# Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose

Felipe Buendia-Kandia, Emmanuel Rondags, Xavier Framboisier, Guillain Mauviel, Anthony Dufour, Emmanuel Guedon

► To cite this version:

Felipe Buendia-Kandia, Emmanuel Rondags, Xavier Framboisier, Guillain Mauviel, Anthony Dufour, et al.. Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose. AMB Express, 2018, 8 (1), pp.9. 10.1186/s13568-018-0615-2. hal-01797977

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Submitted on 11 Feb 2024

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# Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose

Felipe Buendia-Kandia, Emmanuel Rondags, Xavier Framboisier, Guillain Mauviel, Anthony Dufour, Emmanuel Guedon

► To cite this version:

Felipe Buendia-Kandia, Emmanuel Rondags, Xavier Framboisier, Guillain Mauviel, Anthony Dufour, et al.. Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose. AMB Express, 2018, 8 (1), pp.9. 10.1186/s13568-018-0615-2. hal-01797977

HAL Id: hal-01797977

https://hal.science/hal-01797977v1

Submitted on 11 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

ORIGINAL ARTICLE

Open Access

<figure><img src="image_3.png" /></figure>

CrossMark

# Diauxic growth of *Clostridium acetobutylicum* ATCC 824 when grown on mixtures of glucose and cellobiose

Felipe Buendia-Kandia<fnref n="1" />,², Emmanuel Rondags<fnref n="1" />, Xavier Framboisier<fnref n="1" />, Guillain Mauviel², Anthony Dufour² and Emmanuel Guedon<fnref n="1" />*

**Abstract**

*Clostridium acetobutylicum*, a promising organism for biomass transformation, has the capacity to utilize a wide variety of carbon sources. During pre-treatments of (ligno) cellulose through thermic and/or enzymatic processes, complex mixtures of oligo saccharides with beta 1,4-glycosidic bonds can be produced. In this paper, the capability of *C. acetobutylicum* to ferment glucose and cellobiose, alone and in mixtures was studied. Kinetic studies indicated that a diauxic growth occurs when both glucose and cellobiose are present in the medium. In mixtures, *β*-glucose is the preferred substrate even if cells were pre grown with cellobiose as the substrate. After the complete consumption of glucose, the growth kinetics exhibits an adaptation time, of few hours, before to be able to use cellobiose. Because of this diauxic phenomenon, the nature of the carbon source deriving from a cellulose hydrolysis pre-treatment could strongly influence the kinetic performances of a fermentation process with *C. acetobutylicum*.

**Keywords:** Biomass, Cellulose, Cellobiose, Diauxie, Fermentation, *Clostridium acetobutylicum*

**Introduction**

Lignocellulosic biomass represents an interesting alternative to fossil carbon resources (McKendry 2002; Mosier et al. 2005; Wyman et al. 2005; Briens et al. 2008; Wettstein et al. 2012; Nanda et al. 2014).

Indeed, lignocellulosic biomass can be transformed into energy and fuels through a variety of chemical, thermo-chemical and biological conversion processes. Among biological conversion processes, anaerobic digestion and more specifically fermentation processes are the most implemented in industries (Lin and Tanaka 2005; Azman et al. 2015), because it offers the most important biological conversion ways to transform a wide variety of organic materials mainly coming from agroindustry such as by-products into fuels and chemical products with higher values (McKendry 2002; Briens et al. 2008, Turon et al. 2016). However, fermentation processes need an

efficient monitoring and control to ensure optimal conditions for productions and yields (Lin and Tanaka 2005; Azman et al. 2015).

Bioethanol, one of the main fermentation products, is predominantly produced using yeast through the fermentation of easily degradable carbohydrate substrates, such as corn starch and sugar cane (Henstra et al. 2007). Nonetheless, the utilization of these kinds of substrates to produce biofuels threatens food supplies and biodiversity. For this reason, the production of new generation biofuels using bacteria may offer an interesting alternative to this problem (Ranjan and Moholkar 2012; Morone and Pandey 2014). As an alternative, some *Clostridium* species can produce acetone-butanol-ethanol (ABE) from renewable resources, such as biomass and derivatives (Lee et al. 2008; Tracy et al. 2012; Jang et al. 2012; Gu et al. 2014). Butanol can be blended with gasoline or used directly as a fuel for transportation (Qureshi and Ezeji 2008; Pfromm et al. 2010; Ranjan and Moholkar 2012). For this purpose, a diversity of *Clostridium* strains coming from the butyric and butylic groups has been

<figure><img src="image_4.png" /></figure>

<footnote>*Correspondence: Emmanuel.Guedon@univ-lorraine.fr</footnote>

<footnote n="1">¹ LRGP, UMR CNRS 7274, Université de Lorraine, 2 Avenue de la Forêt de Haye TSA 40602, 54518 Vandoeuvre lès Nancy, France
Full list of author information is available at the end of the article</footnote>
<figure><img src="image_2.png" /></figure>

ORIGINAL ARTICLE

Open Access

<figure><img src="image_3.png" /></figure>

CrossMark

# Diauxic growth of *Clostridium acetobutylicum* ATCC 824 when grown on mixtures of glucose and cellobiose

Felipe Buendia-Kandia<fnref n="1" />,², Emmanuel Rondags<fnref n="1" />, Xavier Framboisier<fnref n="1" />, Guillain Mauviel², Anthony Dufour² and Emmanuel Guedon<fnref n="1" />*

**Abstract**

*Clostridium acetobutylicum*, a promising organism for biomass transformation, has the capacity to utilize a wide variety of carbon sources. During pre-treatments of (ligno) cellulose through thermic and/or enzymatic processes, complex mixtures of oligo saccharides with beta 1,4-glycosidic bonds can be produced. In this paper, the capability of *C. acetobutylicum* to ferment glucose and cellobiose, alone and in mixtures was studied. Kinetic studies indicated that a diauxic growth occurs when both glucose and cellobiose are present in the medium. In mixtures, *β*-glucose is the preferred substrate even if cells were pre grown with cellobiose as the substrate. After the complete consumption of glucose, the growth kinetics exhibits an adaptation time, of few hours, before to be able to use cellobiose. Because of this diauxic phenomenon, the nature of the carbon source deriving from a cellulose hydrolysis pre-treatment could strongly influence the kinetic performances of a fermentation process with *C. acetobutylicum*.

**Keywords:** Biomass, Cellulose, Cellobiose, Diauxie, Fermentation, *Clostridium acetobutylicum*

**Introduction**

Lignocellulosic biomass represents an interesting alternative to fossil carbon resources (McKendry 2002; Mosier et al. 2005; Wyman et al. 2005; Briens et al. 2008; Wettstein et al. 2012; Nanda et al. 2014).

Indeed, lignocellulosic biomass can be transformed into energy and fuels through a variety of chemical, thermo-chemical and biological conversion processes. Among biological conversion processes, anaerobic digestion and more specifically fermentation processes are the most implemented in industries (Lin and Tanaka 2005; Azman et al. 2015), because it offers the most important biological conversion ways to transform a wide variety of organic materials mainly coming from agroindustry such as by-products into fuels and chemical products with higher values (McKendry 2002; Briens et al. 2008, Turon et al. 2016). However, fermentation processes need an

efficient monitoring and control to ensure optimal conditions for productions and yields (Lin and Tanaka 2005; Azman et al. 2015).

Bioethanol, one of the main fermentation products, is predominantly produced using yeast through the fermentation of easily degradable carbohydrate substrates, such as corn starch and sugar cane (Henstra et al. 2007). Nonetheless, the utilization of these kinds of substrates to produce biofuels threatens food supplies and biodiversity. For this reason, the production of new generation biofuels using bacteria may offer an interesting alternative to this problem (Ranjan and Moholkar 2012; Morone and Pandey 2014). As an alternative, some *Clostridium* species can produce acetone-butanol-ethanol (ABE) from renewable resources, such as biomass and derivatives (Lee et al. 2008; Tracy et al. 2012; Jang et al. 2012; Gu et al. 2014). Butanol can be blended with gasoline or used directly as a fuel for transportation (Qureshi and Ezeji 2008; Pfromm et al. 2010; Ranjan and Moholkar 2012). For this purpose, a diversity of *Clostridium* strains coming from the butyric and butylic groups has been

<figure><img src="image_4.png" /></figure>

<footnote>*Correspondence: Emmanuel.Guedon@univ-lorraine.fr</footnote>

<footnote n="1">¹ LRGP, UMR CNRS 7274, Université de Lorraine, 2 Avenue de la Forêt de Haye TSA 40602, 54518 Vandoeuvre lès Nancy, France
Full list of author information is available at the end of the article</footnote>
studied (*C. saccharoperbutylacetonicum*, *C. acetobutylicum*, *C. beijerinckii*, *C. butyricum*) (Lee et al. 2008; Jang et al. 2012; Gu et al. 2014).

