Robustness of Clostridium saccharoperbutylacetonicum for acetone-butanol-ethanol production: Effects of lignocellulosic sugars and inhibitors

Fuel 208 (2017) 549–557

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Full Length Article

Robustness of Clostridium saccharoperbutylacetonicum for acetonebutanol-ethanol production: Effects of lignocellulosic sugars and inhibitors Dunfan Yao a,b, Sheng Dong a, Pixiang Wang a, Tianhu Chen b, Jin Wang b, Zheng-Bo Yue b, Yi Wang a,c,⇑ a b c

Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA School of Resource and Environmental Engineering, Hefei University of Technology, Hefei 230009, China Center for Bioenergy and Bioproducts, Auburn University, Auburn, AL 36849, USA

h i g h l i g h t s  C. saccharoperbutylacetonicum can utilize most lignocellulosic sugars efficiently.  The consumption rate is sugar specific, with glucose is the most preferable. 

q-Coumaric acid is the most toxic out of the tested phenolic inhibitors.

 Furfural and HMF can be completely converted to their corresponding alcohols.  Cells can adapt to inhibitory conditions and produce more ABE than the control.

a r t i c l e

i n f o

Article history: Received 17 May 2017 Received in revised form 29 June 2017 Accepted 1 July 2017

Keywords: Acetone-butanol-ethanol (ABE) Butanol Carbon catabolite repression (CCR) Carbon sources Clostridium saccharoperbutylacetonicum N14 Fermentation inhibitor

a b s t r a c t Clostridium saccharoperbutylacetonicum N1-4 has great potentials for acetone-butanol-ethanol (ABE) production from lignocellulosic carbon sources. However, fundamental information about its metabolism of lignocellulosic sugars and tolerance to fermentation inhibitors is not available. Here, we systematically evaluated effects of representative sugars and lignocellulosic inhibitors on ABE fermentation by C. saccharoperbutylacetonicum. Results indicated that C. saccharoperbutylacetonicum can use glucose, cellobiose, xylose, arabinose and mannose efficiently, while degrade galactose slowly and incompletely. Glucose was the most preferable carbon source that has shown carbon catabolite repression (CCR) on the degradation of other sugars. However, the actual sugar preference is highly dependent on the composition (including sugar types and concentrations). Ferulic acid, syringaldehyde and q-coumaric acid are potent phenolic inhibitors, with q-coumaric acid as the most toxic. C. saccharoperbutylacetonicum can tolerate up to 0.8, 0.8 and 0.4 g/L of ferulic acid, syringaldehyde and q-coumaric acid, respectively. Furfural and hydroxymethylfurfural (HMF) are inhibitory but not as toxic as phenolic inhibitors. C. saccharoperbutylacetonicum can tolerate up to 3 g/L furfural or HMF individually or as a mixture (1.5 g/L for each). Both furfural and HMF can be completely converted into their corresponding alcohols, with furfural is more rapidly transformed than HMF. Comparing to other prominent solventogenic clostridia, C. saccharoperbutylacetonicum can tolerate higher or at least comparable levels of inhibitors; it can adapt to inhibition conditions and produce more ABE than the control. Therefore, our results testified that C. saccharoperbutylacetonicum is a robust workhorse for biofuel production. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Butanol (n-butanol) can not only be used as a valuable fuel that has various advantages over ethanol, but also be applied as a ⇑ Corresponding author at: Department of Biosystems Engineering, Auburn University, 215 Tom E. Corley Building, Auburn, AL 36849, USA. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.fuel.2017.07.004 0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.

chemical feedstock in many industries [1,2]. While the traditional butanol production through petrochemical approach is highly energy-demanding and generates various environmental pollutants, bio-butanol production from lignocellulosic carbon sources through the clostridial acetone-butanol-ethanol (ABE) fermentation has attracted tremendous attention recently due to its renewable and environmentally-friendly features. The substrate cost is a large part out of the overall cost of ABE fermentation [3]. Therefore,

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low-value substrates from sustainable feedstocks are desirable for ABE fermentation to make the bioprocess economically viable. Lignocellulose is the most abundant renewable resource on the planet. Fermentable sugars can be obtained from the lignocellulosic biomass through pretreatment and enzymatic hydrolysis [4]. Thus, the lignocellulosic biomass is a promising feedstock for renewable ABE production [5,6]. During biomass pretreatment and enzymatic hydrolysis, various simple sugars are generated in different amounts depending on the type of the feedstocks and the pretreatment conditions. With a regular biomass pretreatment and enzymatic hydrolysis, glucose is the primary sugar generated from the cellulosic materials, with a concentration of as high as 76.8 g/ L [7]. The concentration of other sugars including xylose, arabinose, mannose, galactose and cellobiose (disaccharide of glucose) are generally low. However, high concentration of xylose (42.1 g/L) has been reported for the dilute acid prehydrolysis with enzymatic saccharification of corn stover [8]. Mannose is the leading component in the hemicellulose of softwood [9]. Up to 7.2 g mannose out of 100 g of pretreated softwood can be obtained [9]. High concentrations of galactose can be found in the industrial feedstock sources such as the cheese whey [10]; meanwhile galactose is also the primary sugar that can be obtained from the hydrolysis of some marine biomass, such as the red seaweed [11]. During the enzymatic hydrolysis, the enzymatic cocktail generally contains extra b-glucosidase (besides the fungal cellulase) to fully convert cellobiose into glucose. However, when a host strain for efficient cellobiose consumption is available, b-glucosidase is not necessary for the hydrolysis (and thus can save the enzyme cost). In this case, as high as 80 g/L cellobiose can be obtained from the biomass pretreatment and hydrolysis process [12]. Along with the sugar production, phenolic compounds such as syringaldehyde, ferulic acid and q-coumaric acid can also be generated from the degradation of lignin, while furfural and hydroxymethylfurfural (HMF) from the degradation of hexose and pentose sugars [13,14]. It has been reported that 1.2 g/L ferulic acid and 2.2 g/L q-coumaric acid can be generated from the pretreatment of corn cobs [15]. During a hydrothermolysis-pretreatment of switchgrass, 0.36 g/L of syringaldehyde was generated [16]. Furfural and HMF are common furan aldehydes derived from dehydration of pentoses and hexoses during pretreatment and hydrolysis processes. Depending on the source of biomass and the type of pretreatment method employed, up to 5.0 g/L furfural and 5.9 g/L HMF can be obtained [7,17]. All these compounds are potential microbial inhibitors during ABE fermentation [6,18]. Clostridium saccharoperbutylacetonicum N1-4 was isolated in 1959, and was first described by Motoyoshi in a patent [19]. The strain was stopped for industrial use in early 1960s due to phage problems [20]. It represents the type strain of one very important spieces out of four (C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. saccharobutylicum) that can perform efficient ABE production [21]. Particularly, C. saccharoperbutylacetonicum N1-4 possess various interesting features for ABE production and can naturally produce very high levels of ABE [22,23]. Therefore, C. saccharoperbutylacetonicum has great potentials for economic ABE production from low-value lignocellulosic carbon sources. However, compared to the extensively studied C. acetobutylicum and C. beijerinckii, the research on the fermentation characteristics of C. saccharoperbutylacetonicum is apparently lagging. Especially, the fundamental information about its metabolism of different lignocellulosic sugars and tolerance to various inhibitors is not available until now. Therefore, in this study we set out to systematically evaluate the effects of representative sugars and various inhibitors that might be present in lignocellulosic hydrolysates on the ABE fermentation by C. saccharoperbutylacetonicum. Our results demonstrated that C. saccharoperbutylacetonicum can efficiently utilize

