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Chen, T., Zhang, L., Luo, G., and Yuan, W. (2017). "Butyric acid production by Clostridium tyrobutyricum in sugar mixtures and corncob hydrolysate containing arabinose," BioRes. 12(4), 7931-7942.


The objective of this study was to understand arabinose effects on butyric acid fermentation by Clostridium tyrobutyricum in sugar-supplemented media and biomass hydrolysate. Compared to glucose and xylose, the conversion of arabinose to butyric acid was less efficient with a longer lag phase (4 d) and fermentation period (6 d), and a lower butyric acid yield of 0.26 g/g and 0.15 g/g in 5 g/L and 10 g/L of arabinose media, respectively. However, the addition of 2 g/L or 5 g/L of arabinose to the baseline medium that contained glucose (15 g/L) and xylose (15 g/L) enhanced butyric acid synthesis, resulting in 16.1 g/L and 20.3 g/L of butyric acid, respectively. Further increase in arabinose to 10 g/L in the medium of 10 g/L of glucose and 10 g/L of xylose showed inhibitory effects on C. tyrobutyricum, which suggested that high concentrations of arabinose (> 10 g/L) were not desirable. Dilute-acid pretreated corncobs that contained xylose (19 g/L) and a small amount of arabinose (2 g/L) were a feasible substrate for butyric acid production by C. tyrobutyricum, and resulted in 10.6 g/L butyric acid, which was slightly lower than in the mimic medium (11.3 g/L).

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Butyric Acid Production by Clostridium tyrobutyricum in Sugar Mixtures and Corncob Hydrolysate Containing Arabinose

Tianren Chen,a Ling Zhang,b Guanghong Luo,a and Wenqiao Yuan c,*

The objective of this study was to understand arabinose effects on butyric acid fermentation by Clostridium tyrobutyricum in sugar-supplemented media and biomass hydrolysate. Compared to glucose and xylose, the conversion of arabinose to butyric acid was less efficient with a longer lag phase (4 d) and fermentation period (6 d), and a lower butyric acid yield of 0.26 g/g and 0.15 g/g in 5 g/L and 10 g/L of arabinose media, respectively. However, the addition of 2 g/L or 5 g/L of arabinose to the baseline medium that contained glucose (15 g/L) and xylose (15 g/L) enhanced butyric acid synthesis, resulting in 16.1 g/L and 20.3 g/L of butyric acid, respectively. Further increase in arabinose to 10 g/L in the medium of 10 g/L of glucose and 10 g/L of xylose showed inhibitory effects on C. tyrobutyricum, which suggested that high concentrations of arabinose (> 10 g/L) were not desirable. Dilute-acid pretreated corncobs that contained xylose (19 g/L) and a small amount of arabinose (2 g/L) were a feasible substrate for butyric acid production by C. tyrobutyricum, and resulted in 10.6 g/L butyric acid, which was slightly lower than in the mimic medium (11.3 g/L).

Keywords: Clostridium tyrobutyricum; Butyric acid; Corncob; Xylose; Lignocellulosic biomass; Fermentation; Arabinose

Contact information: a: Gansu Engineering Research Center for Microalgae, Hexi University, Zhangye, Gansu Province, China; b: Chengdu Zhituo Education Consulting LLC, Chengdu, Sichuan Province, China; c: Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695, USA; *Corresponding author:


Plant biomass is an abundant and relatively inexpensive renewable resource that has gained great attention in the last few decades (Mohan et al. 2006; Burhenne et al. 2013). In addition to biofuels, plant biomass can also be used to produce petrochemical products such as butyric acid (Nhien et al. 2016). Among varieties of biomass, the non-edible lignocellulosic biomass is more favorable because it does not compete with human food (Patel et al. 2015). Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin, with small amounts of pectin, protein, extractives, and ash (Jorgensen et al. 2007). In the microstructure of lignocellulose, cellulose and hemicellulose are tangled together to form a carbohydrate polymer, which is cross-linked with lignin (Jorgensen et al. 2007; Toquero and Bolado 2014). The composition of these three constituents can vary from one plant species to another (Kumar et al. 2008). Hardwoods are known to have greater proportional amounts of cellulose, whereas forage biomass contains higher levels of hemicelluloses (Sun and Cheng 2002).