*C. acetobutylicum* is one of the most studied species thanks to its capability of producing butanol and hydrogen by anaerobic fermentation in high yields, from a wide range of substrates (Qureshi and Ezeji 2008; Survase et al. 2011b; Napoli et al. 2011; Li et al. 2012; Jurgens et al. 2012; Gao et al. 2014; Aristilde et al. 2015; Raganati et al. 2015). This strain was largely used in the ABE fermentation process during the 1910s until the end of 1950s, because the petrochemical industry development led to the decline of this fermentation as an industrial process (Jones and Woods 1986). An important feature of the ABE fermentation is its biphasic development leading to two distinct groups of metabolic products. The first phase is the acidogenesis, which is characterized by acids (mainly butyric, acetic and lactic acid) and hydrogen production. During acidogenesis, the cells will usually display an active growth including an exponential growth phase (Andersch et al. 1983; Hartmanis and Gatenbeck 1984). The second phase is the solventogenesis, which is characteristized by organic acids re-assimilation and solvent production, with butanol, acetone and ethanol as the major products (Monot et al. 1984). The transition between the two phases is the result of strong gene expression changes (Grupe and Gottschalk 1992; Girbal et al. 1995).Nowadays, with the depletion of oil, renewable processes to replace fossil carbon need to be developed. But, a major factor that determines the viability of this process is the cost of feedstock. In this regard, lignocellulosic biomasses are considered as interesting feedstocks for fermentation (Ezeji et al. 2007; Jang et al. 2012), Whereas its complex structure composed of cellulose embedded in a complex hemicellulose and lignin matrix may hinder biological conversion due to a high resistance to most chemical and biological pretreatments (Wyman et al. 2005; Kumar et al. 2009; Rinaldi and Schüth 2009; Hendriks and Zeeman 2009; Alvira et al. 2010; Jurgens et al. 2012).

To enhance the fermentability of cellulose a preliminary treatment is frequently used. Chemical, thermal and enzymatic pre-treatments allow to produce a mixture of carbohydrates and other organic compounds that are soluble and accessible to bacteria (Kumar et al. 2009; Rinaldi and Schüth 2009; Alvira et al. 2010; Ibrahim et al. 2015). The conversion of each one of these compounds into added-value products and building blocks is crucial to increase the efficiency of an integrated bio-refinery, and the understanding the metabolism of individual carbohydrates and mixtures is very important to lower the cost of fermentation process. This and the ability of saccharolytic clostridia to use a wide range of carbohydrates

have prompted research dedicated to the production of a cheaper substrates (Jones and Woods 1986; Lee et al. 2008; Tracy et al. 2012; Gu et al. 2014).

In addition, the growth of *C. acetobutylicum* on a mixture of substrates has been already studied using different combinations of carbon sources (glucose and xylose, glucose and mannose, D-glucose and glycerol, ...) and different metabolic responses were obtained according to the nutritional environment (Mes-Hartree and Saddler 1982; Ounine et al. 1985; Fond et al. 1986; Vasconcelos et al. 1994; Mitchell et al. 1995; Survase et al. 2011a). Recently, the interest in *C. acetobutylicum* as an effective biofuel producer of butanol instead of ethanol from lignocellulose derivated substrates (glucose, cellobiose and xylose) has increased, resulting in numerous studies privileging mainly butanol production from xylose alone or in mixture with glucose rather than with cellobiose (Patakova et al. 2013; Nogué and Karhumaa 2015; Raganati et al. 2015; Zhao et al. 2016) whereas this last substrate with glucose and other cello-oligosaccharides in mixture would be also expected to be major products of lignocellulose degradation.

Indeed, despite a recent proteomic study describing the influence of lignin in the metabolic behavior of *C. acetobutylicum* ATCC 824 with cellobiose as the substrate (Raut et al. 2016), the capability of *Clostridium* strains to ferment mixtures of cellobiose and glucose in synthetic medium is not well studied yet. Furthermore, bacteria have developed mechanisms that allow them to use selectively mixtures of different carbon sources (Mitchell et al. 1995).

In this study, kinetics of *C. acetobutylicum* cultivated with glucose and/or cellobiose, two substrate models representatives of cellulose hydrolysis products were compared. Only the acidogenic phase in which the active growth phase and substrate consumptions could be studied was considered. Transitions and re-assimilation of acids mechanisms were excluded from this work.

## Materials and methods

### Microorganism and media

Spores of *Clostridium acetobutylicum* ATCC 824 were maintained in Difco™ Reinforced Clostridial Medium (RCM) at ambient temperature. Whereas precultures were different for each culture experiment, they were prepared rigorously in a similar way. Each experiment was started with the spores of *C. acetobutylicum*. The spore culture was diluted to a concentration of 10% in 10 mL of RCM fresh media (Hungate tubes) and then heat shocked at 80 °C for 20 min to induce germination. Reactivated cultures were incubated in fresh RCM medium at 37 °C for 12 h and then transferred into 30 mL (pre-culture tubes) of a synthetic medium. In fact, the
studied (*C. saccharoperbutylacetonicum*, *C. acetobutylicum*, *C. beijerinckii*, *C. butyricum*) (Lee et al. 2008; Jang et al. 2012; Gu et al. 2014).

*C. acetobutylicum* is one of the most studied species thanks to its capability of producing butanol and hydrogen by anaerobic fermentation in high yields, from a wide range of substrates (Qureshi and Ezeji 2008; Survase et al. 2011b; Napoli et al. 2011; Li et al. 2012; Jurgens et al. 2012; Gao et al. 2014; Aristilde et al. 2015; Raganati et al. 2015). This strain was largely used in the ABE fermentation process during the 1910s until the end of 1950s, because the petrochemical industry development led to the decline of this fermentation as an industrial process (Jones and Woods 1986). An important feature of the ABE fermentation is its biphasic development leading to two distinct groups of metabolic products. The first phase is the acidogenesis, which is characterized by acids (mainly butyric, acetic and lactic acid) and hydrogen production. During acidogenesis, the cells will usually display an active growth including an exponential growth phase (Andersch et al. 1983; Hartmanis and Gatenbeck 1984). The second phase is the solventogenesis, which is characteristized by organic acids re-assimilation and solvent production, with butanol, acetone and ethanol as the major products (Monot et al. 1984). The transition between the two phases is the result of strong gene expression changes (Grupe and Gottschalk 1992; Girbal et al. 1995).Nowadays, with the depletion of oil, renewable processes to replace fossil carbon need to be developed. But, a major factor that determines the viability of this process is the cost of feedstock. In this regard, lignocellulosic biomasses are considered as interesting feedstocks for fermentation (Ezeji et al. 2007; Jang et al. 2012), Whereas its complex structure composed of cellulose embedded in a complex hemicellulose and lignin matrix may hinder biological conversion due to a high resistance to most chemical and biological pretreatments (Wyman et al. 2005; Kumar et al. 2009; Rinaldi and Schüth 2009; Hendriks and Zeeman 2009; Alvira et al. 2010; Jurgens et al. 2012).

To enhance the fermentability of cellulose a preliminary treatment is frequently used. Chemical, thermal and enzymatic pre-treatments allow to produce a mixture of carbohydrates and other organic compounds that are soluble and accessible to bacteria (Kumar et al. 2009; Rinaldi and Schüth 2009; Alvira et al. 2010; Ibrahim et al. 2015). The conversion of each one of these compounds into added-value products and building blocks is crucial to increase the efficiency of an integrated bio-refinery, and the understanding the metabolism of individual carbohydrates and mixtures is very important to lower the cost of fermentation process. This and the ability of saccharolytic clostridia to use a wide range of carbohydrates

have prompted research dedicated to the production of a cheaper substrates (Jones and Woods 1986; Lee et al. 2008; Tracy et al. 2012; Gu et al. 2014).

In addition, the growth of *C. acetobutylicum* on a mixture of substrates has been already studied using different combinations of carbon sources (glucose and xylose, glucose and mannose, D-glucose and glycerol, ...) and different metabolic responses were obtained according to the nutritional environment (Mes-Hartree and Saddler 1982; Ounine et al. 1985; Fond et al. 1986; Vasconcelos et al. 1994; Mitchell et al. 1995; Survase et al. 2011a). Recently, the interest in *C. acetobutylicum* as an effective biofuel producer of butanol instead of ethanol from lignocellulose derivated substrates (glucose, cellobiose and xylose) has increased, resulting in numerous studies privileging mainly butanol production from xylose alone or in mixture with glucose rather than with cellobiose (Patakova et al. 2013; Nogué and Karhumaa 2015; Raganati et al. 2015; Zhao et al. 2016) whereas this last substrate with glucose and other cello-oligosaccharides in mixture would be also expected to be major products of lignocellulose degradation.

Indeed, despite a recent proteomic study describing the influence of lignin in the metabolic behavior of *C. acetobutylicum* ATCC 824 with cellobiose as the substrate (Raut et al. 2016), the capability of *Clostridium* strains to ferment mixtures of cellobiose and glucose in synthetic medium is not well studied yet. Furthermore, bacteria have developed mechanisms that allow them to use selectively mixtures of different carbon sources (Mitchell et al. 1995).

In this study, kinetics of *C. acetobutylicum* cultivated with glucose and/or cellobiose, two substrate models representatives of cellulose hydrolysis products were compared. Only the acidogenic phase in which the active growth phase and substrate consumptions could be studied was considered. Transitions and re-assimilation of acids mechanisms were excluded from this work.