most of the lignocellulosic sugars for efficient ABE production, and can tolerate higher concentrations of inhibitors when compared to other prominent solventogenic clostridial strains. Therefore, C. saccharoperbutylacetonicum can be used as a robust platform strain for ABE production from lignocellulosic carbon sources. 2. Materials and methods 2.1. Microorganism and culture maintenance Laboratory stocks of C. saccharoperbutylacetonicum N1-4 (HMT) (also known as DSM 14923, ATCC 27021, and NCIB 12606) were routinely maintained in 20% glycerol at 80 °C. The culture was first grown in an anaerobic chamber (N2-CO2-H2 with volume ratio of 85:10:5) in tryptone-glucose-yeast extract (TGY) medium containing 30 g/l of tryptone, 20 g/l of glucose, 10 g/l of yeast extract, and 1 g/l of L-cysteine for 17–18 h [24]. Then the active growing culture was transferred at 2% (v/v) to another fresh TGY medium or tryptone-xylose-yeast extract (TXY; 20 g/l of xylose was used instead of 20 g/l of glucose) medium. The culture was further grown in the chamber for 4–5 h until the optical density (OD) at 600 nm reached 0.8–1.0 before it was inoculated into solvent production medium. 2.2. Fermentation All the fermentation was carried out in 250 mL serum bottles with a fermentation volume of 100 mL. P2 medium was used as the fermentation medium [24]. The P2 synthetic medium was originally described by Monot et al. [25]. It was widely used for the ABE fermentation with various solventogenic clostridial strains. The P2 medium contains following compounds (in g/L in the final fermentation broth): KH2PO4, 0.5; K2HPO4, 0.5; CH3COONH4, 2.2; MgSO4
7H2O, 0.2; MnSO4
H2O, 0.01; FeSO4
7H2O, 0.01; NaCl, 0.01; paminobenzoic acid, 0.001; thiamine-HCl, 0.001; and biotin, 0.00001 [24]. The stock solutions (100x concentrated) were prepared as three separate fractions to avoid possible precipitation. The P2 buffer stock solution contains KH2PO4, K2HPO4, and CH3COONH4, the P2 mineral stock solution contains MgSO4
7H2O, MnSO4
H2O, FeSO4
7H2O, and NaCl, and the P2 vitamin stock solution contains p-aminobenzoic acid, thiamine-HCl, and biotin. For the fermentation, a total of 80 g/L sugar (either pure glucose or mixed sugars; for mixed sugars, each sugar was added in the same amount to make a total of 80 g/L) was used as the carbon source. In addition, 2 g/L yeast extract and 6 g/L tryptone was also supplemented. Before the fermentation, the sugar solutions, mixture of yeast extract and tryptone, and inhibitors (when necessary) were all set to pH 6.8, sparged with N2 for 2–3 min and sterilized separately, and then put into the anaerobic chamber and mixed as designed. Meantime, pre-sterilized P2 stock solutions were also added. Active growing preculture (at OD of 0.8–1.0) was inoculated into the fermentation with an inoculum ratio of 5% (v/v). Fermentation was performed at 30 °C with 150 rpm agitation. The pH of the fermentation was not controlled. All fermentations were performed in duplicate. During the course of the fermentation, 1.5 mL samples were collected regularly for the analysis of OD, ABE, acids, furfuryl alcohol and HMF alcohol. 2.3. Analytical procedures Cell density (OD at 600 nm) was measured with an Ultrospec 10 cell density meter (Amersham Biosciences Corp., Piscataway, NJ). Concentrations of ABE, acids, furfuryl alcohol as well as HMF alcohol were determined using an HPLC (Agilent Technologies 1260

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Infinity series, CA) with a refractive index Detector (RID) and a diode array ultraviolet detector (DAD), equipped with a Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). The column was eluted with 0.005 N H2SO4 with a flow rate of 0.6 ml/min at 25 °C. Concentrations of sugars were determined using a Shimadzu HPLC (Tokyo, Japan) equipped with an automatic sampler/injector and a refractive index detector (RID-10A, Milford, MA) and an Aminex HPX-87P column (Bio-Rad). Deionized water was used as the mobile phase at a flow rate of 0.6 mL/min at 65 °C. 3. Results and discussion 3.1. ABE fermentation with individual sugar

