furfural (4, 5, and 6 g/L)-challenged cultures. Error bars represent

standard deviations of means (n = 3). Glc glucose, Gly glycerol. Filled circle, concentration of glucose in glucose alone; inverted triangle, concentration of glucose in glucose-glycerol mixture; open circle, concentration of glycerol in glucose-glycerol mixture

Discussion In situ detoxification of furfural and other lignocellulosederived microbial inhibitory compounds currently attracts

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tremendous attention as a cost-effective strategy for complete bioconversion of biomass-derived sugars to biofuels and fine chemicals (Alriksson et al. 2010; Almeida et al. 2008; Miller et al. 2009; Liu et al. 2008). Although considerable progress has been made with regards to engineering novel strains for furfural and HMF detoxification, particularly with yeast and Escherichia coli, high concentrations of these inhibitory compounds remain recalcitrant to microbial transformation. The competition for a limited pool of redox cofactors (NADH, NAPDH) presents a major challenge when ABE biosynthetic requirements are counterbalanced with the dire need to reduce furaldehydes to less toxic alcohols. Here, we explored supplementation of standard P2 medium with glycerol to enhance NADH generation and thereby facilitate furfural reduction and ABE production in furfural-challenged cultures of C. beijerinckii. Indeed, fermentation of a glucose-glycerol mixture by C. beijerinckii resulted in a significant increase in NADH generation when compared to the cultures grown on glucose alone, and this led to rapid detoxification of furfural, enhanced substrate utilization, and improved ABE production in C. beijerinckii cultures challenged with furfural, particularly at 5 and 6 g/L. To our knowledge, this is the first report of furfural detoxification up to 6 g/L by a solventogenic Clostridium species. The findings of this study, therefore, substantiate the hypothesis that NAD(P)H depletion during furfural reduction is a major metabolic impediment to the transformation of high concentrations of furfural (Sávári et al. 2003; Liu et al. 2008; Liu 2011; Wahlbom and Hähn-Hägerdal 2002; Gorsirch et al. 2006). Cofactor depletion and disruption of redox balance in C. beijerinckii grown in control medium + furfural is evident not only from the excess accumulation of acids but also higher acetone/butanol ratios. In a previous study, we observed that when C. acetobutylicum ATCC 824 was challenged with 3 and 4 g/L furfural, cell growth stalled during furfural reduction and resumed later (Zhang et al. 2012); however, acid production, particularly butyrate, proceeded unaffected. This response was mimicked by C. beijerinckii in this study (Figs. 4 and 5). In both instances, this response is presumably to maintain the energy status of the cell, as the production of acetate and butyrate results in the generation of ATP (Grupe and Gottschalk 1992; Doremus et al. 1985; Witzke and Bahl 2012). In fact, both HMF and furfural are known to cause a sharp decline in ATP and NAD(P)H levels in S. cerevisiae (Ask et al. 2013; Palmqvist et al. 1999). If the extended secretion of acids following challenge with 5 and 6 g/L furfural (Figs. 4 and 5) is indeed a metabolic response to decreasing energy and NADPH charge, then decreased cell growth upon inclusion of 6 g/L furfural in control fermentations might stem from depletion of NAD(P)H, which in turn would disrupt acid reassimilation and progression of solventogenesis.