## Materials and methods

### Microorganism and media

Spores of *Clostridium acetobutylicum* ATCC 824 were maintained in Difco™ Reinforced Clostridial Medium (RCM) at ambient temperature. Whereas precultures were different for each culture experiment, they were prepared rigorously in a similar way. Each experiment was started with the spores of *C. acetobutylicum*. The spore culture was diluted to a concentration of 10% in 10 mL of RCM fresh media (Hungate tubes) and then heat shocked at 80 °C for 20 min to induce germination. Reactivated cultures were incubated in fresh RCM medium at 37 °C for 12 h and then transferred into 30 mL (pre-culture tubes) of a synthetic medium. In fact, the
volume of preculture was prepared depending of the final volume of culture in order to get a ratio of 1/4. The synthetic medium was composed of 20 g/L glucose, 0.5 g/L KH₂PO₄, 1.5 g/L (NH₄)₂SO₄, 1 g/L MgCl₂, 0.15 g/L CaCl₂, 1.5 g/L yeast extract, 0.01 g/L FeSO₄·7H₂O, 0.01 g/L MnSO₄·H₂O, 3 g/L CaCO₃, 4 × 10⁻⁵ g/L biotin. All the Chemicals, yeast extract and biotin were provided by Sigma Aldrich.

## Fermentation

Batch fermentations were carried out in a bioreactor controlled by an Applikon ADI 1030 bio controller (Applikon Biotechnology). Throughout all fermentation experiments, temperature was maintained at 37 °C and pH at 5.5 by the automatic addition of 3 N NaOH, 1 N HCl using the biocontroller and a pH probe (Mettler Toledo). The bioreactor was initially purged with nitrogen to ensure an anaerobic atmosphere and then inoculated with 6% (v/v) active growing pre-cultures into 1.5 L of synthetic media with the same composition of the pre-culture media (without CaCO₃ to prevent interference with DO measurements). The glucose concentrations for the fermentation studies were comprised between 25 and 35 g/L (138–195 mM), unless otherwise indicated. Other experiments with cellobiose were carried out with the same glucose-equivalent mass concentration. However, controlling residual substrate from pre-culture was not possible. For this reason, the initial substrate concentration was not exactly the same. These variations did not alter the study case and the desired results. Each experience was carried out until complete substrate consumption. All the experiments were performed at least in duplicate.

## Analyses

Cell density was measured at 600 nm using a spectrophotometer (HITACHI U-2000). The relationship between the cell dry weight and the optical density was established thanks to a calibration made by triplicate using spectrophotometer at 600 nm. The correlation factor found was 0.346 g/L cell dry weight per unity of absorbance. This value was in agreement with others related in the literature (Kim et al. 1984).

Glucose, cellobiose, acetone, ethanol, butanol, acetic acid, lactic acid and butyric acid concentrations were measured by high-performance liquid chromatography equipped with a refractive index detector and an ultraviolet-visible spectroscopy (HPLC-RID-UV) using a Aminex HPX 87 h column. The samples were filtered with a 0.2 µm filter and the injection volume was 10 µL. The oven kept the column at 45 C; the mobile phase was a 25 mM sulfuric acid (H₂SO₄) solution. The analysis time was 35 min in isocratic mode.

## Calculations

The main products of glucose or cellobiose fermentation by C. cellulolyticum were acetate, Butyrate, ethanol, butanol, acetone, lactate, H₂, and CO₂, as previously described (Vasconcelos et al. 1994). Carbon recoveries were calculated from the production of metabolites, and biomass, present in the supernatant. Biomass was taken into account on the basis of the cell dry weight and a mean biomass formula of C₄H₇O₂N (Guedon et al. 1999). According to the metabolic scheme (Vasconcelos et al. 1994), the conversion of glucose to products can be written as follows:

$$ \text{glucose} + 2 \text{ADP} + 2 \text{NADH} \rightarrow 2 \text{ethanol} + 2 \text{ATP} \\ + 2 \text{NAD}^{+} + 2 \text{CO}_{2} + 2 \text{H}_{2} $$

$$ \text{glucose} + 4 \text{ADP} + 2 \text{NAD}^{+} \rightarrow 2 \text{acetate} + 4 \text{ATP} \\ + 2 \text{NADH} + 2 \text{CO}_{2} + 2 \text{H}_{2} $$

$$ \text{glucose} + 3 \text{ADP} \rightarrow \text{butyrate} + 3 \text{ATP} \\ + 2 \text{NADH} + 2 \text{CO}_{2} + 2 \text{H}_{2} $$

$$ \text{glucose} + 2 \text{ADP} + 2 \text{NADH} \rightarrow \text{butanol} + 2 \text{ATP} \\ + 2 \text{NAD}^{+} + 2 \text{CO}_{2} + 2 \text{H}_{2} $$

$$ \text{glucose} + 2 \text{ADP} + 2 \text{NAD}^{+} \rightarrow \text{acetone} + 3 \text{ATP} \\ + 2 \text{NADH} + 3 \text{CO}_{2} + 2 \text{H}_{2} $$

For example, from the last stoichiometric equation, each acetone molecule produced is associated to the formation of 3 CO₂ molecules. Therefore, the CO₂ production was calculated on the basis of product formation, as the sum of [acetate], 2*[butyrate], [ethanol], 2*[butanol], and 3*[acetone] concentrations.

CO₂ production was calculated as the sum of [acetate], 2*[butyrate], [ethanol], 2*[butanol], and 3*[acetone] concentrations.

## Results

### General growth and metabolic features of Clostridium acetobutylicum ATCC 824 cultivated with glucose or cellobiose

Kinetics of growth and metabolism of C. acetobutylicum with glucose or cellobiose as the carbon and energy source were performed with precultures carried out with either glucose or cellobiose (Fig. 1). Pre cultures and cultures performed with the same carbon source were compared with precultures and cultures performed with the switched substrates. The pH was set at 5.5 in order to favor acidogenesis, resulting in butyric, acetic and lactic acids, as the main products whatever the substrate used.

The carbon balances for these experiments were comprised between 90 and 98%, indicating that most of the carbon substrates and products have been taken
volume of preculture was prepared depending of the final volume of culture in order to get a ratio of 1/4. The synthetic medium was composed of 20 g/L glucose, 0.5 g/L KH₂PO₄, 1.5 g/L (NH₄)₂SO₄, 1 g/L MgCl₂, 0.15 g/L CaCl₂, 1.5 g/L yeast extract, 0.01 g/L FeSO₄·7H₂O, 0.01 g/L MnSO₄·H₂O, 3 g/L CaCO₃, 4 × 10⁻⁵ g/L biotin. All the Chemicals, yeast extract and biotin were provided by Sigma Aldrich.

## Fermentation

Batch fermentations were carried out in a bioreactor controlled by an Applikon ADI 1030 bio controller (Applikon Biotechnology). Throughout all fermentation experiments, temperature was maintained at 37 °C and pH at 5.5 by the automatic addition of 3 N NaOH, 1 N HCl using the biocontroller and a pH probe (Mettler Toledo). The bioreactor was initially purged with nitrogen to ensure an anaerobic atmosphere and then inoculated with 6% (v/v) active growing pre-cultures into 1.5 L of synthetic media with the same composition of the pre-culture media (without CaCO₃ to prevent interference with DO measurements). The glucose concentrations for the fermentation studies were comprised between 25 and 35 g/L (138–195 mM), unless otherwise indicated. Other experiments with cellobiose were carried out with the same glucose-equivalent mass concentration. However, controlling residual substrate from pre-culture was not possible. For this reason, the initial substrate concentration was not exactly the same. These variations did not alter the study case and the desired results. Each experience was carried out until complete substrate consumption. All the experiments were performed at least in duplicate.

## Analyses

Cell density was measured at 600 nm using a spectrophotometer (HITACHI U-2000). The relationship between the cell dry weight and the optical density was established thanks to a calibration made by triplicate using spectrophotometer at 600 nm. The correlation factor found was 0.346 g/L cell dry weight per unity of absorbance. This value was in agreement with others related in the literature (Kim et al. 1984).

Glucose, cellobiose, acetone, ethanol, butanol, acetic acid, lactic acid and butyric acid concentrations were measured by high-performance liquid chromatography equipped with a refractive index detector and an ultraviolet-visible spectroscopy (HPLC-RID-UV) using a Aminex HPX 87 h column. The samples were filtered with a 0.2 µm filter and the injection volume was 10 µL. The oven kept the column at 45 C; the mobile phase was a 25 mM sulfuric acid (H₂SO₄) solution. The analysis time was 35 min in isocratic mode.