3.2. ABE fermentation with mixed sugars Fermentations were carried out with all six sugars (glucose, mannose, xylose, arabinose, galactose and cellobiose) were mixed together in equal amounts (making a total of 80 g/L). As shown in Fig. 2a, all the sugars were concurrently utilized during the fermentation; however, the utilization rate was sugar specific. Glucose was consumed very rapidly from the very beginning with a very short lag phase. It was degraded completely in less than 42 h. For all the other sugars, the consumption was slow in the first 18 h, which might be due to the carbon catabolite repression (CCR)

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First, we set out to investigate the ability of C. saccharoperbutylacetonicum to ferment individual representative sugars that can be obtained from the lignocellulosic biomass. As shown in Fig. 1a, in the initial 18 h, the consumption for most of the sugars was slow except for cellobiose, which demonstrated very rapid consumption starting from 6 h. After 24 h, the fermentation progressed rapidly with most of the carbon sources. By 48 h, the fermentation was completed with glucose, mannose, arabinose or cellobiose as the carbon source, while it took around 72 h to finish with xylose. The fermentation with galactose was rather slow, and only around 30 g/L galactose consumed after 120 h of the fermentation. Corresponding to the sugar consumption, the cell growth profiles were very similar and reached the similar maximum OD when glucose, mannose, or arabinose was used as the carbon source (Fig. 1b). The cell growth was a little bit slower and reached a little lower maximum OD (15% lower than the fermentation with glucose) with xylose as the substrate. Interestingly, when cellobiose was used as the substrate, the cell grew very fast from the beginning of the fermentation, and reached a highest maximum OD of 25.2 at 24 h, which is 50% higher than the fermentation with glucose. After this point the cell density soon declined to a lower level but still comparable to that of the fermentation with glucose, mannose or arabinose. When compared to all other fermentations, the cell growth with galactose was much slower and reached a much lower maximum OD of 6.5 (at 120 h). In the fermentation with glucose as the carbon source, 15.1 g/L butanol and 25.8 g/L ABE was produced within 48 h (Fig. 1c and Table 1), leading to an ABE yield of 0.38 g/g and an ABE productivity of 0.54 g/L/h, respectively. The butanol/acetone ratio was 1.6, which was a little bit lower than the typical value of 2.0 as previously reported for C. acetobutylicum [26]. Fermentations with mannose, arabinose or cellobiose all generated comparable final butanol and ABE (as well as comparable butanol/acetone ratio) as the fermentation with glucose. The

consumption of mannose in C. saccharoperbutylacetonicum is almost as efficient as glucose (Fig. 1a). This is different from the results by Ezeji and Blaschek (2008) that mannose was not a preferreable carbon source for the ABE fermenation with C. beijerinckii BA101 [27]. For most of the biomass feedstocks, b1-4 linked xylose dominates the backbone of hemicelluose structre. However, mannose is the leading component in the hemicellulose of softwood [9]. Therefore, the high efficient consumption of mannose in C. saccharoperbutylacetonicum has remarkable significance, especially when softwood based lignocellulosic biomass is used as the feedstock. Fermentation with xylose produced 12.2 g/L butanol and 17.5 g/L total ABE by the end of the fermentation, which were about 19% and 32% respectively less than that with glucose (Fig. 1c & Table 1). C. saccharoperbutylacetonicum produced the least amount of butanol (10.4 g/L) or ABE (12.9 g/L) when galactose was used. Due to the slow fermentation with galactose, the ABE productivity was only 0.11 g/L/h, while surprisingly the ABE yield reached 0.43 g/g, which was the highest among all the fermentations with individual sugars. Meanwhile, the butanol/acetone ratio in the fermentation with galactose reached 4.5, which is much higher than the fermentation with glucose, and is the highest among all the fermentations with various carbon sources (Table 1). Solventogenic clostridia and many other anaerobic microorganisms transport sugars into the cell using phosphoenolpyruvatedependent phosphotransferase system (PTS) [28,29]. However, the PTS for galactose has never been identified in solventogenic clostridia; the galactose uptake is likely carried out through a non-PTS transferase and the phosphorylation is catalyzed by galactokinase [18]. It looks like the galactose degradation mechanism in C. saccharoperbutylacetonicum is not as potent as the PTS for other carbon sources. We want to indicate that, the fermentation with 80 g/L glucose as discussed here would serve as the control for comparison purpose in the following sections when we discuss the fermentation with mixed sugars or with inhibitor supplemenation.

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G X C A Gl uc ylos Mann rab ala ello os os inos ctos bio e e se e e e

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Fig. 1. Fermentation profiles of C. saccharoperbutylacetonicum when individual sugar was used as the carbon source. (a) Sugar degradation; (b) cell growth (c) solvent production.

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Table 1 Summary of fermentation results for C. saccharoperbutylacetonicum N1-4 with various sugar compositions as the carbon source. Fermentation conditions

Acetone (g/L)

Butanol (g/L)

Ethanol (g/L)

Butanol/Acetone (g/g)

ABE (g/L)

ABE yield (g/g)

ABE productivity (g/L/h)

Glucose Xylose Mannose Arabinose Galactose Cellobiose Mix Sugara G + C-TGYb X + C + G-TGYb X + C + G-TXYb X + G-TGYb X + G-TXYb X + C-TGYb X + C-TXYb

9.7 ± 0.1 4.8 ± 1.2 7.9 ± 0.2 8.3 ± 0.2 2.3 ± 0.4 8.4 ± 0.0 11.1 ± 0.4 12.0 ± 0.1 10.7 ± 0.0 10.8 ± 0.0 9.7 ± 0.7 9.8 ± 0.2 11.8 ± 0.1 10.6 ± 0.2

15.1 ± 0.2 12.2 ± 1.2 14.9 ± 0.1 15.6 ± 0.3 10.4 ± 0.9 15.1 ± 0.1 13.7 ± 0.1 15.3 ± 0.1 14.0 ± 0.2 13.3 ± 0.1 14.5 ± 0.6 13.8 ± 0.0 14.5 ± 0.2 13.3 ± 0.3

1.0 ± 0.0 0.4 ± 0.0 0.8 ± 0.1 0.9 ± 0.0 0.2 ± 0.0 1.3 ± 0.4 1.2 ± 0.1 1.3 ± 0.0 1.2 ± 0.0 1.1 ± 0.2 1.1 ± 0.1 1.0 ± 0.0 1.3 ± 0.1 1.1 ± 0.1