In general, cellulose, hemicellulose, and lignin account for 35% to 50%, 20% to 50%, and 12% to 35%, respectively, of the dry weight of lignocellulosic biomass (Kumar et al. 2008). Cellulose consists of long, crystalline polymers of glucose. After pretreatment and enzymatic hydrolysis of cellulose, glucose can be obtained for biofuel production. Degradation of hemicellulose is the easiest of the three main components of biomass because of its random amorphous structureConverting cellulose and hemicellulose to renewable fuels and chemicals has been extensively studied (Martien and Amador-Noguez 2016).

Some work has been reported to convert cellulose and part of hemicellulose (mainly xylose) to butyric acid by C. tyrobutyricum (Zhu et al. 2002; Zhu and Yang; 2004; Jiang et al. 2009; Mitchell et al. 2009; Luo et al. 2017). Butyric acid is a short-chain fatty acid (C3H7COOH), which has many applications, including use in the production of butanol, ethyl butyrate, and butyl butyrate (Al-Shorgani et al. 2012). It may also be used to treat hemoglobinopathies, cancer, and gastrointestinal diseases (Dwidar et al. 2012). It is also a source material for plastics production, especially cellulose acetate butyrate plastics (Cao et al. 2011; Dwidar et al. 2012). In addition, plasticizers, surfactants, and textile auxiliaries also use butyric acid as an important ingredient (Liu et al. 2013). In the form of esters and salts, butyric acid can be utilized as a fragrance and flavoring agents in foods, beverages, and cosmetics (Dwidar et al.2012; Liu et al. 2013).

Most of the abovementioned work has focused on converting glucose or xylose to butyric acid by various microbes, especially C. tyrobutyricum. As an important component of many agricultural residues, arabinose is also a notable pentose, but less attention has been paid to the fermentation of arabinose. Research exploring arabinose and their effects on fermentation media by C. tyrobutyricum has not been reported to the best of the authors’ knowledge.

In contrast, converting hemicellulose in real biomass, specifically corncobs, to butyric acid has not been reported. Corn is one of the most important plants worldwide. Every part of corn has been used extensively to produce biofuels (mainly ethanol) and bioproducts. Corncobs, which generally contain approximately 45% cellulose, 35% hemicelluloses, and 15% lignin on a dry basis (Sun and Cheng 2002), can be utilized as sustainable feedstock for biofuel and biochemical production because it does not interfere with the food chain of the ecosystem. Many reports are available in the literature dealing with the use of corncobs for the production of animal and poultry hovel (Tsai et al. 2001), energy (Mullen et al. 2010), proteins (Perotti and Molina 1988), carbon adsorbents (Tsai et al. 2001), ethanol (de Carvalho Lima et al. 2002), xylitol (Dominguez et al. 1997; Latif and Rajoka 2001), and biochar (Mullen et al. 2010). However, no report of using corncobs for butyric acid production was found when searching the literature.

Thus, this study had two objectives: to understand the effects arabinose has on butyric acid fermentation by C. tyrobutyricum in either arabinose-alone media or in glucose-xylose mixtures, and to evaluate the performance of corncob hydrolysate (hemicellulose) in butyric acid fermentation by C. tyrobutyricum. The bacteria C. tyrobutyricum is a gram-positive, obligate anaerobe. It is one of the most promising strains for butyric acid production due to its high butyric acid selectivity, yield, and productivity, and great tolerance to high product concentration (Zigova and Sturdik 2000; Liu and Yang 2006; Najafpour 2006; Song et al. 2010).


Bacteria Strain and Growth Media

The C. tyrobutyricum (ATCC 25755) was obtained from American Type Culture Collection (ATCC, Manassas, VA). The synthetic medium (recipe provided by ATCC) with sugar supplements was used for the inoculation and culture experiments. In all of the experiments, D-glucose (CAS 50-99-7) was obtained from Fisher Scientific (Hampton, NH, USA). The D-xylose (CAS 58-86-6) and L-arabinose (CAS 5328-37-0) were obtained from Cascade Analytical Reagents & Biochemicals (Corvallis, OR, USA). They were added to the synthetic medium at varying initial concentrations (Table 1).

Table 1. Experimental Design of Arabinose-supplemented Media Test

In arabinose-alone experiments, media with 5 g/L or 10 g/L of arabinose were tested. The purpose was to understand the effects of arabinose as the sole carbon source in the culture medium on butyric acid fermentation. Glucose- or xylose-alone media with the same sugar concentration were also studied as the controls. In arabinose-supplemented experiments, arabinose of 2 g/L or 5 g/L was added to the glucose and xylose mixture. The control had no arabinose in the medium. The purpose of these experiments was to understand the effect of small amounts of arabinose on glucose/xylose fermentation, but arabinose was not the main carbon source. In sugar mixture experiments, the treatment medium contained 10 g/L each of glucose, xylose, and arabinose. The control contained 15 g/L each of glucose and xylose without arabinose. Thus, the total sugar was 30 g/L for both the control and the treatment. The purpose was to understand the effect of arabinose as a major carbon source in the culture medium along with glucose and xylose.