## Calculations

The main products of glucose or cellobiose fermentation by C. cellulolyticum were acetate, Butyrate, ethanol, butanol, acetone, lactate, H₂, and CO₂, as previously described (Vasconcelos et al. 1994). Carbon recoveries were calculated from the production of metabolites, and biomass, present in the supernatant. Biomass was taken into account on the basis of the cell dry weight and a mean biomass formula of C₄H₇O₂N (Guedon et al. 1999). According to the metabolic scheme (Vasconcelos et al. 1994), the conversion of glucose to products can be written as follows:

$$ \begin{aligned} \text{glucose} + 2 \text{ADP} + 2 \text{NADH} \rightarrow & \text{2 ethanol} + 2 \text{ATP} \\ & + 2 \text{NAD}^{+} + 2 \text{CO}_{2} + 2 \text{H}_{2} \end{aligned} $$

$$ \begin{aligned} \text{glucose} + 4 \text{ADP} + 2 \text{NAD}^{+} \rightarrow & \text{2 acetate} + 4 \text{ATP} \\ & + 2 \text{NADH} + 2 \text{CO}_{2} + 2 \text{H}_{2} \end{aligned} $$

$$ \begin{aligned} \text{glucose} + 3 \text{ADP} \rightarrow & \text{butyrate} + 3 \text{ATP} \\ & + 2 \text{NADH} + 2 \text{CO}_{2} + 2 \text{H}_{2} \end{aligned} $$

$$ \begin{aligned} \text{glucose} + 2 \text{ADP} + 2 \text{NADH} \rightarrow & \text{butanol} + 2 \text{ATP} \\ & + 2 \text{NAD}^{+} + 2 \text{CO}_{2} + 2 \text{H}_{2} \end{aligned} $$

$$ \begin{aligned} \text{glucose} + 2 \text{ADP} + 2 \text{NAD}^{+} \rightarrow & \text{acetone} + 3 \text{ATP} \\ & + 2 \text{NADH} + 3 \text{CO}_{2} + 2 \text{H}_{2} \end{aligned} $$

For example, from the last stoichiometric equation, each acetone molecule produced is associated to the formation of 3 CO₂ molecules. Therefore, the CO₂ production was calculated on the basis of product formation, as the sum of [acetate], 2*[butyrate], [ethanol], 2*[butanol], and 3*[acetone] concentrations.

CO₂ production was calculated as the sum of [acetate], 2*[butyrate], [ethanol], 2*[butanol], and 3*[acetone] concentrations.

## Results

### General growth and metabolic features of Clostridium acetobutylicum ATCC 824 cultivated with glucose or cellobiose

Kinetics of growth and metabolism of C. acetobutylicum with glucose or cellobiose as the carbon and energy source were performed with precultures carried out with either glucose or cellobiose (Fig. 1). Pre cultures and cultures performed with the same carbon source were compared with precultures and cultures performed with the switched substrates. The pH was set at 5.5 in order to favor acidogenesis, resulting in butyric, acetic and lactic acids, as the main products whatever the substrate used.

The carbon balances for these experiments were comprised between 90 and 98%, indicating that most of the carbon substrates and products have been taken
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**Fig. 1** Kinetics of growth and metabolism of glucose or cellobiose as the substrate by *C. acetobutylicum*. a Glucose as the substrate (136 mM) and glucose-pregrown cells as the inoculum. b Cellobiose as the substrate (67 mM) and glucose-pregrown cells as the inoculum. The dashed line represents the beginning of the lag phase after glucose was exhausted. c Cellobiose as the substrate (67 mM) and cellobiose-pregrown cells as the inoculum. d–f are the respective product kinetics of (a–c). Each graph is representative of at least three independent experiments (n = 3)
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<figure><img src="image_10.png" /></figure>

**Fig. 1** Kinetics of growth and metabolism of glucose or cellobiose as the substrate by *C. acetobutylicum*. a Glucose as the substrate (136 mM) and glucose-pregrown cells as the inoculum. b Cellobiose as the substrate (67 mM) and glucose-pregrown cells as the inoculum. The dashed line represents the beginning of the lag phase after glucose was exhausted. c Cellobiose as the substrate (67 mM) and cellobiose-pregrown cells as the inoculum. d–f are the respective product kinetics of (a–c). Each graph is representative of at least three independent experiments (n = 3)
into account. Therefore, resulting kinetics can be fully interpreted.

Figure 1a shows the growth kinetic of *C. acetobutylicum* on glucose inoculated with a preculture grown with the same substrate. Taking into account the residual substrate coming from the preculture, the initial glucose concentration was 136 mM (25 g/L). After approximately 13 h of culture, all the glucose was completely consumed and no lag phase was observed since the culture rapidly entered in exponential phase. In these experimental conditions, a maximal specific growth rate (μmax) of 0.26 h⁻¹ was reached after 3 h of culture whereas a maximal cell concentration of 2.36 g/L (dry cell weight) was obtained when glucose was entirely depleted. Glucose fermentation by *C. acetobutylicum* was accompanied by the accumulation of acidic products (Fig. 1d) such as butyric, acetic and lactic acid up to 74, 40 and 9 mM respectively, whereas butanol was a minor product with a production of only 8 mM.

Figure 1b and c report the growth kinetics of *C. acetobutylicum* with cellobiose (67 and 61 mM respectively) as the carbon and energy source, and with glucose or cellobiose as the respective preculture substrates.

When inoculated with a preculture cultivated with glucose, an immediate and short growth phase occurred during the 3 first hours of culture (Fig. 1b). During this phase, no cellobiose was used, whereas glucose coming from preculture (7.2 mM) was completely exhausted. Then, the consumption of cellobiose by *C. acetobutylicum* occurred until exhaustion and was associated with an active cell growth.

Interestingly, as indicated by the dashed line (Fig. 1b), the use of cellobiose was accompanied by a short lag phase during approximately 2 h after glucose exhaustion. Then the growth of *C. acetobutylicum* started and reached a μmax of 0.21 h⁻¹ and maximal biomass concentration of 3.7 g/L after 12 h of culture.

As observed during fermentation kinetics performed with glucose in Fig. 1a, the product pattern obtained with cellobiose as the carbon source and with glucose as the preculture substrate (Fig. 1e) is similar since butyric (76 mM), acetic (35 mM) and lactic acids (5.5 mM) were the main products at the end of the culture whereas butanol accumulated at a low concentration.

Figure 1c displays the growth kinetic of *C. acetobutylicum* with cellobiose as the substrate (61 mM) after an inoculation with a preculture grown with cellobiose. In less than 11 h, all the cellobiose was fully consumed by *C. acetobutylicum*, resulting in an active cell growth without apparent lag phase. In these conditions, a μmax of 0.23 h⁻¹ and a maximal cell concentration of 3.4 g/L after 8 h of culture were observed. Similarly to the previous kinetics (Fig. 1a, b), the product pattern obtained

with cellobiose as the carbon source in the fermentation broth and in precultures (Fig. 1f) are similar (Fig. 1d, e), indicating that neither the carbon source in precultures, nor the carbon source in cultures have an influence on the carbon distribution during fermentation.

However, when pre-cultures were grown with glucose as the substrate (Fig. 1b), an adaptation stage of *C. acetobutylicum* seems to be important before to be able to use cellobiose as the main carbon and energy source.

## Fermentation of glucose and cellobiose mixtures by C. acetobutylicum

In order to get a better understanding of the lag phase observed during the cellobiose culture inoculated with pre-cultures performed with glucose (Fig. 1b), experiments using mixtures of glucose and cellobiose were performed with either glucose and cellobiose pre-cultures as the inoculum. These experiments were carried out in similar conditions to the previous ones. Glucose and cellobiose were entirely consumed and typical acidogenesis product profiles were observed in batch fermentations. In these experimental conditions, the carbon recoveries were a bit lower than previously and comprised between 85 and 87%.

Figure 2a reports sequential consumption of glucose and cellobiose as well as growth kinetics observed when pre-cultures were performed with glucose. In this experiment, the culture medium contained a glucose and cellobiose mixture with respective initial concentrations of 47 and 43.3 mM.

During this fermentation process, no lag phase was observed and glucose was the first substrate to be consumed by cells. In these experimental conditions, a μmax of 0.28 h⁻¹ was measured; resulting in a cell concentration of 2 g/L. Cellobiose began to be consumed by *C. acetobutylicum* in a second time and only when glucose was completely exhausted after 4.5 h of culture. During the first 2 h of cellobiose utilization, a temporary growth cessation was observed. Then, cellobiose was fully consumed, in association with a second growth, resulting in a maximal cell concentration of 3.2 g/L, and a μmax of 0.22 h⁻¹. However and as initially observed in Fig. 1b, a lag phase was observed when glucose was exhausted suggesting that an adaptation phase may be required before cellobiose utilization by *C. acetobutylicum*.

Figure 2b reports sequential consumption of glucose and cellobiose as well as growth kinetics observed when the fermentation process was inoculated with cellobiose pregrown cells. The respective initial concentrations of glucose and cellobiose in the bioreactor were 58.7 and 38.8 mM. Interestingly, a lag phase of 4 h without any glucose or cellobiose consumption was observed. Then cells started to use glucose until exhaustion. Then cellobiose
into account. Therefore, resulting kinetics can be fully interpreted.