1.6 ± 0.0 2.6 ± 0.4 1.9 ± 0.1 1.9 ± 0.1 4.5 ± 0.3 1.8 ± 0.0 1.2 ± 0.0 1.3 ± 0.0 1.3 ± 0.0 1.2 ± 0.0 1.5 ± 0.2 1.4 ± 0.0 1.4 ± 0.2 1.3 ± 0.1

25.8 ± 0.2 17.5 ± 2.4 23.6 ± 0.1 24.8 ± 0.2 12.9 ± 1.3 24.9 ± 0.6 25.9 ± 0.6 28.6 ± 0.1 25.9 ± 0.2 25.1 ± 0.2 25.2 ± 0.1 24.5 ± 0.1 27.6 ± 0.2 25.0 ± 0.1

0.38 ± 0.01 0.35 ± 0.01 0.41 ± 0.00 0.39 ± 0.01 0.43 ± 0.00 0.42 ± 0.01 0.43 ± 0.01 0.43 ± 0.00 0.38 ± 0.01 0.38 ± 0.01 0.38 ± 0.01 0.35 ± 0.01 0.40 ± 0.01 0.39 ± 0.01

0.54 ± 0.00 0.24 ± 0.03 0.49 ± 0.00 0.52 ± 0.01 0.11 ± 0.01 0.52 ± 0.01 0.54 ± 0.01 0.68 ± 0.00 0.54 ± 0.00 0.53 ± 0.01 0.53 ± 0.00 0.51 ± 0.00 0.38 ± 0.03 0.35 ± 0.01

a

Mix Sugar means the mixed sugar of glucose, xylose, mannose, arabinose, galactose and cellobiose at equal amounts (making a total of 80 g/L). The carbon source composition and the preculture type were used to demonstrate the fermentation condition. For example, ‘G + C-TGY’ indicates that the fermentation was carried out with mixed glucose and cellobiose (at equal amounts to make a total of 80 g/L) as carbon source and the preculture was prepared with TGY medium. G: glucose; X: xylose; C: cellobiose. TGY: tryptone-glucose-yeast extract; TXY: tryptone-xylose-yeast extract. b

Glucose Xylose Mannose Arabinose Galactose Cellobiose

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from glucose. Interestingly, cellobiose, which could be consumed very efficiently (even has a much shorter lag phase than glucose) when it was used as the single carbon source (Fig. 1a), experienced the severest repression with almost no consumption in the first 18 h. However, once it started to be consumed, the consumption rate was very fast (even faster than the glucose consumption); all the cellobiose was consumed in less than 24 h (from 18 h to 42 h). It has been reported that cellobiose is uptaken directly by PTS and hydrolyzed to glucose by the intracellular b-glucosidases for further metabolism in C. acetobutylicum ATCC 824, while glucose is uptaken by different PTSs [30]. This is likely also the case in C. saccharoperbutylacetonicum. There are multiple PTSs annotated for cellobiose and glucose consumption respectively in the genome of C. saccharoperbutylacetonicum. Therefore, the cellobiose transportation is likely repressed by the presence of glucose in the medium. However, the intracellular degradation mechanism for cellobiose seems very efficient and thus lead to speedy cellobiose degradation once it is transported into the cell. This was consistent with the results from the fermentation with cellobiose as sole carbon source as discussed above. Active consumption for all the sugars stopped after 48 h; there were more or less residual sugars left unconsumed for the other four (xylose, arabinose, mannose and galactose, in the order of less to more that was unconsumed). In this sense, we can conclude that the order of sugar preference by C. saccharoperbutylacetonicum is glucose > cellobiose > xylo

se > arabinose > mannose > galactose. The cell growth profile with the mixed sugars was very similar to the control (when glucose was used as the sole carbon source); a similar maximum OD of 16.3 (vs. 16.8 in the control) was obtained at 42 h. For the ABE production, after 48 h of fermentation, 13.7 g/L butanol and 25.9 g/L ABE was produced (Fig. 2b), with an ABE yield of 0.43 g/g, and an ABE productivity of 0.54 g/L/h (Table 1). The simultaneous uptake and metabolism of mixed sugars for ABE production in C. saccharoperbutylacetonicum is a very desirable feature especially when lignocellulosic hydrolysates is used as the fermentation substrate. We further tested the fermentation performance with two- or three-sugar mixtures, including the mixture of 1) glucose and cellobiose, 2) glucose, xylose and cellobiose, 3) glucose and xylose, and 4) xylose and cellobiose (Fig. S1). More detailed results and discussion are presented in the Supplementary materials. One interesting aspect worth mentioning is that, in the fermentation with the mixture of all six sugars, cellobiose exhibited a very fast consumption rate (after a remarkable lag phase) and was the only sugar besides glucose that was completely consumed (in another words, the cellobiose consumption was more efficient than xylose) (Fig. 2a); while in fermentations with either three-sugar mixture (glucose/xylose/cellobiose; Figs. S1b and S1c) or two-sugar mixture (xylose/cellobiose; Figs. S1f and S1g), cellobiose was consumed slower than xylose and also had more left unconsumed