In biomass experiments, corncobs were hydrolyzed to obtain sugars from hemicellulose. The corncobs were obtained from Kaytee Products Inc. (Chilton, WI) and ground using a Retsch SM2000 rotary cutting mill (Retsch Inc., Newtown, PA) with a 1.0 mm screen. The hydrolysate was prepared by a diluted acid treatment of 50 g of corncobs in 50 mL of 5% sulphuric acid (vol/vol) and autoclaved at 121 ºC for 30 min. The solid and liquid phases were separated by filtration (Fisherbrand™, Coarse cellulose fiber papers remove particles > 25 μm). The hydrolysate was stored at 4 ºC until use. The corncob mimic medium was prepared to match corncob hydrolysates and contained xylose (19 g/L), arabinose (2 g/L), and glucose (2 g/L).

All of the media also contained yeast extract (5 g/L), ammonium sulfate ((NH4)2SO4, 3 g/L), dipotassium phosphate (K2HPO4, 1.5 g/L), magnesium sulfate heptahydrate (MgSO4· 7H2O, 0.6 g/L), and ferrous sulfate heptahydrate (FeSO4· 7H2O, 0.03 g/L), and were sterilized at 121 °C for 25 min before inoculation. All chemicals were purchased from Fisher Scientific.

Fermentation process

All media were adjusted to a pH of 6.4 daily by adding ammonium hydroxide (NH4OH, Fisher Scientific). All experiments were performed in 500-mL Erlenmeyer flasks containing 270 mL of culture medium and 30 mL of bacteria seeds. The flasks were capped with air-lock valves. After inoculation, the bottles were flushed with N2 gas to ensure anaerobic conditions. The culture was maintained at 37 °C on a reciprocating shaker (Fisher Scientific) at 150 rpm with three replicates.


The cell density was analyzed by measuring the optical density (OD) of a cell suspension at the wavelength of 620 nm (OD620) with a spectrophotometer (Model 340, Sequoia-Turner Corp., Santa Clara, CA, USA). A high-performance liquid chromatography (HPLC) system (Agilent Technologies 1200 Series, Agilent Technologies, Santa Clara, CA, USA) was used to analyze the organic compounds, including xylose, glucose, arabinose, lactate, butyrate, and acetate in the fermentation broth. The HPLC system consisted of an auto-sampler, an ion-exclusion rezex organic acid column (Phenomenex, Torrance, CA, USA) in a column oven at 45 °C, a diode array detector, and an evaporative light scattering detector. The mobile phase was 5 mM sulfuric acid (H2SO4, HPLC grade, Fisher Scientific) at a flow rate of 0.6 mL/min.

A statistical analysis was performed to determine the level of significance. Multiple one-way analyses of variance (ANOVA) tests were conducted to evaluate the effect of the initial sugar concentration on butyric acid production. The concentration of the initial sugar was used as the independent variable, while the butyric acid peak concentration, yield, or productivity was used as the dependent variable. All of the results were expressed as the mean ± standard deviation (n ≤ 3). Tukey’s adjustment was applied to the general linear model for the determination level of significance (P < 0.05) among various treatments. The software SAS Version 9.1.3 (SAS Institute Inc., Cary, NC) was used for all statistical analysis testing.


Fermentation in Arabinose-alone Media

When C. tyrobutyricum was cultured in low concentrations of arabinose (5 g/L or 10 g/L), it grew very slowly and had a long lag phase of approximately 4 d, and arabinose was not used until after day 3 (Fig. 1A). In contrast, the lag phase in the glucose media was 1 day, and in the xylose media was 2 d (Luo et al. 2017). It was also apparent that cell growth increased with an increased arabinose concentration, but butyric acid synthesis was approximately the same for 5 g/L and 10 g/L of arabinose (Fig. 1B).

Compared to glucose or xylose media, arabinose conversion to butyric acid was much less efficient (Table 2). The conversion and effects of glucose and xylose on butyric acid production by C. tyrobutyricum was reported in a previous study (Luo et al. 2017). In both 5 g/L and 10 g/L sugar-supplemented media, xylose resulted in the highest butyric acid concentration and yield, and arabinose was the poorest. In terms of butyric acid productivity, glucose was the best sugar because of its much shorter fermentation time (2 d in glucose vs.4 d in xylose and 6 d in arabinose media to reach peak butyric acid concentration), and arabinose was again the poorest. The results suggested that arabinose alone at concentrations higher than 5 g/L in the medium was not suitable for butyric acid fermentation by C. tyrobutyricum. However, when other sugars are present in the culture medium, the bacteria is capable of converting arabinose to butyric acid (see next section).