Figure 1a shows the growth kinetic of *C. acetobutylicum* on glucose inoculated with a preculture grown with the same substrate. Taking into account the residual substrate coming from the preculture, the initial glucose concentration was 136 mM (25 g/L). After approximately 13 h of culture, all the glucose was completely consumed and no lag phase was observed since the culture rapidly entered in exponential phase. In these experimental conditions, a maximal specific growth rate (μmax) of 0.26 h⁻¹ was reached after 3 h of culture whereas a maximal cell concentration of 2.36 g/L (dry cell weight) was obtained when glucose was entirely depleted. Glucose fermentation by *C. acetobutylicum* was accompanied by the accumulation of acidic products (Fig. 1d) such as butyric, acetic and lactic acid up to 74, 40 and 9 mM respectively, whereas butanol was a minor product with a production of only 8 mM.

Figure 1b and c report the growth kinetics of *C. acetobutylicum* with cellobiose (67 and 61 mM respectively) as the carbon and energy source, and with glucose or cellobiose as the respective preculture substrates.

When inoculated with a preculture cultivated with glucose, an immediate and short growth phase occurred during the 3 first hours of culture (Fig. 1b). During this phase, no cellobiose was used, whereas glucose coming from preculture (7.2 mM) was completely exhausted. Then, the consumption of cellobiose by *C. acetobutylicum* occurred until exhaustion and was associated with an active cell growth.

Interestingly, as indicated by the dashed line (Fig. 1b), the use of cellobiose was accompanied by a short lag phase during approximately 2 h after glucose exhaustion. Then the growth of *C. acetobutylicum* started and reached a μmax of 0.21 h⁻¹ and maximal biomass concentration of 3.7 g/L after 12 h of culture.

As observed during fermentation kinetics performed with glucose in Fig. 1a, the product pattern obtained with cellobiose as the carbon source and with glucose as the preculture substrate (Fig. 1e) is similar since butyric (76 mM), acetic (35 mM) and lactic acids (5.5 mM) were the main products at the end of the culture whereas butanol accumulated at a low concentration.

Figure 1c displays the growth kinetic of *C. acetobutylicum* with cellobiose as the substrate (61 mM) after an inoculation with a preculture grown with cellobiose. In less than 11 h, all the cellobiose was fully consumed by *C. acetobutylicum*, resulting in an active cell growth without apparent lag phase. In these conditions, a μmax of 0.23 h⁻¹ and a maximal cell concentration of 3.4 g/L after 8 h of culture were observed. Similarly to the previous kinetics (Fig. 1a, b), the product pattern obtained

with cellobiose as the carbon source in the fermentation broth and in precultures (Fig. 1f) are similar (Fig. 1d, e), indicating that neither the carbon source in precultures, nor the carbon source in cultures have an influence on the carbon distribution during fermentation.

However, when pre-cultures were grown with glucose as the substrate (Fig. 1b), an adaptation stage of *C. acetobutylicum* seems to be important before to be able to use cellobiose as the main carbon and energy source.

## Fermentation of glucose and cellobiose mixtures by C. acetobutylicum

In order to get a better understanding of the lag phase observed during the cellobiose culture inoculated with pre-cultures performed with glucose (Fig. 1b), experiments using mixtures of glucose and cellobiose were performed with either glucose and cellobiose pre-cultures as the inoculum. These experiments were carried out in similar conditions to the previous ones. Glucose and cellobiose were entirely consumed and typical acidogenesis product profiles were observed in batch fermentations. In these experimental conditions, the carbon recoveries were a bit lower than previously and comprised between 85 and 87%.

Figure 2a reports sequential consumption of glucose and cellobiose as well as growth kinetics observed when pre-cultures were performed with glucose. In this experiment, the culture medium contained a glucose and cellobiose mixture with respective initial concentrations of 47 and 43.3 mM.

During this fermentation process, no lag phase was observed and glucose was the first substrate to be consumed by cells. In these experimental conditions, a μmax of 0.28 h⁻¹ was measured; resulting in a cell concentration of 2 g/L. Cellobiose began to be consumed by *C. acetobutylicum* in a second time and only when glucose was completely exhausted after 4.5 h of culture. During the first 2 h of cellobiose utilization, a temporary growth cessation was observed. Then, cellobiose was fully consumed, in association with a second growth, resulting in a maximal cell concentration of 3.2 g/L, and a μmax of 0.22 h⁻¹. However and as initially observed in Fig. 1b, a lag phase was observed when glucose was exhausted suggesting that an adaptation phase may be required before cellobiose utilization by *C. acetobutylicum*.

Figure 2b reports sequential consumption of glucose and cellobiose as well as growth kinetics observed when the fermentation process was inoculated with cellobiose pregrown cells. The respective initial concentrations of glucose and cellobiose in the bioreactor were 58.7 and 38.8 mM. Interestingly, a lag phase of 4 h without any glucose or cellobiose consumption was observed. Then cells started to use glucose until exhaustion. Then cellobiose
<figure><img src="image_11.png" /><figcaption>**Fig. 2** Kinetics of growth and metabolism of d-glucose/cellobiose mixtures by *C. acetobutylicum*. a glucose-pregrown cells as the inoculum, initial glucose or cellobiose concentrations were 46.97 and 43.30 mM respectively. b cellobiose-pregrown cells as the inoculum, initial glucose or cellobiose concentrations were 58.7.97 and 38.8 mM respectively. c, d are the respective product kinetics of (a, b). Each graph is representative of at least three independent experiments (n = 3)</figcaption></figure>

utilization started about 2 h after glucose was entirely depleted. However, the maximal growth rate was barely slower with cellobiose (μmax of 0.22 h⁻¹), compared to glucose (0.27 h⁻¹). Product patterns (Fig. 2c, d) obtained with mixtures of glucose and cellobiose were similar to those previously observed in Fig. 1e–g whatever the culture conditions.

In this study, results strongly suggest that glucose is definitely the preferred substrate, since it is always used in first place, even if pre-cultures were performed with cellobiose are exposed to a mixture of glucose and cellobiose. However, glucose consumption does not start immediately a lag phase is always observed when carbon substrates in pre cultures and in cultures are different, suggesting that a physiological adaptation may occur, resulting in a growth cessation. In fact, glucose and

cellobiose are able to influence the consumption of each other by *C. acetobutylicum*.

## Discussion

In this study, fermentations of glucose and/or cellobiose by *C. acetobutylicum* were performed in batch mode. Mono-substrate cultures showed that *C. acetobutylicum* is able to grow with cellobiose as efficiently as with glucose.

With glucose as the substrate, kinetics of growth, substrate consumption and products formation were in good agreement with previous studies (Jones and Woods 1986; Girbal et al. 1995). Interestingly, no major difference was observed when cellobiose was the sole carbon substrate: specific growth rates measured with both substrate were almost similar, and no significant change in fermentation
<figure><img src="image_11.png" /><figcaption>**Fig. 2** Kinetics of growth and metabolism of d-glucose/cellobiose mixtures by *C. acetobutylicum*. a glucose-pregrown cells as the inoculum, initial glucose or cellobiose concentrations were 46.97 and 43.30 mM respectively. b cellobiose-pregrown cells as the inoculum, initial glucose or cellobiose concentrations were 58.7.97 and 38.8 mM respectively. c, d are the respective product kinetics of (a, b). Each graph is representative of at least three independent experiments (n = 3)</figcaption></figure>

utilization started about 2 h after glucose was entirely depleted. However, the maximal growth rate was barely slower with cellobiose (μmax of 0.22 h⁻¹), compared to glucose (0.27 h⁻¹). Product patterns (Fig. 2c, d) obtained with mixtures of glucose and cellobiose were similar to those previously observed in Fig. 1e–g whatever the culture conditions.

In this study, results strongly suggest that glucose is definitely the preferred substrate, since it is always used in first place, even if pre-cultures were performed with cellobiose are exposed to a mixture of glucose and cellobiose. However, glucose consumption does not start immediately a lag phase is always observed when carbon substrates in pre cultures and in cultures are different, suggesting that a physiological adaptation may occur, resulting in a growth cessation. In fact, glucose and

cellobiose are able to influence the consumption of each other by *C. acetobutylicum*.

## Discussion

In this study, fermentations of glucose and/or cellobiose by *C. acetobutylicum* were performed in batch mode. Mono-substrate cultures showed that *C. acetobutylicum* is able to grow with cellobiose as efficiently as with glucose.

With glucose as the substrate, kinetics of growth, substrate consumption and products formation were in good agreement with previous studies (Jones and Woods 1986; Girbal et al. 1995). Interestingly, no major difference was observed when cellobiose was the sole carbon substrate: specific growth rates measured with both substrate were almost similar, and no significant change in fermentation
patterns (acidogenic phase) was observed. In fact, little was known about the cellobiose metabolism by *C. acetobutylicum* and such a study was never deeply investigated so far. Indeed, only studies dedicated to enzymatic activities expressed by *C. acetobutylicum* during the fermentation of cellobiose were reported in the literature (Allcock and Woods 1981; Mes-Hartree and Saddler 1982; Lee et al. 1985; López-Contreras et al. 2000).

However, and contrary to *C. thermocellum* (Weimer and Zeikus 1977), cultures grown on glucose/cellobiose mixtures demonstrated that *C. acetobutylicum* was unable to co-utilize both substrates at the same time and glucose was consistently the preferred carbon source. In the present work, growth cessations were always observed after glucose exhaustion and before cellobiose utilization, resulting in a second growth phase.