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than xylose at the end. This indicated that the sugar consumption in C. saccharoperbutylacetonicum is actually composition dependent, in terms of both sugar types and concentrations. 3.3. Effects of phenolic inhibitors Phenolic compounds (syringaldehyde, ferulic and q-coumaric acids) are produced from the degradation of lignin during the biomass pretreatment process [13,14]. They are potential inhibitors for microbial fermentations. Therefore, we further investigated the impact of phenolic compounds on ABE fermentation with C. saccharoperbutylacetonicum. Various concentrations ranging from 0.2 to 1.0 g/L of ferulic acid, q-coumaric acid or syringaldehyde were added into the P2 medium containing 80 g/L glucose as substrate. As shown in Fig. 3a, the cell growth was negligibly inhibited compared to the control in the presence of 0.2 or 0.4 g/L ferulic acid

in that the cell grew to the same maximum OD as the control in a similar time frame (at 48 h). When the ferulic acid was increased to 0.6 g/L, an obvious delay of the cell growth was observed, and reached a slightly lower maximum OD at 48 h compared to the control. At 0.8 g/L ferulic acid, there was negligible cell growth in the first 18 h; afterwards, the cell slowly grew up to a maximum OD of 12.9 (vs. 16.8 of the control) at 72 h. When the ferulic acid was increased to 1.0 g/L, the fermentation was fully inhibited and no cell growth was observed. Corresponding to the cell growth, the ABE production profiles at 0.2 and 0.4 g/L ferulic acid were similar to that of the control and reached the maximum at 48 h (Fig. 3b). While at 0.6 and 0.8 g/L ferulic acid, the ABE production has been delayed and the fermentation reached the maximum at 72 h and 96 h respectively. There was no ABE production when 1.0 g/L ferulic acid was present in the fermentation. Surprisingly, the fermentations with 0.2, 0.6

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Fig. 3. Effects of phenolic inhibitors on ABE fermentation with C. saccharoperbutylacetonicum. (a) cell growth profiles with supplementation of various concentrations of ferulic acid; (b) solvent production profiles with supplementation of various concentrations of ferulic acid; (c) cell growth profiles with supplementation of various concentrations of syringaldehyde; (d) solvent production profiles with supplementation of various concentrations of syringaldehyde; (e) cell growth profiles with supplementation of various concentrations of q-coumaric acid; (f) solvent production profiles with supplementation of various concentrations of q-coumaric acid.

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Table 2 Summary of the ABE fermentation results for C. saccharoperbutylacetonicum N1-4 in the presence of various inhibitors. Fermentation conditionsa

Acetone (g/L)

Butanol (g/L)

Ethanol (g/L)

ABE (g/L)

ABE yield (g/g)

ABE productivity (g/L/h)

Corresponding alcoholsd (g/L)

Controlb Ferulic-0.2 Ferulic-0.4 Ferulic-0.6 Ferulic-0.8 Syringaldehyde-0.2 Syringaldehyde-0.4 Syringaldehyde-0.6 Syringaldehyde-0.8 Coumaric-0.2 Coumaric-0.4 Furfural-1 Furfural-2 Furfural-3 HMF-1 HMF-2 HMF-3 1:1c 1.5:1.5c

9.7 ± 0.1 11.5 ± 0.2 9.8 ± 1.0 10.2 ± 0.1 11.5 ± 0.2 13.1 ± 0.1 12.7 ± 0.2 12.9 ± 0.9 12.7 ± 0.7 12.3 ± 0.1 10.9 ± 1.0 12.2 ± 0.9 12.8 ± 0.9 9.6 ± 0.8 13.6 ± 0.2 13.2 ± 0.2 12.4 ± 0.1 13.3 ± 0.3 13.3 ± 1.0

15.1 ± 0.2 14.5 ± 0.4 14.0 ± 0.7 16.1 ± 0.7 16.2 ± 0.1 15.8 ± 0.1 15.1 ± 0.6 15.3 ± 0.6 15.0 ± 0.2 16.0 ± 0.4 16.8 ± 0.6 15.3 ± 0.7 13.1 ± 0.6 13.4 ± 0.6 15.3 ± 0.2 15.1 ± 0.7 16.1 ± 0.9 15.5 ± 0.6 14.7 ± 0.3

1.0 ± 0.0 2.0 ± 0.2 1.9 ± 0.3 2.0 ± 0.0 1.4 ± 0.0 1.2 ± 0.0 1.9 ± 0.3 1.6 ± 0.4 1.7 ± 0.1 1.1 ± 0.2 2.8 ± 0.4 0.8 ± 0.4 0.8 ± 0.4 0.8 ± 0.3 0.4 ± 0.0 1.8 ± 0.0 1.7 ± 0.1 1.5 ± 0.1 1.3 ± 0.1

25.8 ± 0.2 27.9 ± 0.0 25.7 ± 1.2 28.3 ± 0.7 29.1 ± 0.1 30.0 ± 0.2 29.7 ± 0.5 29.8 ± 1.4 29.3 ± 0.9 29.4 ± 0.5 30.5 ± 1.3 28.3 ± 1.5 26.8 ± 1.4 23.8 ± 1.2 29.3 ± 0.4 30.1 ± 0.9 30.1 ± 0.9 30.2 ± 0.2 29.4 ± 1.4

0.38 ± 0.01 0.41 ± 0.00 0.40 ± 0.01 0.40 ± 0.01 0.41 ± 0.01 0.40 ± 0.00 0.40 ± 0.00 0.41 ± 0.01 0.41 ± 0.01 0.42 ± 0.00 0.41 ± 0.01 0.45 ± 0.01 0.44 ± 0.01 0.38 ± 0.01 0.41 ± 0.00 0.41 ± 0.01 0.41 ± 0.01 0.42 ± 0.00 0.42 ± 0.01

0.54 ± 0.00 0.58 ± 0.00 0.54 ± 0.07 0.39 ± 0.01 0.30 ± 0.00 0.42 ± 0.00 0.31 ± 0.00 0.31 ± 0.07 0.24 ± 0.01 0.61 ± 0.01 0.64 ± 0.05 0.59 ± 0.02 0.56 ± 0.07 0.20 ± 0.08 0.61 ± 0.01 0.42 ± 0.11 0.25 ± 0.01 0.42 ± 0.00 0.24 ± 0.01