Fig. 1. Growth and sugar consumption (A) and butyric acid concentration (B) of C. tyrobutyricum in arabinose-alone media

Table 2. Final Butyric Acid Concentration, Yield, and Productivity in Single-sugar Media

#Butyric acid concentration was adjusted by deducting the initial butyric acid coming from the seed inoculum; different letters following the numbers indicate significant differences.

Fermentation in Arabinose-supplemented Media

In this experiment, the mixture of 15 g/L of glucose and 15 g/L of xylose was chosen as the baseline medium because such a mixture resulted in efficient butyric acid production (Luo et al. 2017). When supplemented with arabinose of 2 g/L or 5 g/L, C. tyrobutyricum reached the maximum OD of approximately 1.8 on day 4. This was similar to the baseline medium without arabinose (Fig. 2A), which implied that the addition of small amounts of arabinose (2 g/L or 5 g/L) to the glucose and xylose mixture did not significantly affect bacterial growth (Tukey’s test, p > 0.05).

The butyric acid production was quite different among the three media, especially when the medium was supplemented with 5 g/L arabinose. The butyric acid concentration increased with no significant change (Tukey’s test, p > 0.05) in the fermentation time (Fig. 2B). As shown, the medium with 2 g/L or 5 g/L arabinose produced 16.1 g/L and 20.3 g/L butyric acid, respectively, while the baseline resulted in only 14.5 g/L butyric acid. This increase in butyric acid was possible only when arabinose stimulated or enhanced butyric acid synthesis from glucose and xylose. This phenomenon has not been reported before and is yet to be fully understood. The authors’ previous work showed that mixtures of glucose and xylose all had better results than the single sugar media having the same total sugar concentration (Luo et al. 2017). This suggested that mixing the two sugars could promote bacteria growth and butyric acid generation. It seems a similar mechanism might also have an important function when adding small amounts of arabinose to the glucose and xylose mixture, which could promote butyric acid synthesis by C. tyrobutyricum.


Fig. 2. Growth (A) and butyric acid synthesis (B) of C. tyrobutyricum in arabinose-supplemented media

The sugar consumption in the three media was as expected. Glucose was used first, followed by xylose. Arabinose was not used until glucose and xylose were almost depleted (Fig. 3A). This was consistent with previous studies (Hahn-Hägerdal 2007; Luo et al. 2017). It has been known that the affinity of the hexose transporters for xylose is one to two orders of magnitude lower than the affinity for glucose (Kotter and Ciriacy 1993). Therefore, in sugar mixtures, xylose is usually consumed only after the depletion of hexose sugars.


Fig. 3. Sugar consumptions in 2 g/L (A) and 5 g/L (B) arabinose-supplemented media

Although arabinose and xylose are both pentoses from hemicellulose, it is known that L-arabinose has a different metabolic pathway from D-xylose in bacteria (Fig. 4). L-arabinose and D-xylose are isomerized to L-ribulose and D-xylulose, which are then phosphorylated to L-ribulose-5-phosphate and D-xylulose-5-phosphate, respectively. It takes another step to convert L-ribulose-5-phosphate to D-xylulose-5-phosphate. This extra step may require some extra energy and not occur when glucose or xylose are present.

Fig. 4. The metabolic pathway of xylose and arabinose by bacteria (Hahn-Hagerdal 2007). ATP = adenosine triphosphate.

Fermentation in Glucose-xylose-arabinose Mixtures

A logical idea was to see if higher arabinose concentrations in a glucose/xylose mixture would also support butyric acid synthesis. The C. tyrobutyricum was cultured in the baseline medium with 15 g/L glucose and 15 g/L xylose, and in the treatment the medium contained 10 g/L glucose, 10 g/L xylose, and 10 g/L arabinose. Thus, the total amount of sugars was kept the same in both media. Interestingly, the treatment reached maximum OD similarly to the control (Fig. 5A vs. 3A); however, the OD did not increase after day 5 when glucose and xylose were both exhausted.