This phenomenon was reported for the first time by (Monod 1942), and was called "diauxie". It is nowadays better known under the generic term of catabolic repression since some substrates have the ability to repress the expression of genes encoding catabolic enzymes and/or protein transporters, as already described in bacterial species (Magasanik 1961; Brückner and Titgemeyer 2002; Deutscher et al. 2006; Deutscher 2008; Görke and Stülke 2008) but these mechanisms are not the same for each strain and their complete characterization is still being studied.

Besides, the effect of carbon source on cell growth and fermentation products by *C. acetobutylicum* has been previously studied for different mixtures (Vasconcelos et al. 1994). In continuous cultures, *C. acetobutylicum* grown with glucose at neutral pH, produced only acids. In the same conditions, but with substrate mixtures of glucose and glycerol, alcohols were produced in higher yields but no catabolic repression was observed with these carbon sources. However, the repression of lactose transport system by glucose (Yu et al. 2007) as well as diauxic growths of *C. acetobutylicum* cultivated on mixtures of xylose and glucose (Jiang et al. 2014) have been reported, demonstrating that these mechanisms are of a great importance and participate to the control of carbon catabolic fluxes of cells depending on the nature of carbon sources.

In this study, similar mechanisms could be involved according to our results. Indeed, two putative PTSs (phosphotransferase systems) operon genes, strongly induced by cellobiose with functions connected to cellobiose metabolism, were reported by Servinsky et al. (2010), suggesting that *C. acetobutylicum* displays multiple mechanisms to import, phosphorylate and hydrolyse B-glucosides for entry into glycolyse. Besides, the similarity of the *C. acetobutylicum* PTS to PTSs found in other well characterized low GC gram positive bacteria, has led

to the suggestion that they may also play a role in carbon catabolite repression (Behrens et al. 2001; Saier and Reizer 1992; Singh et al. 2008; Tangney et al. 2003). This hypothesis was reinforced by the fact that a recent proteomic study revealed that such PTS operons, especially those involved in cellobiose uptake in *C. acetobutylicum* was shown to be down regulated in the presence of lignin residues, in mixture with cellobiose (Raut et al. 2016). In this study, our results suggests that an analogous phenomenon may exist that was never reported for *C. acetobutylicum* when cultivated with mixtures of glucose and cellobiose, and therefore is an original contribution to the comprehension of carbohydrate metabolism by *Clostridial* sp.

Besides, diauxic growths observed during the kinetic studies may have a great importance for ABE fermentation processes of pretreated (ligno) cellulosic biomass. From a technological point of view, this observation may be of critical importance if industrial substrates mixes were to be used for hydrogen and/or other chemical precursor productions by *C. acetobutylicum*, since substrate consumption discontinuities could occur and may affect production performances and productivities of processes.

More studies with other complex mixtures of cellulosic derivatives as well as with various operating conditions will help to understand how *C. acetobutylicum* can manage its carbohydrate metabolism in order to perform an efficient ABE production process.

**Abbreviations**

ATCC: American Type Culture Collection; ABE: acetone-butanol-ethanol; PTS: phosphotransferase system; RCM: Reinforced Clostridial Medium.

**Authors' contributions**

All authors have contributed to this research work. All authors read and approved the final manuscript.

**Author details**

¹ LRGP, UMR CNRS 7274, Université de Lorraine, 2 Avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre lès Nancy, France. ² LRGP, UMR CNRS 7274, Université de Lorraine, 1 Rue Grandville, 54000 Nancy, France.

**Acknowledgements**

Felipe Buendia-Kandia was supported by the ICÉEL CARNOT institution. The authors thank to Fabrice Blanchard for his intensive help in the preparation of the bioreactors.

**Competing interests**

The authors declare that they have no competing interests.

**Availability of data and materials**

All data and materials are available on request.

**Consent for publication**

Not applicable.

**Ethics approval and consent to participate**

Not applicable.
patterns (acidogenic phase) was observed. In fact, little was known about the cellobiose metabolism by *C. acetobutylicum* and such a study was never deeply investigated so far. Indeed, only studies dedicated to enzymatic activities expressed by *C. acetobutylicum* during the fermentation of cellobiose were reported in the literature (Allcock and Woods 1981; Mes-Hartree and Saddler 1982; Lee et al. 1985; López-Contreras et al. 2000).

However, and contrary to *C. thermocellum* (Weimer and Zeikus 1977), cultures grown on glucose/cellobiose mixtures demonstrated that *C. acetobutylicum* was unable to co-utilize both substrates at the same time and glucose was consistently the preferred carbon source. In the present work, growth cessations were always observed after glucose exhaustion and before cellobiose utilization, resulting in a second growth phase.

This phenomenon was reported for the first time by (Monod 1942), and was called “diauxie”. It is nowadays better known under the generic term of catabolic repression since some substrates have the ability to repress the expression of genes encoding catabolic enzymes and/or protein transporters, as already described in bacterial species (Magasanik 1961; Brückner and Titgemeyer 2002; Deutscher et al. 2006; Deutscher 2008; Görke and Stülke 2008) but these mechanisms are not the same for each strain and their complete characterization is still being studied.

Besides, the effect of carbon source on cell growth and fermentation products by *C. acetobutylicum* has been previously studied for different mixtures (Vasconcelos et al. 1994). In continuous cultures, *C. acetobutylicum* grown with glucose at neutral pH, produced only acids. In the same conditions, but with substrate mixtures of glucose and glycerol, alcohols were produced in higher yields but no catabolic repression was observed with these carbon sources. However, the repression of lactose transport system by glucose (Yu et al. 2007) as well as diauxic growths of *C. acetobutylicum* cultivated on mixtures of xylose and glucose (Jiang et al. 2014) have been reported, demonstrating that these mechanisms are of a great importance and participate to the control of carbon catabolic fluxes of cells depending on the nature of carbon sources.

In this study, similar mechanisms could be involved according to our results. Indeed, two putative PTSs (phosphotransferase systems) operon genes, strongly induced by cellobiose with functions connected to cellobiose metabolism, were reported by Servinsky et al. (2010), suggesting that *C. acetobutylicum* displays multiple mechanisms to import, phosphorylate and hydrolyse B-glucosides for entry into glycolyse. Besides, the similarity of the *C. acetobutylicum* PTS to PTSs found in other well characterized low GC gram positive bacteria, has led

to the suggestion that they may also play a role in carbon catabolite repression (Behrens et al. 2001; Saier and Reizer 1992; Singh et al. 2008; Tangney et al. 2003). This hypothesis was reinforced by the fact that a recent proteomic study revealed that such PTS operons, especially those involved in cellobiose uptake in *C. acetobutylicum* was shown to be down regulated in the presence of lignin residues, in mixture with cellobiose (Raut et al. 2016). In this study, our results suggests that an analogous phenomenon may exist that was never reported for *C. acetobutylicum* when cultivated with mixtures of glucose and cellobiose, and therefore is an original contribution to the comprehension of carbohydrate metabolism by *Clostridial* sp.

Besides, diauxic growths observed during the kinetic studies may have a great importance for ABE fermentation processes of pretreated (ligno) cellulosic biomass. From a technological point of view, this observation may be of critical importance if industrial substrates mixes were to be used for hydrogen and/or other chemical precursor productions by *C. acetobutylicum*, since substrate consumption discontinuities could occur and may affect production performances and productivities of processes.

More studies with other complex mixtures of cellulosic derivatives as well as with various operating conditions will help to understand how *C. acetobutylicum* can manage its carbohydrate metabolism in order to perform an efficient ABE production process.

**Abbreviations**

ATCC: American Type Culture Collection; ABE: acetone-butanol-ethanol; PTS: phosphotransferase system; RCM: Reinforced Clostridial Medium.

**Authors' contributions**

All authors have contributed to this research work. All authors read and approved the final manuscript.

**Author details**

¹ LRGP, UMR CNRS 7274, Université de Lorraine, 2 Avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre lès Nancy, France. ² LRGP, UMR CNRS 7274, Université de Lorraine, 1 Rue Grandville, 54000 Nancy, France.

**Acknowledgements**

Felipe Buendia-Kandia was supported by the ICÉEL CARNOT institution. The authors thank to Fabrice Blanchard for his intensive help in the preparation of the bioreactors.

**Competing interests**

The authors declare that they have no competing interests.

**Availability of data and materials**

All data and materials are available on request.

**Consent for publication**

Not applicable.