Although different factors are involved in the shift from acidogenesis to solventogenesis, high intracellular accumulation of NAD(P)H pre-solventogenesis plays a crucial role in this transition, as butanol and ethanol biosyntheses (upon acid reassimilation) are both cofactor dependent (Grupe and Gottschalk 1992; Wang et al. 2012). However, with high concentrations of furfural, NAD(P)H is expended during reduction of furfural to furfuryl alcohol, thus making the shift to solventogenesis less favored. Unlike butanol and ethanol, however, acetone production is not cofactor-linked. Therefore, depletion of NAD(P)H with increasing furfural concentrations causes the metabolic flux to shift in favor of acetone biosynthesis. Clearly, the presence of glycerol dampens the shift to acetone and favors butanol production, which we attribute to increased availability of reducing power. This result is consistent with previous studies that reported significant steady-state increases in the production of butanol/ ethanol relative to acetone by C. acetobutylicum grown on a mixture of glucose and glycerol and attributed the same to increased NADH/NAD+ ratios (Girbal and Soucaille 1994; Vasconlelos et al. 1994). Even when taking the necessary precautions, we and others have noted that it is difficult to establish perfect synchrony in these fermentation cultures, thus rendering cross-culture comparisons difficult. Despite this caveat, some key inferences can be drawn from our cofactor analysis. Foremost, the trends we observed underscore consumption of NADH and NADPH in glucose-only cultures for combating furfural stress as evidenced by the increases in NAD+ and NADP+ (oxidation of reduced cofactors to oxidized forms). This is a vicious cycle as reflected in the delayed transformation of furfural in glucose-grown cultures (24 h, relative to 16 h for cultures grown on glycerolsupplemented medium). Progressively less metabolically active cells (in glucose-only cultures), as indicated by a more rapid decrease in optical density (Fig. 2c), sharp accumulation of acids (Figs. 4c and 5c), and minimal increases in ABE production post-furfural addition (at 5 and 6 g/L, Fig. 3c, d; Table 1), are likely to only accentuate the redox crisis by decreasing reduced cofactor generation. In contrast, the redox surpluses from glycerol metabolism are best illustrated in the ratios of the reduced/oxidized forms (Fig. 6c, f). Since NAD+ serves as both the precursor for NADP+ biosynthesis and as an allosteric effector of the ATP-NAD+ kinase that generates NADP+ (Moat and Foster 1987; Heuser et al. 2007; Lundquist and Olivera 1971), it is not surprising that glycerol-mediated payoffs for NADH are mirrored in NADPH. The dual gains in NADH and NADPH are likely critical in detoxifying furanic aldehydes. Transcriptomic analysis of the response of C. beijerinckii to furfural revealed the upregulation of both NADH- and NADPH-dependent aldo/keto reductases (Zhang and Ezeji 2013). In fact, a recent study has also exploited the ability of

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an endogenous NADH-dependent oxidoreductase to accept furfural as a substrate (in addition to its native palette of substrates) to increase furfural tolerance (Wang et al. 2011). Due to some intrinsic limitations (e.g., glycerol utilization only as a mixed substrate), the ameliorating effects of glycerol are not unlimited. The intrinsic substrate bias of C. beijerinckii for glucose over glycerol is difficult to override. Despite glucose being the preferred substrate, the furfural-mediated oxidative stress affects glycolytic enzymes and thereby decreases glycolytic flux (Banerjee et al. 1981); stress mitigation strategies cause a shift to glycerol catabolism and NADH generation, as evidenced by the increased glycerol/glucose utilization ratios at 6 g/L furfural. Albeit unproven, glycerol uptake might be influenced by physiological needs (e.g., redox crisis); this idea is consistent with the absence of a dramatic change in the acetone/butanol ratio in the presence of glycerol and the absence of furfural (Fig. 3e). If indeed redox crises trigger a spike in glycerol assimilation, changes in expression and activity of the glycerol transporter as a function of cellular redox merit further investigation. We emphasize that the decisive advantage of glycerol as a medium additive must be guided by the yield of fermentation products rather than the comparative fold increases in ABE and butanol titers. To illustrate, although the glycerol-supplemented cultures perform more favorably compared to the control at 6 g/L furfural with respect to fold increases in ABE and butanol, the overall ABE yield is significantly diminished. Thus, two goals must be balanced—the need to generate end products while effectively removing an inhibitor before it damages the fermenting microbe (e.g., glycerol-supplemented culture with 5 g/L furfural). In conclusion, supplementation of P2 medium with glycerol was explored as a strategy to increase the generation of cofactors required for furfural reduction by C. beijerinckii. Our results demonstrate that glycerol catabolism indeed increases NADH/NAD+ and NAD(P)H/NAD(P)+ ratios, thereby improving furfural detoxification and ABE production. Given the various microbial inhibitory compounds in lignocellulosic hydrolysates and the crude glycerol glut caused by the expanding biodiesel industry, our findings are expected to motivate efforts to improve glycerol uptake by C. beijerinckii and to design metabolic engineering approaches for enhanced co-bioconversion of lignocellulosic biomass and glycerol to bio-butanol and other bulk chemicals.

Acknowledgments Salaries and research support were provided in part by State of Ohio funds appropriated to the Ohio Plant Biotechnology Consortium by The Ohio State University, Ohio Agricultural Research and Development Center (OARDC), Western Region Sungrant (Prime award No. 2010-38502-21839), and the Hatch grant (Project No. OHO01222).

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