Fig. 5. Growth and sugar consumption (A) and butyric acid synthesis (B) of C. tyrobutyricum in glucose/xylose/arabinose mixtures

The sugar consumption curves showed that glucose was exhausted in 4 d and xylose in 5 d, while arabinose was not used at all until after day 6. This again suggested that arabinose at high concentrations (≥10 g/L) was not a desirable sugar for C. tyrobutyricum growth in the presence of glucose or xylose.

The bacteria in the 30 g/L three-sugar mixture medium produced 9.5 g/L butyric acid in 9 d, whereas bacteria in the medium with 15 g/L glucose and 15 g/L xylose produced 14.5 g/L butyric acid in 7 d (Fig. 5B). It was apparent that arabinose at 10 g/L was much less efficient than glucose and xylose in the conversion to butyric acid. The beneficial effects similar to 2 g/L or 5 g/L of arabinose added into the butyric acid synthesis were not observed. Instead, adverse effects were evident. It suggested that arabinose at 10 g/L or higher in a glucose and xylose mixture had inhibitory effects on butyric synthesis by C. tyrobutyricum, similar to in an arabinose-alone media.

Fermentation in Corncob Hydrolysate and Mimic Medium

The main sugar in the corncob hydrolysate was xylose, with small amounts of arabinose and glucose (Table 1). The synthetic mimic medium had the same sugar amounts as the hydrolysate. Compared to the mimic medium, C. tyrobutyricum in the corncob hydrolysate had a longer lag phase (3 d vs. 1 d in the mimic medium). This occurred because the acid-pretreated lignocellulose not only released sugar monomers in the hemicellulose, but it also generated other substances, such as phenolic compounds, furan derivatives, and aliphatic acids (Larsson et al. 2000; Ranatunga et al. 2000; Nilvebrant et al. 2003), which all could adversely affect bacterial growth during the initial stage of cultivation. Cell growth at the end of the cultivation was similar in the two media, which indicated that the bacteria adapted to the inhibitors in approximately 3 d (Fig. 6A). Although the same amount of bacteria inoculum was added to the two culture media, the initial OD value of corncob hydrolysate was higher than that of the mimic medium due to the darker color of the hydrolysate at the beginning.


Fig. 6. Growth (A) and xylose consumption and butyric acid synthesis (B) of C. tyrobutyricum in corncob hydrolysate and its mimic medium

Sugar consumption in the hydrolysate was slower than in the mimic medium (Fig. 6B), which was consistent with cell growth. On day 9, all of the xylose in the mimic medium was exhausted, while 4.6 g/L of xylose still remained in the corncob hydrolysate. The main product of both media was butyric acid. At the end of the cultivation, the concentration of butyric acid in the corncob hydrolyses reached 10.6 g/L, whereas the mimic medium reached 11.3 g/L (Fig. 6B). Although the hydrolysate resulted in slightly lower butyric acid production, its xylose to butyric acid conversion efficiency was actually higher than in the mimic medium if xylose was assumed to be the sole carbon source for butyric acid synthesis. It is hypothesized that some of the non-xylose compounds that were generated during the pretreatment process might have been metabolized by the bacteria and converted to butyric acid. This was in accordance with the growth curve (Fig. 6A) that the bacteria was initially inhibited but caught up in the later phase. These results indicated that the acid-treated corncob hydrolysate was an effective substrate for butyric acid fermentation by C. tyrobutyricum, and no further treatment of the hydrolysate was necessary.


  1. Arabinose alone was not an effective carbon source for butyric acid synthesis by C. tyrobutyricum. Compared to glucose- or xylose-alone with the same concentration, xylose alone resulted in the lowest butyric acid concentration, yield, and productivity.
  2. Arabinose addition at low concentrations (2 g/L or 5 g/L) to glucose and xylose mixtures resulted in enhanced butyric acid production by C. tyrobutyricum.
  3. A high concentration of arabinose (10 g/L) in the glucose and xylose mixture had inhibitory effects on butyric acid synthesis by C. tyrobutyricum.
  4. Acid-pretreated corncob hydrolysate was an effective substrate for butyric acid fermentation by C. tyrobutyricum, and no further treatment of the hydrolysate was necessary.


The authors would like to thank Dr. Donghai Wang at Kansas State University for sharing the HPLC for this work. The research was also supported by the “2017 Microalgae Biotechnology Research and Application Innovation Team Project of Gansu Province, China” and USDA National Institute of Food and Agriculture, Hatch Project NC02613.


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Article submitted: April 20, 2017; Peer review completed: July 22, 2017; Revised version received and accepted: September 2, 2017; Published: September 12, 2017.

DOI: 10.15376/biores.12.4.7931-7942