**Ethics approval and consent to participate**

Not applicable.
Funding

Not applicable/no funding was received for this study.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 30 April 2018 Accepted: 12 May 2018

Published online: 22 May 2018

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Ibrahim MF, Abd-Aziz S, Yusoff MEM, Phang LY, Hassan MA (2015) Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel. *Renew Energy* 77:447-455. https://doi.org/10.1016/j.renene.2014.12.047

Jang Y-S, Malaviya A, Cho C, Lee J, Lee SY (2012) Butanol production from renewable biomass by clostridia. *Bioresour Technol* 123:653-663. https://doi.org/10.1016/j.biortech.2012.07.104

Jiang W, Zhao J, Wang Z, Yang S T (2014) Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations. *Bioresour Technol* 163:172-179

Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. *Microbiol Rev* 50:484-524

Jurgens G, Survase S, Berezina O, Sklavounos E, Linnekoski J, Kurkijärvi A, Väkevä M, van Heiningen A, Granström T (2012) Butanol production from lignocellulosics. *Biotechnol Lett* 34:1415-1434. https://doi.org/10.1007/s10529-012-0926-3

Kim BH, Bellows P, Datta R, Zeikus JG (1984) Control of carbon and electron flow in *Clostridium acetobutylicum* fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. *Appl Environ Microbiol* 48:764-770

Kumar P, Barrett DM, Delwiche MJ, Stroeve P (2009) Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. *Ind Eng Chem Res* 48:3713-3729. https://doi.org/10.1021/ie801542g

Lee SF, Forsberg CW, Gibbins LN (1985) Cellulolytic activity of *Clostridium acetobutylicum*. *Appl Environ Microbiol* 50:220-228

Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. *Biotechnol Bioeng* 101:209-228. https://doi.org/10.1002/bit.22003

Li X, Li Z, Zheng J, Shi Z, Li L (2012) Yeast extract promotes phase shift of butanol fermentation by *Clostridium acetobutylicum* ATCC824 using cas-sava as substrate. *Bioresour Technol* 125:43-51. https://doi.org/10.1016/j.biortech.2012.08.056

Lin Y, Tanaka S (2005) Ethanol fermentation from biomass resources: current state and prospects. *Appl Microbiol Biotechnol* 69:627-642. https://doi.org/10.1007/s00253-005-0229-x

López-Contreras AM, Claassen PA, Mooibroek H, De Vos WM (2000) Utilisation of saccharides in extruded domestic organic waste by *Clostridium acetobutylicum* ATCC 824 for production of acetone, butanol and ethanol. *Appl Microbiol Biotechnol* 54:162-167

Magasanik B (1961) Catabolite repression. Cold Spring Harb Symp Quant Biol 26:249-256

McKendry P (2002) Energy production from biomass (part 2): conversion technologies. *Bioresour Technol* 83:47-54

Mes-Hartree M, Saddler JN (1982) Butanol production of *Clostridium acetobutylicum* grown on sugars found in hemicellulose hydrolysates. *Biotechnol Lett* 4:247-252. https://doi.org/10.1007/BF00132395

Mitchell WJ, Albasheri KA, Yazdanian M (1995) Factors affecting utilization of carbohydrates by clostridia. *FEMS Microbiol Rev* 17:317-329

Monod J (1942) Recherches sur la croissance des cultures bactériennes. Human et Cie, Paris

Monot F, Engasser JM, Petitdemange H (1984) Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. *Appl Microbiol Biotechnol* 19:422-426

Morone A, Pandey RA (2014) Lignocellulosic biobutanol production: gridlocks and potential remedies. *Renew Sustain Energy Rev* 37:21-35. https://doi.org/10.1016/j.rser.2014.05.009

Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. *Bioresour Technol* 96:673-686. https://doi.org/10.1016/j.biortech.2004.06.025
Funding

Not applicable/no funding was received for this study.

Publisher's Note

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Received: 30 April 2018 Accepted: 12 May 2018

Published online: 22 May 2018

References

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Ibrahim MF, Abd-Aziz S, Yusoff MEM, Phang LY, Hassan MA (2015) Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel. *Renew Energy* 77:447–455. https://doi.org/10.1016/j.renene.2014.12.047

Jang Y-S, Malaviya A, Cho C, Lee J, Lee SY (2012) Butanol production from renewable biomass by clostridia. *Bioresour Technol* 123:653–663. https://doi.org/10.1016/j.biortech.2012.07.104

Jiang W, Zhao J, Wang Z, Yang S T (2014) Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations. *Bioresour Technol* 163:172–179

Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. *Microbiol Rev* 50:484–524

Jurgens G, Survase S, Berezina O, Sklavounos E, Linnekoski J, Kurkijärvi A, Väkevä M, van Heiningen A, Granström T (2012) Butanol production from lignocellulosics. *Biotechnol Lett* 34:1415–1434. https://doi.org/10.1007/s10529-012-0926-3

Kim BH, Bellows P, Datta R, Zeikus JG (1984) Control of carbon and electron flow in *Clostridium acetobutylicum* fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. *Appl Environ Microbiol* 48:764–770

Kumar P, Barrett DM, Delwiche MJ, Stroeve P (2009) Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. *Ind Eng Chem Res* 48:3713–3729. https://doi.org/10.1021/ie801542g

Lee SF, Forsberg CW, Gibbins LN (1985) Cellulolytic activity of *Clostridium acetobutylicum*. *Appl Environ Microbiol* 50:220–228

Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. *Biotechnol Bioeng* 101:209–228. https://doi.org/10.1002/bit.22003

Li X, Li Z, Zheng J, Shi Z, Li L (2012) Yeast extract promotes phase shift of butanol fermentation by *Clostridium acetobutylicum* ATCC824 using cas-sava as substrate. *Bioresour Technol* 125:43–51. https://doi.org/10.1016/j.biortech.2012.08.056

Lin Y, Tanaka S (2005) Ethanol fermentation from biomass resources: current state and prospects. *Appl Microbiol Biotechnol* 69:627–642. https://doi.org/10.1007/s00253-005-0229-x

López-Contreras AM, Claassen PA, Mooibroek H, De Vos WM (2000) Utilisation of saccharides in extruded domestic organic waste by *Clostridium acetobutylicum* ATCC 824 for production of acetone, butanol and ethanol. *Appl Microbiol Biotechnol* 54:162–167

Magasanik B (1961) Catabolite repression. Cold Spring Harb Symp Quant Biol 26:249–256

McKendry P (2002) Energy production from biomass (part 2): conversion technologies. *Bioresour Technol* 83:47–54

Mes-Hartree M, Saddler JN (1982) Butanol production of *Clostridium acetobutylicum* grown on sugars found in hemicellulose hydrolysates. *Biotechnol Lett* 4:247–252. https://doi.org/10.1007/BF00132395

Mitchell WJ, Albasheri KA, Yazdanian M (1995) Factors affecting utilization of carbohydrates by clostridia. *FEMS Microbiol Rev* 17:317–329

Monod J (1942) Recherches sur la croissance des cultures bactériennes. Human et Cie, Paris

Monot F, Engasser JM, Petitdemange H (1984) Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. *Appl Microbiol Biotechnol* 19:422–426

Morone A, Pandey RA (2014) Lignocellulosic biobutanol production: gridlocks and potential remedies. *Renew Sustain Energy Rev* 37:21–35. https://doi.org/10.1016/j.rser.2014.05.009

Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. *Bioresour Technol* 96:673–686. https://doi.org/10.1016/j.biortech.2004.06.025
Nanda S, Mohammad J, Reddy SN, Kozinski JA, Dalai AK (2014) Pathways of lignocellulosic biomass conversion to renewable fuels. *Biomass Convers Biorefinery* 4:157–191. https://doi.org/10.1007/s13399-013-0097-z

Napoli F, Olivieri G, Russo ME, Marzocchella A, Salatino P (2011) Continuous lactose fermentation by *Clostridium acetobutylicum*—Assessment of acidogenesis kinetics. *Bioresour Technol* 102:1608–1614. https://doi.org/10.1016/j.biortech.2010.09.004

Nogué VS, Karhumaa K (2015) Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals. *Biotechnol Lett* 37:761–772

Ounine K, Petitdemange H, Raval G, Gay R (1985) Regulation and butanol inhibition of *D*-xylose and *D*-glucose uptake in *Clostridium acetobutylicum*. *Appl Environ Microbiol* 49:874–878

Patakova P, Linhova M, Rychtera M, Paulova L, Melzoch K (2013) Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: *Clostridium* metabolic diversity, tools for process mapping and continuous fermentation systems. *Biotechnol Adv* 31:58–67

Pfromm PH, Amanor-Boadu V, Nelson R, Vadlani P, Madl R (2010) Bio-butanol vs. bio-ethanol: a technical and economic assessment for corn and switchgrass fermented by yeast or *Clostridium acetobutylicum*. *Biomass Bioenergy* 34:515–524. https://doi.org/10.1016/j.biombioe.2009.12.017

Qureshi N, Ezeji TC (2008) Butanol, "a superior biofuel" production from agricultural residues (renewable biomass): recent progress in technology. *Biofuels Bioprod Biorefining* 2:319–330. https://doi.org/10.1002/bbb.85

Raganati F, Olivieri G, Götz P, Marzocchella A, Salatino P (2015) Butanol production from hexoses and pentoses by fermentation of *Clostridium acetobutylicum*. *Anaerobe* 34:146–155. https://doi.org/10.1016/j.anaerobe.2015.05.008

Ranjan A, Moholkar VS (2012) Biobutanol: science, engineering, and economics. *Int J Energy Res* 36:277–323. https://doi.org/10.1002/er.1948