ND ND ND ND ND ND ND ND ND ND ND 1.0 ± 0.0 1.9 ± 0.0 2.9 ± 0.0 1.0 ± 0.0 2.0 ± 0.1 2.8 ± 0.2 0.9/1.1e 1.5/1.5f

ND: not determined. Also, the unsuccessful fermentations (with no ABE production under highly inhibitory conditions) were not included in the table. a The inhibitor type and the concentration was used. For example, ‘Ferulic-0.2’ represents 0.2 g/L ferulic acid was supplemented in the fermentation. b This is exactly the same fermentation as shown in the first row of Table 1. It was put here repeatedly for easy comparison purposes. c Mixture of furfural and HMF. ‘1:1’ represents 1 g/L furfural and 1 g/L HMF, ‘1.5:1.5’ represents 1.5 g/L furfural and 1.5 g/L HMF. d Corresponding alcohols: furfuryl alcohol (converted from furfural) and/or HMF alcohol (converted from HMF). e 0.9 ± 0.0 g/L furfuryl alcohol and 1.1 ± 0.1 g/L HMF alcohol. f 1.5 ± 0.0 g/L furfuryl alcohol and 1.5 ± 0.0 g/L HMF alcohol.

and 0.8 g/L ferulic acid all generated higher final ABE titers (27.9– 29.1 g/L vs. 25.8 g/L in the control) and yields than the control (Fig. 3b and Table 2). There were several previous reports concerning the toxicity of ferulic acid on solventogenic clostridia. Ezeji et al. [18] reported that only very low amount of ABE was produced by C. beijerinckii BA101 in the presence of 0.3 g/L ferulic acid. In another study, it was reported that the butanol production and cell growth of C. beijerinckii NCIMB 8052 were significant repressed when 0.5 g/L of ferulic acid was added for the fermentation [31]. In comparison, our results indicated that the wild type C. saccharoperbutylacetonicum N1-4 strain demonstrated decent growth with supplementation of ferulic acid up to 0.8 g/L. Although extended lag phases were observed with the addition of ferulic acid, the strain can adapt to the inhibitory condition and grew well once adapted. It was previously reported that 0.5 g/L of ferulic acid can hampered the ability of C. beijerinckii to convert acids to solvents and thus significantly decreased ABE production [32]. While in this study, C. saccharoperbutylacetonicum produced even more ABE than the control in the presence of up to 0.8 g/L ferulic acid (Fig. 3b and Table 2). With 0.2 g/L syringaldehyde added into the fermentation, the cell growth profile of C. saccharoperbutylacetonicum was very similar to the control, and it actually reached a higher maximum OD of 17.8 (vs. 16.8 for the control) at 42 h (Fig. 3c). With the increase of syringaldehyde to 0.4, 0.6 or 0.8 g/L, the cell growth experienced a lag phase (when the OD < 0.15) of 18, 24 and 30 h, respectively. But the cell can still grow slowly to about the same maximum OD as the control within 96 h. When the concentration of syringaldehyde was further increased to 1.0 g/L, the cell growth was completed inhibited. For the ABE production, with the increase of syringaldehyde from 0.2 to 0.8 g/L, corresponding extended lag phase was observed. At 0.8 g/L syringaldehyde, the ABE production was even not detected until 72 h (Fig. 3d). However, with prolonged fermentation time, all the fermentations produced 29.3–30.0 g/L of ABE, which is around 15% higher than the control (Table 2). Ezeji et al. [18] reported ‘un-coupled’ inhibition of syringaldehyde on the cell growth and ABE production for C. beijerinckii

BA101. While 1.0 g/L syringaldehyde had very little inhibition on C. beijerinckii BA101 growth, especially before the solventogenic growth phase, as low as 0.3 g/L syringaldehyde demonstrated very severe inhibition on ABE production (the ABE production was less than 2 g/L under this condition). Guo et al. [31] reported that 0.5 g/ L syringaldehyde in the fermentation inhibited the cell growth and butanol production of C. beijerinckii IB4 by 0.5 and 2%, respectively. In this study, the presence of syringaldehyde (from 0.2 to 0.8 g/L) significantly increased the lag time for both cell growth and ABE production. However, C. saccharoperbutylacetonicum can adapt to the inhibitory condition, and grow to comparable maximum OD and produced even higher ABE than the control. When 0.2 g/L q-coumaric acid was added to the fermentation, the cell growth profile of C. saccharoperbutylacetonicum was very similar to the control and reached a similar maximum OD of 16.7 at 42 h (Fig. 3e). When q-coumaric acid was increased to 0.4 g/L, the cell growth was slightly inhibited and a maximum OD of 14.0 was reached at 48 h. However, when the concentration of qcoumaric acid was further increased to 0.6 and 0.8 g/L, the cell growth was severely inhibited and the OD was <1.5 over the whole fermentation process. At 1.0 g/L of q-coumaric acid, the cell growth was completely inhibited. For the ABE fermentation, at 0.2 and 0.4 g/L q-coumaric acid, a slight increase of the lag time (before the production of detectable ABE) was observed when compared to the control (Fig. 3f). But the ABE production still increased very rapidly and finished in 48 h (at 0.2 g/L q-coumaric acid) and 72 h (at 0.4 g/L q-coumaric acid), respectively. As seen for the ABE production in the presence of ferulic acid or syringaldehyde, the ABE production also increased compared to the control at both 0.2 and 0.4 g/L q-coumaric acid (Table 2). The maximum ABE produced in the presence of 0.4 g/L q-coumaric acid was 30.5 g/L, which was 18% higher than the control. Furthermore, it is also interesting that at the presence of q-coumaric acid (0.2 or 0.4 g/L), the ABE productivity increased by 13% and 19% respectively compared to the control. It was reported that when 0.3 g/L q-coumaric acid was introduced into the fermentation, cell growth and ABE production by C. beijerinckii BA101 significantly decreased [18]. Guo et al. [31]

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OD

15

10

5

0

40

30

20

10

0 0

24

48

72

96

120

144

0

24

48

72

2

1

0

144

0

24

48

96

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96

120

144

Time (h)

(f)

HMF

(e)

30

20

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0 48

120

HMF alcohol (g/L)

OD

5

ABE Concentration (g/L)

HMF

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96

3

4

40

(d)

15

0 0

72

Furfural

(c)

Time (h)

Time (h) 20

4

Furfural

(b)

Furfuryl alcohol (g/L)

Furfural

(a)

ABE Concentration (g/L)

20

0

24

48

Time (h)