Raut MP, Couto N, Pham TK, Evans C, Noirel J, Wright PC (2016) Quantitative proteomic analysis of the influence of lignin on biofuel production by *Clostridium acetobutylicum* ATCC 824. *Biotechnol Biofuels* 9:113

Rinaldi R, Schüth F (2009) Acid hydrolysis of cellulose as the entry point into biorefinery schemes. *ChemSuschem* 2:1096–1107. https://doi.org/10.1002/cssc.200900188

Saier MH Jr, Reizer J (1992) Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. *J Bacteriol* 174:1433

Servinsky MD, Kiel JT, Dupuy NF, Sund CJ (2010) Transcriptional analysis of differential carbohydrate utilization by *Clostridium acetobutylicum*. *Microbiology* 156:3478–3491

Singh KD, Schmalisch MH, Stülke J, Görke B (2008) Carbon catabolite repression in *Bacillus subtilis*: quantitative analysis of repression exerted by different carbon sources. *J Bacteriol* 190:7275–7284

Survase SA, van Heiningen A, Granström T (2011a) Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized *Clostridium acetobutylicum* DSM 792. *Appl Microbiol Biotechnol* 93:2309–2316. https://doi.org/10.1007/s00253-011-3761-x

Survase SA, Sklavounos E, Jurgens G, van Heiningen A, Granström T (2011b) Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce. *Bioresour Technol* 102:10996–11002. https://doi.org/10.1016/j.biortech.2011.09.034

Tangney M, Galinier A, Deutscher J, Mitchell WJ (2003) Analysis of the elements of catabolite repression in *Clostridium acetobutylicum* ATCC 824. J Mol Microbiol Biotechnol 6:6–11

Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET (2012) Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. *Curr Opin Biotechnol* 23:364–381. https://doi.org/10.1016/j.copbio.2011.10.008

Turon V, Trably E, Fouilland E, Steyer J-P (2016) Potentialities of dark fermentation effluents as substrates for microalgae growth: a review. *Process Biochem.* https://doi.org/10.1016/j.procbio.2016.03.018

Vasconcelos I, Girbal L, Soucaille P (1994) Regulation of carbon and electron flow in *Clostridium acetobutylicum* grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. *J Bacteriol* 176:1443–1450

Weimer PJ, Zeikus JG (1977) Fermentation of cellulose and cellobiose by *Clostridium thermocellum* in the absence of Methanobacterium thermoautotrophicum. *Appl Environ Microbiol* 33:289–297

Wettstein SG, Alonso DM, Gürbüz EI, Dumesic JA (2012) A roadmap for conversion of lignocellulosic biomass to chemicals and fuels. *Curr Opin Chem Eng* 1:218–224. https://doi.org/10.1016/j.coche.2012.04.002

Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY (2005) Coordinated development of leading biomass pretreatment technologies. *Bioresour Technol* 96:1959–1966. https://doi.org/10.1016/j.biortech.2005.01.010

Yu Y, Tangney M, Aass HC, Mitchell WJ (2007) Analysis of the mechanism and regulation of lactose transport and metabolism in *Clostridium acetobutylicum* ATCC 824. *Appl Environ Microbiol* 73:1842–1850

Zhao X, Condruz S, Chen J, Jolicoeur M (2016) A quantitative metabolomics study of high sodium response in *Clostridium acetobutylicum* ATCC 824 acetone-butanol-ethanol (ABE) fermentation. *Sci Rep* 6:28307

Submit your manuscript to a SpringerOpen journal and benefit from:

► Convenient online submission

► Rigorous peer review

► Open access: articles freely available online

► High visibility within the field

► Retaining the copyright to your article

Submit your next manuscript at ► springeropen.com
Nanda S, Mohammad J, Reddy SN, Kozinski JA, Dalai AK (2014) Pathways of lignocellulosic biomass conversion to renewable fuels. *Biomass Convers Biorefinery* 4:157–191. https://doi.org/10.1007/s13399-013-0097-z

Napoli F, Olivieri G, Russo ME, Marzocchella A, Salatino P (2011) Continuous lactose fermentation by *Clostridium acetobutylicum*—Assessment of acidogenesis kinetics. *Bioresour Technol* 102:1608–1614. https://doi.org/10.1016/j.biortech.2010.09.004

Nogué VS, Karhumaa K (2015) Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals. *Biotechnol Lett* 37:761–772

Ounine K, Petitdemange H, Raval G, Gay R (1985) Regulation and butanol inhibition of D-xylose and D-glucose uptake in *Clostridium acetobutylicum*. *Appl Environ Microbiol* 49:874–878

Patakova P, Linhova M, Rychtera M, Paulova L, Melzoch K (2013) Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: *Clostridium* metabolic diversity, tools for process mapping and continuous fermentation systems. *Biotechnol Adv* 31:58–67

Pfromm PH, Amanor-Boadu V, Nelson R, Vadlani P, Madl R (2010) Bio-butanol vs. bio-ethanol: a technical and economic assessment for corn and switchgrass fermented by yeast or *Clostridium acetobutylicum*. *Biomass Bioenergy* 34:515–524. https://doi.org/10.1016/j.biombioe.2009.12.017

Qureshi N, Ezeji TC (2008) Butanol, "a superior biofuel" production from agricultural residues (renewable biomass): recent progress in technology. *Biofuels Bioprod Biorefining* 2:319–330. https://doi.org/10.1002/bbb.85

Raganati F, Olivieri G, Götz P, Marzocchella A, Salatino P (2015) Butanol production from hexoses and pentoses by fermentation of *Clostridium acetobutylicum*. *Anaerobe* 34:146–155. https://doi.org/10.1016/j.anaerobe.2015.05.008

Ranjan A, Moholkar VS (2012) Biobutanol: science, engineering, and economics. *Int J Energy Res* 36:277–323. https://doi.org/10.1002/er.1948

Raut MP, Couto N, Pham TK, Evans C, Noirel J, Wright PC (2016) Quantitative proteomic analysis of the influence of lignin on biofuel production by *Clostridium acetobutylicum* ATCC 824. *Biotechnol Biofuels* 9:113

Rinaldi R, Schüth F (2009) Acid hydrolysis of cellulose as the entry point into biorefinery schemes. *ChemSuschem* 2:1096–1107. https://doi.org/10.1002/cssc.200900188

Saier MH Jr, Reizer J (1992) Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. *J Bacteriol* 174:1433

Servinsky MD, Kiel JT, Dupuy NF, Sund CJ (2010) Transcriptional analysis of differential carbohydrate utilization by *Clostridium acetobutylicum*. *Microbiology* 156:3478–3491

Singh KD, Schmalisch MH, Stülke J, Görke B (2008) Carbon catabolite repression in *Bacillus subtilis*: quantitative analysis of repression exerted by different carbon sources. *J Bacteriol* 190:7275–7284

Survase SA, van Heiningen A, Granström T (2011a) Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized *Clostridium acetobutylicum* DSM 792. *Appl Microbiol Biotechnol* 93:2309–2316. https://doi.org/10.1007/s00253-011-3761-x

Survase SA, Sklavounos E, Jurgens G, van Heiningen A, Granström T (2011b) Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce. *Bioresour Technol* 102:10996–11002. https://doi.org/10.1016/j.biortech.2011.09.034

Tangney M, Galinier A, Deutscher J, Mitchell WJ (2003) Analysis of the elements of catabolite repression in *Clostridium acetobutylicum* ATCC 824. J Mol Microbiol Biotechnol 6:6–11

Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET (2012) Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. *Curr Opin Biotechnol* 23:364–381. https://doi.org/10.1016/j.copbio.2011.10.008

Turon V, Trably E, Fouilland E, Steeyer J-P (2016) Potentialities of dark fermentation effluents as substrates for microalgae growth: a review. *Process Biochem.* https://doi.org/10.1016/j.procbio.2016.03.018

Vasconcelos I, Girbal L, Soucaille P (1994) Regulation of carbon and electron flow in *Clostridium acetobutylicum* grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. *J Bacteriol* 176:1443–1450

Weimer PJ, Zeikus JG (1977) Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum. *Appl Environ Microbiol* 33:289–297

Wettstein SG, Alonso DM, Gürbüz EI, Dumesic JA (2012) A roadmap for conversion of lignocellulosic biomass to chemicals and fuels. *Curr Opin Chem Eng* 1:218–224. https://doi.org/10.1016/j.coche.2012.04.002

Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY (2005) Coordinated development of leading biomass pretreatment technologies. *Bioresour Technol* 96:1959–1966. https://doi.org/10.1016/j.biortech.2005.01.010

Yu Y, Tangney M, Aass HC, Mitchell WJ (2007) Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824. *Appl Environ Microbiol* 73:1842–1850

Zhao X, Condruz S, Chen J, Jolicoeur M (2016) A quantitative metabolomics study of high sodium response in *Clostridium acetobutylicum* ATCC 824 acetone-butanol-ethanol (ABE) fermentation. *Sci Rep* 6:28307

Submit your manuscript to a SpringerOpen journal and benefit from:

► Convenient online submission

► Rigorous peer review

► Open access: articles freely available online

► High visibility within the field

► Retaining the copyright to your article

Submit your next manuscript at ► springeropen.com