72

96

120

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HMF

3

2

1

0

0

24

Control

1 g/L

2 g/L

48

72

96

120

144

Time (h)

Time (h)

3 g/L

4 g/L

Fig. 4. Effects of furfural and hydroxymethylfurfural (HMF) on ABE fermentation with C. saccharoperbutylacetonicum. (a) cell growth profiles with supplementation of various concentrations of furfural; (b) solvent production profiles with supplementation of various concentrations of furfural; (c) profiles for the conversion of furfural to furfuryl alcohol in the fermentation with supplementation of various concentrations of furfural; (d) cell growth profiles with supplementation of various concentrations of HMF; (e) solvent production profiles with supplementation of various concentrations of HMF; (f) profiles for the conversion of HMF to HMF alcohol in the fermentation with supplementation of various concentrations of HMF.

reported that 0.5 g/L q-coumaric acid can inhibit the cell growth by 80% and butanol production by 90% in C. beijerinckii 8052; while for the mutant strain C. beijerinckii IB4, which was screened by lowenergy ion implantation, 0.5 g/L q-coumaric acid inhibited the cell growth by 35% and butanol production by 17%. Here, C. saccharoperbutylacetonicum can tolerate up to 0.4 g/L q-coumaric acid, with good cell growth and ABE production. However, when qcoumaric acid was increased to 0.6 g/L, the cell metabolism was severely suppressed and no successful ABE production was achieved. Comparison of the toxicity of different phenolic compounds for ABE fermentation has been conducted previously. Ezeji et al. [18] reported that ferulic acid was the most toxic compound tested, followed by q-coumaric acid. Both compounds completely inhibited the growth of C. beijerinckii BA101 when their concentrations were >1.0 g/L. In another study, Ezeji and Blaschek [27] demonstrated that q-coumaric acid was the most toxic compound tested for ABE fermentation, followed by ferulic acid. Cho et al. [33] showed that among the tested phenolic compounds (vanilin, syringaldehyde, 4-hydroxybenzoic acid (4-HBA), vanilic, ferulic and qcoumaric acids), q-coumaric acid displayed the most toxic effects. In this study, among the three tested phenolic compounds, qcoumaric acid was also the most toxic. Comparing effects of the three phenolic compounds, C. saccharoperbutylacetonicum has a more robust resilience to adapt to syringaldehyde. At 0.8 g/L syringaldehyde, cells could recover from a lag time of as long as 72 h and produce high concentration of ABE. While with ferulic acid or q-coumaric acid, cells were also able to adapt to the inhibitory condition, but if they cannot recover within a short time frame (<24 h), they are likely killed. This phenomenon might represent very different inhibition mechanisms among these three inhibitors. On the other hand, during all the fermentations, as long as C. sac-

charoperbutylacetonicum can recover from the inhibitory environment and achieve a decent growth, it can produce high level of ABE which is often even higher than that from the control. This demonstrated that C. saccharoperbutylacetonicum has a very robust solventogenesis mechanism that can sustain the impairing impact from phenolic inhibitors. 3.4. Effects of furfural and HMF Furfural and HMF are degraded products from hexose and pentose sugars during the biomass pretreatment process [13,14]. In this study, we conducted experiments to evaluate their impacts on C. saccharoperbutylacetonicum for ABE fermentation. As shown in Fig. 4a, in the presence of 1 g/L furfural, the cell growth rate was actually stimulated (with faster growth) and reached the maximum OD of 15.6 at 30 h (vs. 16.8 at 42 h). At 2 g/L furfural, the cell growth was slightly inhibited, but still reached a maximum OD of 16.6 at 42 h. When the concentration of furfural increased to 3 g/L, a long lag phase of 42 h was observed for the cell growth, but the cell was still able to recover and reached a similar maximum OD as the control after 96 h. The cell growth was completely inhibited at 4 g/L furfural. For the ABE production, the kinetic profiles at 1 or 2 g/L furfural was very similar to that of the control and reached 10% and 4% respectively higher final titers than the control. At 3 g/L furfural, ABE started to be detected from 48 h, and reached a final titer of 8% lower than the control (Fig. 4b). Meanwhile, almost all the furfural in the fermentation media (for the fermentation with 1, 2, or 3 g/L furfural) was transformed into the furfuryl alcohol (Fig. 4c and Table 2). With 1 g/L HMF added to the fermentation, the cell grew very similarly to the control, but reached a slightly lower maximum OD at 42 h (Fig. 4d). When the level of HMF was increased to 2

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or 3 g/L, the cell growth was delayed with a lag phase (24 h was observed at 3 g/L HMF), but it was still able to reach the comparable maximum OD at a later time (72 h). Consistent with the cell growth, the ABE production at 1 g/L HMF started early and followed very similar kinetics as the control (Fig. 4e). While at 2 or 3 g/L HMF, the ABE production was delayed and was detected from 24 h and 48 h, respectively. However, for all the fermentations with HMF (1–3 g/L), the final ABE titer was higher (by 14–17%) than the control. Furthermore, as for the furfural supplemented fermentation, all the HMF (from 1-3 g/L) was transformed into HMF alcohol by the end of the fermentation (Fig. 4f and Table 2). Due to the similar property between furfural and HMF, we carried out further experiments with both furfural and HMF to investigate whether they can exert additive inhibition on the ABE fermentation with C. saccharoperbutylacetonicum. More details can be find in the ‘Results and Discussion’ section in the Supplementary material section. Biotransformation of furfural and HMF into their corresponding alcohols has been previously reported for yeast, archaea, and bacteria including clostridial species [14,18,34]. This biotransformation has been inferred to be achieved by using aldehyde as an electron acceptor and converting furfural and HMF to furfuryl alcohol and HMF alcohol by 2e reduction [14]. Cofactors (NADH and NADPH) are involved in the detoxification of furans by microbes [34]. These cofactors are also involved in butanol synthesis and play important roles during the transition from acidogenesis to solventogenesis [35]. Therefore, there is a potential competition of cofactors for the detoxification of furans and the production of solvents. However, interestingly, the final ABE titer and yield by C. saccharoperbutylacetonicum in the presence of furfural and/or HMF (except for 3 g/L furfural) was generally higher than the control (Table 2). The mechanism behind this phenomenon warrants further investigation. Actually, for all the fermentations carried out in the presence of inhibitors (phenolic, furfural or HMF) in this study, as long as the C. saccharoperbutylacetonicum culture can adapt to the condition, comparable or higher ABE production was observed (Table 2). Especially the acetone production was significantly increased. For example, >12.5 g/L acetone (vs. 9.7 g/L in the control) has been observed in many of these fermentations. This led to a decreased butanol/acetone ratio of close to 1:1 (Table 2). This might be because, in order to sustain and survive from the inhibitory conditions, the cell managed to generate more energy (ATP) to support the cell growth. The generation of energy needs to be achieved through the acid production pathway. Thus more acids (especially acetic acid, since the acetate production pathway is more efficient for ATP generation than the butyrate production pathway) were generated and re-assimilated, resulting in more acetone production. This from another angle testified that C. saccharoperbutylacetonicum has a very robust solventogenesis metabolism to ensure the potent ABE production under unfavorable conditions. The strain can tolerate to sufficiently high levels of both phenolic inhibitors and furan aldehyde inhibitors (furfural and HMF), and thus is a very desirable host for ABE production using low value lignocellulosic biomass hydrolysates as the substrate.

4. Conclusions C. saccharoperbutylacetonicum, as a robust workhorse for ABE production, can use glucose, cellobiose, xylose, arabinose and mannose efficiently, but degrade galactose slowly and incompletely. All sugars as a mixture could be utilized concurrently, but degradation rate is sugar specific. q-Coumaric acid, ferulic acid and syringaldehyde are potent phenolic inhibitors, and q-coumaric acid is the most toxic. Furfural and HMF are not as toxic as phenolic inhibi-

tors, and both furfural and HMF can be completely converted into the corresponding alcohols, with furfural is more rapidly converted than HMF. The C. saccharoperbutylacetonicum culture can adapt to inhibition conditions and produce more ABE than the control. The results from this study testify the robustness of C. saccharoperbutylacetonicum for ABE production from lignocellulosic carbon sources. Acknowledgements This work was supported by the Auburn University Intramural Grants Program (IGP), the Hatch program of the USDA National Institute of Food and Agriculture (NIFA), and the USDA-NIFA Southeastern SunGrant. Dunfan Yao is a recipient of a scholarship offered by the China Scholarship Council (CSC). We thank Dr. Max Bangs for his assistance with the English language of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fuel.2017.07.004. References [1] Dürre P. Fermentative production of butanol-the academic perspective. Curr Opin Biotechnol 2011;22:331–6. [2] Green EM. Fermentative production of butanol-the industrial perspective. Curr Opin Biotechnol 2011;22:337–43. [3] Qureshi N, Blaschek H. Economics of butanol fermentation using hyperbutanol producing Clostridium beijerinckii BA101. Food Bioprod Process 2000;78:139–44. [4] Cheng J. Biomass to renewable energy processes. CRC Press; 2009. [5] Qureshi N, Ezeji TC. Butanol, ‘a superior biofuel’production from agricultural residues (renewable biomass): recent progress in technology. Biofuel Bioprod Biorefin 2008;2:319–30. [6] Qureshi N, Saha BC, Hector RE, Dien B, Hughes S, Liu S, et al. Production of butanol (a biofuel) from agricultural residues: Part II-Use of corn stover and switchgrass hydrolysates. Biomass Bioenergy 2010;34:566–71. [7] Liu K, Atiyeh HK, Pardo-Planas O, Ezeji TC, Ujor V, Overton JC, et al. Butanol production from hydrothermolysis-pretreated switchgrass: quantification of inhibitors and detoxification of hydrolyzate. Bioresour Technol 2015;189:292–301. [8] Humbird D, Davis R, Tao L, Kinchin C, Hsu D, Aden A et al. National Renewable Energy Laboratory (NREL), Golden, CO. 2011. [9] Stenberg K, Tengborg C, Galbe M, Zacchi G. Optimisation of steam pretreatment of SO2-impregnated mixed softwoods for ethanol production. J Chem Technol Biotechnol 1998;71:299–308. [10] Siso MIG. The biotechnological utilization of cheese whey: a review. Bioresour Technol 1996;57:1–11. [11] Yoon JJ, Kim YJ, Kim SH, Ryu HJ, Choi JY, Kim GS, Shin MK. Advanced Materials Research, Trans Tech Publ; 2010, p. 463-66. [12] Ha SJ, Galazka JM, Rin Kim S, Choi JH, Yang X, Seo JH, et al. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc Natl Acad Sci USA 2011;108:504–9. [13] Mussatto SI, Roberto IC. Chemical characterization and liberation of pentose sugars from brewer’s spent grain. J Chem Technol Biotechnol 2006;81:268–74. [14] Zhang Y, Han B, Ezeji TC. Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. New Biotechnol 2012;29:345–51. [15] Torre P, Aliakbarian B, Rivas B, Domingue JM, Converti A. Release of ferulic acid from corn cobs by alkaline hydrolysis. Biochem Eng J 2008;40:500–6. [16] Liu K, Atiyeh HK, Pardo-Planas O, Ramachandriya KD, Wilkins MR, Ezeji TC, et al. Process development for biological production of butanol from Eastern redcedar. Bioresour Technol 2015;176:88–97. [17] Larsson S, Reimann A, Nilvebrant NO, Jonsson LJ. Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce. Appl Biochem Biotechnol 1999;77:91–103. [18] Ezeji T, Qureshi N, Blaschek HP. Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol Bioeng 2007;97:1460–9. [19] Motoyoshi H. Process for producing butanol by fermentation. US Patent 2,945,786A; July 1960. [20] Jones D. The strategic importance of butanol for Japan during WWII: a case study of the butanol fermentation process in Taiwan and Japan. In: Dürre P, editor. Systems biology of Clostridium. London: Imperial College Press; 2014. p. 220–72. [21] Keis S, Shaheen R, Jones DT. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium

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